REGULATION OF PITUITARY FUNCTION BY la,25-DIHYDROXYVITAMIN D3
by
Stanley David Rose
B.A.
Cornell University
(1978)
Submitted to the Department of
Applied Biological Sciences
in Partial Fulfillment of the
Requirements of the
Degree of
DOCTOR OF PHILOSOPHY
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June 1985
(
Massachusetts Institute of Technology 1985
Signature redacted
Signature of Autho r
Desartment of Applied Biological Sciences
May 3, 1985
This doctoral thesis has been examined by a Committ
Applied Biological Sciences as follows:A
'of the Department of
Signature redacted
Dr. A. North
Signature redacted
Chairman
Dr. M.F. Holick
Thesis Supervisor
Signature redacted
Dr. H. Munro
Signature redacted
Dr. R. Franceschi
Signature redacted
Dr. S. Nussbaum
Accepted by
Signature redacted
;.SA,"HUSFTT3
OFTCNNLOWVn R.
MAY 3 11985
Archives
Tannenbaum,
Chairman, Commi-tee on
Graduate Students
REGULATION OF PITUITARY FUNCTION BY la,25-DIHYDROXYVITAMIN D3
by
STANLEY DAVID ROSE
Submitted to the Department of Applied Biological Sciences
on May 3, 1985 in partial fulfillment of the
requirements for the Degree of Doctor of Philosophy in
Neural and Endocrine Regulation
ABSTRACT
A specific pituitary binding protein for la,25-dihydroxyvitamin D3
(1,25-(OH) 2 D 3 ) has recently been identified and characterized in several
species. The physical characteristics of this binding protein indicate that it
is similar, if not identical, to the receptor for 1,25-(OH) 2 D3 present in
rat intestinal cells. This protein has also been detected in several strains of
clonal rat pituitary tumor cells. Although biological responses have been
observed in cultures of these transformed rat pituitary cells following
administration of 1,25-(OH)2D3, no attempt has been made to determine
whether the binding protein present in normal pituitary cells functions as a
true receptor. In addition, there is data which suggests that the responses
detected in the transformed pituitary cells may be artifactual.
.
The presence of a 3.5-3.7 S specific binding protein for 1,25-(OH) 2 D3 in
both cytosolic and nuclear extracts of rat anterior pituitary was confirmed.
The dissociation constant (Kd) for the binding of 1,25-(OH)2D3 to this
protein was determined to be 6.0 x 10-10M, with a Bmax of
approximately 36 fmol/mg protein. The effects of 1,25-(OH) 2 D 3 on various
parameters of the function of non-transformed pituitary cells was examined in
primary cultures of enzymatically dispersed rat anterior pituitary cells. These
cells in culture were demonstrated to retain both viability and functionality
over the time course employed for studies on the effects of 1,25-(OH) D
3
No effects of 1,25-(OH)2D3 were observed on the growth of these cells 2 (in
terms of DNA content, protein content, or gross cellular morphology), regardless
of the time of the test incubation period, or the dose of 1,25-(OH) 2 D3 used.
No sustained significant effects of 1,25-(OH) 2 D 3 were observed on either
secretion or synthesis of prolactin. Both secretion and synthesis of
thyrotropin, however, were stimulated by treatment with 1,25-(OH) 2 D 3. These
observations represent the first demonstration that the specific binding protein
for 1,25-(OH) 2 D3 in rat anterior pituitary functions as a receptor, in the
classical pharmacological sense. In addition, they are consistent with previous
reports in the literature indicative of a biologically significant relationship
between the vitamin D and thyrotropin/thyroid hormone endocrine systems.
Finally, these results suggest that events which occur in tumor cells (in this
case, alterations in prolactin secretion induced by 1,25-(OH) 2 D3 ) may not
always mimic the situation observed in their non-transformed counterparts.
Thesis Supervisor:
Dr. Michael F. Holick, Associate Professor of Nutritional
Biochemistry and Endocrinology
2
ACKNOWLEDGEMENTS
The contributions of the following individuals and insititutions to this thesis
warrant acknowledgement:
Dr. Michael F. Holick, for introducing me to vitamin D, for his constant support
and guidance, for the respect and independence he gave me, and most of all for
the fine example he set for an aspiring scientist;
Dr. Alan North, Dr. Sam Nussbaum, Dr. Rene Franceschi, and Dr. Hamish Munro, for
their guidance and advice as members of my thesis committee;
The NIDA and the NIMH, for granting me the fellowships which enabled me to
pursue my doctorate at M.I.T.;
The NIADDK, for supplying me with the materials required for the
radioimmunoassays I performed;
Dr. Fukase, for kindly taking the time to teach me how to grow cells in culture;
On a more personal level, I especially thank:
Elise, for being with me throughout, and for enhancing my work and my life in
countless ways;
Dad and Betty, Mom and Jack, Diane and her family, and Audrey, for their
constant love and support, and for the lessons they've taught me about life and
how to live it;
Howard, Brian, and Christina, for being the best friends a person could desire;
Maxwell and Nicole, for always being there when I needed them; and
My friends at the Muddy Charles Pub, for the intermittent relief they provided
from an otherwise grueling pace.
'The object of war is to survive it.'
Which struck me as the object of
graduate school... Such comparisons struck me hard in those days.
-
John Irving
-
Lawrence Berra
"It ain't over till it's over."
3
DEDICATION
To Elise, for all she's given me.
5
4
TABLE OF CONTENTS
page
Title Page ...........
1
Abstract ..........................
2
........... 0 0
Acknowledgements
Dedication ........................
...............
.......
0
.......
*
Table of Contents .................
List of Figures ...................
.......
0
.......
0
.......
0
List of Tables ....................
......
0
Introduction ........*0*..
.
Literature Survey
Metabolism of Vitamin D ......
Function of 1,25-(OH) 2 D3 ----Mechanism of Action ..........
Parathyroid Gland as a Target for 1,25-(O)2D3 ..................
la-Hydroxylase ..................... . . . . . . . . . . . .
.
Effects of Anterior Pituitary Hormones on the Renal
Pituitary Gland as a Target for 1,25-(OH)2D3 --------------------
Materials and Methods ................. ..
. ..
..
..
. ..
..
.
Rationale and Specific Aims ......
Drugs and Chemicals ............................................
Animals .........................................................
Dissection of Rat Anterior Pituitary for Use in Binding Studies
Preparation of Cytosolic and Nuclear Extracts of Rat Anterior
Pituitary .....................................................
5
page
Sucrose Density Gradient Sedimentation Analysis
.................
46
Determination of Binding Characteristics ........................
47
Rat Anterior Pituitary Cell Culture ...........................
48
Determination of Cell Number and Viability .....................
50
Cell Photography .............................................. 51
Determination of Cellular Protein Content .......................
51
Determination of Cellular DNA Content ...........................
51
Radioimmunoassay for Rat Prolactin ..............................
52
Iodination of rTSH for Use in Radioimmunoassay ..................
55
Radioimmunoassay for Rat Thyroid Stimulating Hormone
60
Double Antibody Immunoprecipitation of
............
35 s-Methionine
Labelled rTSH ...............................................
Gel Electrophoresis of Immunoprecipitated
Labelled rTSH .....
..
..
....
Double Antibody Immunoprecipitation of
Labelled rPRL .........
62
35 S-Methionine
. . ..
. .
. . ..
..
64
35 S-Methionine
. ... ....... .... ........ .. .. .. . 66
Trichloroacetic Acid Precipitation of Proteins ..................
Sta t ist ics ......................................................
Results ................
*...........*..
67
0000000006 7
..................
*
69
Detection of Specific Binding Protein for 1,25-(OH) 2 D 3 in
Rat Anterior Pituitary .....000...0000000000............
69
Determination of Binding Characteristics ........................
71
Recovery of Viable Cells in Culture .............................
73
.Viability and Functionality of Cells in Culture
76
6
.................
page
Effects of TRH on Secretion of TSH
...
....................
78
Effects of 1,25-(OH)2D3 on the Growth of Rat Anterior
Pituitary Cells in Primary Culture ............................
83
Effects of 1,25-(OH) 2 D3 on Content of rPRL in Cells and
in Media ......................................................
87
Effects of 1,25-(OH)2D3 on Content of rTSH in Cells and
in Media ..
. . . . . . . . . . . . . . . . . . . . . . .
. ..
Effects of 1,25-(OH) 2 D3 on Synthesis of rTSH ....................
98
109
.
Discussion
88
Summary and Conclusions .......................................... 116
116
Conclusions ................
119
Suggestions for Future Research ................................
.
Summary of Results
References
... 121
. ....
................................
.... ..
120
141
Biographical Note...........................
7
LIST OF FIGURES
Number
Title
Page
1.
Structures of the D Vitamins .................................
12
2.
Typical Standard Curve for Rat Prolactin Radioimmunoassay 0...
56
3.
Sephadex G-50 Gel Chromatographic Purification of
Radioiodinated Rat Thyroid Stimulating Hormone
4.
.............
Typical Standard Curve for Rat Thyroid Stimulating Hormone
Radioimmunoassay ...........................................
5.
59
63
Sucrose Density Gradient Sedimentation Analysis of Rat
Anterior Pituitary Cytosolic and Nuclear Extracts
6.
3 H-1,25-(OH)2D3
-.- 00.------
Competitive Binding Assay Employing
70
...--.-------0
3 H-1,25-(OH)
2 D3
,
Incubated with
1,25-(OH) 2 D3, and Rat Anterior Pituitary Cytosol
...........
72
7.
Scatchard Analysis of Binding Data ...........................
74
8.
Photograph of Cells in Culture 1 Hour Subsequent to Seeding
..
75
9.
Basal Secretion of Rat Prolactin from Enzymatically
Dispersed Rat Anterior Pituitary Cells in Primary Culture ..
77
10.
Effects of Increased Media Potassium Concentration on
rPRL Secretion .....
11.
*..................................... 79
Effect of Increased Media Potassium Concentration on rTSH
Secretion .................................................
80
12.
Effects of TRH on Secretion of TSH ..........................
81
13.
Effects of 1,25-(OH)2D3 on Cellular Protein Content
.........
84
14.-
Effects of 1,25-(OH) 2D3 on Cellular DNA Content
..............
85
8
Title
Number
15.
Effects of 1,25-(OH)2D3 on Gross Cellular Morphology .........
16.
Effects of 1,25-(OH)2 D3 on Cellular rPRL Content
(ng/ug protein) ............................................
17.
95
96
Effects of 1,25-(OH)2D3 on Media rTSH Content
(% control) ................................................
24.
94
Effects of 1,25-(OH) 2 D 3 on Media rTSH Content
(ng/ug protein) ............................................
23.
92
Effects of 1,25-(OH)2D3 on Cellular rTSH Content
(% control) .............................................
22.
91
Effects of 1,25-(OH)2D3 on Cellular rTSH Content
(ng/ug protein) ............................................
21.
90
Effects of 1,25-(OH) 2 D3 on Media rPRL Content
(% control) ................................................
20.
89
Effects of 1,25-(OH) 2 D 3 on Media rPRL Content
(ng/ug protein) ............................................
19.
86
Effects of 1,25-(OH)2D3 on Cellular rPRL Content
(% control) ................................................
18.
Page
97
Effects of 48 Hour Treatment with Various Doses of
1,25-(OH)2D3 on Media rTSH Content ........................
99
25.
Effects of 1a,25-(OH) 2 D 3 on Media and Cellular rTSH Content .. 100
26.
Gel Electrophoresis of Immunoprecipitated
3 5 S-Methionine-Labelled
27.
rTSH ...............................
103
Effects of 10,25-(0H) 2D3 on Incorporation of
3 5 S-Methionine
Into Newly Synthesized
Immunoprecipitatable rTSH .................................. 108
9
LIST OF TABLES
1.
Page
Title
Number
Sedimentation Coefficients of Well-Characterized Proteins
Which Specifically Bind 1,25-(OH)2D3
2.
18
Physical Characteristics of Specific Pituitary Binding Sites
Detected for 1,25-(OH)2 D3
3.
26
Physical Characteristics of Specific Binding Sites for
1,25-(OH)2D3 Detected in Clonal Pituitary Tumor Cells
29
4.
Effects of TRH on Secretion of TSH ............................
5.
Effects of 1,25-(OH)2 D3 on Incorporation of
35 S-Methionine
into Newly Synthesized
Immunoprecipitatable rTSH
6.
....
..............
Effects of 1,25-(OH)2D3 on Incorporation of
102
Effects of 1,25-(OH) 2 D3 on Incorporation of
106
35 S-Methionine
into Newly Synthesized TCA-Precipitatable Proteins
10
104
35 S-Methionine
into Newly Synthesized Immunoprecipitatable rPRL
8.
.
Recovery of Counts Added to Gel Following Electrophoresis of
35 S-Methionine-Labelled-rTSH ...........
7.
82
107
LITERATURE SURVEY
Introduction
The history of vitamin D can be traced back to ancient tales of- bony
diseases which may have been due to vitamin D deficiency (1).
During the 20th
century, from the first assignment of a scientific basis to rickets (2,3), to
the eventual demonstration of la,25-dihydroxyvitamin D 3 (1,25-(OH)2D3)
as
the active hormonal form of vitamin D3 (4,5), consistent progress has been
made in elucidating the nature of the vitamin D endocrine system (6-18).
As a
consequence of this work, and due to the fortification of milk with vitamin D,
nutritional rickets and osteomalacia have been virtually eliminated as public
health problems in the United States and other complying countries.
Metabolism of Vitamin D
An important concept in the study of this endocrine system is that there is
not a single vitamin D.
There exists a family of compounds which exhibit
vitamin D activity (see fig.1).
The most important of these are vitamin D 2
and vitamin D3 . Vitamin D2 is produced via ultraviolet irradiation of
ergosterol (pro-D 2 ), a fungal sterol.
Vitamin D3 is produced in skin via
ultraviolet irradiation of 7-dehydrocholesterol (pro-D 3 )(19).
Pre-vitamin
D2 and pre-vitamin D3 are first produced, following photolysis of their
respective 5,7-diene sterol precursors (ergosterol and 7-dehydrocholesterol).
The pre-vitamin Ds then exist in thermal equilibrium with their respective forms
of vi-tamin D (D2 and D3).
When consumed in the diet, both forms of vitamin
D may be absorbed in the intestine (20-22).
11
Being fat soluble, they are most
.N CN
12
C
D
1
14
1
N
5
CN2
A
D,
N
H
CHI
CN2
N 0'
N 0e
0O
D5
Figure 1.
CMN
CMH
Structures of the D vitamins
12
CHZCHS
likely absorbed with other neutral lipids, and thus appear in the lymphatic
system as chylomicrons (23,24).
Although vitamin D is viewed as an essential
dietary factor, the production of vitamin D3 (D3; also known as
cholecalciferol) in the skin (following exposure to ultraviolet radiation)
requires that it be regarded as a pro-hormone rather than as a vitamin.
The majority of vitamin D present in blood is transported bound to a
specific a-globulin (24-27).
Vitamin D is rapidly taken up by the liver
(20,21,23,28, 34), but is not stored specifically in this organ.
It appears
that the major storage site for vitamin D is actually in the fat depots (30).
In the liver, vitamin D is hydroxylated at the 25-C position by specific
microsomal enzymes (29,31-33), yielding 25-hydroxyvitamin D (25-OH-D).
25-hydroxylated form of vitamin D3 (25-OH-D3)
D3 (34,35).
The
is 2-5 times more active than
Despite this increased activity, it is now clear that 25-OH-D3
is inactive at physiological concentrations, and is not the hormonal form of
vitamin D (36-38).
25-OH-D produced in the liver is transported in blood bound
to the same vitamin D-transport protein described above, and is the major
circulating form of vitamin D (39-41).
Although several routes of metabolism have been described for 25-OH-D3,
the path of major interest to this thesis involves the formation of
1,25-(OH) 2 D3.
la-Hydroxylation of 25-OH-D3 occurs primarily in the kidney
(42-44), via the action of a mitochondrial 25-OH-D3-la-hydroxylase (42,49).
The placenta has also been reported to possess la-hydroxylase activity (45-48).
This dihydroxylated form of cholecalciferol is now recognized as the hormonal
13
form of vitamin D3 (37,51-53).
1,25-(OH)2D3 is transported to target
tissues bound to the same vitamin D-transport protein described above.
The renal la-hydroxylase has been studied extensively (54-57).
Parathyroid
honnone (PTH) is the most potent known stimulator of this enzyme (58-62).
Low
plasma phosphorous concentration is also a major signal for its activation
0
(63,64).
The role of calcium as a direct stimulator of this enzyme is unclear,
as decreases in plasma calcium concentration lead to increased secretion of PTH.
0
However, there is some evidence indicating that decreased plasma calcium
concentration may be a direct signal for activation of the renal la-hydroxylase,
independent of PTH (65-67).
I
There is also evidence suggesting a role for growth
hormone (GH) and prolactin (PRL) in the regulation of 25-OH-D3-la-hydroxylase
activity.
The studies examining effects of these hormones will be described in
detail in a separate section below.
Estrogens (68,69) and thyroxine (70,71)
have also been reported to influence the activity of this renal hydroxylase.
*
Finally, 1,25-(OH) 2 D3 appears to exert direct negative feedback effects on
2 5 -OH-D3-la-hydroxylase,
-
in addition to stimulating 24-hydroxylation of
25-OH-D 3 (72).
Side-chain cleavage to form calcitroic acid appears to be the major route
of inactivation of 1,25-(OH)2D3 (73,74).
t
However, additional hydroxylations
also yield less active metabolites of vitamin D 3 (75-78).
All of these
vitamin D metabolites eventually appear in the bile and feces (79).
The
The half life of 1,25-(OH) 2 D3 in plasma has been estimated at between 2 and
6 hours (80).
14
Function of 1,25-(OH)2D3
In a broad sense, the most well-characterized function of 1,25-(OH)
2D3
is to maintain plasma calcium and phosphorous at levels sufficient to support
the physiological processes for which they are required.
Acting alone, and in
conjunction with other hormones, 1,25-(OH) 2 D3 initiates responses at a
variety of target tissues.
The common factor in all of these actions of
1,25-(OH)2D3 is that they lead to a maintainance of calcium and phosphorous
homeostasi s.
1,25-(OH)2D3, via a variety of mechanisms, functions to maintain plasma
calcium and phosphorous at levels that support normal mineralization of bone
(76,81,82).
It is the only known hormone to stimulate active intestinal calcium
absorption (83).
In addition, 1,25-(OH)2D3 stimulates active intestinal
transport of phosphate (76).
