AMERICAN JOURNAL OF PHYSIOLOGY
Vol. 222, No. 2, February
1972. Printed
in
U.S.A.
Effects of vasoactive agents on isolated
umbilical
arteries and veins
BURTON
M. ALTURA,
D. MALAVIYA,
CHARLES
Departments of Anesthesiology and Physiology, Albert Einstein
Bronx, New York 10461
ALTURA, BURTON M., D. MALAVIYA, CHARLES F. REICH, AND
Effects of vasoactive agents on isolated human umLOUIS R. ORKIN.
bilical arteries and veins. Am. J. Physiol.
222(Z):
345-355.
1972.In vitro
experiments,
using
both
helically
and longitudinally
cut human
umbilical
arteries
and veins (HUAV),
were designed
to determine:
I) whether
any of the known
circulating
vasoactive
substances
can induce
contraction
of these vessels in low (physiologic?) concentrations;
2) the relative
potency
of these substances;
3) the existence
of specific
drug receptors
which subserve
contraction; and 4) the relative
contribution
each type of smooth
muscle
layer (i.e., longitudinal
vs. circular)
may play in response
to vasoactive agents
and in development
of spontaneous
mechanical
activity.
The results
indicate
: 1) 10 different
vasoactive
agents
(amines,
polypeptides,
prostaglandins,
and
potassium
ions)
induce varying
degrees
of unequal
contractile
responses
(serotonin = maximum);
2) only serotonin
( <O. 1 pg/liter),
bradykinin (0.5-4 pg/liter),
and oxytocin
(l-5
mU /liter)
induce
contractile responses
in very
low
(physiologic?)
concentrations;
3) specific
drug
receptors
appear
to exist for all 10 agents
in
HUAV;
4) dose-response
curves
for longitudinally
cut strips
do not always parallel
those of the helically
cut strips;
and 5)
circular
smooth
muscle
appears
to play a greater
role than longitudinal smooth muscle
in spontaneous
mechanical
activity
of
HUAV.
vascular
smooth
ous activity;
drug
muscle; bradykinin;
umbilical
cord; spontanereceptors;
neonatal circulation;
neurohypophyseal hormones; prostaglandins;
serotonin; catecholamines
CONTROL
OF UMBILICAL
CORD
BLOOD
FLOW
IN MAN
is, at
present, largely speculative ( 14). Although
it is well known
that the primary factor contributing
to the low fetal systemic
vascular resistance is the umbilicoplacental
circulation
(9),
the exact site or sites of this resistance is not known. At birth,
interruption
and eventual obliteration
of the umbilical
cord
vessels take place. The exact mechanism
responsible
for the
latter is, however, not known ( 15).
Recently, several investigators
have perfused intact umbilical cords and segments of umbilical
arteries in vitro in
order to assess the pharmacologic
behavior of these vessels to
various drugs and humoral
substances ( 11, 13, 17, 19, 24,
25). But as early as 1938, von Euler (35) noticed that it is
extremely dificult,
if not impossible,
to obtain physiologic
flow values in such perfused preparations
even if perfused at
normal physiologic
pressures. In 1965 gtembera and his colleagues (33) measured flow in vivo in the umbilical
vein by
a local thermodilution
technique
just after birth and re-
human
F. REICH,
AND LOUIS
R. ORKIN
College of Medicine of Yeshiva University,
ported a mean of 75 ml of blood per minute per kilogram
of
baby’s weight or approximately
248 ml/min
in an average
fetus weighing
3.3 kg. Yet the highest reported flows in perfused preparations
are 60-80 ml/min
(25, 35). Furthermore,
in 1967 Gokhale and his co-workers
( 19) working
with intact human umbilical
cords in vitro, in which only one artery was perfused, reported
a tremendous
variability
from
one preparation
to another
in response to drugs. None of
these previous studies with perfused umbilical
arteries and
veins clearly demonstrates,
quantitatively,
the relative sensitivity of either human umbilical
arteries or veins to vasoactive substances. Attempts
in this direction
have recently
been made by Somlyo et al. (30) using helically cut umbilical artery and vein strip preparations
as well as by Eltherington and his colleagues ( 17), using perfused segments and
isolated rings of human
umbilical
artery. Although
these
latter two studies, when taken together, have demonstrated
a high sensitivity
of these vessels to serotonin
and bradykinin, studies on quantitative
dose-response
relationships
and the existence of specific drug receptors have not been
reported
in detail.
In addition,
since, histologically,
human
umbilical
arteries and veins contain two layers of smooth muscle (27,
3 1, 32), an inner one oriented longitudinally,
and the outer
one circularly,
it is impossible
to determine
from perfused
preparations
or helical strip preparations
alone the relative
contribution
or role each type of oriented
smooth muscle
layer may play in response to vasoactive substances. In vitro
using both helically
and longitudinally
cut
experiments,
human umbilical
arteries and veins, were therefore designed
to determine:
1) whether any of the known circulating
vasoactive substances can induce contraction
of these vessels in
low (physiologic?)
concentrations;
2) the relative potency
of these substances; 3) the existence of specific drug receptors
which subserve contraction;
and 4) the relative contribution
each type of smooth muscle layer may play in response to
vasoactive agents.
METHODS
Collection and preparation of human umbilical arteries and veins
for in vitro study. A total of 90 human umbilical
cords from
normal
full-term
spontaneous
deliveries
were utilized
for
this study (Caesarean
sections were not utilized).