As dietary intake of calcium varies considerably,
intestinal absorption may not always provide sufficient amounts of calcium to
support another of its major roles, namely neuromuscular function.
In order to
maintain normal neuromuscular function, calcium may be mobilized from bone,
which acts as a virtual reservoir of this mineral.
This mobilization is
primarily under the control of PTH, working in concert with 1,25-(OH)2D3
(76).
1,25-(OH) 2 D 3 also acts in the distal renal tubule, again in
conjunction with PTH, to cause reabsorption of calcium (76).
The kidney
normally reabsorbs 99% of the filtered load of calcium, but the residual 1% is
under the control of these two hormones.
Vitamin D deficiency may produce a variety of clinical disorders.
young, growing, terrestrial vertebrate, rickets will ensue.
In the
In this disease,
the collagen fibrils fail to mineralize, the bones become soft, and the stress
of weight and muscle operation causes overt structural deformities.
15
Ultimately,
death occurs as a result of impairment of internal organ function by the
collapse of the bones which protect the internal viscera (76,84).
In adults,
when normal bone is constantly being remodeled, osteomalacia will result from
vitamin D deficiency.
In this disease, the entire remodelling process is slowed
down, with the mineralization of new organic matrix elaborated by osteoblasts
being particularly affected (85).
Finally, in the absence of sufficient amounts
of extracellular calcium, neuromuscular function will be affected (76,82).
A
convulsive state of hypocalcemic tetany will be produced, leading to death.
In reviewing the current literature concerning 1,25-(OH) 2 -D3, it has
become clear that there is yet another important function of this seco-sterol.
Although the conception of 1,25-(OH)2-D3 as a calcium homeostatic hormone
has been amply justified, several recent reports have provided evidence
supporting a more basic biological role for this hormone.
These experiments
have examined the effects of 1,25-(OH)2-D3 on the replication and
differentiation of a variety of cells which possess the classical receptor for
this hormone.
The reports indicate that 1,25-(OH)2-D3 has potent effects on
cell replication and differentiation.
These effects have been observed in both
normal and transformed receptor-positive cells, including fibroblasts,
intestinal cells, bone marrow cells, mammary cancer cells, monocytes, melanoma
cells, and ovarian cells (186-196).
In most of these studies, the data
demonstrates an inhibition of cell proliferation, and a concommitant stimulation
of differentiation.
The possibility that 1,25-(OH)2-D3 acts as one of many
circulating factors which influence the maturation of specific cell types is of
major biological significance.
It may also provide an explanation as to the
16
relevance of a specific receptor for 1,25-(OH)2-D3 existing in such a wide
array of cell types.
Mechanism of Action
The only significant work performed to date on the mechanism of action of
1,25-(OH)2D3 has been in relation to the hormone's effects on intestinal
calcium absorption.
Given the structural similarity of vitamin D to steroid
hormones, it has been assumed that 1,25-(OH)2D3 might function in a manner
similar to the mechanism proposed for steroid action (86).
This would involve
association with a cytoplasmic receptor, translocation to the nucleus,
alteration of transcription of mRNA, and subsequent alteration in the synthesis
of specific proteins.
Several proteins have been described which specifically bind
1,25-(OH)2D3 (see table 1).
The 4.1 S protein is the plasma transport
protein for vitamin D and its metabolites, displaying highest affinity for
25-OH-D.
The 6.0 S cytosolic protein has been isolated from a large number of
tissues (41), and appears to be specific for 25-OH-D (87).
Furthermore, this
6.0 S protein is not found in cytosol preparations isolated free of plasma
protein (88).
It now appears that this 6.0 S cytosolic binding protein is an
artifactual product resulting from the interaction of the 4.1 S plasma transport
protein and an unidentified nonspecific cytosolic protein (88).
The 3.7 S
protein isolated from rachitic chick cytosol, and the 3.2 S protein obtained
from rat cytosol, appear to be highly specific for 1,25-(OH)2D3 (87,89,90).
It is currently believed that the 3.7 S protein is the cytosolic receptor
responsible for initiating the effects of 1,25-(OH) 2 D3 on chick intestinal
calcium transport.
The theory is that 1,25-(OH) 2D3, being transported on
17
Source
Sucrose Density Gradient Sedimentation Coefficient
3.7 S
chick intestinal cytosol
rat intestinal cytosol
3.2-3.3 S
rat plasma
4.0-4.1 S
rat intestinal cytosol
5.8-6.0 S
Table 1. Sedimentation coefficients of well-characterized proteins which
specifically bind 1,25-(OH)qDi
18
the 4.1 S plasma protein, is somehow transferred to the villus cell.
There is
currently no evidence that 1,25-(OH)2D3 is actively transported across the
cell membrane.
Once inside the cell, 1,25-(OH) 2 D3 binds to the 3.7 S
cytosolic protein.
This modified receptor then enters the nucleus, interacting
with the nuclear chromatin in an unknown fashion.
The result is transcription
of specific genes that code for the vitamin D-dependent calcium binding protein
(91-96).
Although synthesis of other proteins may also be induced by
1,25-(OH) 2 D3 , this has yet to be demonstrated.
Furthermore, while the
vitamin D-dependent calcium binding protein is induced by 1,25-(OH) 2 D3 (97),
and its presence has been correlated with active intestinal calcium absorption,
its exact role in calcium transport remains unclear (98).
Thus, the effect of 1,25-(OH)2D3 on intestinal calcium absorption
appears to be due, at least in part, to a nuclear mechanism;
The dissociation
constant for 1,25-(OH) 2 D3 and the 3.7 S binding protein is 5 x 10-11
M.
This binding protein is not found in non-target tissues such as smooth
muscle, skeletal muscle, and liver.
However, it has been found in tissues
previously not considered to be targets for 1,25-(OH) 2 D3, such as skin,
parathyroid glands, stomach mucosa, pituitary glands, and mammary glands (99).
Recent evidence suggests that 1,25-(OH)2D3 may also cause increased
intestinal calcium absorption via a mechanism which does not involve de novo
protein synthesis (100,101).
In these reports, the primary effect of the sterol
was thought to be a modification of membrane phospholipids, leading to an
I
increased permeability of the brush border membrane to Ca++.
Subsequent
steps in' the absorption of Ca++ were considered to be secondary to this
event (102,103).
In line with this theory, it has been reported that the
19
administration of a single dose of 1,25-(OH)2D3 to vitamin D-deficient
animals stimulates intestinal calcium absorption prior to the induction of
synthesis of the vitamin D-dependent calcium-binding protein (the synthesis of
which is induced by the genomic action of 1,25-(OH) 2 D3) (101,104,105).
Although others have not observed this temporal discrepancy, it has been
reported that when minimal amounts of vitamin D-dependent calcium-binding
protein are already present in the intestine, 1,25-(OH) 2 D3 can stimulate
calcium absorption prior to the synthesis of additional vitamin D-dependent
calcium-binding protein (106).
Therefore, it appears that 1,25-(0H)2D3 may
exert its effects on intestinal calcium absorption through more than one
molecular mechanism.
Parathyroid Gland as a Target for 1,25-(OH)2D3
The parathyroid gland produces PTH, a powerful stimulator of the renal
25-OH-D3-la-hydroxylase.
In addition, some evidence indicates that the
parathyroid gland may be a target tissue for vitamin D.
1,25-(0H) 2 D3 has
been shown to associate with nuclei of the parathyroid gland in vivo (107,108).
Furthermore, a specific cytosol receptor for 1,25-(OH)2D3 has been
characterized in parathyroid glands from several species (109).
The
parathyroids also contain a calcium-binding protein similar to that induced by
1,25-(OH)2 D3 in the duodenum (110).
Of particular interest is the fact that
1,25-(OH)2D3 has been reported to exert feedback inhibition on PTH
production in vivo (111) and in vitro (112), causing a decreased response of the
parathyroids to low plasma calcium concentrations.
Although some investigators
havebeen unable to observe such an effect, this evidence has been cited as
20
providing a precedent for feedback effects of vitamin D on endocrine organs
which produce regulators of 1,25-(OH)2D3 synthesis (113,114).
Effects of Anterior Pituitary Hormones on the Renal la-Hydroxylase
Controversy exists over whether GH and/or PRL stimulate, or have no effect
on, 25-OH-D3-l-hydroxylase activity.
It appears that if these hormones do
influence circulating levels of 1,25-(OH) 2 D3, their role is limited to
periods of "calcium stress".
would stimulate
Teleologically, it makes sense that these hormones
2 5 -OH-D3-la-hydroxylase,
since pregnancy, lactation, and
growth are associated with increased demands for calcium.
However, in man it
appears that there are either no changes, or only small changes, in plasma
1,25-(OH) 2 D3 levels associated with changes in the levels of GH and PRL.
Spencer and Tobiassen showed that in hypophysectomized rats, GH promoted
the conversion of 25-OH-[ 3 H]-D 3 to 1,25-(OH)2-[ 3 H]-D3 (115,116).
However, GH was ineffective in increasing la-hydroxylase activity in intact rats
(115).
Spanos, et al. (117) reported that GH produced increased levels of
1,25-(OH)2D3 in hypophysectomized rats which had lower 1,25-(OH)2D3
levels than intact controls.
Pahuja and DeLuca (118) confirmed this effect.
However, Kumar, et al. (119) found little or no change in plasma
1,25-(OH) 2 D 3 in man during periods of chronically increased or decreased
plasma GH.
Likewise, Gertner, et al. (120) found that GH administration to
GH-deficient dwarfs produced little or no change in plasma levels of
1,25-(OH)2D3.
In contrast, Eskildsen, et al.
plasma levels of 1,25-(OH) 2 D3 in acromegalics.
(121) reported elevated
Furthermore, this elevation
was normalized following treatment with bromocriptine.
21
In mammals, increased levels of 1,25-(OH)2D3 have been reported during
pregnancy and lactation (122-127), periods associated with increased plasma
levels of PRL.
CB-154, a dopamine agonist used to inhibit PRL secretion, was
successful in blocking the rise in 1,25-(OH) 2D3 during lactation (128).
In
hens, increased levels of 1,25-(OH) 2 D3 have been reported during egg-laying
(129).
Furthermore, this increase in plasma 1,25-(0H)2D3 has been produced
by injecting PRL into non-laying hens (129).
PRL has been reported to stimulate
production of 1,25-(OH) 2 D3 in a renal tubule preparation (130), and
25-OH-D3-la-hydroxylase activity in kidney homogenates was found to be
increased following injection of PRL (131).
In contrast, Matsumoto, et al.
(132) failed to find a stimulatory effect of PRL on 25-OH-D3-la-hydroxylase
activity in vitamin D-deficient rats.
In man, an effect of PRL on
25-OH-D3-la-hydroxylase activity has yet to be clearly demonstrated.
PRL does
not appear to alter plasma levels of 1,25-(OH) 2 D3 in non-pregnant women
(133,134).
In addition, no significant changes in intestinal calcium absorption
have been found in hyperprolactinemic states other than lactation (134).
Finally, while levels of 1,25-(OH)2D3 are increased in lactating women
during the first few weeks post-partum (135), it
is still unclear that PRL is
directly responsible for this effect.
Although most studies on effects of anterior pituitary hormones on renal
la-hydroxylase activity have focused on the effects of GH or PRL, there is also
evidence for effects of both thyroid stimulating hormone (TSH), and the thyroid
hormones whose secretion is stimulated by TSH.
It has recently been reported
that TSH has a direct effect on renal 25-OH-D3 metabolism in vitro.
Using a
perfused rat kidney system, Kano and Jones (197) demonstrated that TSH inhibited
22
synthesis of 1,25-(OH)2D3 from 25-OH-D3.
Along with this inhibition of
renal 25-(OH)-D3-la-hydroxylase activity, these investigators observed a
stimulation of
2 5 -(OH)-D3-24-hydroxylase
activity induced by TSH.
In
addition, the thyroid hormones T 3 and T4 were found to affect renal
25-OH-D 3 metabolism in the same fashion as did TSH.
Thyrotropin releasing
hormone (TRH), on the other hand, had no effect.
Pituitary Gland as a Target for 1,25-(OH)2D3
Prior to 1979, a role for 1,25-(OH)2D3 in the regulation of pituitary
function had not been considered.
However, results from several lines of
experimentation have recently led investigators to speculate that the pituitary
gland might be a target organ for the active form of vitamin D.
This
speculation was based on the following observations:
1.
as discussed above, there have been reports in the literature indicating
that the anterior pituitary hormones GH, PRL, and TSH may affect the
production of 1,25-(OH)2D3;
2.
other steroids (e.g., cortisol) whose synthesis is regulated by
pituitary hormones have been found to have receptors in the pituitary,
and exert feedback effects on the pituitary cells which synthesize these
hormones (136); and
3.
it has already been reported that 1,25-(OH)2D3 may be involved in
a feedback system to regulate the production of PTH by the parathyroid
gland (111,112).
23
The first report of a specific pituitary binding protein ("receptor") for
1,25-(OH)2D3 came from Stumpf, et al. (107).
Using autoradiographic
techniques, these investigators found that when 1,25-(OH) 2 -[3H]-D
3
was
injected into vitamin D-deficient rats, the label subsequently appeared in the
nuclei of certain anterior pituitary cells (107,138).
With combined
autoradiography and immunohistochemistry, this group later demonstrated that
radioactivity appears in the nuclei of pituitary thyrotropes following injection
of 1,25-(OH)2-[3H]-D 3 in the normal rat (140).
Furthermore, there was no
label apparent in pituitary cells stained by anti-PRL, anti-GH, or anti- LH.
The presence of a specific binding protein for 1,25-(OH)2D3 in normal
rat pituitary was subsequently confirmed by Haussler, et al. (113).
This group
characterized a cytosolic binding protein for 1,25-(OH) 2 D3 via sucrose
density gradient sedimentation, saturation analysis, and DNA-cellulose
chromatography.
Non-pregnant female rats were used in this study.
investigators looked for specific binding of 1,25-(OH) 2 -[3H]-D
3
The
in
pituitary sections, dispersed anterior pituitary cells, whole pituitary cytosol,
and kidney cytosol.
DNA-cellulose chromatography revealed a pituitary cytosolic
binding protein which co-eluted with the kidney cytosol receptor.
However, this
protein represented only a small amount of the bound ligand which eluted from
the column.
Subsequent analysis of whole pituitary cytosol by sucrose density
gradient sedimentation revealed a minor portion of label which migrated at 3.3
S, with the "vast majority" binding to a 5.8 S macromolecule.
This 5.8 S
protein did not absorb to DNA-cellulose, and the authors concluded that it was
an artifactual complex of the serum vitamin D-binding protein with an
intracellular protein which is present in all nucleated cells.
24
This 5.8 S
species had a higher affinity for 25-OH-D3 than for 1,25-(OH)2D3.
When
pituitary quarters were assayed in a similar fashion, there was a more selective
association of 1,25-(OH)2D3 with the 3.3 S species.
still present in sizeable amounts.
The 5.8 S protein was
It appeared that the 3.3 S protein was
specific for 1,25-(OH)2 D3, while 25-OH-D3 bound exclusively to the 5.8 S
compound.
Finally, when dispersed anterior pituitary cells were examined,
1,25-(OH)2D3 was found to bind uniquely to the 3.3 S binding protein.
The
5.8 S species was still present, but not in high enough concentration to compete
with the 3.3 S protein for binding of 1,25-(OH)2D3.
The dispersed cell preparation was subjected to saturation analysis.
Saturation with 1,25-(OH) 2 D3 occurred at approximately 1 nM, with a Kd of
5x10-10M.
The concentration of the receptor in cytosol was approximately
4 fmole/ mg protein.
No attempt was made to study the functional sequellae of
binding of 1,25-(OH) 2D3 to the 3.3 S binding protein.
Although specific
binding was demonstrated, and the physical characteristics of the binding
protein determined, no biological response was shown.
This binding protein,
therefore, could not yet be characterized unequivocably as a receptor, in the
classical pharmacological sense.
Similar specific binding sites for 1,25-(OH)2D3 in pituitary
preparations have now been demonstrated in several other species.
The physical
characteristics of these sites are all similar, and are described in table 2.
Gelbard, et al. (142,143) have described specific binding of 1,25-(OH)2D3 in
crude nuclear and cytosolic fractions of bovine pituitary homogenates.
al. .(144)
Pike, et
injected tritiated 1,25-(OH)203 into vitamin D-deficient chickens,
sacrificed them several hours later, and found label in the pituitary.
25
This
Animal
Preparation
Rat
cytosol
5
Calf
nuclear fr.
Chick
Eel
!1 a Bb
Specc
SDGd
DNAe
Reference
4
1,25>25
3.3
+
113
1
104
1,25>25
ND
ND
142
cytosol
ND
ND
ND
3.3
+
144
cytosol
1
76
1,25>25
3.7
+
145
Table 2. Physical characteristics of specific pituitary binding sites detected
for 1,25-(OH)D
a. Dissociation constant (multiply values by 10-10 M).
b. Number of binding sites (units = fmol/mg protein).
c. Specificity: rank order affinity of vitamin D-metabolites for binding
protein (1,25 = 1,25-(OH)2D3; 25 = 25-OH-D3).
d. Sucrose density gradient sedimentation coefficient (units = S).
e. Affinity for DNA-cellulose (+ if hormone-binding protein complex binds to
DNA-cellulose column under low salt conditions and is eluted under high salt
conditions).
ND. Not done.
26
group also prepared rachitic chick pituitary cytosol and directly labelled it
with 10-9M 1,25-(OH)2 -[3 H1-D3-
DNA-cellulose chromatography and
sucrose density gradient sedimentation revealed a sterol-protein complex with
the same characteristics as that obtained from chick intestinal cytosol, and
similar to the complex found in normal rat pituitary cytosol.
Freake, et al.
(145) have recently described a pituitary cytosolic binding protein for
1,25-(OH)2D3 in the common eel (A. arguilla).
The characteristics of this
protein are similar to those already described in other species.
At this point
in time, no biological response has been demonstrated as a consequence of
1,25-(OH)2D3 binding to the specific pituitary protein(s) described above.
Several groups have attempted to study the interaction between
1,25-(OH)2D3 and the pituitary using clonal cell lines obtained from either
rat or mouse pituitary tumors.
Cultured cell lines provide an easily
manipulated homogeneous cell population in which one may identify and
characterize the components of an endocrine response.
However, many of the
studies performed to date have used different strains of cells and/or different
growth conditions.
The inconsistencies in cell type, media composition,
duration of cell growth, and experimental design (doses of drugs administered,
time points for examination, method of assay, method of preparation of cytosol
and/or nuclear fractions) make contradictory results difficult to objectively
evaluate.
Furthermore, the cultured pituitary tumor cells differ from normal
pituitary cells in several respects.
Some investigators have found that
concentrations of 1,25-(OH)2 D3 required for "receptor" occupancy are
significantly higher than the in vitro Kd.
This may be due to binding of
1,25-(OH) 2 D3 to media proteins, but this has not been confirmed.
27
More
importantly, tumor cells in culture show greater fluctuations in phenotypic
expression than commonly seen in non-transformed cells.
This is a key point to
be kept in mind as the results from the following studies are reviewed.
In all
of these studies, if any biological response was examined, it was synthesis
and/or secretion of PRL and/or GH.
These parameters were no doubt chosen
because of the reported effects of these hormones on the renal
25-(OH)-D-la-hydroxylase.
In addition, of the commercially available pituitary
tumor cell lines, identification of a specific 1,25-(OH)2D3 binding protein
has been reported only in GH cells (which secrete only PRL and GH).
The
putative receptor was not found in a cell line (AtT20) which secretes
metabolites of pro-opiomelanocorticotropin (185).
No cell line secreting TSH is
currently commercially available, and no attempts have been made to establish a
clonal TSH-secreting cell line for the purposes of examining effects of
1,25-(OH)2D3.
In spite of their drawbacks, the tumor cell studies are of interest because
several of the investigators have attempted to determine the biological
response(s) which might result from the interaction of 1,25-(OH)2D3 with its
putative receptor.
Since the physical characteristics of this protein appear to
be identical to those reported for the specific binding protein detected in
normal pituitary (see table 3), one might speculate that receptor activation
would produce similar effects in both GH cells and normal anterior pituitary.
However, such a speculation is risky since the presence of the specific binding
protein in these tumor cells may be due to abnormal phenotypic expression of the
genome.
If this binding protein is not normally expressed in pituitary
somatotrophs or lactotrophs, then the responses initiated by 1,25-(OH) D3
2
28
Cell
Preparation
GH 4
cytosol
1-2
(8-10,000)
1,25>25
GH 3
cytosol
2.9
55,(8500)
1,25>25
AtT20
DNAe
f
Ref
3.7
ND
114
3.3
+
185
cytosol
NO SPECIFIC BINDING PROTEIN DETECTED
185
GH 3
cytosol
0.47
(10,500)
1,25>25
3.3
+
148
GH 1
cytosol
0.31
(10,500)
1,25>25
3.3
+
148
GH 3
nuclear fr.
1.1
59
1,25>25
ND
ND
141
Cpecc
SDGd
Table 3. Physical characteristics of specific binding sites detected for
1,25-(OH)?D3 in clonal pituitary tumor cells
a. Dissociation constant (multiply values by 10-10 M).
b. Number of binding sites (units = fmol/mg protein, or receptors/cell if
displayed in parentheses).
c. Specificity: rank order affinity of vitamin D-metabolites for binding
protein (1,25 = 1,25-(0H)2D3i 25 = 25-OH-D3).
d. Sucrose density gradient sedimentation coefficient (units = S).
e. Affinity for DNA-cellulose (+ if hormone-binding protein complex binds to
DNA-cellulose column under low salt conditions and is eluted under high salt
conditions).
ND. Not done.
29
binding to GH cell receptors must be considered artifactual.
In light of the
evidence obtained by Sar, et al. (140), using autoradiography and
immunohistochemistry, this possibility must be seriously considered.
Acknowledging that the effects of 1,25-(OH)2 D3 binding to GH cell
receptors may not be a true reflection of the sterol's actions in normal
pituitary, these tumor cell cultures are still the only preparation in which
biological responses to 1,25-(OH)2D3 have been examined.
The results
obtained to date have been contradictory and difficult to compare.
However,
they may provide insight into some of the biological responses which would
warrant further examination in normal anterior pituitary cells.
Murdoch and Rosenfeld (114,146) were the first to report both detection of
a specific binding protein for 1,25-(OH)2D3 in a pituitary cell line, and a
biological response of these cells following administration of the sterol.
In
this study, GH 4 cells were grown in Ham's F10 medium, 12.5% horse serum, 2.5%
fetal calf serum, and subcultured at 5x10 5 cells/ 30 mm 2 Falcon plate.
Four
days after subculture, the medium was changed to Ham's F12 plus serum, and
supplemented with 0.01 mM Ca++ (their medium contained 0.41 mM Ca++ in
the absence of added calcium).
1,25-(OH) 2 D3 and/or TRH was added to the
medium to begin an experiment.
The medium was removed 48 h later and assayed
for PRL.
They found that 1,25-(OH) 2 D3 decreased basal PRL secretion, and
augmented TRH-stimulated PRL secretion.
Both effects varied directly with the
concentration of calcium in the medium.
Furthermore, they found that in
addition to altering PRL synthesis, 1,25-(OH) 2 D3 induced the synthesis of
three unidentified cellular proteins.
Finally, TRH was found to block basal
synthesis of these proteins along with their induction by 1,25-(OH)2D3.
30
The
ED50 for the effect of 1,25-(OH)2D3 on PRL secretion was 6x10- 9 M.
The authors state that this concentration is higher than they expected, and
speculate that this finding is merely a reflection of the medium's capacity to
bind 1,25-(OH)2D3.
Under these conditions, they claim that only 2.75% of
the secosteroid is unbound.
If the ED 50 is adjusted for this factor, then
it appears to be quite close to the Kd of the receptor.
The ED50 for the
effect of 1,25-(OH) 2 D3 on protein induction was similar (3 x 10- 9 M).
Finally, the ED 50 for TRH-inhibition of this protein induction was the same
as the ED 5 0 for induction of PRL by TRH alone.
Wark and Tashjian (139) also examined effects of 1,25-(OH)2D3 on PRL
secretion using a rat pituitary tumor cell line.
However, they used the
GH4 C1 strain, and they did not demonstrate that a specific binding protein
is expressed by these cells.
Furthennore, their experiments. were carried out
under serum-free conditions.
Consequently, it
is difficult to evaluate the
discrepancy between the effects they detected and the results reported by
Murdoch and Rosenfeld (114,146).
F10 medium, 17.5% serum.
Wark and Tashjian grew their cells in Ham's
Four or five days after subculturing, they replaced
the serum-supplemented medium with a serm-free medium (minimal essential medium
with non-essential amino acids, Earle's balanced salts without CaCl 2 , and 10%
"hormone-free serum substitute").
This serum-free medium contained calcium at a
concentration of only 28 + 1 UM (well below physiological levels).
Cells were
-
incubated for 24 h prior to treatment with vitamin D metabolites and/or CaCl 2
Hormone production was then measured at 24 h intervals for up to four days.
Concentration of PRL in the medium was determined by RIA, and GH was measured by
microcomplement fixation immunoassay.
31
1,25-(OH)2D3 (1x1O-9 M) and CaCl2 (0.15 mM) had no effect on
release of GH into the medium at any time point.
This combined treatment also
failed to alter PRL release during the first 24 h of incubation.
However,
during the second 24 h period an increase in PRL release was observed (163% of
control).
Following 96 h of combined treatment, PRL release into the medium was
stimulated greater than five-fold, as compared with untreated controls.
These investigators next examined hormone release in the presence of
various concentrations of CaC1 2 in the medium.
Hormone levels were measured
only during the second 24 h period subsequent to administration of
1,25-(OH) 2 D3 (1x10-9 M).
In the absence of added CaCl 2 , PRL release
was unaffected by 1,25-(OH) 2 D3, while GH release diminished to 60% of
control (no 1,25-(0H) 2 D3 present).
When the medium contained 0.1mM CaCl2,
PRL release rose to 300% of control, with GH release remaining at 60% of
control.
1,25-(OH)2D3 produced a further increase in PRL release when the
medium contained 0.2mM CaCl 2 (to 400% of control), while bringing GH release
back to control values.
In the presence of 0.4 mM CaCl 2 , the ED50 for the effect of
1,25-(OH)2D3 on PRL release during the second 24 hours of treatment was
reported as 2x10-10M.
1-OH-D 3 was found to be 30-fold less potent.
25-OH-D 3 and 24,25-(OH)2D3 were 1000-fold less potent in stimulating PRL
release under these conditions.
Cell growth in the presence of 1,25-(OH)2D3 was observed following
plating at three different densities.
No effect of 1,25-(0H) 2 D 3 on cell
number was seen, regardless of the initial plating density.
32
Finally, the effect
of 1,25-(OH)2D3 on PRL release was also determined to be independent of
initial plating density.
Thus, in contrast to the results reported by Murdoch and Rosenfeld
(114,146), Wark and Tashjian concluded that 1,25-(OH)2D3 stimulates PRL
release from cultured pituitary tumor cells, and that this effect is
calcium-dependent.
Which of the results (if
either) represents a physiological
effect of the active form of vitamin D in normal pituitary tissue is unclear.
Wark and Tashjian, however, have recently extended their studies by examining
the effects of 1,25-(OH)2D3 on accumulation of specific rPRL mRNA in
GH4 C 1 cells, determined via cytoplasmic dot hybridization (198).
Using
complementary DNA (cDNA) probes, these authors very elegantly confirmed their
earlier findings.
Although they state that no consistent effect of
1,25-(OH) 2 D3 could be observed in the first 24 hours of treatment, they
found that stimulation of rPRL mRNA accumulation was 163% of control after 48
hours, and 200% of control after 96 hours.
The effect was once again determined
to be calcium dependent, and no change in growth hormone mRNA accumulation (also
measured using a cDNA probe) was observed in response to treatment with
1,25-(OH)2D3.
Two other groups have investigated the effects of 1,25-(OH) 2 D3 on the
activity of clonal rat pituitary tumor cells, using the GH 3 and GH1 cell
lines.
These studies further cloud the issue.
Haug, et al. (147) report a
parallel and dose-dependent decrease in both PRL and GH synthesis induced by
1,25-(OH) 2 D3 in GH3 cells.
Ham's F10 medium.
These cells were grown in serum-supplemented
The effects on hormone production were significant at
10-11M and 10-10M respectively.
In this study, hormone production
33
was calculated as the amount released into the medium during the last 24 h of a
six day incubation with 1,25-(OH) 2 D3.
Although maximal inhibition of
hormone release was found following six days of treatment, the effect was
detectable after only two days.
25-OH-D3 and la-OH-D3 also inhibited
hormone release, but in both cases the ED50 was 1000-fold higher than for
1,25-(OH) 2 D3.
No effect of 24,25-(OH)2D3 was observed over a reasonable
range of concentrations (o-11M
to 10- 6M).
affect cell growth (ug protein/ well).
1,25-(OH)2D3 failed to
When 1,25-(OH)2D3 was
co-administered with either 25-OH-D 3 , la-0H-D3, or 24,25-(OH)2D3 the
effect of 1,25-(OH)2D3 on hormone release was only slightly counteracted.
TRH and DES (a synthetic analog of estrogen) both were capable of
increasing secretion of PRL from GH 3 cells under these incubation conditions.
1,25-(0H) 2 D 3 was found to partially inhibit the effects of these factors on
PRL release, without affecting binding of TRH or 170-estradiol to their
respective receptors.
It was thus concluded that the effect of 1,25-(0H) 2 D3
on PRL release in this cell line was not due to a direct action on receptors for
TRH or estrogen.
Haussler, et al. (148) also examined the effect of 1,25-(OH) D
2 3
(10-11M to 1o- 7M) on hormone production by GH 3 cells.
In contrast
to the findings of Haug, et al. (147), no effect was seen on secretion of GH.
This group did not measure PRL release, but confirmed the lack of effect of
1,25-(OH)2D3 on GH3 cell growth (in terms of cell number and cellular
protein content).
They further examined the same parameters in GH 1 cells, and
again-found no effect of 1,25-(OH) 2 D 3 on GH release.
34
The studies performed on rat pituitary tumor cell lines have yet to
elucidate a consistent effect of 1,25-(OH)2D3 on pituitary function.
In
light of the autoradiographic evidence for exclusive localization of label from
1,25-(0H) 2 -[3 H]-D3 in nuclei of normal rat pituitary thyrotropes, along
with the knowledge of large fluctuations in phenotypic expression of tumor cells
in culture, any direct physiological effect of 1,25-(OH) 2 D3 on PRL or GH
secretion must, at this point in time, be viewed with skepticism.
remains unclear what role, if
function.
It thus
any, 1,25-(OH)2D3 may play in normal pituitary
A physiological effect of 1,25-(OH) 2 D3 binding in normal
pituitary, whether it be alteration of cell growth, hormone synthesis, or
hormone secretion, remains to be clearly demonstrated.
In conclusion, some mention must be made of the in vivo evidence which is
suggestive of an effect of 1,25-(OH) 20 3 on pituitary function.
Stumpf, et
al. published an abstract (159) describing effects of 1,25-(OH)2D3 on
circulating TSH levels in intact and thyroidectomized vitamin D-deficient rats.
In both cases, vitamin D-deficient rats treated with 1,25-(OH) 2 D3 exhibited
elevated serum levels of TSH.
calcium orally to healthy
Czechoslovakian women and measured serum thyroxine,
TSH, calcium, and magnesium.
treatment, and it
Zafkova, et al. (160) administered 25-OH-D 3 and
Thyroxine was the only parameter affected by such
was elevated.
Blumberg, et al. (161) examined several
endocrine parameters before and after administration of 1,25-(OH) 2 D3 to
patients undergoing maintenance hemodialysis.
elevated.
Prior to treatment, serum PRL was
Following treatment, serum calcium was elevated, serum PTH was
decreased, testosterone was slightly elevated in men, and serum PRL was
moderately decreased in both men and women.
35
Suzuki, et al. (162) reported
effects of la-OH-D3 in a case study of a woman being treated for hypocalcemia
due to pseudohypoparathyroidism.
Endocrine tests in the hypocalcemic state
revealed an exaggerated response of TSH to TRH, and a blunted GH response to
arginine-HCl.
Both of these abnormalities were rectified following restoration
of normocalcemia by giving la-OH-D3.
Finally, Tornquist and Lamberg-Allardt
recently presented an abstract in which they describe effects of
1,25-(OH) 2 D3 on the regulation of TSH secretion in rats (199).
These
investigators injected rats with 1,25-(OH)2D3 (5 ug/kg/day) for 3 days, and
then injected i.v. TRH 8 hours after the last dose.
At various times
thereafter, samples of blood were withdrawn from carotic artery cannulae.
It
was found that 1,25-(OH)2D3 significantly increased the response of TSH to
TRH.
Although all of these studies demonstrate changes in pituitary function
following administration of vitamin D-metabolites, none of them demonstrate
conclusively a direct effect of 1,25-(OH) 2 D 3 on pituitary function.
36
RATIONALE AND SPECIFIC AIMS
The current conception of 1,25-(OH) 2 D3 as a calcium homeostatic hormone
has gained wide acceptance (202,203).
This notion is justified by the fact that
decreases in plasma calcium concentration stimulate secretion of parathyroid
hormone (PTH) (204), and PTH acts as the most potent known stimulator of the
renal 25-OH-D3-la-hydroxylase (205-207).
Furthermore, at target tissues in
which the effects of the hormone have been best characterized (intestine and
bone), the ultimate effect is to direct calcium into the circulation (208,209).
Given the critical role of calcium in a vast array of biological phenomena
(211), any information concerning the overall mechanisms by which the body
maintains calcium homeostasis is of potential physiological, pathological, and
pharmacological significance.
It is clear that calcium is involved in many aspects of pituitary function.
These include normal pituitary cell growth (211), basal and stimulated hormone
release (200,201), and mediation of many target tissue responses induced by
pituitary hormones (212-218).
These facts, coupled with the possibility that
1,25-(OH)2D3 may affect intracellular free calcium concentration, suggest
that the activation of pituitary receptors for 1,25-(OH) 2 D3 might produce
changes in the secretion of specific pituitary hormones.
Evidence has been presented indicating that the pituitary may be a target
site for 1,25-(OH) 2 D3.
Several groups have now demonstrated the existence
of a specific pituitary binding protein for 1,25-(OH) 2 D3.
A physiological
role for this putative receptor in normal pituitary function has yet to be
determined.
A receptor, by definition, must specifically bind a ligand, with
37
the receptor-ligand interaction initiating a biological response.
In the
absence of a response, the specific binding protein cannot be classified as a
receptor (specific transport proteins and storage proteins are examples of
specific binding proteins which are not receptors).
The biological -response
initiated by the interaction of 1,25-(OH) 2D 3 with a specific binding protein
in normal mammalian pituitary cells, and the mechanism underlying such a
response, remain unclear.
In light of the research already performed concerning effects of
1,25-(OH)2D3 on the activity of neoplastic pituitary cells, it was important
to determine how this sterol might affect the activity of non-transformed
mammalian anterior pituitary cells which possess the putative receptor.
Due to
the increased frequency of abnormal genetic expression in pituitary tumor cells,
the effects of 1,25-(OH)2D3 receptor activation detected in these cells may
be artifactual.
It cannot be concluded that the responses observed following
binding of 1,25-(OH)2D3 to the expressed receptor are necessarily indicative
of the sterol's action(s) in normal pituitary.
The variability in responses
which have been reported by different groups following administration of
1,25-(OH)2D3 to cultures of transformed pituitary cells is further support
for this conclusion.
Although dispersed anterior pituitary cells maintained in
culture may not be "normal", they are less likely to be expressing abnormal
characteristics than are transformed cells.
While it
is possible that
biological responses may be either masked or de-repressed in cultures of
dispersed anterior pituitary cells, the information gained from the use of this
preparation may provide valuable insights into the responses induced by
1,25-(OH)2D3 in vivo.
38
Given our knowledge of vitamin D and the pituitary, it was possible to
speculate several possible roles for 1,25-(OH) 2D 3 in the regulation of
pituitary function.
Based on the studies with rat pituitary tumor cells, and
the reports of alterations in 25-OH-D3-la-hydroxylase activity induced by PRL
and GH, one might expect 1,25-(OH) 2 D 3 to alter the synthesis and/or release
of these hormones.
The autoradiographic evidence for exclusive localization of
1,25-(OH)2D3 in nuclei of pituitary thyrotropes would lead one to suspect an
effect of vitamin D on TSH synthesis and/or release.
may in some manner alter growth of pituitary cells.
Finally, 1,25-(OH)2 D3
Although no effects of
1,25-(OH)2D3 have yet been reported on growth of GH cells, this form of
vitamin D has been found to alter the growth of several other types of cells
(163-167).
The purpose of this thesis work was to examine a variety of biological
responses which would be indicative of an effect of 1,25-(OH) 2 D 3 on the
activity of non-transformed anterior pituitary cells obtained from normal rats.