Cords from
mothers
exhibiting
eclampsia,
hypertension,
diabetes,
an
Rh factor problem, or other overt diseases were not included
345
346
in this study. Cords from mothers on medications
during the
last two months of pregnancy,
such as antihistamines,
morphine, adrenergic
blockers, or anticholinergic
drugs were
also excluded. Fifteen- to 25-cm segments were cut from the
cords midway
between
the placentas
and infants as this
area is nerve free ( 13, 30, 35, 37). Initially,
in our early studies, we immediately
flushed the severed cords with KrebsRinger bicarbonate
solution, the composition
of which has
been given previously
( 1, 6), in order to expel the blood.
This procedure,
however, was soon abandoned
for two reasons: I) the cords would expand and bulge under the forced
Ringer solution;
and 2) we found that this drastically
cut
down on the reactivity of the tissues to drugs. In subsequent
studies we found that if one gently squeezed the cords, in
order to expel blood, the preparations
would retain a very
high sensitivity to drugs. Therefore,
this procedure
was employed in all studies. The arteries and veins were then carefully dissected out of the cords, using microdissecting
instruments. The cord and the dissected umbilical
vessels were
continuously
bathed in cold Krebs-Ringer
bicarbonate
buffer ( 1, 6). Approximately
385 helical and longitudinal
arterial and venous strip preparations
were utilized
for the
present in vitro study. The arteries were cut, helically and
longitudinally,
1.3-1.5 mm in width by 25 mm in length
while the veins were cut, helically and longitudinally,
2.5-3
mm in width by 25 mm in length. The four types of preparations were then simultaneously
set up, isometrically
in four
separate ZO-ml muscle baths, using procedures
and instrumentation
previously
described
elsewhere
( 1, 6), and allowed to equilibrate
for 2 hr in Krebs-Ringer
bicarbonate
under a resting tension of 2 g. The loaded tensions were
maintained
and periodically
adjusted throughout
incubation and the experiments.
The Krebs-Ringer
solutions were
oxygenated
continuously
with a 95 % 02-5 % CO2 mixture
and kept at 37 C (pH 7.2-7.4).
Drug-induced
contractions. Complete
cumulative
dose-response curves ( 1, 6) as well as single-dose stimulations
were
obtained on the human umbilical
arteries and veins for serotonin,
histamine,
bradykinin,
potassium,
prostaglandins,
oxytocin,
vasopressin, norepinephrine,
epinephrine,
acetylcholine,
and angiotensin
in the presence and absence of
specific drug antagonists. Each vascular strip was stimulated
to contract by serotonin (in maximal
or supramaximal
concentrations
or complete
dose-response
curves) periodically
throughout
the experiments
since this vasoactive substance
induces the greatest maximum
response in isolated human
umbilical
arterial
and venous strip preparations
(see RESULTS).
All drugs and vasoactive substances were randomized in the experiments,
i.e., all of the drugs, including
serotonin, were added to the isolated organ baths in a different sequence from one preparation
to another. In other experiments,
paired
longitudinal
and helical
venous and
arterial
umbilical
strips were placed in preweighed
Pyrex
beakers for dry (oven at 110 C for 18-24 hr) tissue weight
determinations.
Drugs and chemicals. The chemicals used to make up the
Krebs-Ringer
bicarbonate
solutions, as well as the potassium
chloride (KCl), were all American
Chemical
Society certified reagent grade. The following vasoactive agents and drug
antagonists
were utilized : epinephrine
(Adrenalin
chloride, Parke, Davis & Co.), norepinephrine
(Levophed
ALTURA,
MALAVIYA,
REICH,
AND
ORKIN
Bitartrate,
Winthrop
Laboratories),
histamine
(histamine
dihydrochloride,
Nutritional
Biochemicals
Corp.), serotonin
(5-HT)
(serotonin
creatinine
sulfate, Nutritional
Biochemicals Corp.), angiotensin
(angiotensin
amide, Hypertensin,
Ciba Pharmaceuticals),
acetylcholine
(acetylcholine
chloride,
Nutritional
Biochemicals
Corp.),
bradykinin
(BRS 640,
Sandoz Pharmaceuticals),
prostaglandin
FZa, prostaglandin
Al, oxytocin (synthetic oxytocin, preservative
free, 400 IU/
ml, Sandoz
Pharmaceuticals),
vasopressin
(synthetic
8arginine
vasopressin, approx. 450 IU/mg,
Sandoz Pharmaceuticals),
phenoxybenzamine
(Dibenzyline
hydrochloride,
Smith, Kline & French Laboratories),
atropine
(atropine
sulfate, Mann Research Laboratories),
UML-49
1 (Methysergide, Sandoz Pharmaceuticals),
and pyrilamine
(pyrilamine maleate, K and K Laboratories).
All of the vasoactive
drug stimulants
and pharmacologic
antagonists,
except for
the prostaglandins,
were made up fresh in triply distilled,
deionized
water, and concentrated
stock solutions so that
the total volumes added to the ZO-ml muscle baths never
exceeded 1.0 ml. The prostaglandins
were dissolved in a solution of 0.095 % ethyl alcohol and 0.00 18 % sodium carbonate in triply distilled,
deionized
water. All of the vasoactive
drug concentrations
(or doses) expressed below are in terms
of the salt, except for angiotensin,
bradykinin,
prostaglandins, oxytocin, and vasopressin which are given a .sfree bases.