The general goal of this thesis was to determine whether or not 1,25-(OH)2D3
is capable of affecting the activity of non-transformed mammalian anterior
pituitary cells, at least as far as the parameters to be investigated are
concerned.
If it was to be found that 1,25-(OH)2D3 affects any of these
parameters, then it would be apparent that the specific pituitary binding
protein present in normal mammalian anterior pituitary is in fact a receptor.
If no effects could be demonstrated, then the question of whether or not the
specific binding protein detected in non-transformed mammalian anterior
pituitary cells is a true receptor would have remained unanswered.
case, it could only be concluded that 1,25-(OH) 2 D3 was incapable of
39
In this
affecting the specific parameters of pituitary function examined.
However,
since some of these parameters have been reported to be altered following
binding of 1,25-(OH)2D3 to receptors in clonal rat pituitary tumor cells, a
negative thesis would raise serious doubts about the physiological relevance of
receptor responses detected in transformed cells.
It is possible that 1,25-(OH)2D3 may affect neuroendocrine activity
either directly, via the putative pituitary receptor, or indirectly, via effects
on calcium metabolism in bone, kidney, and intestine.
The importance of calcium
to neural, endocrine, and muscular function cannot be overstated.
The
clarification of the relationship between 1,25-(OH)2D3 and the pituitary
would further our understanding of the mechanisms by which the body maintains
calcium homeostasis.
In addition, the speculation of feedback effects of the
hormonal form of vitamin D, on an endocrine organ which produces a regulator of
1,25-(OH) 2 D3 synthesis, is of general endocrine interest.
The results of
these studies would also help to clarify the effects of 1,25-(OH)2D3 on PRL
secretion (a response which has been inconsistently affected in tumor cell
studies).
In the process, they would provide insight into the relevance of
receptor responses detected in tumor cells to events which occur in
non-transformed cells.
In addition, if it is found that 1,25-(OH)2D3
affects the growth of pituitary cells in culture, this would raise the
possibility that genesis or progression of specific types of pituitary tumors
might be effectively inhibited by 1,25-(OH)2D3.
Finally, the results would
add to our understanding of the physiological interplay that exists between
vitamin D and other endocrine systems, and the regulation of pituitary
function.
40
Given this rationale, the specific aims of this thesis work were as
follows:
1.
Verify the presence of a specific binding protein for 1,25-(OH)2D3 in
rat anterior pituitary;
2.
Determine the binding characteristics of this putative pituitary receptor
for 1,25-(OH)2 D3;
3.
Establish primary cultures of viable enzymatically dispersed rat anterior
pituitary cells which possess the putative receptor for 1,25-(OH)2D3;
4.
Demonstrate the ability to maintain cell viability and functionality in
culture for periods of time sufficient to examine the effects of
1,25-(OH) 2 D 3 on the activity of these cells;
5.
Determine whether these cells in primary culture retain normal
responsiveness to a known secretogogue (TRH) throughout the time period of
interest in subsequent studies on the effects of 1,25-(OH)2D3;
6.
Examine the effects of treatment with 10- 8 M 1,25-(OH) 2 D3 for various
periods of time on several parameters of the function of rat anterior pituitary
cells in primary culture, including:
A.- synthesis and secretion of rPRL;
B.' synthesis and secretion of rTSH; and
41
C.
7.
cell growth;
Determine whether the responses observed following treatment with
1,25-(OH) 2 D3 are dose-dependent;
8.
Determine the structural specificity of 1,25-(OH) 2 D3 required for
inducing any observed effects by treating cultures with 10,25-(OH)2D3, a
structural analog of 1,25-(OH)2D3.
This analog has been shown to be
biologically inactive with other well-characterized receptors for
1,25-(OH) 2 D 3 , and does not compete with 1,25-(OH)2D3 for binding to its
receptor.
42
MATERIALS AND METHODS
Drugs and Chemicals
la,25-Dihydroxyvitamin D3 was kindly provided by Dr. M. Uskokovic,
Hoffman-LaRoche, Nutley, New Jersey.
25-Hydroxyvitamin D3 was kindly provided by Dr. J. Babcock, Upjohn, Kalamazoo,
Michigan.
18,25-Dihydroxyvitamin D3 was kindly provided by Dr. S. Holick, M.I.T.,
Cambridge, Massachusetts.
1,25-Dihydroxy-[26,27- 3 H]-vitamin D3 (158-170 or 91 Ci/mmol) was purchased
from New England Nuclear, Boston, Massachusetts.
Thryotropin Releasing Hormone (TRH; p-Glu-His-Pro-NH 2 ) was purchased from
United States Biochemical Corporation (catalog #22371; lot #21977), Cleveland,
Ohio.
3 5 S-methionine
(Methionine, L-[ 3 5S]; 1058 Ci/mmol; 1 mCi/O.1 ml) was
purchased from New England Nuclear (catalog #NEG-009T; lot #1013-210), Boston,
Massachusetts.
43
Immediately prior to their use, all vitamin D metabolites were re-checked for
purity and concentration via ultraviolet spectroscopy and high pressure liquid
chromatography.
Sources and descriptions of any other chemicals used in these
studies, other than standard laboratory reagents, are provided in the sections
describing their use.
Animals
Adult male Sprague Dawley CD-derived rats, weighing 250-300 grams, were
obtained from Charles River Breeding Laboratories, Inc. (Wilmington,
Massachusetts).
Prior to their use, rats were housed in facilities maintained
by the M.I.T. Department of Laboratory Animal Medicine.
Rats were kept in
hanging cages, in pairs, in a climate-controlled room, under a 12:12 light:dark
schedule, and fed normal rat chow (Charles River Rat Chow, Charles River
Laboratories, Wilmington, Massachusetts).
Dissection of Rat Anterior Pituitary for use in Binding Studies
Anterior pituitary tissue was obtained from 250-300 g male Sprague Dawley
CD-derived rats.
Rats were sacrificed by rapid decapitation.
opened, and the brain was removed.
The calvarium was
The membrane covering the pituitary gland
was then separated from the underlying tissue, thus exposing the hypophysis.
The pituitary was then lifted from the sella turcica, and immediately placed in
ice-cold isotonic buffer containing 50 mM Tris-HCl (pH 7.4), 1.5 mM EDTA, and 5
mM dithiothreitol.
The posterior pituitary was carefully dissected away from
44
the anterior pituitary and discarded.
removed.
Any extraneous connective tissue was also
The isotonic buffer was then discarded, and the remaining anterior
pituitary was washed three times with hypertonic buffer (10 mM Tris-HCl, pH 7.4,
1 mM EDTA, 5 mM dithiothreitol, 300 mM KCl, and 10 mM sodium molybdate) to
remove as much plasma as possible.
When nuclear extracts of anterior pituitary
were to be prepared, this last step was omitted, and the glands were left in
isotonic buffer.
Preparation of Cytosolic and Nuclear Extracts of Rat Anterior Pituitary
Cytosolic and nuclear extracts were prepared from pooled anterior pituitary
glands of 20 rats.
Isolated anterior pituitary glands were washed in ice-cold
hypertonic buffer and minced into 1 mm 2 fragments with sterile razor blades.
The tissue fragments were then suspended in 1.0 ml of fresh hypertonic buffer
and transferred to a 1.5 ml Eppendorf tube.
sonicated for 15 seconds on ice.
The tissue suspension was then
The resulting homogenate was transferred to a
polycarbonate tube, and centrifuged at 100,000 x g for 1 hour, at 40C, in a
Beckman 50Ti rotor.
This treatment yielded a cytosolic supernatant.
The
cytosol extract was either used immediately, or kept frozen at -700C prior to
assay.
Crude nuclear extracts were prepared from isolated rat anterior pituitary
glands in isotonic buffer.
The anterior pituitaries were minced into 1 mm 2
fragments and suspended in 1.0 ml of fresh isotonic buffer.
The tissue
suspension was transferred to a 1.5 ml Eppendorf tube and sonicated for 15
seconds on ice.
The resulting homogenate was transferred to a polycarbonate
45
tube, and centrifuged at 12,000 x g, for 10 minutes, at 40C, using a Beckman
50Ti rotor.
The supernatant was then removed, and the crude nuclear pellet was
resuspended in 4 volumes of ice-cold isotonic buffer.
This tissue suspension
was then transferred to a 1.5 ml Eppendorf tube and washed 4 times With isotonic
buffer.
Each wash involved addition of ice-cold isotonic buffer, vortexing,
centrifugation for 5 minutes in a Beckman Microfuge B at 40C, and removal of
the supernatant.
After the final wash, the pellet was resuspended in 0.5 ml
hypertonic buffer and vortexed vigorously for 2 minutes.
The tissue suspension
was then transferred to a polycarbonate tube, and centrifuged for 1 hour at
22,000 x g, at 40C, in a Beckman 50Ti rotor.
the crude nuclear extract.
The resulting supernatant was
This nuclear extract was either used immediately, or
kept frozen at -700C prior to assay.
Sucrose Density Gradient Sedimentation Analysis
Sucrose density gradient sedimentation analysis was performed essentially
as described by Clemens, et al. (180).
0.2 ml aliquots of either rat anterior
pituitary cytosolic or nuclear extracts were incubated on ice for 4 hours in the
presence of 4 nM
3 H-1,25-(0H)2D3
(91 Ci/mmol; 80,000 cpm), alone or
together with a 50-fold excess of unlabelled 1,25-(OH)2D3.
75 ul of a
Dextran-coated charcoal solution was then added to each tube to remove any
unbound sterol.
The samples were incubated with charcoal for 20 minutes on ice,
and then centrifuged at 3,000 x g, for 10 minutes, at 40C.
Following
centrifugation, 0.2 ml of the supernatant was layered onto 4.8 ml sucrose
density gradients (4%-20% sucrose, prepared in hypertonic buffer) in Beckman
46
Ultraclear tubes.
The gradients were then centrifuged for 16-18 hours, at
257,000 x g, at 20C, in a Beckman SW50.1 rotor.
Gradients were fractionated
from the bottom of the tube (5 drops/ fraction) using a peristaltic pump.
fraction was placed in a scintillation vial.
Each
10 ml of a scintillation cocktail
(Instagel; United Technologies/ Packard) was then added to each vial, and the
radioactivity in each fraction was determined by liquid scintillation counting
in a Packard Tri-Carb 460C liquid scintillation counter.
Parallel gradients
run on cytosol prepared from intestine of vitamin D-deficient chicks, or from
intestine of mice, were used as positive controls.
Protein markers (bovine
serum albumin, 4.4 S; ovalbumin, 3.7 S) were also used to estimate sedimentation
coefficients.
Determination of Binding Characteristics
Aliquots of rat anterior pituitary cytosol were used in a competitive
binding assay in order to determine the receptor equilibrium dissociation
constant (Kd) and the number of binding sites (Bmax).
The method used
was a modification of the method described by Haussler, et al. (113).
0.1 ml
aliquots of cytosol were added to duplicate tubes containing various
concentrations of
3 H-1,25-(0H)
2D3
(0.25 nM to 4.0 nM), alone or together
with a 200-fold excess of unlabelled 1,25-(OH) 2 D3 (to determine non-specific
binding).
water-bath.
The samples were incubated for 30 minutes, at 250C, in a shaking
The samples were placed on ice, and 0.15 ml of an ice-cold
Dextran-coated charcoal solution was added to remove any unbound sterol.
After
addition of the charcoal solution, the samples were incubated on ice -for 10
47
minutes, and then centrifuged at 5,000 x g, for 10 minutes, at 40C.
0.2 ml
aliquots of the resulting supernatants were transferred to small scintillation
vials.
4 ml of a scintillation cocktail (Instagel; United Technologies/
Packard) was added to each vial, and the radioactivity in each sample was
determined by liquid scintillation counting in a Packard Tri-Carb 460C liquid
scintillation counter.
Specific binding was calculated by subtracting the
non-specific from the total binding.
The data was then subjected to Scatchard
analysis (181) to determine the Kd, Bmax, and presence of singular or
multiple classes of binding sites (e.g., high affinity and low affinity).
Rat Anterior Pituitary Cell Culture
Cultures of enzymatically dispersed rat anterior pituitary cells were
prepared and maintained essentially as described by Vale, et al. (168).
equipment and procedures were used throughout.
Sterile
Anterior pituitaries were
obtained from 250-300 g adult male Sprague Dawley CD-derived rats.
Rats were
sacrificed via rapid decapitation, the calvarium opened, the brain removed, and
the pituitary lifted from the sella turcica.
The pituitaries were immediately
placed in a HEPES buffer (137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 25 mM HEPES
(n-2-hydroxyethylpiperazine ethanesulfonic acid), 10 mM glucose, 0.36 mM
CaCl 2 , pH 7.2) containing 1% (v/v) of an antibiotic solution (penicillin,
Sigma PEN-NA, 448 mg/ 100 ml ddH 20; streptomycin, Sigma S-6501, 667 mg/ 100 ml
ddH 2 0.).
The posterior lobes were then dissected free and discarded.
The
remaining adenohypophyses were then sliced into 1 mm 2 fragments, and washed
three times with HEPES buffer.
The washed anterior pituitary fragments were
48
then transferred to a 25 ml Erlenmeyer flask containing a teflon coated magnetic
stirring bar, the HEPES buffer was removed, and the tissue fragments were
resuspended in 10 ml HEPES buffer supplemented with 3% bovine serum albumin (RIA
grade; Sigma), 0.1% hyaluronidase (550 U/mg, ovine testis, type III; Sigma), and
0.35% collogenase (146 U/mg; Worthington).
The tissue fragments were then
incubated in this enzyme solution at 370C, for 45 minutes, with stirring.
Enzymatic dispersion of the cells was assisted by periodic gentle lifting and
releasing of the cell suspension with a siliconized Pasteur pipette.
Following
complete dispersal of the cells, the suspension was transferred to a 15 ml
plastic tube and centrifuged at 500 x g for 8 minutes.
The supernatant enzyme
solution was discarded, and the pelleted cells were resuspended in 10 ml culture
medium (85% Ham's F10, M.A. Bioproducts; 12.5% donor horse serum, M.A.
Bioproducts; 2.5% fetal bovine serum, M.A. Bioproducts; supplemented with 1%
(v/v) of the antibiotic solution described above, filtered through a 0.2 um
membrane (Millipore), and heated to 370C).
The cells were then washed six
times via centrifugation for 2 minutes at 500 x g, removal of the supernatant,
and resuspension in fresh culture medium.
Following the last wash, 100 ul of
the final cell suspension was removed, mixed with an equal volume of a trypan
blue dye solution (0.1% trypan blue dye in 0.9% NaCl), and examined
microscopically on a hemocytometer to determine the concentration of viable
cells present in the suspension.
Sufficient culture medium was then added to
the cell suspension to achieve a concentration of 3-5 x 105 viable cells/ ml.
1.5 ml of this cell suspension was then added to each of the desired number of
35 mm x 14 mm tissue culture wells (Falcon #3046; approximately 9.6 cm 2 growth
area/ well), and the cell cultures were placed in an incubator (NAPCO 6300).
49
Cultures were incubated at 370C, in a humidified atmosphere containing 95% air
and 5% C0 2 , for 4 days prior to initiation of experiments.
Before beginning
an experiment, the serum-supplemented culture medium was removed, and the cells
were washed with Ham's F10 (which had been previously filtered through a 0.2 um
membrane (Millipore) and heated to 370C).
The wash was then discarded, 1.5 ml
of fresh Ham's F10 was added to each well, and the cultures were returned to the
incubator for a 1 hour pre-incubation period.
The desired chemicals were then
added to each culture, and the cells were returned to the incubator for the
duration of the test period.
of each test period.
Media and cell samples were collected at the end
The media from each culture was removed, placed in an
individual 1.5 ml Eppendorf tube, and centrifuged at 1,000 x g for 5 minutes to
pellet any floating cells.
Each supernatant was then transferred to a new
Eppendorf tube, and stored at -200C prior to assay.
1.5 ml of a Tris buffer
(50 mM Tris-HCl, pH 7.4, 140 mM NaCl) containing 0.1% Triton X-100 was added to
each well, and the attached cells were gently scraped off the dish using a
rubber policeman.
The scraped cell solution was then combined with the pelleted
floating cells, and the total cell sample stored at -200C.
Just prior to the
initial assay, cell samples were thawed and sonicated for 15 seconds, on ice,
using a Tekmar sonic cell disrupter.
Determination of Cell Number and Viability
The number of viable cells was determined by the trypan blue dye exclusion
test '(176).
Typically, 100 ul of a cell suspension was mixed with an equal
volume of a trypan blue dye solution (0.1% trypan blue dye in 0.9% NaCl).
50
A
drop of this mixture was immediately applied to a hemocytometer, and examined
microscopically.
Duplicate cell counts were performed, each time counting at
least 5 regions of the hemocytometer.
Concentration of viable cells was
calculated as follows:
(total number of viable cells counted) / (number of regions examined) x 2 x
104 = (number of viable cells) / ml.
Cell Photography
Cells in culture were photographed under a variety of treatment conditions
using an Olympus microscope coupled with an Olympus camera system.
Determination of Cellular Protein Content
Protein content of cell samples was determined by the method of Lowry, et
al. (178), using bovine serum albumin (RIA grade; Sigma) as standard.
Determination of Cellular DNA Content
DNA content of cell samples was determined by the method of Burton (179),
based on the diphenylamine reaction.
Calf thymus DNA (Sigma) was used as
standard.
51
Radioimmunoassay for Rat Prolactin
The concentration of rPRL in media and cell samples was determined by
double-antibody radioimmunoassay, using standards, primary antibodies, and
procedures supplied by the NIADDK.
Material s:
Samples:
Samples were typically assayed in either duplicate or triplicate, and
occasionally aliquots of different volume fron the same sample were assayed in
parallel as a further control for accuracy of results.
Assay Buffer:
0.01 M P0 4 , 0.5% bovine serum albumin (RIA grade; Sigma), 0.88% NaCl,
0.05 M EDTA, 0.1% sodium azide, pH 7.6.
1st Antibody Buffer:
0.05% normal rabbit serum (Pel-Freez Biologicals, Rogers, Arkansas) in
assay buffer.
1st Antibody:
NIADDK-anti-rPRL-S-9 (AFP-131581570), rat prolactin antiserum (rabbit),
dissolved in 2% normal rabbit serum in phosphosaline buffer, was diluted 1:10
with 1st Antibody Buffer and stored in 0.5 ml aliquots at -200C.
Prior to
each assay, an aliquot of this solution was thawed and diluted 1:210 with 1st
52
Antibody Buffer, yielding a final dilution of 1:2100, and a "final tube
dilution" of 1:12,600.
2nd Antibody:
Goat anti-rabbit IgG (heavy and light chains; Cappel Laboratories,
catalog #0112-0081, lot #17808), dissolved in 0.02 M phosphosaline (pH 7.3)
containing 0.05% sodium azide, was stored in 500 ul aliquots at -200C.
Prior to each assay, an aliquot of this solution was thawed and diluted 1:100
with assay buffer.
Tracer:
1251-rPRL (New England Nuclear, catalog #NEX-108; 16.3 uCi total
activity; 36.4 uCi/ug specific activity) was diluted with assay buffer to
obtain a solution containing approximately 10,000 cpm/100 ul.
Standards:
NIADDK-rPRL-RP-3 (AFP-4459B) was used as the standard reference
preparation.
Reconstitution of 1 vial with 1 ml dH 2 0 yielded a solution
containing 10 ug rPRL/ml, in 1% BSA in phosphosaline.
100 ul aliquots of this
solution were stored in Eppendorf tubes at -200C, and used within 5 months
subsequent to solubilization.
was used with each assay.
A range of standards, prepared in triplicate,
This involved diluting an aliquot of the stock
reference preparation with assay buffer to achieve a final concentration of
5.0 ng/50 ul, and then serially diluting this solution 1:1 with assay buffer
53
to obtain a series of standards ranging in concentration from 5.0 ng/50 ul to
0.01 ng/50 ul.
Procedure:
Sufficient assay buffer was added to 12 x 75 mm glass tubes, on ice, such
that the combined volume of assay buffer and sample or standard totaled 300 ul.
Standards and samples were then added to the appropriate tubes.
Six tubes were
always prepared containing no sample or standard in order to determine maximum
binding and non-specific binding.
100 ul of the 1st antibody was then added to
all tubes except for the three tubes used to determine non-specific binding.
These three tubes received 100 ul of 1st antibody buffer instead.
All tubes
were then vortexed gently, and allowed to incubate at room temperature for 24
hours.
100 ul of
125 1-rPRL
(10,000 cpm/100 ul) was then added to each
tube, followed by gentle vortexing.
The tubes were then corked and allowed to
incubate for an additional 24 hours at room temperature.
During this incubation
period, the tubes were pre-counted in a Gamma 4000 gamma counter (Beckman).
ul of the 2nd antibody was then added to each tube.
and incubated for 24 hours at 40C.
100
The tubes were vortexed,
The tubes were then centrifuged for 15
minutes, at 40C, at 6,500 x g, in a Beckman J2-21 centrifuge.
The supernatant
was aspirated off, and the tubes were counted in a Gamma 4000 gamma counter.
The percent bound (%B) in each tube was calculated by comparing the final count
(B) with the pre-count (BO).
This percentage was then corrected for
non-specific binding (NSB) according to the following equation:
corrected %B = [(%B-%NSB)/(100-%NSB)] x 100
54
The corrected %B for each standard concentration was then plotted on semi-log
paper to obtain a standard curve (see figure 2).
The amount of rPRL in each
sample aliquot was then determined by comparing each sample's corrected %B with
the standard curve.
lodination of rTSH for Use in Radioimmunoassay
Radioiodination of rTSH was achieved through use of the chloramine T
reaction, essentially as described by Greenwood, et al. (218).
The
radioiodinated rTSH was then purified by Sephadex gel chromatography.
Iodination Reaction:
Reagents:
0.5 M P04, pH 7.4; Phosphate buffered saline (PBS: 0.05 M P04, 0.88%
NaCl, pH 7.4); PBS containing 0.2% sodium azide; rTSH (NIADDK-Rat TSH-I-6,
1 ug/2 ul PBS); Sodium Metabisulfite (12.5 mg/10 ml PBS); 2% Bovine serum
albumin (BSA) in PBS; 1% BSA in PBS; Chloramine-T (10.0 mg/10 ml PBS);
125 1-Sodium
Iodide (New England Nuclear, catalog :#NEZ-033; 1 mCi/100 ul,
in 0.1 N NaOH; specific activity 17.4 Ci/mg).
Procedure:
The iodination reaction was performed in the vial containing the
12 5I-Sodium iodide, in a glove box authorized for use of high levels of
radioactivity.
After venting the vial, the following reagents were added via
55
12-
zD
0
o.
6-
3-
.020
.039
.078
.158
.3125
.625
1.25
2.5
NANOGRAMS RAT PRL- RP-3
Figure 2. Typical Standard Curve for Rat Prolactin Radioimmunoassay
Each point is the mean of triplicate determinations.
56
5.0
Hamilton syringes:
10 ul 0.5 M P04, 5 ul rTSH, and 10 ul Chloramine-T
(prepared just before use, kept on ice, and protected from exposure to light).
The vial was then gently shaken for 30 seconds.
The reaction was then stopped
by the addition of 10 ul sodium metabisulfite and 200 ul 2% BSA in PBS.
The
entire reaction mixture was then transferred onto a Sephadex G-50 column for
isolation of purified
12 5 1-rTSH.
Purification of Radioiodinated rTSH:
Preparation of Gel:
Sephadex G-50 (Pharmacia) was swelled in PBS containing 0.2% sodium
azide, overnight, at room temperature.
The gel was then poured into a 20 cm
high x 0.7 cm diameter plastic Econocolumn (Beckman).
bed was 17.5 cm, and the column volume was 6.75 ml.
The height of the gel
Five column volumes of
PBS containing 0.2% sodium azide were immediately run through the column to
settle the gel.
A few drops of 10% BSA in PBS were then applied to the gel,
followed by elution with another 5 column volumes of PBS containing 0.2%
sodium azide.
A few drops of a Dextran Blue solution (Dextran Blue 2000, 2
mg/ml PBS; Pharmacia) were then run through the column in order to determine
uniformity of flow through the column, and the void volume of the column.
gel was then eluted with 5 more column volumes prior to application of the
iodination reaction mixture.
57
The
Gel Chromatography:
The iodination reaction mixture was applied to the column and eluted with
PBS containing 0.2% sodium azide.
Five drop fractions (approximately 200 ul)
were collected in 1.5 ml Eppendorf tubes.
1 ul of each fraction was then
counted in a Gamma 4000 gamma counter (Beckman) (see figure 3).
The
fractions representing the first peak in radioactivity were assumed to be
1251-rTSH, and were subsequently checked for binding activity using
antiserum to rTSH.
Verification of Binding of Radioactive Fractions to rTSH-anti-serum:
Aliquots of each fraction comprising the first peak in radioactivity
eluted from the Sephadex gel were diluted to 10,000 cpm/100 ul with the assay
buffer used in the rTSH radioimmunoassay.
Nine diluted 100 ul samples from
each fraction were then assayed for binding activity using the same
methodology described below for the rTSH radioimmunoassay.
For each fraction,
triplicate samples were used to determine maximal binding (no competing
standard present), non-specific binding (no competing standard and no 1st
antibody present), and displacement by rTSH (co-incubation of 1251-rTSH
with 100 ng rTSH standard reference preparation).
*
The fractions demonstrating
high maximal binding, low non-specific binding, and ability to be displaced by
non-radioactive rTSH were then pooled and used in the rTSH radioimmunoassay.
The pooled
12 5 1-rTSH
was stored at 40C, and used within 7 days.
58
30-
t:
.J
0
25
20
0
()
15-
x
10
I-
0-01
5
15
10
20
FRACTION
Figure 3. Sephadex G-50 Gel Chromatographic Purification of Radioiodinated Rat
Thyraid Stimulating Hormone
Each fraction contains 5 drops. 1 ul of each fraction was counted. The first
peak represents 12 5 1-labelled-rTSH. Each fraction in this peak was
subsequently tested for binding activity to antibodies against rTSH, and for
displaceability from binding by non-radioactive rTSH. The most active fractions
were then pooled for use in the radioimmunoassay.
59
Radioimmunoassay for Rat Thyroid Stimulating Hormone
The concentration of rTSH in media and cell samples was determined by
double-antibody radioimmunoassay, using standards, primary antibodies, rTSH for
iodination, and procedures supplied by the NIADDK.
Materials:
Samples:
Samples were typically assayed in either duplicate or triplicate, and
occasionally aliquots of different volume from the same sample were assayed in
parallel as a further control for accuracy of results.
Assay Buffer:
0.01 M P0 4 , 0.1% Bovine serum albumin (RIA grade; Sigma), 0.14 M NaCl,
0.025 M EDTA, 0.1% sodium azide, pH 7.5.
1st Antibody Buffer:
0.5% Normal rabbit serum (Pel-Freez Biologicals) in assay buffer.
1st Antibody:
NIADDK-anti-rTSH-S-5 (C21381), rat thyroid stimulating hormone antiserum
(rabbit), dissolved in 2% normal rabbit serum in phosphosaline buffer, was
diluted 1:10 with 1st antibody buffer and stored in 0.5 ml aliquots at
-200C.
Prior to each assay, an aliquot of this solution was thawed and
diluted 1:150 with 1st antibody buffer, yielding a final dilution of 1:1,500,
and a "final tube dilution" of 1:10,500.
60
2nd Antibody:
Goat anti-rabbit IgG (heavy and light chains; Cappel Laboratories,
catalog #9112-0081, lot #17808), dissolved in 0.02 M phosphosaline (pH 7.3)
containing 0.05% sodium azide, was stored in 500 ul aliquots at -200C.
Prior to each assay, an aliquot of this solution was thawed and diluted 1:40
with assay buffer.
Tracer:
125 I-rTSH,
prepared from NIADDK-rTSH-I-6 as described above, was
diluted with assay buffer to obtain a solution containing approximately 5,000
cpm/100 ul.
Standards:
NIADDK-rTSH-RP-2 (AFP-5153B) was used as the standard reference
preparation.
Reconstitution of 1 vial with 1 ml dH2 0 yielded a solution
containing 5 ug rTSH/ml, in 1% BSA in phosphosaline.
50 ul aliquots of this
solution were stored in Eppendorf tubes at -200C, and used within 5 months
subsequent to solubilization.
was used with each assay.
A range of standards, prepared in triplicate,
This involved diluting an aliquot of the stock
reference preparation with assay buffer to achieve a final concentration of 25
ng/100 ul, and then serially diluting this solution 1:1 with assay buffer to
obtain a series of standards ranging in concentration from 25 ng/100 ul to
0.0.5 ng/100 ul.
61
Procedure:
Sufficient assay buffer was added to 12 x 75 mm glass tubes, on ice, such
that the combined volume of assay buffer and sample or standard totaled 400 ul.
Standards and samples were then added to the appropriate tubes.
Six tubes were
always prepared containing no sample or standard in order to determine maximal
binding and non-specific binding.
100 ul of the 1st antibody was then added to
all tubes except for the three tubes used to determine non-specific binding.
These three tubes received 100 ul of 1st antibody buffer instead.
All tubes
were then vortexed gently, and allowed to incubate at 40C for 3 days.
of
12 5 1-rTSH
100 ul
(5,000 cpm/100 ul) was then added to each tube, followed by
gentle vortexing.
The tubes were then corked and allowed to incubate for an
additional 3 days at 40C.
On the last day of this incubation, the tubes were
pre-counted in a Gamma 4000 gammma counter (Beckman).
antibody was then added to each tube.
for 24 hours at 40C.
100 ul of the 2nd
The tubes were vortexed, and incubated
The tubes were then centrifuged for 15 minutes, at
40C, at 6,500 x g, in a Beckman J2-21 centrifuge.
aspirated off, and the tubes were re-counted.
The supernatant was then
The percent bound in each tube,
and the amount of rTSH in each sample, were determined as described above in the
procedure for the rPRL radioimmunoassay. A typical standard curve for the rTSH
radioimmunoassay is displayed in figure 4.
Double Antibody Immunoprecipitation of
35 S-Methionine-Labelled-rTSH
'Other than the exceptions noted below, the methodology and reagents used
for the double antibody immunoprecipitation of
35 S-methionine-labelled-rTSH
were identical to those employed in the rTSH radioimmunoassay.
62
The
z
0
10-
.025
.05
.10
.20
.39
.8
.iBs
3.125
NANOGRAMS RAT TSH-RP-2
Figure 4. Typical Standard Curve for Rat Thyroid Stimulating Hormone
Radioirmmunoassay
Each point is the mean of triplicate determinations.
63
ebs
concentrations of both antibodies used in this assay were increased 10-fold over
those employed in the corresponding radioimmunoassay.
It was unnecessary to use
standards in this procedure, and 100 ul of assay buffer were added to each tube
instead of 100 ul of
125 1-rTSH.
Following aspiration of the final
supernatant, the pelleted immunoprecipitated rTSH was resuspended in assay
buffer instead of being counted.
to glass scintillation vials.
Aliquots of each sample were then transferred
10 ml of a scintillation cocktail (Instagel;
United Technologies/ Packard) was then added to each vial, and the vials were
vortexed.
The samples were then allowed to incubate for 24 hours at 40C prior
to counting in a Packard Tri-Carb 460C liquid scintillation counter.
When
samples were to be run on polyacrylamide-SDS gels, the pelleted
immunoprecipitated rTSH was resuspended in the gel electrophoresis buffer
(described below) instead of the radioimmunoassay buffer.
3 5 S-Methionine-Labelled-rTSH
Gel Electrophoresis of Immunoprecipitated
Gradient SDS polyacrylamide gel electrophoresis of immunoprecipitated
3 5 S-methionine-labelled-rTSH
was performed using a modification of
procedures described by Chin, et al. (219).
Reagents:
1) 30% Acrylamide, 0.8% Bis (N'N'Methylene-bisacrylamide);
2) 20% SDS (sodium lauryl sulfate/ sodium dodecyl sulfate);
3)10% Ammonium persulfate;
4) TEMED (N' ,N' ,N',N',Tetramethylethylenediamine);
64
80 mM Tris-HCl, pH 6.8, 10% glycerol, 0.0024% bromophenol
5) Sample buffer:
blue, 2% SDS, 100 mM dithiothreitol;
6) Running buffer:
0.19 M glycine, 0.025 M Tris-base, 0.1% SDS;
7) Running gels:
20% polyacrylamide
10% polyacrylamide
Acrylamide/Bis (30%/0.8%)
3.33 ml
6.67 ml
Tris-HCl, pH 8.7 (1.5 M)
2.50 ml
2.50 ml
SDS (20%)
0.05 ml
0.05 ml
H20
4.12 ml
0.78 ml
8) Stacking gel (5% polyacrylamide):
2.51 ml acrylamide/Bis (30%/0.8%); 1.88
ml Tris-HCl, pH 6.8 (1.0 M); 0.075 ml SDS (20%); 10.5 ml H20;
9) Standards:
BRL Pre-stained low molecular weight standards:
insulin
(3000), bovine trypsin inhibitor (6200), cytochrome C (12300), lysozyme
(14300), beta-lactoglobulin (18400), alpha-chymotrypsinogen (25700), and
ovalbumin (43000) (Bethesda Research Laboratories).
Procedure:
Immediately before pouring the running gel and the stacking gel, ammonium
persulfate (5 ul/ml of the gel) and TEMED (0.5 ul/ml of the gel) were added to
the gel mixtures.
The 10%-20% polyacrylamide gradient running gel was poured
first and allowed to polymerize, and then the stacking gel was poured.
A
plastic comb was inserted in the top of the stacking gel, prior to
polymerization, in order to form wells for the samples.
Following
polymerization of the stacking gel, the comb was removed, the gel was attached
65
to a vertical gel box, and running buffer was added to the upper and lower
chambers of the box.
Samples and standards were prepared in sample buffer, and
incubated for 5 minutes in boiling H20 prior to placement into the wells of
the gel (via Hamilton syringes).
The gel was run at 150 V (constant voltage)
until the tracking dye reached approximately 1 cm from the bottom of the gel
(usually 4.5-5.0 hours).
Evaluation of Gels:
Following electrophoresis, the gel was removed from the gel box, and sliced
into individual lanes.
The positions of the pre-stained standards were
recorded, and the sample lanes were cut into 1 mm slices.
Each slice was then
placed in a scintillation vial, to which 1 ml of 30% hydrogen peroxide was
added.
The vials were then incubated at 500C, in a shaking water bath, for 24
hours, to dissolve the gel slices.
10 ml of a scintillation cocktail (Instagel)
was then added to each vial, and the vials were allowed to incubate for 24 hours
at 40C.
The vials were then counted in a Packard Tri-Carb 460C liquid
scintillation counter.
Double Antibody Immunoprecipitation of
3 5S-Methionine-Labelled-rPRL
Other than the exceptions noted below, the methodology and reagents used
for the double antibody immunoprecipitation of
3 5S-methionine-labelled-rPRL
were identical to those employed in the rPRL radioimmunoassay.
The
concentrations of both antibodies used in this assay were increased 10-fold over
those employed in the corresponding radioimmunoassay.
66
It was unnecessary to use
standards in this procedure, and 100 ul of assay buffer were added to each tube
instead of 100 ul of
125 1-rPRL.
The treatment of pelleted
immunoprecipitated rPRL following aspiration of the final supernatant was
identical to that described above in the procedure for double antibody
immunoprecipitation of
3 5 S-methionine-labelled-rTSH.
Trichloroacetic Acid Precipitation of Proteins
Incorporation of
35S-methionine
into newly synthesized proteins was
examined through scintillation counting of trichloroacetic acid (TCA)
precipitated proteins.
tube on ice.
100 ul of each sample was placed in a 1.5 ml Eppendorf
900 ul of ice-cold 5.56% TCA was added to each tube.
were then incubated on ice for 30 minutes.
The samples
The samples were then applied to
individual 2.3 cm Whatman 3MM filter papers (or to Schleicher
& Schuell#
34
glass fiber filter papers) in a multiple sample filtration device (Millipore).
The samples were then washed twice with 5 ml of ice-cold 5% TCA, and twice with
2.5 ml of absolute ethanol.
The filters were then removed, allowed to dry
overnight, and placed in scintillation vials.
10 ml of a scintillation cocktail
(Instagel) was then added to each vial, and the vials were counted in a Packard
Tri-Carb 460C liquid scintillation counter.
Statistics
Statistical significance of differences between treatment groups was
determined by either Student's t-test (for differences between 2 groups) or
67
analysis of variance (for differences between more than 2 treatment groups), as
described elsewhere (184).
68
RESULTS
Detection of Specific Binding Protein for 1,25-(OH)7D3 in Rat Anterior
Pituitary
Prior to examining the regulation of pituitary function by
1,25-(OH)2D3, it
was first necessary to verify the presence of the putative
receptor for this hormone in rat anterior pituitary.