RESULTS
S’ontaneous mechanical activity of human artery and venous str$s
in vitro. Initially
upon in vitro incubation
(i.e., the first Z-10
min), the umbilical
arterial and venous strips contract from
20 to 60 % over the base-line resting tension of 2 g. Thereafter (i.e., usually within
20-40 min), 90 % of the venous
helical and longitudinal
strips developed
spontaneous
mechanical activity. Only 20-30 % of the corresponding
artetial preparations
would,
however,
develop
spontaneous
mechanical
activity, and this usually occurred
20-30 min
after the veins exhibited
signs of spontaneous
contractions,
and this in turn usually lasted for only 1-2 hr while the spontaneous rhythmic
mechanical
activity seen in the veins usually persisted, to different
degrees, for 5-10 hr. Figure 1
demonstrates
the typical spontaneous
contractile
activity
patterns seen in paired (taken from same umbilical
cord)
helical and longitudinal
umbilical
arteries and veins. Two
things become apparent
from such tracings:
I) the amplitudes of the helical preparations
are at least 2-4 times larger
than their corresponding
longitudinal
preparations;
and 2)
4
/
HELICAL
ARTERY
II,
LONGITUDINAL
HELICAL
ARTERY
VEIN
min
bml d
LONGITUDINAL
FIG. 1. Spontaneous
contractile
activity
in helically
dinally cut human
isolated umbilical
arteries and veins.
are taken from same umbilical
cord.
VEIN
and longi tuAll 4 tracings
REACTIVITY
OF
HUMAN
UMBILICAL
347
VESSELS
the frequency
of spontaneous
contractions
in the longitudinal preparations
is greater than in their corresponding
helical preparations.
Occasionally
both the longitudinal
and
helical arteries and veins exhibited
single, spontaneous
contractions which, in magnitude,
were 70-85 % of a serotonininduced maximal
response. This, however,
did not occur
during the first 2 hr of incubation.
This phenomenon
could,
however, be induced in most preparations
by quick stretch
or addition
of cold Krebs-Ringer
bicarbonate
solution.
Reproducibility of cumulative dose-response curues to 5HT.
In
1962 Panigelc (25) observed that perfused umbilical
vessels
exhibited a tremendous
variability
in response to serotonin,
but which was clearly not tachyphylaxis.
In view of these
findings, we thought
it advisable
to determine
the reproducibility
of helically
and longitudinally
cut vessels to repetitive serotonin
stimulation.
Figure 2 demonstrates
that
although there is variability
in response to serotonin,
as noted
by the large bars representing
the standard
errors of the
means, the cumulative
dose-response
curves on helically
cut umbilical
arteries are not significantly
different from one
another over a period of 8-9 hr. Similar observations
were
noted on the corresponding
longitudinally
cut arteries. Figure 3, however,
demonstrates
that the maximum
5-HT
responses on the veins during the first 3.5 hr were less than
40
35
N-10
3c
1o-9
FIG. 2.
duced by
lative log
that were
1o-8
SEROTON
lo-’
IN (Molar
1o-6
1o-5
cont.)
Effects of incubation
in Krebs-Ringer
on contractions
inserotonin
in helically
cut human
umbilical
arteries.
Cumudose-isometric
response curves. N = number
of preparations
obtained
from different
umbilical
cords. Brackets
= SEM.
those seen during
the subsequent
stimulations.
This, however, is not unusual since incubation
for 2-4 hr is usually
required
for excised blood vessels to fully recover from surgical procedures
(5). It is of interest to note that serotonin in
a concentration
as little as lo-lo M can induce threshold contractile responses on these umbilical
vessels (Fig. 2).
Relative sensitivity of human umbilical arteries and veins to uasoactiue substances. Figures 4-7 demonstrate
that of all the vasoactive
substances
examined
for contractile
properties
on helically
and longitudinally
cut umbilical
arteries and
veins serotonin
exhibits the greatest relative
sensitivitv
as
well as potency in that it elicits the maximal
contractile
response. Although
all of the vasoactive substances that we
have examined
can induce contractile
responses on these
vessels, they produce different degrees of unequal
maximal
contractile
responses (Figs. 4-7). Although
there are slight
quantitative
differences
in threshold
concentrations,
ED50
dose levels and maximal
responses, the relative sensitivity
of the paired arterial and venous preparations
to vasoactive
substances is similar.
In addition,
it should be noted that: I) not every arterial
or venous strip would always respond to acetylcholine,
angiotensin,
epinephrine,
norepinephrine,
and vasopressin ;
and 2) these latter substances in many cases would not elicit
graded,
cumulative
dose-dependent
contractile
responses;
that is to say, these drugs produced
all-or-none
type contractile responses in some arterial and venous strip preparations. For example, 4 out of 41 arteries and 7 out of 26 veins
responded in an all-or-none
manner to acetylcholine.
Of the
five agents capable of evoking this type of response, angiotensin had the highest
frequency
of all-or-none
type
responses, occurring
in approximate1 .y 50 % of the arterial
and venous strips that we examined.
Vasopressin
produced
all-or-none-type
contractile
responses in 10 of 22 vein preparations.
Drug-induced
unequal maximal responses. Since the above
cumulative
dose-response
data suggested that all of the vasoactive drugs can induce
different
degrees
of unequal
maximal contractile
responses, it was of interest to determine
whether
single supramaximal
doses of these vasoactive substances would
yield similar
unequal
maximal
responses.
Figures 8 and 9 show that such unequal maximal
contractile
responses are, indeed, obtained
on helical and longitudinal
arterial
strips, using supramaximal
drug doses. It is interesting
to note that serotonin,
as well
as the other
drugs, quantitatively
induces comparable
maximal
contractile responses on both types of arterial preparations.
Although
unequal maximal
contractile
responses are also
obtained on helically and longitudinally
cut umbilical
veins
using supramaximal
drug doses, serotonin,
as well as most
of the other drugs, induces, quantitatively,
a significantly
contractile
re(P < 0.01, Student t test) greater maximal
sponse on the helical umbilical
vein when compared
to the
longitudinal
vein (Fig. 9). However,
norepinephrine
is
approximately
equal in magnitude
while oxytocin,
vasopressin, and angio tensin developed
greater tensions on the
longitudinally
cut venous strips.