Detection and preliminary
characterization of a specific binding protein for 1,25-(OH)2D3 in rat
anterior pituitary was achieved through the use of sucrose density gradient
sedimentation analysis.
Anterior pituitaries were dissected from 20 male
Sprague Dawley CD rats (275-300 g) following rapid decapitation.
nuclear extracts were then prepared from the pooled tissue.
Cytosolic and
Cytosolic extracts
of mouse intestine and chick intestine were also prepared for use as positive
controls.
Aliquots of each extract were incubated with 4 nM
3 H-1,25-(0I)
2 D3,
1,25-(OH) 2D3.
either alone or in the presence of 200 nM
Subsequent to this incubation, unbound sterols were removed
by treatment with dextran-coated charcoal.
The solutions containing bound
hormone were then subjected to sucrose density gradient sedimentation analysis,
as described in the Methods section.
The results of sucrose density gradient sedimentation analysis of rat
anterior pituitary cytosolic and nuclear extracts incubated with
3H-1,25-(OH)
2D 3
are displayed in figure
5.
A 3.5-3.7 S specific binding
protein for 1,25-(OH) 2 D3 was detected in both cytosolic and nuclear extracts
of rat anterior pituitary.
Two peaks of radioactivity (3.5-3.7 S and 6.0 S)
69
logo,
K
A
3.73
C
1.058 3.73
I I
lit
4
433
A
1133'
.0
~0
.0
.0
0
S
1233!
33,
0
411'
0
M.34
zig,
0
*
A.
0
V
00
C)
&
0
0
*
*
4
:
4W
q
S.
3.33
B
- I.
D
3.73
1M'
433
33m
-
33.
331
43.
0
.0
.
.'.!.| ..
.... A.
1
I
10
15
20
25
38
S
is
15
20
25
31
FRACTION
Figure 5. Sucrose Density Gradient Sedimentation Analysis of Rat Anterior
Pituitary Cytosolic and Nuclear Extracts Incubated with 3H-1,25-(OHbD3
(A) Chick intestinal cytosol (positive control); (B) mouse intestina cytosol
(positive control); (C) rat anterior pituitary cytosolic extract; (D) rat
anterior pituitary nuclear extract. In each case (A through D), aliquots from
the same sample were incubated with 3H-1,25-(OH) 2 D 3 , alone (solid line) or
together with a 50-fold excess of non-radioactive 1,25-(OH) 2 D3 (broken
line). Sedimentation coefficients assigned to peaks were estimated from known
protein standards (bovine serum albumin, 3.7 S; ovalbumin, 4.4 S) which were run
in parallel gradients.
70
were observed in the sedimentation profile of rat anterior pituitary cytosolic
extract incubated with
3H-1,25-(OH)
2D3 .
The 3.5-3.7 S peak was not
apparent when a 50 fold excess of non-radioactive 1,25-(OH) 2 D3 was included
in the incubation, thus demonstrating the specificity of this binding protein
for 1,25-(OH) 2D3.
Binding of
3 H-1,25-(OH)
2 D3
to the 6.0 S protein
could not be inhibited by similar treatment with excess 1,25-(OH)2D3.
This
6.0 S non-specific binding protein has been detected previously in many receptor
positive tissues (41,87,88), and is believed to be a complex of the 4.0 S plasma
vitamin D binding protein and some unknown cytosolic protein.
Determinination of Binding Characteristics
Binding studies were performed in order to determine whether the binding
characteristics of the specific binding protein for 1,25-(OH) 2 D3 in rat
anterior pituitary were similar to those previously reported for the
1,25-(OH) 2 D3 receptor present in other target tissues.
The binding
characteristics of the putative rat anterior pituitary cytosolic receptor for
1,25-(OH) 2 D3 were examined via a competitive binding assay.
The resulting
data was subjected to Scatchard analysis (181) in order to determine the
dissociation constant (Kd), number of binding sites (Bmax), and presence
of singular or multiple classes of binding sites (e.g., high affinity and low
affinity).
The results of the competitive binding assay employing 1,25-(OH)2 D3 and
its tritiated analog are displayed in figure
6.
The curve representing
specific binding indicates that binding was saturable.
71
Scatchard analysis of
Total
-
30
'.4
0
0
25
4J
0
20
'4
'.4
9-4
Non-Specific
15
Specific
10
rn'
5
9
1
2
3
4
2
3
4
2
a
TOTAL 3H-1,25-(OH) 2 D 3 (nM)
Figure 6. Competitive Binding Assay Employing 3 H-1,25-(OH)D3.
1,25-(H)D3, and Rat Anterior Pituitary Cytosol
The curve representing specific binding was determined by subtracting values for
non-specific bound from those for total bound at each point in the curve.
72
this data is displayed in figure
7.
The observed Kd, calculated as the
negative reciprocal of the slope (slope
=
-1/Kd), was 6.0 x 10-10M.
The
approximate Bmax, as indicated by the x-intercept, was 37 fmol/mg protein.
Linear regression analysis of the data yielded a correlation coefficient of
-0.9777, indicating the presence of a single class of receptors.
All of this
data is in good agreement with previous reports by others concerning the binding
characteristics of the rat anterior pituitary receptor for 1,25-(OH)2D3
(113,142,145).
Recovery of Viable Cells in Culture
The number and percentage of viable cells recovered from the enzymatic
dispersion procedure, and subsequently plated in culture, were examined in order
to assess the efficacy of this method for obtaining viable enzymatically
dispersed rat anterior pituitary cells for use in primary culture.
Immediately
prior to seeding cultures, an aliquot of the final cell suspension was diluted
with a trypan blue dye solution, and examined under a microscope.
Cell number
was determined using a hemocytometer.
Cell viability was determined by the
trypan blue dye exclusion test (176).
The percentage of viable cells recovered
from the enzymatic dispersion procedure was then calculated.
The enzymatic dispersion method used to establish primary cultures of rat
anterior pituitary cells yielded greater than 95% viable cells.
Figure 8 is a
photograph of these cells in culture 1 hour subsequent to seeding at a
concentration of 3-5 x 105 cells/ml.
73
.24.22-
Ken 6.0 x 10',
M
.20B~8 5' 37 S..l I.meprotein
.18
0
.14
8
F
0
.12
q
.06
4
.04.021
20
40
60
80
8
100
120
140
180
(pM)
Figre 7. Scatchard Analysis of Binding Data
The data presented in figure 13 was subjected to Scatchard analysis. B = bound
ligand; F = free ligand. Bmax was calculated from the x-intercept, taking
into account the protein content of the cytosol used in this assay (as
determined by the method of Lowry). Kd was calculated as -1/slope.
74
Figure 8. Photograph of Cells in Culture 1 Hour Subsequent to Seeding
Cel s were seeded at a concentration of 3-5 x IOD cells/mi. Greater than 95%
of the cells were viable, as determined by the trypan blue dye exclusion test.
75
Viability and Functionality of Cells in Culture
The viability and functionality of enzymatically dispersed rat anterior
pituitary cells in primary culture was examined in order to determine the
efficacy of the cell culture method employed to study regulation of pituitary
function by 1,25-(OH) 2 D3.
Cell viability under the culture conditions
employed in these studies was determined by trypan blue dye exclusion.
The
number and percentage of viable cells was assayed in triplicate cultures at 24
hour intervals for six days.
cell suspension.
All cultures were seeded from the same initial
Cultures were maintained in serum-supplemented medium for the
first four days, and in serum-free medium for the last two days.
There was no
significant change in the number or percentage of viable cells at any of the
time points examined.
In addition to being viable, it was important to determine that the
cultured cells were functionally intact.
This was achieved by examining basal
hormone secretion over the first four days in culture, and responsiveness to
known secretogogues during the final two day serum-free test period.
Radioimmunoassay of media samples from day 0 and day 4 revealed that basal
hormone secretory capacity was intact (figure 9).
Elevation of media potassium concentration is known to stimulate hormone
secretion by functionally intact pituitary cells in culture (168).
Enzymatically dispersed rat anterior pituitary cells were therefore maintained
for four days in a serum-supplemented medium, and then incubated for 3 hours in
either the control serum-free medium (Ham's F10), or a high potassium test
medium (Ham's F10 supplemented with sufficient potassium to yield a 10 fold
76
>100
20
'
c5
16
z12
DAY
Fligure 9. Basal Secretion of Rat Prolactin From Enzymatically Dispersed Rat
Anterior Pituitary ueiis in Primary Culture
Aliquots of conditioned media were removed from cultures after 4 days of
incubation, and assayed for rPRL content via radioimmunoassay.
values
were compared with those obtained from aliquots of culture mediumThese
taken just
prior to addition to cultures (day 0).
77
increase in concentration).
The cells incubated in the high potassium medium
responded to the treatment by secreting significantly increased amounts of both
rPRL (figure 10) and rTSH (figure 11) into the medium.
Effects of TRH on Secretion of TSH
Since elevation of medium potassium concentration is a relatively
non-specific secretory stimulus, and since the function of rat thyrotropes was
to be a major focus of this thesis, it was important to determine that the
sub-population of TSH-secreting cells present in these primary cultures were
capable of responding normally to a more specific secretogogue.
This was
achieved by examining the effects of thyrotropin-releasing hormone (TRH) on TSH
secretion.
Cultures of enzymatically dispersed rat anterior pituitary cells
were maintained for four days in a serum-supplemented medium, which was then
replaced with a serum-free test medium.
Sets of cultures were pre-incubated in
the test medium for one hour, and then incubated for either 1 hour, 12 hours, 24
hours, or 48 hours.
During the last hour of incubation prior to each of the
time points, sets of cultures were treated with either TRH (at a final
concentration of 0.1 uM) or vehicle.
At each of the time points examined, 1
hour of treatment with 0.1 uM TRH resulted in significantly increased
concentrations of TSH in the medium, as determined by radioimmunoassay (figure
12, table 4).
points.
The amplitude of this effect was similar at all of the time
These results demonstrated that the sub-population of TSH-secreting
cells present in these heterogeneous primary cultures were capable of responding
normolly to a well-characterized specific secretogogue.
78
1.4
1.2
S1.0
cc
IL
0.8
0.6
0
z
0.4
z
0.2-
CONTRSL
g.5 V x*
n - 3;
a:p <0.05
Figure 10. Effect of Increased Media
Potassium Concentration on rPRL Secretion
Cultures of enzymatically dispersed
rat
anterior pituitary cells seeded from-ttie
same initial cell suspension were maintained
for 4 days in a serum-supplemented
medi.uni, which was then replaced with
a
serum-free
medium containing. either
normal (control) or 10-fold increased
(0.5M
K+)
potassium
concentration.
Following a 3 hour test incubation
period, media samples were removed
cultu're and assayed for rPRL content
from each
by radioimmunoassay.
79
1.41
1.2
1.0
0.8
S0.60-
~0
0
a Q4
O .40.2-
0 -.
n -
ME
3;
a: p < 0.05
Figure 11. Effect of Increased Media Potassium Concentration
on rTSH Secretion
Cultures of enzymatically dispersed rat anterior pituitary
cells
seeded from the
same initial cell suspension were maintained for 4 days
in
a
serum-supplemented
medium, which was then replaced with a serum-free medium
normal (control) or 10-fold increased (0.5M K+) potassium containing either
Following a 3 hour test incubation period, media samples concentration.
culture and assayed for rTSH content by radioimmunoassay. were removed from each
80
0
z0.4-
0.1 uM TRH
0 VEHICLE
0.3.
0.2
0.1-
01
12
24
48
TIME (HOURS)
each value is mean + SEM;
2 way ANOVA:
effect of TRH:
p <.005
effect of time:
interaction:
p < .005
NS
Figure 12. Effects of TRH on Secretion of TSH
Following 4 days of incubation in a serum-supplemented medium, sets of cultures
were pre-incubated for 1 hour in a serum-free medium, followed by test
incubations lasting for the indicated durations.
One hour prior to each time
point, sets of cultures were treated with either TRH, at a final concentration
of 0.1 uM (solid circles), or vehicle (open circles). Media samples were
removed at the end of each test period, and subsequently assayed for rTSH
content via radioimmunoassay. Cellular protein content was determined by the
method of Lowry. 4-6 cultures were examined at each point in the figure.
81
rTSH IN MEDIUM (ng/ug protein)
VEHICLE
0.1 uM TRH
0
12 hr
24 hr
48 hr
0.10 + 0.02
0.13 + 0.02
0.20 + 0.03
0.21 + 0.05
(5)
(4)
(5)
(6)
0.18 + 0.04
0.19 + 0.03
0.29 + 0.04
0.30 + 0.03
(5)
(6)
(6)
(5)
each value is mean + SEH;
2 way ANOVA:
n in parentheses.
effect of TRH:
effect of time:
p < .005
p < .005
interaction NS
Table 4.. Effects of TRH on Secretion of TSH
FollOwing 4 days of incubation in a serum-supplemented medium, sets of cultures
were, pre-incubated for 1 hour in a serum-free medium, followed by test
incubations lasting for the indicated durations. One hour prior to each time
point, sets of cultures were treated with either TRH, at a final concentration
of 0.1 uM, or vehicle. Media samples were removed at the end of each test
period, and subsequently assayed for rTSH content via radioimmunoassay.
Cellular protein content was determined by the method of Lowry.
82
Effects of 1,25-(OH)2D3 on the Growth of Rat Anterior Pituitary Cells in
Primary Culture
Given the lack of any previous information concerning the effects of
1,25-(OH) 2 D 3 on the function of non-transformed rat anterior pituitary cells
in primary culture, and the numerous recent reports of effects of this
seco-steroid on the growth of other receptor-positive cell types in culture (186
-196), it was of interest to determine whether the active hormonal form of
vitamin D might similarly affect the growth of receptor-positive rat anterior
pituitary cells.
Several gross parameters of cell growth were examined.
Cultures of enzymatically dispersed rat anterior pituitary cells were maintained
for four days in a serum-supplemented medium, which was then replaced with a
serum-free test medium (Ham's F10).
Following a one hour pre-incubation period,
sets of cultures were treated with either 1,25-(OH) 2 D3 (at final
concentrations ranging from 1O- 7M to 10-1 3M) or vehicle.
The cultures
were then incubated for test periods ranging from 6 hours to 48 hours.
At
various time points, sets of cultures were removed from the incubator,
photographed, and separated into cellular and media samples.
The samples were
kept frozen at -200C prior to being assayed for protein content and DNA
content.
No generalized quantitative effect of 1,25-(OH)2D3 was observed on
cellular protein content (figure 13), cellular DNA content (figure 14), or gross
cellular morphology (figure 15), regardless of the dose of 1,25-(OH)2 D3
administered, or the time of incubation.
Although these results fail to support
the hypothesis that 1,25-(OH)2D3 might affect the growth of
83
o
107% 1,25-() 2-D
0 VEHICLU
so
3
46
~42a
34 a
0
6
9
12
is
24
49
TIME (hours)
Figu'e 13. Effects of 1,25-(OH)2D3 on Cellular Protein Content
A-time course experiment was performed with sets of cultures being incubated in
the presence of either 10- 8 M 1,25-(OH) 2 D3 (open circles) or vehicle
(closed circles). Cell samples were assayed for protein content by the method
of Lowry. Each point represents the mean + SEM for six cultures. Values for
each time point were compared using Student's t-test. No significant
differences were observed at any time point.
84
6.812 HOURS
5.2-
4.4 a
6 HOURS
3.6
2.8
C
-13
-11
-9
LOG [1,25-(OH) 2-D
-8
3
-7
(M)
Figure 14. Effects of 1,25-(OH)2D3 on Cellular DNA Content
Sets of cultures were incubated for either 6 hours (open circles) or 12 hours
(closed circles) in the presence of various doses of 1,25-(OH)2 D3 or
vehicle. Cell samples were assayed for DNA content using the diphenylamine
method of Burton. Each point represents the mean + SEM for six cultures.
Regardless of the dose of 1,25-(OH) 2 D3 employed, no statistically
significant difference in cellular DNA content was observed at either time
point (as determined by analysis of variance).
85
............
.....
.......
-
-
"
r _
8
C
E
F
G
I
J
K
A
I
I
a
I
H
Figure 15.
Effects of 1,25-(OH)2D3 on Gross Cellular
M~rpholog)
Sets ot cult-ures were maintained in tne presence of Mom~5
I,5-uH) 2D3
(+) or vehicle (0) for periods of time lasting as long as 24 hours. Photographs
of the cells were taken at various time points. No obvious morphological
differences were observed in treated vs. control cultures at any time point.
(A) Immediately following 1 hour pre-incubation in serum-free medium (to);
6 hours, 0; (C) 6 hours, +; (D) 9 hours, 0; (E) 9 hours, +; (F) 12 hours, 0;(B)
(G)
12 hours, +; (H) 18 hours, 0; (I) 18 hours, +; (J) 24 hours, 0; (K) 24 hours, +.
86
receptor-positive rat anterior pituitary cells, neither do they contradict such
a possibility.
The problem with interpreting these results lies in the fact
that the experimental model employed in these studies is poorly suited for
observing effects on cell growth which are not gross or generalized.- Most
studies of cell growth employ cultures which are homogeneous in nature.
The
heterogeneous nature of the cell population used in the present studies may have
thus seriously confounded the results.
Possible effects on the growth of
specific sub-populations of cells may have been masked by examining the entire
heterogeneous population simultaneously.
In addition, the possibility of
qualitative changes in either protein synthesis or DNA synthesis induced by
treatment of these cells with 1,25-(OH)2D3 cannot be ruled out.
Such
changes could only be detected, however, by using a homogeneous cell population
and assays designed to reveal qualitative changes in DNA or protein content.
Similarly, possible effects of 1,25-(OH)2D3 on movement of cells through
different phases of the cell cycle could not be examined with a heterogeneous
cell population, except in the unlikely event that all sub-populations of cells
present were similarly affected.
Effects of 1,25-(OH)7D3 on Content of rPRL in Cells and in Media
The only previous investigations of responses induced by treatment of rat
anterior pituitary cells with 1,25-(OH)2D3 involved use of clonal rat
pituitary tumor cells (114,139,146-148,198).
As discussed earlier, these
transformed cells differ from non-transformed rat anterior pituitary cells in a
number of significant ways.
It was therefore important to determine whether
87
non-transformed rat anterior pituitary cells in primary culture respond to
1,25-(OH) 2 D3 in a similar fashion as do their transformed counterparts.
This issue was of further interest due to the contradictory results reported
from various laboratories, which most likely resulted from the use of different
strains of clonal rat pituitary tumor cells, and different cell culture
conditions.
In different laboratories, treatment of rat pituitary tumor cells
with 1,25-(OH)2D3 had thus been reported to both stimulate (139) and inhibit
(114) secretion of rPRL from different strains of GH cells, under different cell
culture conditions.
Cultures of enzymatically dispersed rat anterior pituitary cells were
maintained for four days in a serum-supplemented medium, which was then replaced
with a serum-free test medium (Ham's F10).
Following a one hour pre-incubation
period, sets of cultures were treated with either 1,25-(OH)2D3 (at a final
concentration of 10- 8 M) or vehicle.
A time course experiment was then
initiated, with sets of treated and control cultures being incubated for either
6,9,12,18,24, or 48 hours.
Media and cell samples were collected at each time
point, separated, and frozen at -200C prior to determination of rPRL content
via radioirnmunoassay.
Although some early transient effects were observed,
there was no sustained significant effect of 10-8M 1,25-(OH)2D3 on
either media or cellular content of rPRL (figures 16-19).
Effects of 1,25-(OH)2D3 on Content of rTSH in Cells and in Media
. Several reports in the literature suggested that 1,25-(OH) 2 D3 might
somehow affect the function of TSH-secreting cells in the rat anterior
88
0
T03
.3
-
10M 1,25-(OH) 2 -D 3
VEHICLE
,
0.26-b
0.22a
0.18
0.14-
0.10-
0.06
1
0
6
9
12
7
is
24
49
TIME (hours)
a: p < 0.025
b: p < 0.005
Figure 16. Effects of 1,25-(OH)2D3 on Cellular rPRL Content (ng/ug protein)
Sets of cultures were incubated for various periods of time in the presence of
either 10-8M 1,25-(OH) 2 D3 (open circles) or vehicle (solid circles).
The content of rPRL in cell samples from each time point was determined by
radioimmunoassay.
Cellular protein content was determined by the method of
Lowry. Each value represents the mean + SEM of six cultures. Values from
treated and control cultures at each time point were compared using Student's
t-test.
89
o 10M
0
0.30
1.25-(OH) 2 -D
3
VEHICL3
C
0.25-
0.200
0.15-
0.10-
0.050
6
9
12
01
24
.4
TIME (hours)
a: p < 0.05
Figure 18.
Effects of 1,25-(OH)203 on Media rPRL Content (ng/ug protein)
Sets of cultures were incubated for various periods of time in the presence of
either 10- 8M 1,25-(OH) 2D3 (open circles) or vehicle (solid circles).
The content of rPRL in media samples from each time point was
radieimmunoassay. Cellular protein content was determined by
Lowry. Each value represents the mean + SEM of six cultures.
treated and control cultures at each time point were compared
t-test.
91
determined by
the method of
Values from
using Student's
a
1800
1600
140.
8
AVT
120
dP
100
80
60
40
I
I
0
6
I
9
I
12
I-
18
24
I
48
TIME (hours)
a: p < 0.05
Figure 19. Effects of 1,25-(OH)2D3 on Media rPRL Content (" control)
Sets of cultures were incuDated Tor various periods of time in the presence of
either 10- 8 M 1,25-(OH) 2 D3 (open circles) or vehicle (solid circles).
The content of rPRL in media samples from each time point was determined by
radioimmunoassay. Cellular protein content was determined by the method of
Lowry. Each value represents the mean + SEM of six cultures. Values from
treated and control cultures at each time point were compared using Student's
t-test.
pituitary.
Most notably, a study employing combined autoradiography and
immunohistochemistry (140) demonstrated that
3 H-1,25-(OH)
2 D3
localizes
exclusively in the nuclei of rat thyrotropes, and not in rat anterior pituitary
cells staining positively for PRL, growth hormone, or leutinizing hormone.
It
was therefore of great interest to determine whether 1,25-(OH)2D3 was
capable of altering rTSH secretion in primary cultures of enzymatically
dispersed rat anterior pituitary cells.
In addition, this was an experiment
which could not be performed using commercially available clonal rat pituitary
tumor cells, because these cells only secrete PRL and/or growth hormone.
Cultures of enzymatically dispersed rat anterior pituitary cells were
maintained for four days in a serum-supplemented medium, which was then replaced
with a serum-free test medium (Ham's F10).
Following a one hour pre-incubation
period, sets of cultures were treated with either 1,25-(OH) 2 D 3 (at a final
concentration of 10- 8 M) or vehicle.
A time course experiment was then
initiated, with sets of treated and control cultures being incubated for either
6, 9, 12, 18, 24, or 48 hours.
In a separate experiment, sets of cultures were
pre-incubated for one hour in the serum-free test medium, and then treated with
either vehicle or various doses of 1,25-(OH)2D3, with final concentrations
ranging from 10-1 2M to 10- 8 M.
for 48 hours.
These latter cultures were incubated
Media and cell samples were collected at the end of each
incubation period, separated, and frozen at -200C prior to determination of
rTSH content via radioimmunoassay.
Treatment with 10- 8 M 1,25-(0H)2D3 resulted in a sustained
significant increase in media rTSH content (figures 20-23).
As was the case
with' rPRL content, early transient alterations in rTSH content of media and cell
93
W
2.01.
C
-.4
o
10
*
VEHICLE
m 1,25-(OH) 2-D3
1.
-
C6 1.4
1.2..
1.00.80
0.6 a
0
6
9
12
1I
24
-A
U
46
TIME (hours)
Figure 20. Effects of 1,25-(OH)2D3 on Cellular rTSH Content (ng/ug protein)
Sets 'of cultures were incubated for various periods of time in the presence of
either 10- 8 M 1,25-(OH) 2 D3 (open circles) or vehicle (solid circles)
The content of rTSH in cell samples from each time point was determined by
radioimmunoassay. Cellular protein content was determined by the method of
Lowry. Each value represents the mean + SEM of six cultures. Values from
treated and control cultures at each time point were compared using Student's
t-test.
94
0-4f
.
160
140
*1
8
:B
120
100
80
-r
TA
.I
60
0u
6
9
12
18
TIME
Figure 21.
24
4
48
(hours)
Effects of 1,25-(OH)2D3 on Cellular rTSH Content (% control)
Sets of cultures were incubated Tor various periods of time in the presence of
either 10-8M 1,25-(OH) 2 D3 (open circles) or vehicle (solid circles).
The content of rTSH in cell samples from each time point was determined by
radioimmunoassay. Cellular protein content was determined by the method of
Lowry. Each value represents the mean + SEM of six cultures. Values from
treated and control cultures at each time point were compared using Student's
t-test.
95
a
0 1o-aH 1,2S-(On) -0
2 3
C 3.0
VEHICLZ
,
b
S2.0
0
1.0
0
q
12
is
24
TIME
48
(hours)
a: p < 0.0005
b: p < 0.025
Figure 22. Effects of 1,25-(OH)2D3 on Media rTSH Content (ng/ug protein)
Sets of cultures were incubated for various periods of time in the presence of
either 10- 8 M 1,25-(OH) 2 0 3 (open circles) or vehicle (solid circles).
The content of rTSH in media samples from each time point was determined by
radioimmunoassay. Cellular protein content was determined by the method of
Lowry. -Each value represents the mean + SEM of six cultures. Values from
treated and control cultures at each time point were compared using Student's
t-test.
96
180.
0
U
b
a
160
140
120-
8
100 -L
E-
8060.
I
I
-
0
6
I
9
12
I
U
18
24
-
T
48
TIME (hours)
a:p <
0.0005
b:p < 0.025
Figure 23. Effects of 1,25-(OH)2D3 on Media rTSH Content (% control)
Sets of cultures were incubated for various periods of time in the presence of
either 10- 8 M 1,25-(OH) 2 D3 (open circles) or vehicle (solid circles).
The content of rTSH in media samples from each time point was determined by
radioimmunoassay. Cellular protein content was determined by the method of
Lowry. Each value represents the mean + SEM of six cultures. Values from
treated and control cultures at each time point were compared using Student's
t-test.
97
samples were also observed.
Following longer periods of incubation, however,
media rTSH content remained significantly elevated, unlike the effects observed
for rPRL.
The effect of various doses of 1,25-(OH)2D3 on media rTSH content
was examined, and no significant effect was observed at doses lower than
10-8M (figure 24).
In order to determine the structural specificity of the effect of
1,25-(OH)2D3 on rTSH secretion, sets of cultures prepared according to the
same protocol used in the previous experiment were incubated for 24 hours in the
presence of either 10- 8 M 1,25-(OH) 2 D3, vehicle, or a variety of
concentrations of 10,25-(OH)2D3 (at final concentrations of 10- 8 M,
1o-7M, or 10- 6 M), an analog of 1,25-(0H) 2 D3 which had been
demonstrated to be biologically inactive in other target tissues.
Media and
cell samples were collected, and rTSH content determined via radioimmunoassay.
As shown in figure 25, all doses of 1 ,25-(OH)2 D3 failed to alter either
cellular or media content of rTSH.
These results further supported the
hypothesis that 1,25-(OH)2D3 is capable of stimulating rTSH secretion via a
direct receptor-mediated effect.
Effects of 1,25-(OH)2D3 on Synthesis of rTSH
Although cellular content of rTSH was not statistically significantly
elevated by treatment with 10- 8 M 1,25-(OH) 2 D3, it remained possible that
98
-
140
120
-
:.
100
8
80
..
19
I
C
1
-12
-
60
-11
-10
-9
-8
LOG [1,25-(OH) 2-D3 1 (M)
a: p < 0.05
Figure 24. Effects of 48 Hour Treatment with Various Doses of 1,25-(OH)2D3
on Media rTSH Content
Sets of cultures were incubated for 48 hours in the presence of a variety of
concentrations of 1,25-(OH)2D3 or vehicle (C). The content of rTSH in media
samples from each culture was determined by radioimmunoassay. Cellular protein
content was determined by the method of Lowry. Content of rTSH was calculated
in terms of ng rTSH/ug protein, and then converted into % control. Each value
represents the mean + SEM of four cultures.
99
5.0
a
.,3
4.0"
0
3.0
@
2.0"
1.0T
V
-8
-8
A
B
-7
B
-6
V
B
-8
A
MEDIA
-8
B
-7
B
-6
B
CELLS
V: Vehicle; A: 1,25-(OH) D ; B: 1,25-(OH)2
2 3
D3
Numbers are -log of the concentration of the compound
(M).
a: p < 0.005.
Figure 25. Effects of 1s,25-(OH)2D3 on Media and Cellular rTSH Content
Sets of cultures were incubated for 24 hours in the presence of either_10-8 M
1,25-(OH)2D3, vehicle, or a variety of concentrations of 1$,25-(OH)2D3.
The content of rTSH in media and cell samples was determined by
radioimmunoassay. Cellular protein content was determined by the method of
Lowry. Each value represents the mean + SEM of six cultures. Statistical
significance of differences between eaci treatment group and control was
determined using Student's t-test.
100
1,25-(OH)2D3 was capable of stimulating synthesis of new rTSH, an effect
whose magnitude might be too small to be observed against the background of
total cellular rTSH content.
To investigate this possibility, the effects of
1,25-(OH) 2 D 3 on incorporation of
3 5S-methionine
into newly synthesized
rTSH was examined.
Cultures of enzymatically dispersed rat anterior pituitary cells were
maintained for four days in a serum-supplemented culture medium, which was then
replaced with a serum-free test medium (Ham's F10).
Following a one hour
pre-incubation period, sets of cultures were treated with
35 S-methionine
(70
uCi/well) and either 1,25-(OH) 2 D3 (at a final concentration of 10- 8 M) or
vehicle.
The cultures were then incubated for 24 hours, after which time media
and cell samples were collected, separated, and frozen at -200C prior to
assays.
Cell samples were subsequently assayed for content of
3 5 S-methionine-labelled-rTSH
procedure.
using a double-antibody immunoprecipitation
This assay revealed that 24 hours treatment with 10- 8 M
1,25-(OH) 2 D 3 had resulted in significantly increased incorporation of
3 5 S-methionine
into newly synthesized immunoprecipitatable rTSH (table 5).
In order to confirm that the immunoprecipitated material was in fact rTSH, both
treated and control samples were subjected to gel electrophoresis on 10-20%
gradient polyacrylamide gels containing 0.1% SDS.
Two peaks of radioactivity
were observed in the gels, corresponding to the 18,000 MW alpha and 22,000 MW
beta subunits of rTSH.
The radioactivity in both of these peaks was increased
in samples from cultures which had been treated with 1,25-(OH) 2 D 3 (figure
26,..t'able 6).
101
IMMUNOPRECIPITATED 35S-METHIONINE-rTSH
CPM/SAMPLE
VEHICLE
CPM/ ug PROTEIN
4110 + 197
208 +
(7)
10- 8 m 1,25-(OH) 2 -D
3
12
(7)
6041 + 685 a
319
(7)
+
44
b
(7)
each value is mean + SEM; n in parentheses.
a) Student's t-test:
p <.005
b) Student's t-test:
p <.025
Table 5. Effects of 1,25-(OH)2D3 on Incorporation of 35 S-Methionine
into'Newly Synthesized Immunoprecipitatable rISH
Sets of cultures were incubated for 24 hours in the presence of
3 5 S-methionine (70
uCi/culture) and either 10- 8 M 1,25-(OH) 2 0 3 or
vehicle. Cellular content of 35S-methionine-labelled-rTSH was determined by
liquid scintillation counting of double-antibody immunoprecipitated material.
102
C
0
I.
Sn
I
600.
-
5000
10-8M 1,25-(OH)
2 -D
0
CONTROL
3
400-
300.
200.
100
5
10
15
20
25
30
35
GEL SLICE (1 mm)
Figure 26.
Gel Electrophoresis of Immunoprecipitated
abJS-MKethionine-Labelled-rTSH
Typical profiles of radioactivity observed in 10%-20% gradient polyacrylamide
gels containing 0.1% SDS following electrophoresis of double-antibody
immunoprecipitated material from cellular samples of cultures treated for 24
hours with either 10-8M 1,25-(OH) 2D3 (solid circles) or vehicle (open
circles).
103
CPM ADDED
TOTAL CPN
IN CEL
RECOVERY
CPM IN
TSH PEAKS
2 OF RECOVERED
CPM IN TSH PEAKS
CONTROL
4100
1714
41.82
1351
78.82
1,25-(OH) 2-D3
6000
2538
42.3Z
2102
82.82
*
cpm in duplicate innunoprecipitates resolubilized in buffer and counted.
Table 6. Recovery of Radioactivity Added to Gel Following Electrophoresis of
Immunoprecipitated JbS-Methionine- Label led-rTSH
Typical profile of recovery and distribution of radioactivity added to gel.
104
In order to determine whether this effect was specific for rTSH, aliquots
of the same samples were assayed for content of
35 S-methionine-labelled-rPRL.
A similar double-antibody immunoprecipitation
procedure was employed for this assay.
Treatment with 10- 8 M
1,25-(OH)2D3, in this case, failed to produce any significant difference in
incorporation of
35 S-methionine
into newly synthesized rPRL (table 7).
As an additional check on the specificity of the effect, the incorporation
of
35 S-methionine
into TCA-precipitatable proteins was examined.
Once
again, treatment with 10- 8 M 1,25-(OH) 2 D3 failed to produce any
significant difference in incorporation of
3 5 S-methionine
into newly
synthesized TCA-precipitatable protein, thus ruling out any generalized
non-specific effect of the seco-steroid on protein synthesis (table 8).
Finally, in order to determine the structural specificity of
1,25-(OH)2D3 for stimulation of rTSH synthesis, sets of cultures prepared
according to the same protocol described above were incubated for 24 hours with
3 5 S-methionine
and either 10- 8 M l,25-(OH) 2 D 3 , vehicle, or a variety
of doses of 1,25-(OH)2 D3 (ranging in final concentration from 10- 8 M to
.
10- 6 M), a biologically inactive structural analog of
1,25-(OH) 2 D 3
Although this analog differs only slightly in structure from 1,25-(OH)
2 D3
(the 1-hydroxyl group being in the beta position instead of alpha), it has been
found to not compete for binding to the 1,25-(OH) 2 D3 receptor, and to lack
biological activity in other well-characterized 1,2 5 -(OH)2D3-receptor
systems.
As shown in figure 27, double-antibody immunoprecipitation of newly
synthesized
3 5 S-methionine-labelled-rTSH
revealed that all concentrations of
10,25-(OH)2D3 were incapable of stimulating synthesis of rTSH.
105
IMMUNOPRECIPITATED 35S-METHIONINE IABELED rPRL
CPM/SAMPLE
VEHICLE
107 M 1,25-(OH) 2-D3
each value is
mean
Student's t-test:
CPM/ug PROTEIN
570 + 20
58.4 + 3.6
(6)
(6)
625 + 50
64.2 + 6.6
(6)
(6)
+ SEM; n In parentheses.
NS
Table 7.. Effects of 1,25-(OH) D3 on Incorporation of 35 S-Methionine
into Newly Synthesized Immunoprecipitatable rPRL
Sets-of cultures were incubated for 24 hours in the presence of
3 5 S-methionine
(70 uCi/culture) and either 10-8M 1,25-(0H) 2 D3 or
vehicle. Cellular content of 35 S-methionine-labelled-rPRL was determined by
liquid scintillation counting of double-antibody immunoprecipitated material.
106
TCA-PRECIPITATED 3SS-METHIONINE LABELED PROTEINS
CLASS FIBER FILTERS
3MN FILTERS
CPM/SAMPLE
2010 + 200
VEHICLE
(6)
2277 + 181
10OH 1,25-(OH)2-D3
(6)
CPM/ug PROTEIN
CPM/SAMPLE
CPM/ug PROTEIN
1048 + 148
808 + 172
397 + 70
(6)
(6)
(6)
1210 + 142
(6)
918 +
(6)
180
498 + 116
(6)
each value is mean + SEM; n In parentheses.
Student's t-test:
HS
Table 8. Effects of 1,25-(OH)2D3 on Incorporation of 3 5 S-Methionine
into Newly Synthesized TCA-Precipitatable Proteins
Sets of cultures were incubated for 24 hours in the presence of
3 5 S-methionine (70 uCi/culture)
and either 10- 8 M 1,25-(OH) 2 D 3 or
5
vehicle. Cellular content of 3 S-methionine-labelled-proteins was
determined by liquid scintillation counting of TCA-precipitated material. Two
different filter systems were used in the precipitation procedure, both
producing similar results.
107
4000
a
3500"
3000
2500
V
-8
A
-8
B
-7
B
-6
B
V: Vehicle; A: 1,25-(OH) 2 D3 ; B: lU,25-(OH) 2 D 3 ;
Numbers are -log of the concentration of the compound (M).
a:p <0.025.