Serotonin-induced contractile responses on helical and longitudinal
umbilical strips. When a comparison
of the dose-response
relationships
for serotonin
is made for all four types of
umbilical
preparations,
it becomes apparent
that the dose-
ALTURA,
MALAVIYA,
REICH,
AND ORKIN
TT T
T
T
FIG.
3. Effects of incubation
in KrebsRinger
on contractions
induced
by serotonin
in longitudinally
cut human
umbilical
veins. Cumulative
log doseisometric
response curves. N = number
of preparations
that were obtained
from
different
umbilical
cords. Brackets
=
SEM.
lo-lo
1O-9
lo+
SEROTONI
1O-7
N (MOLAR
10+
CONC.)
1O-5
1o-4
. -.
HISTAMINE
(23)
/
10-10
10-9
10
I
10-S
-6
AGONIST
(Molar
10-4
10-3
10-2
10"
cont.)
FIG.
4. Relative
sensitivity
of helically
cut human umbilical
arteries
to vasoactive
substances.
Cumulative
log dose-isometric
response
curves. Brackets
= SEM. All data are presented
in terms of percent
5-HT maximal
responses.
100% response to 5-HT
= 3.42 g ZJZ 0.32
SEM.
Numbers
in parentheses
denote
number
of different
vascular
strips, obtained
from different
cords, utilized
response
curve.
ANG
= angiotensin;
VP
NE = norepinephrine;
OXY
= oxytocin;
Al ; PGFz,
= prostaglandin
Ffa! ; and ACH
response
relationships
for the helical
and longitudinal
arteries as well as the helical vein are almost identical,
while
the dose-response
relationship
for the corresponding
longitudinal vein is distinctly
different (Fig. 10).
Neurohypophyseal hormone action on human umbilical arteries and
veins. The phenomenon
illustrated
in Fig. 10 is not related
exclusively to serotonin-induced
contractions.
A similar dissimilarity-in
dose-response
relationships
is illustrated
in Fig.
11 for two other vasoactive materials, namely oxytocin and
vasopressin,
but with one important
difference.
This time
the helical umbilical
veins, instead of the longitudinal
veins,
show a different dose-response
relationship
for oxytocin and
vasopressin. Another point of interest shown in Fig. 11 is the
relatively greater affinity of oxytocin over vasopressin for the
neurohypophyseal
peptide receptor in human umbilical
arteries and veins. It should be noted that oxytocin and vasopressin were not tested together on the same preparation
since we found early in our studies that these substances
exhibited
profound
cross tachyphylaxis,
similar to that reported previously
by others (30).
Speczjkty of drug receptors which subserve contraction in human
umbilical arteries and veins. Figures 12-14, when taken together with the data summarized
in Table
1, reveal that
four different
types of pharmacologic
antagonists,
namely
an antihistamine,
pyrilamine;
an a-adrenergic
blocking drug,
phenoxybenzamine;
an anticholinergic
blocker, atropine;
for each cumulative
dose
= arginine
vasopressin;
PGAl
= prostaglandin
= acetylcholine.
REACTIVITY
OF HUMAN
UMBILICAL
VESSELS
349
AGONIST
5. Relative
sensitivity
of longitudinally
arteries to vasoactive
substances.
Cumulative
sponse curves. All data are presented
in terms
FIG.
human
umbilical
log dose-isometric
reof percent 5-HT maxicut
(Molar
cont.)
ma1 responses.
100% response to 5-HT
= 3.21 g rf= 0.36 SEM. EPI =
epinephrine.
All other symbols
and conventions
are similar
to those
used in Fig. 4.
HISTAMINE (7)
KCI (IO)
I
10-10
i
10’9
10-8
10-7
10-b
AGONIST
10-S
(MOLAR
CONC
I
10-4
f
10-X
10-2
I
lo-’
)
FIG. 6. Relative
sensitivity
of helically
cut human
umbilical
veins
to vasoactive
substances.
Cumulative
log dose-isometric
response
curves. All data are presented
in terms of percent
5-HT
maximal
responses.
100% response
symbols
and conventions
to 5-HT
are similar
= 3.30 g zt 0.85 SEM. All
to those used in Fig. 4.
other
and the antiserotonin,
UML-491,
rather specifically
and
selectively antagonize
histamine,
norepinephrine,
acetylcholine, and serotonin-induced
contractions,
respectively,
in these isolated blood vessels.
Contribution of tissue mass to drug-induced unequal maximal
2 indicate
that
contractile responses. The data in Table
although there might be a trend for the longitudinally
cut
arteries and veins to be somewhat larger in total mass than
the corresponding
helically cut arteries and veins, the total
dry tissue weights for the paired umbilical
arteries and veins
are not statistically different from one another. It is apparent
from these results, and the developed,
maximal
isometric
tensions shown in Figs. 8 and 9, that a simple relationship
between mass of smooth muscle cells1 and the contractile
responses to the various drugs cannot account completely
for the relative differences in magnitudes
of unequal maximal contractile
responses in longitudinally
vs. helically cut
strips.
DISCUSSION
The present findings which
of the spontaneous
mechanical
indicate
activity
1 We have used the dry tissue weight
mass of smooth
muscle cells responsible
metric tension.
that the magnitudes
found in helically cut
as a rough indicator
for the development
of the
of iso-
350
ALTURA,
1o-lO
1o-9
-
lo-8
lo-7
- lo-6
AGON
FIG. 7. Relative
sensitivity
of longitudinally
veins to vasoactive
substances.
Cumulative
sponse curves.