Figure 27. Effects of 10,25-(0H)ZD3 on Incorporation of 35 S-Methionine
into Newly Synthesized Immunoprecipitatable rTSH
Sets of cultures were incubated for 24 hours in the presence of
3 5 S-methionine
(70 uCi/culture) and either 10-8M 1,25-(OH) 2D3,
vehicle, or a variety of concentrations of 10,25-(0H) 2 D3. Cellular content
of 3 5 S-methionine-labelled-rTSH was determined by liquid scintillation
counting of double-antibody immunoprecipitated material. Each value represents
the mean + SEM of six cultures. Statistical significance of differences between
each treatment group and control was detennined using Student's t-test.
108
DISCUSSION
Prior to the studies described in this thesis, no investigations had been
performed concerning the direct effects of 1,25-(OH)2D3 on the function of
non-transformed anterior pituitary cells.
Several in vivo studies had been
conducted revealing changes in pituitary hormone secretion following
administration of 1,25-(OH)2D3 or other vitamin D metabolites, but none had
unequivocably demonstrated a direct receptor-mediated response induced following
activation of anterior pituitary receptors for 1,25-(OH) 2 D3 . According to
the classical pharmacological definition, a receptor macromolecule must
specifically bind a ligand, with this interaction producing a biological
response.
Although a specific binding protein for 1,25-(OH) 2 D3 had been
identified in anterior pituitaries of several species, there had been no reports
of responses induced by the ligand-binding protein interaction.
Many specific
binding proteins have previously been identified which are not receptors.
Transport proteins and storage proteins are two examples.
receptors are usually tissue-specific.
Furthermore,
It is thus impossible to conclude that a
specific binding protein is a receptor simply because it specifically binds a
ligand.
The data described in this thesis thus represents the first conclusive
evidence that the specific binding protein for 1,25-(OH) 2D3 present in
non-transformed rat anterior pituitary cells is a receptor.
The existence of a
receptor for 1,25-(OH)2D3 had been previously demonstrated in various
strains of clonal rat pituitary tumor cells.
For several reasons, however, it
was not possible to conclude that the specific binding protein for
109
1,25-(OH)2D3 present in non-transformed rat anterior pituitary cells was a
receptor on the basis of these reports.
Since receptors are often
tissue-specific, the existence of a receptor protein in clonal pituitary tumor
cells does not necessarily imply that this receptor is expressed in
non-transformed cells of similar origin.
Neoplastic cells, by nature, express
many proteins not seen in their non-transformed counterparts.
In addition, the
particular tumor cells used in these studies differ from non-transformed rat
anterior pituitary cells in several significant respects.
The distribution of
calcium among intracellular compartments, and the size of the pool of stored
intracellular calcium, have been reported to be different in GH cells as
compared with non-transformed rat anterior pituitary cells (238).
It has also
been found that, unlike normal pituitary cells, GH cells do not store hormones
following synthesis (239).
GH cells.
Secretion is thus completely linked to synthesis in
Furthermore, while normal pituitary cells only synthesize and secrete
individual types of hormones, GH cells are capable of synthesizing several
different hormones simultaneously (an indication of inappropriate expression of
proteins)(240).
Finally, and of particular interest to the work described
herein, GH cells do not secrete TSH.
Sar, et al.
This is significant due to the report by
(140), demonstrating through combined autoradiography and
immnunohistochemistry that radioactivity from
3 H-1,25-(OH)
2 D3
can only be
detected in the nuclei of rat anterior pituitary cells staining positively for
rTSH.
Even if
these significant differences were to be ignored, one must still
view any attempts to extend the findings in tumor cells to non-transformed cell
physiology with skepticism simply on the basis of the lack of consistency in the
110
responses observed in different laboratories, using different strains of cells
and different cell culture conditions.
It was therefore reasonable to speculate
that although the same receptor protein may have been expressed in both clonal
rat pituitary tumor cells and their non-transformed counterparts, the
differences in expression of other cellular proteins might lead to significant
differences in the responses observed following activation of the receptor in
these cells.
Activation of the receptor for 1,25-(OH) 2 D3 present in a sub-population
of non-transformed rat anterior pituitary cells was observed to result in
stimulation of both synthesis and secretion of rTSH.
The specific nature of
the effect was indicated by the lack of any effect of this seco-steroid on
synthesis or secretion of rPRL, and on synthesis of TCA-precipitatable proteins.
The fact that a close structural analog of 1,25-(OH)2D3 (1,25-(OH)2D3)
was incapable of similarly stimulating rTSH synthesis and/or secretion supported
the hypothesis that this effect was not a non-specific effect which might be
induced by any seco-sterol, and is probably receptor-mediated.
The observed effect of 1,25-(OH)2D3 on the function of cells
synthesizing and secreting rTSH was consistent with many previous reports in the
literature implying a possible relationship between these two endocrine systems.
As mentioned above, the combined autoradiographic and immunohistochemical study
of Sar, et al. (140) suggested that 1,25-(OH) 2 D3 might somehow affect the
function of TSH-secreting cells in the rat anterior pituitary.
Stumpf, et al. published an abstract (159) describing effects of
1,25-(OH)2D3 on circulating TSH levels in intact and thyroidectomized
rachi'tic rats.
In both cases, vitamin D-deficient rats treated with
111
1,25-(OH)2D3 exhibited elevated serum levels of TSH.
administered 25-OH-D 3 and calcium orally to healthy
Zafkova, et al. (160)
Czechoslovakian women and
measured serum thyroxine, TSH, calcium, and magnesium.
Thyroxine was the only
parameter affected by such treatment, and it was elevated.
Suzuki, et al. (162)
reported effects of la-OH-D 3 in a case study of a woman being treated for
hypocalcemia due to pseudohypoparathyroidism.
Endocrine tests in the
hypocalcemic state revealed an exaggerated response of TSH to TRH, and a blunted
GH response to arginine-HCl.
Both of these abnormalities were rectified
following restoration of normocalcemia by giving la-OH-D 3 .
Finally, Tornquist
and Lamberg-Allardt recently presented an abstract in which they describe
effects of 1,25-(OH)2D3 on the regulation of TSH secretion in rats (199).
These investigators injected rats with 1,25-(OH) 2 D 3 (5 ug/kg/day) for 3
days, and then injected TRH i.v. 8 hours after the last dose.
At various times
thereafter, samples of blood were withdrawn from carotic artery cannulae.
It
was found that 1,25-(OH)2 D3 significantly increased the response of TSH to
TRH.
Although all of these studies suggest changes in the function of pituitary
thyrotropes following administration of vitamin D-metabolites, none of them
demonstrate conclusively a direct effect of 1,25-(OH)2D3 in the anterior
pituitary.
In addition, there have been several reports of changes in thyroid status
resulting in physiological conditions which might be attributable to alterations
in the activity or metabolism of 1,25-(OH) 2D3.
For example, it has been
well documented that hyperthyroidism results in increased bone turnover,
osteoporesis, reduced intestinal calcium absorption, and hypercalciuria
(221-226).
It has further been shown (227-229) that elevated levels of
112
24,25-dihydroxyvitamin D3 and reduced levels of 1,25-(OH)2D3 are present
in hyperthyroidism, and that the opposite effects on vitamin D metabolism are
seen in hypothyroidism.
Finally, it has been demonstrated that TSH and the
thyroid hormones T3 and T4 are potent inhibitors of 25-OH-D3-la
-hydroxylase activity in vitro, using a perfused rat kidney system (197).
There is thus ample evidence suggesting that a physiologically significant
relationship might exist between the vitamin D and thyroid endocrine systems.
The present data suggest that 1,25-(OH) 2 D3 may be one of many circulating
factors involved in the complex physiological regulation of rTSH secretion in
vivo.
The implied existence of a feedback system involving effects of
1,25-(OH) 2 D 3 on the activity of cells which produce a factor (rTSH) which is
capable of regulating 1,25-(OH) 2D3 synthesis is not unique.
Such a system
has been suggested for regulation of parathyroid hormone secretion by
1,25-(OH) 2 D3 (111,112).
A more general analogy can be seen with the
feedback system whereby adrenal corticosteroids inhibit the synthesis of ACTH in
the anterior pituitary, with ACTH being involved in the regulation of
corticosteroid synthesis in the adrenal cortex (136).
The failure to observe any changes in either synthesis or secretion of rPRL
is also of significance.
As discussed above, the only previous reports
concerning effects of 1,25-(OH) 2 D 3 on the activity of pituitary cells
demonstrated alterations in rPRL synthesis in clonal rat pituitary tumor cells.
The lack of any effects of 1,25-(OH)2D3 on rPRL synthesis or secretion by
non-transformed rat anterior pituitary cells in primary culture supports the
speculation that the effects observed in tumor cells do not necessarily mimic
events occurring in their non-transformed counterparts.
113
While such tumor cell
studies are of great value in elucidating the biochemical mechanisms involved
in hormone secretion, they may not always be of as much value for determining
the ultimate physiological responses induced by receptor activation.
In the
present case, it is possible to speculate that the anterior pituitary receptor
for 1,25-(OH)2D3, like many other cellular proteins, may be inappropriately
expressed in these PRL-secreting tumor cells.
The various strains of GH cells
may, however, still represent a powerful tool for investigating the biochemical
mechanism by which 1,2 5 -(OH)2D3-receptor activation results in hormone
synthesis.
Finally, the studies described herein concerning effects of
1,25-(OH)2D3 on anterior pituitary cell growth deserve further attention.
In primary cultures of enzymatically dispersed rat anterior pituitary cells, no
generalized quantitative effect of 1,25-(OH)2D3 was observed on protein
synthesis, DNA synthesis, or the morphological distribution of cells.
the heterogeneous nature of the cell population, however, it
Due to
is not possible to
rule out effects of this seco-steroid on the growth of particular
sub-populations of cells.
In addition, qualitative changes in DNA synthesis and
protein synthesis were not adequately examined.
Possible effects on movement of
cells through different phases of the cell cycle were also not addressed.
Unfortunately, none of these experiments can be performed appropriately using a
heterogeneous cell population.
A different experimental model would be
required.
The possibility of such a selective effect of 1,25-(OH) 2 D3 on pituitary
cell growth remains of great interest.
It appears clear that 1,25-(OH) 2 -D
affects its target tissues through a classical steroid mechanism of action
114
3
(86,98).
This involves binding of the hormone to a cytosolic receptor,
translocation to the nucleus, and interaction of the hormone-receptor complex
with the chromatin.
In the past, most research in this area has been concerned
with the effects of this interaction on the synthesis of proteins which are
presumed to be involved in calcium transport (230,231).
If one considers that
alteration of calcium transport might not be the ultimate effect of
1,25-(OH) 2 -D3, then it
is possible to speculate that these same proteins, or
other as yet unidentified induced proteins, could be involved in the regulation
of target cell maturation.
In support of this possibility, there is evidence
that intracellular calcium content, or calcium fluxes, may be involved in the
commitment of cells towards an end-differentiated state (232-234).
There is
also evidence for a role of calcium binding proteins in the regulation of cell
differentiation (235-237).
1,25-(OH) 2 -D
3
Furthermore, it is possible to speculate that
might be capable of affecting cellular development through a
mechanism unrelated to induced protein synthesis.
It is conceivable, for
example, that the mere binding of 10,000 occupied receptors to a cell's
chromatin might be sufficient to alter the dynamics of cell replication and
differentiation.
Such an effect might be dependent upon the particular target
tissue, the stage of development of a particular cell, and the stage of the cell
cycle during which the binding occurs.
An interesting possibility is that the
production of specific hormones by pituitary cells in culture could be
influenced through changes in the state of differentiation of immature cells, or
the rate of replication of proliferating cells.
115
SUMMARY AND CONCLUSIONS
Summary of Results
1.
The presence of a specific binding protein for 1,25-(OH) 2 D3 in both
cytosolic and nuclear extracts of rat anterior pituitary was confirmed.
This
protein exhibited a sucrose density gradient sedimentation coefficient of
3.5-3.7 S.
The dissociation constant (Kd) for the binding of 1,25-(OH)2D3
to this protein was determined to be 6.0 x 10-10M, with a Bmax of
approximately 36 fmol/mg protein.
2.
A previously described cell culture methodology was employed to study the
possible receptor-mediated effects of 1,25-(OH)2D3 on the activity of
enzymatically dispersed rat anterior pituitary cells in primary culture.
The
enzymatic dispersion procedure yielded greater than 95% viable cells at the time
of seeding of the cultures.
When these cells were incubated for 4 days in a
serum-supplemented culture medium, followed by 2 days of incubation in a
serum-free medium, the number and percentage of viable cells remained unchanged.
3.
Radioimmunoassay of conditioned medium from cultures incubated for the
initial 4 day period revealed that basal hormone secretory capacity was intact.
These cells were capable of responding normally to both non-specific (increased
media potassium concentration) and receptor-mediated (TRH) secretory stimuli.
116
4.
Studies were performed to determine the effects of 1,25-(OH)2D3 on
several parameters of enzymatically dispersed rat anterior pituitary cell
function in primary culture.
A.
Regardless of the time of incubation or the dose of 1,25-(OH)2D3
administered, no significant effects were observed on any of the indices of
cell growth examined.
These included cellular protein content, cellular DNA
content, cell number, and gross cellular morphology.
B.
When cultures were incubated with 10- 8 M 1,25-(OH) 2D3 for periods
of time ranging from 6 hours to 48 hours, there was no consistent significant
alteration in either cellular or media content of rPRL.
C.
When cultures were incubated with 10-8M 1,25-(OH)2D3 for periods
of time ranging from 6 hour to 48 hours, media content of rTSH was found to be
significantly elevated at both the 24 hour and 48 hour time points.
This
effect was found to be dose dependent, with an ED 50 between 10- 8 M and
10- 9 M at 48 hours.
Although cellular content of rTSH was not
statistically significantly altered during the time course experiment, a trend
towards increased cellular rTSH content was observed, suggestive of a possible
stimulatory effect on rTSH biosynthesis.
1.
The structural specificity of 1,25-(OH)2D3 for stimulating secretion
of rTSH was determined by incubating cultures for 24 hours in the presence
Qf a variety of concentrations of 10,25-(OH)2D3.
117
The only difference in
structure between 1,25-(OH)2D3 and this analog is the sterochemical
configuration of the 1-hydroxyl group.
This experiment was performed to
rule out any non-specific steroid effect of 1,25-(OH) 293 , since it had
been previously demonstrated that a change in the sterochemistry of the
1-hydroxyl group from alpha to beta completely abolished its biological
activity and its binding to the receptor.
All concentrations of this analog
failed to have any significant effect on either media or cellular content of
rTSH.
D.
Possible effects of 1,25-(OH)2D3 on rTSH biosynthesis were examined by
measuring incorporation of
3 5S-methionine
into newly synthesized
immunoprecipitatable rTSH following 24 hours of incubation in the presence of
10- 8 M 1,25-(OH)2D3.
Using this methodology, it was found that
treatment with 1,25-(OH)2D3 resulted in a significant increase in
incorporation of
35S-methionine
into newly synthesized
immunoprecipitatable rTSH, suggesting that 1,25-(OH)2D3 is capable of
stimulating synthesis of rTSH.
1.
of
The specificity of this effect was examined by measuring incorporation
35 S-methionine
into both TCA precipitatable proteins and
immunoprecipitatable rPRL following 24 hours of incubation in the presence
of 10- 8 M 1,25-(OH) 2 D3.
Neither of these parameters were
significantly affected.
118
2.
The structural specificity of 1,25-(OH)2D3 for stimulating rTSH
biosynthesis was determined by measuring incorporation of
35 S-methionine
into newly synthesized immunoprecipitatable rTSH following 24 hours of
incubation in the presence of a variety of concentrations of
10,25-(OH)2D3.
All concentrations of this analog failed to
significantly alter rTSH biosynthesis.
The effect was thus shown to be
specific for 1,25-(OH) 2 D3, and not a non-specific effect which might be
induced by any seco-sterol.
Conclusions
1.
A receptor for 1,25-(OH) 2 D3 is present in rat anterior pituitary.
2.
1,25-(OH)2D3 stimulates both synthesis and secretion of rTSH by the
sub-population of rat thyrotropes present in primary cultures of enzymatically
dispersed rat anterior pituitary cells.
3.
1,25-(OH) 2 D 3 has no sustained effect on either synthesis or secretion of
rPRL by the sub-population of rat mammotrophs present in primary cultures of
enzymatically dispersed rat anterior pituitary cells.
4.
1,25-(OH) 2 D3 has no effect on the parameters of cell growth examined in
this.thesis, although selective effects on the growth of specific
sub-populations of cells present in rat anterior pituitary cannot be ruled out.
119
SUGGESTIONS FOR FUTURE RESEARCH
Although the work described in this thesis has shed some light on the
function of the pituitary receptor for 1,25-(OH) 2 D 3 , it also raises a number
of interesting questions which might be studied in the future.
Some of these
include:
1.
What is the molecular mechanism involved in the effects of 1,25-(OH)2 D3
on rTSH biosynthesis and secretion?
Is calcium transport involved?
Is the
vitamin D-dependent calcium binding protein involved?
2.
Does 1,25-(OH) 2 D3 affect the growth of specific sub-populations of rat
anterior pituitary cells? Is 1,25-(OH) 2 D3 perhaps capable of altering the
growth of receptor-positive anterior pituitary cells at certain defined times,
such as in the young developing pituitary?
3.
Are the effects of 1,25-(OH)2D3 dependent on other aspects of endocrine
status, or on the age of the pituitary tissue being studied ?
4.
Is there a similar receptor for 1,25-(OH)2D3 present in human pituitary'?
If so, does 1,25-(OH) 2 D3 similarly affect the activity of receptor-positive
human anterior pituitary cells ?
5.
I's 1,25-(OH) 2 D3 capable of altering the growth and/or activity of
neoplastic anterior pituitary cells which secrete TSH ?
120
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BIOGRAPHICAL NOTE
Stanley David Rose was born on February 2, 1956, in New York, New York.
raised in Greenburgh, New York, and attended Woodlands High School.
He was
During his
senior year, he was awarded his high school's math award, and a New York State
Regents Scholarship.
In September, 1974, he enrolled at Cornell University.
was awarded the Bachelor of Arts degree in psychology in May, 1978.
He
The
following year he worked at Harvard Medical School, as a research assistant for
Dr. Peter Dews in the Psychobiology Laboratory.
In September, 1979, he enrolled
in the graduate program in Neural and Endocrine Regulation at the Massachusetts
Institute of Technology.
While pursuing his doctorate, he was awarded
fellowships by the National Institute of Drug Abuse and the National Institute
of Mental Health.
He was also a National Institutes of Health graduate trainee.
141