All data are presented
in
HELICAL
cut human
umbilical
log dose-isometric
reterms of percent
5-HT
~~~
LONGITUDINAL
5-HT- SEROTONIN
(N-43)
H - HISTAMINE
(N-23)
BK- BRADYKININ
(N- 8)
Fza - PROSTACLANDIN
F2a (N- 5)
A, - PROSTAGLANDIN
A, IN- 5)
K - POTASSIUM
CHLORIDE(N- 24)
AN6 - ANCIOTENSINIi
(N- I I)
NE - NOREPINEPHRINE (N- 18)
ACH- ACETYLCHOLINE (N-21)
OXY- OXYTOCIN
(W- 12)
VP - VASOPRESSIN
IN- 12)
T
5-HT (N=36)
F20 (N= 5)
H (N=21)
A, (N= 5)
BK (N= 7)
K (N=24)
OXY (N=
9)
ACH (N= 19)
NE (N= 14)
ANG(N=
8)
VP (N= 11)
T
5-tiTFza H A, BK K OXYACHNEANCVP
HELICALLY
CUT STRIPS
LONGITUDINALLY
CUT STRIPS
8. Unequal
maximal
contractile
responses of various
agonists
on isolated
human
umbilical
arteries.
Vertical
bars with brackets
represent
mean isometric
response
=I= SEM induced
by supramaximal
doses of various
vasoactive
substances
(5-HT
= serotonin,
5 lug/n-J;
H = histamine,
40 &ml;
BK = bradykinin,
5 ,ug/ml;
Feat = prostaglandin
F 2a, 20 pg/n-d;&
A1 20 &ml;
K = po= prostaglandin
tassium
chloride,
80 mu;
ANG
= angiotensin,
5 pg/rnl;
NE =
norepinephrine,
20 pg/rn.l; ACH
= acetylcholine,
40 pg/m.l; OXY
=
oxytocin,
0.1 U/ml;
and VP = arginine
vasopressin,
0.2 U/ml).
of different
cords utilized.
N = number
FIG.
10-S
I ST
(Molar
maximal
All other
MALAVIYA,
lo-4
REICH,
AND
ORKIN
‘0”
cont.)
responses.
100% response
to 5-HT
symbols and conventions
are similar
= 1.80 g =t 0.25 SEM.
to those used in Fig. 4.
umbilical
arteries and veins are much greater than their
corresponding
longitudinally
cut counterparts
can be correlated to previous histological
findings which indicate that
the outer layer of circularly
arranged
smooth muscle cells,
in the walls of these human umbilical
vessels, is much larger
in muscle mass than the inner longitudinal
layers (3 1, 32).
The rhythmical
behavior
of these helically
and longitudinally
cut arteries and veins suggests that: I) cell-to-cell
conduction
in both layers of smooth muscle is probably quite
good; and 2) there are probably
numerous
regions of close
membrane
apposition
or numerous nexus areas which function as low-resistance
pathways ( 10). The finding that the
frequencies of spontaneous activity in the longitudinally
prepared
umbilical
vessels are slightly
greater than their
corresponding
helically prepared
counterparts
is suggestive
of more rapid cell-to-cell
conduction
in the longitudinal
than
in the latter circular smooth muscle cell layers. One could
also entertain
the possibility
that conduction
in the circular
smooth muscle layers is naturally
slower and may be functionally
important
in the in vivo situation.
Electronmicrographic
and electrophysiological
studies should
throw
considerable
light on the characterization
of the individual
smooth muscle layers. Although
it is always difficult, if not
dangerous,
to extrapolate
in vitro findings to the in vivo
situation, one must entertain the possibility
that these strong
(as great as 75-80 % maximum
response in certain cases),
spontaneous,
and rhythmic
contractions
of the individual
layers of smooth muscle cells may play an important
role in
propelling
blood to and away from the fetus in utero.
The data presented here indicate that a variety of vasoactive substances (amines, polypeptides,
prostaglandins,
and
potassium
ions) induce unequal
maximal
contractile
responses in human umbilical
arteries and veins, similar to
that noted previously
for various somatic arteries and veins
in animals ( 1, 4, 6, 29). The present quantitative
in vitro
data, employing
a wide variety of vasoactive agonists, suggest that the relative number of potential
drug receptors or
REACTIVITY
OF
HUMAN
UMBILICAL
351
VESSELS
4.c
HELICAL
5-HT=
H =
BK =
K =
ACH=
NE =
OXY=
VP=
ANG=
LONGITUDINAL
SEROTONIN (N=18)
HISTAMINE (N= 7)
BRADYKININ (N= 5)
POTASSIUM (N=lO)
ACETYLCHOLINE (N=9)
NOREPINEPHRINE
(N =8)
OXYTOCIN
(N= 5)
VASOPRESSIN (N=6)
ANGIOTENSIN
(N=5)
5-HT(N=37)
H (N=20)
BK(N4 1)
ACH(N= 17)
K(N=18)
NE(N=15)
OXY(N= 17)
VP(N= 5)
ANG(N=
6)
3.5
(42)
(20)
3.0
(36)
25
xii
$y, 2.0
z
Lu
b
(36)
:
g
1.5
s1
-
1.0
0.5
0
,,L_,o
HELICALLY
CUT STRIPS
LONGITUDINALLY
-
10-9
I
I
I
10-7
10-6
1o-5
SEROTONIN
CUT STRIPS
FIG. 9. Unequal
maximal
contractile
responses of various
agonists
on isolated
human
umbilical
veins. Symbols
and conventions
are
similar to those used in Fig. 8. Supramaximal
drug doses were utilized
(5-HT
= 20 pg/ml; H = 80 pg/ml;
BK = 5 ,ug/ml;
K = 80
mM; ACH
= 40 pg/ml;
NE = 20 pg/ml;
OXY
= 0.1 U/ml;
VP = 0.2 U/ml;
and ANG
= 5 pg/ml).
N = number
of different
cords utilized.
I
10-8
(Molar
cod
10. Comparison
of contractile
responses
induced
by 5-HT
on isolated
longitudinally
and helically
cut human
umbilical
arteries
and veins. Cumulative
log dose-isometric
response
curves. HUA
=
helical
umbilical
artery;
HUV
= helical
umbilical
vein; LUA
=
longitudinal
umbilical
artery;
and LUV
= longitudinal
umbilical
vein. Numbers
in parentheses
denote the number
of different
cords
utilized.
Values
are means 3~ SEM.
FIG.
T
T
A-OXY
(12) LUA
TT
5 0.8FIG. 11. Neurohypophyseal
hormone
action
on isolated
longitudinally
and
helically
cut human
umbilical
arteries
and
veins.
Cumulative
log
doseisometric
response curves. OXY
= oxytocin; VP = arginine
vasopressin.
Other
symbols
and conventions
are similar
to
those used in Fig. 10. Values are means
is
z
Z
:"
z
Oh-
5
0
2 0.4-
=t: SEM.
--OXY
i
NEUROHYPOPHYSEAL
HORMONE
(U/L)
\
\ A -VP(7)
(IO) HUV
HUV
ALTURA,
352
t t t
5-HT
H
PY.
BK
+
H + 5-HT
PY
+
H
+
H
OiY
PY
BK
H + 5-HT
PY + H b OXY
PY + H t BK
.1,
min
I
I
I
~~
CONTROLS
EXPERIMENTALS
t t t
NE
5-HT
t t
Ach
BK
PBZ
~~
-1
PBZ + NE + Ach PBZ + BK
mm
ARTERY
t
t KCIt
NE
t
5-HT
PBZ
Ach
+
NE
+ 5-HT
PBZ +NE
+ KCI
PBZ + Ach
t-J-4
mln
CONTROLS
EXPERIMENTALS
AND ORKIN
(PYRILAMINE)
t tt t tt t
NE +5-HT
+
REICH,
FIG.
12. Influence
of pyrilamine
on
drug-induced
contractions
in longitudinally cut human
umbilical
vein and artery. Note that preincubation
(15 min)
with the antihistamine
pyrilamine
(0.5
pg/ml)
completely
blocks contractions
induced
by histamine
(H) (20 &ml)
but does not affect contractions
induced
by 5-HT
(0.5 pg/ml),
bradykinin
(BK)
(0.5 pg/ml),
or oxytocin
(OXY)
(0.05
U/ml).
Antihistamine
remained
in bath
in contact
with vascular
strips during
addition
of drug agonists.
BK
ARTERY
5-HT
MALAVIYA,
FIG.
13. Influence
of phenoxybenzamine on drug-induced
contractions
in
helically
cut human
umbilical
vein and
artery.
Note
that
preincubation
(15
rnin) with a+adrenergic
antagonist
phenoxybenzamine
(0.05
pg/ml)
completely
blocks contractions
induced
by
norepinephrine
(NE)
(1.0 pg/ml)
but
does not affect contractions
induced by
5-HT
(0.5 pg/rn.l),
acetylcholine
(Ach)
(10 pg/ml),
bradykinin
(BK)
(0.5 pg/
ml), or KC1 (40 mM). Phenoxybenzamine remained
in bath in contact with
vascular
strips during
addition
of drug
agonists.
(PHENOXYBENZAMINE)
VEIN
t
5-HT
t t J-i
H
Ach
AT + A;h
min
+ S-HT
AT
+ Ach + H
FIG.
14. Influence
of atropine
on drug-induced
contractions
in longitudinally
cut human
umbilical
vein and artery.
Note that preincubation
(15 min)
with
cholinergic
antagonist
atropine
completely
blocks contractions
induced
by acetylcholine
(Ach)
(10 pg/ml)
but does not affect contractions
induced
by 5-HT
(0.5 lug/ml)
or histamine
(H) (20 pg/rnl).
Atropine
remained
in bath in contact with vascular
strips during
addition
of drug agonists.
ARTERY
lgmC
t Lt t --,+I
t t t -tt f t
5-HT
H
Ach
AT + Ach
A
min
+ S-HT
AT
+ Ach + H
I I
1 I
I
CONTROLS
i
EXPERIMENTALS (ATROPINE)
reactive smooth muscle cells for different drugs is not necessarily the same for the longitudinal
and circular
smooth
muscle layers, even though the maximal responses for a parFor
titular
agonist
may be quantitatively
equivalent.
example,
serotonin
elicits comparable
degrees of isometric
tension development
in longitudinal
and helical
arterial
preparations
(Fig. 8), but the relative decreasing
order of
unequal maximal contractile
responses elicited by the other
vasoactive substances is not the same. Furthermore,
previous
histological
studies (3 1, 32) indicate that the outer circular
layer of smooth muscle cells in both the human umbilical
artery and vein is many times thicker (and thus much richer
in total numbers of smooth muscle cells) than the inner longitudinal
smooth muscle layer, yet: a) the maximal isometric
REACTIVITY
TABLE
OF HUMAN
UMBILICAL
VESSELS
1. Influence of various pharmacologic
353
antagonists on drug-induced
contractions in human umbilical
arteries and veins
Agonist
Antagonist
p
Ax,
Ach*,
10 ccg/ml
Pyrilamine,
0.5 pg/
ml
Phenoxybenzamine,
0.05 j&ml
Atropine,
0.5 pg/ml
UML-491,
0.5 Irg/ml
- t
w
(4)
+
w
-
0.05
/%/ml
>
(3)
* Ach
= acetylcholine;
Vp
OXY = oxytocin;
$ Number
of different
to respond
to agonist
Ang
= 8-arginine
preparations
in presence
=
0.5 tk/rnl
(3)
(37.5)
(4)
-
(5)
-
(2)
(3)
angiotensin
vasopressin;
(arteries
of antagonist.
EpL
Hist,
20 pg/ml
1.0 erg/ml
(3)
+
w- >
(3)
(2) -
(8)
(3)
-
amide;
Bk = bradykinin;
PGAr
= prostaglandin
and veins).
8 Number
TABLE
2. Dry tissue weights of helically and longitudinally
cut human umbilical arteries and veins
Helical
Artery
Weight*
6.31
=I= 0.54t
V)$
Longitudinal
Artery
7.18
&
(7)
Helical
Vein
0.355.39
=t
Longiizinal
1.17t6.47
(7)
See METHODS
for dimensions
of various
preparations.
values
(mg * s~hi).
t Not
significantly
different
sponding
longitudinal
artery
and vein
(P > 0.05).
different
preparations
from
different
cords.
=t
0.48
(7)
* Mean
from
corre$ Number
of
tensions (i. e., induced
by 5-HT)
obtained
in the present
experiments, in the paired longitudinally
and helically cut
human umbilical
vessels are either approximately
equal (e.
g., arteries) (Fig. 8) or quite different in magnitude
(e. g.,
veins) (Fig. 9); and b) the total masses of smooth muscle
cells, on the basis of dry weight determinations,
in the paired
umbilical
arterial
and venous strip preparations
are not
statistically different from one another. It would, therefore,
be very difficult to account for the differences in developed
tensions in the longitudinal
vs. helical preparations
solely
on the basis of the amount of contractile
substance present
in the longitudinal
vs. circular layers. Although
this suggests
that heterogeneity
of drug receptors exists in the circular and
longitudinal
smooth muscle layers, the various individual
drugs activate specific receptors since a variety of pharmacologic antagonists
rather
selectively
inhibited
only the
specific drug agonists they were designed to counteract.
Serotonin
and bradykinin
appear to exhibit the greatest
affinity for their respective receptors in these vessels; on a
molar basis, serotonin is more potent than bradykinin
on the
umbilical arteries as well as veins. Although
these findings,
in relation
to potency,
are the reverse of those found
by Eltherington
et al. ( 17) for these same two substances on
human umbilical
arteries, one must keep in mind that these
latter investigators
used perfused arterial
segments. Our
isolated helical and longitudinal
arterial strip preparations
appear to be 6-30 times more sensitive to serotonin than are
the perfused
arterial
segments
in the experiments
of
Eltherington
et al. It is of some interest to note that the levels of bradykinin
needed
to elicit threshold
contractile
responses (0.5-4.0 rig/ml)
are in the range of maternal
plas-
0%
KCl,
40InM
(8)
(6)
(4)
(5)
Epi
=
Al ; 5-HT
of different
1 .OT:)rnl
0.05 U/ml
VP9
0.05 U/ml
-
-
-
(5)
+
(8)
-
(f-0
-
(3)
-
(3)
-
(3)
-
(4)
-
@/8
>
(3-4)
(5)
epinephrine;
= serotonin.
preparations
(4)
(4)
PGAI
SHT,
0.5 rg/ml
,
10 pgg/ml
(3)
(2)
(3)
(3)
,
/
11
= histamine;
NE = norepinephrine;
t Minus
= no effect;plus
= blockade.
out of total
number
examined
that failed
Hist
ma bradykinin
concentrations
(22). But probably,
more
importantly,
the umbilical
cord venous and arterial plasma
kinin concentrations
found by Melmon
et al. (22) at birth
were significantly
higher than those found in maternal
venous plasma, reaching levels 45 times those of adult maternal
venous plasma. An extrapolation
of these umbilical
venous
and arterial plasma kinin levels (22) to our work would result in contractile
responses which are equivalent
to 25-60 %
of a serotonin-induced
maximal
response. Although,
to our
knowledge,
umbilical
cord plasma serotonin
levels are not
known, it should be noted that: I) an increase of 5-HT metabolism takes place in the last months of pregnancy
( 18);
and 2) recent findings of Yuwiler et al. (28, 36) indicate that,
at least for whole blood, young children
have significantly
higher serotonin blood levels than do adults (mean values:
0.241 =t 0.053 vs. 0.161 & 0.047 pg/ml). It would not be too
unreasonable
to suggest that the 5HT
levels in umbilical
cord plasma may even be higher since previous findings of
others have shown that the 5-HT content of the rat fetus
increases from undetectable
amounts early in pregnancy
to
0.2 pg/g at term (16), which might suggest a feedback of
5-HT into the umbilical
cord vessels. But even if one considers the blood level of Yuwiler
et al. (i. e., 0.241 pg/ml)
and extrapolates
this to our experiments,
this serotonin level
(if it is mainly free, unbound
5-HT) could effect contractile
responses which are 85-95 % of the maximum
responses observed in the present experiments.
Furthermore,
even if the
actual umbilical
cord plasma levels of free 5-HT were only
10 % of 0.241 pg/ml,
they would result in contractile
responses which
are 65-80%
of the maximum
responses
observed in the present experiments.
At the very least, the
possibility
must be entertained
that bradykinin
and serotonin may be of importance
in regulation
of fetal systemic
vascular resistance via a tonic constrictor
action on the umbilical cord vessels.
Although
this study demonstrates
that histamine
and two
prostaglandins
(A, and Fgar) can produce near maximal contractile responses in isolated human umbilical
arteries and
veins, their possible physiologic
role seems to be highly
questionable,
if not precluded,
in view of our data which
indicate
that comparatively
high concentrations
of these
substances are needed for threshold
effects. In both cases,
that is for histamine
(4.0-5.0 rig/ml)
and the prostaglandins
354
(.05-0.5
pg/ml),
the effective concentrations
necessary for
threshold
contractile
responses are higher than either the
known assayed maternal
plasma levels of these substances or
those found at birth (2 1, 23, 26). Even though actual circulating levels of the prostaglandins
are far below those needed
for threshold contractile
effects, one must take into consideration the recent findings of Karim (20) which indicate that
the walls of the umbilical
cord vessels may contain concentrations of prostaglandins
which, if freed or released during
pregnancy
and/or
birth, could induce umbilical
vessels to
contract.
Our studies indicate that angiotensin,
acetylcholine,
and
the catecholamines
can also elicit contractile
responses on
human umbilical
arteries and veins. The physiologic
role
that these latter substances play in regulating
umbilic al cord
blood flow in man is probably negligible
or minimal
for several reasons : I) the in vitro
concentrations
of these
substances which are necessary for even threshold
contractile effects are higher than those found in man; 2) only some
preparations
were found to respond to these substances confirming
previous findings of others ( 19, 30, 34); 3) many
preparations
did not respond in a dose-dependent
(graded)
fashion but in an all-or-none
manner;
and 4) these vasoactive substances, in those instances where they can produce
graded contractions,
can effect only a small portion of the
maximal response (see Figs. 4-9).
oxytocin and
Although
the neurohypophyseal
hormones
vasopressin can effect only a small portion of the serotonininduced maximal
contractile
responses on human umbilical
arteries and veins, the dose-response
relationships
observed
in the present experiments
are, nevertheless,
important
for
several reasons and should be discussed here. First, oxytocin
induces threshold
contractile
effects in both human umbilical arteries
and veins in very low (physiologic?)
concentrations
(e. g., l-5 mu/liter),
confirming
and extending
previous findings of Somlyo et al. (30). A recent report by
Chard and his co-workers
( 12), using a very sensitive radioimmunoassay
technique
for oxytocin, indicates that human
umbilical
cord venous and arterial
plasma oxytocin
levels
(obtained
immediately
after delivery)
are on the average
between 24 and 45 mu/liter,
concentrations
which, on extrapolation,
are capable of eliciting
20-30 % of maximal
oxytocin contractile
responses (see Fig. 11). Second, oxytotin consistently
(unlike vasopressin)
produced
contractions
in all of the human umbilical
arterial and venous preparations that we examined,
and oxytocin
always
elicited
a greater portion of the maximal contractile
response when
ALTURA,
MALAVIYA,
REICH,
AND
ORKIN
compared
to vasopressin.
Third,
the dose-response
curves
for oxytocin are shifted to the left of those for vasopressin on
each type of preparation
(see Fig. 11). These pharmacologic
phenomena
exhibited
by oxytocin and vasopressin are very
different
from that seen for either of other large somatic
mammalian
blood vessels 1,3a, 6a) or the small mammalian
microscopic
blood vessels (3,6a, 7, and unpublished
observations) and strongly suggest that the neurohypophyseal
peptide receptor
in human
umbilical
arteries and veins is
probably different from that in other mammalian
blood vessels. These data could, therefore, be marshaled
to support
our previous suggestion that the neurohypophyseal
receptor
may not be identical
in all mammalian
vascular smooth
muscle ( l-3).
In view of the data presented here, the possibility
must be
entertained
that oxytocin,
serotonin,
and bradykinin
may
play an important
role in human umbilical
cord blood flow
in vivo. Quantitative
assay of these and other endogenous
vasoactive substances in plasma from women, during various
stages of pregnancy,
should
aid in the clarification
of
the physiologic
role for these substances. Since previous,
recent work of others has indicated
that kinin (22) as well as
oxytocin
( 12) umbilical
cord vessel plasma
levels are
markedly
increased at birth, one must consider the distinct
possibility
that these two polypeptides,
acting in concert
with
increased
oxygen
tensions
( 15) and
serotonin,
may effect closure of human umbilical
cord vessels at birth.
The authors
acknowledge
the many helpful
discussions
held with
Dr. Bella T. Altura.
The authors
are deeply indebted
to the nursing
and resident
staffs of the Departments
of Anesthesiology
and Obstetrics of the Bronx Municipal
Hospital
Center and the College Hospital of the Albert
Einstein
College of medicine,
and to Miss Gertie
Marx,
Professor
of Anesthesiology,
without
whose assistance
these
studies would not have been possible.
The authors
are also indebted
to the late Dr. R. Bircher
and Dr. B. Berde, Sandoz
Pharmaceuticals, for generously
supplying
the pure, synthetic
neurohypophyseal
polypeptides
used in this study. The authors
also thank Dr. Richard
Weiner,
New York Medical
College and Dr. John Pike, The UpJohn
Co., for generously
supplying
the pure, synthetic
prostaglandins
used
in this study.
A preliminary
account
of portions
of this work was presented
to
the Spring
1971 Meeting
of The American
Society of Pharmacology
and Experimental
Therapeutics,
Inc.,
April
14, 197 1, Chicago,
Ill. (8).
This work was supported
by Research
Grants
HE-12462
and HE11391 from the National
Heart
and Lung Institute.
B. M. Altura
is a recipient
of Public
Health
Service
Research
Career
Development
Award
5-K3-GM,
38, 603.
Received
for publication
25 June
197 1.
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