Proc. Nat. Acad. Sci. USA
Vol. 72, No. 10, pp. 3883-3887, October 1975
Biochemistry
Oxygen and cyclic nucleotides in human umbilical artery
(cGMP/cAMP/calcium)
RONALD I. CLYMAN, ADAM S. BLACKSIN, VINCENT C. MANGANIELLO, AND MARTHA VAUGHAN
Laboratory of Cellular Metabolism, National Heart and Lung Institute, NIH, Bethesda, Maryland 20014
Communicated by E. R. Stadtman, July 14,1975
ABSTRACT
In the human umbilical artery 02 has a direct contractile effect and is also required for induction of
contraction by several other agents. Agonists that cause contraction (bradykinin, histamine, and serotonin) cause accumulation of guanosine 3':5'-monophosphate (cGMP) without
altering adenosine 3':5'-monophosphate (cAMP). They appear
to act through two different mechanisms: one Ca++-dependent, the other Ca++-inhibited. 02 increased the cGMP content of the artery in a Ca++-dependent manner without affecting the cAMP content. Inhibitors of oxidative phosphorylation (oligomycin and 2,4-dinitrophenol) did not diminish
this effect of 02.02 was required for demonstration of the
Ca++-dependent accumulation of cGMP in response to bradykinin, histamine, and ionophore A23187. The effect of the
phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine on
basal cGMP content and on the bradykinin-induced accumulation was also dependent on the presence of 02. Methylene
blue and sodium ascorbate caused cGMP accumulation in
02-deprived arteries. Their effects were not diminished in
Ca++-depleted arteries and, in fact, seemed to be inhibited
when 2.7 mM Ca++ was present in the medium. The effects
of these agents and of serotonin on cGMP, which were inhibited by Ca++, were also inhibited by 02. These non Ca++-,
non 02-dependent agents (methylene blue, ascorbate, and
serotonin) did not, however, permit demonstration of the effects of the Ca++- and Ordependent agonists on Ordeprived
arteries. It appears that there are in the umbilical artery (and
probably in other tissues also) at least two separate mechanisms for control of cGMP synthesis that are influenced differently by Ca++- and 02-linked processes.
There is considerable evidence that in human umbilical artery an increase in 02 tension induces contraction and a decrease leads to relaxation (1-3). Humoral agents that cause
contraction of the artery in vitro induce accumulation of guanosine 3':5'-monophosphate (cGMP) and those that initiate
relaxation cause accumulation of adenosine 3':5'-monophosphate (cAMP) (4). Regulation of cGMP content appears to
be effected through two distinct mechanisms: one that requires Ca and one that is inhibited by Ca++ (5). In the
experiments reported here the effects of 02 on cyclic nucleotide metabolism in human umbilical artery were investigated. It was found that 02 caused accumulation of cGMP
in a Ca++-dependent manner, without affecting the cAMP
content of the artery. Further, agonists that required Ca++
in order to bring about an increase in cGMP content also required 02, whereas the effects of the agonists that were inhibited by Ca++ were also inhibited by 02METHODS AND MATERIALS
Human umbilical cords from full-term deliveries, obtained
within 30 min of delivery, were dissected and prepared for
incubation as previously described (4). For these procedures
Hanks' medium without Ca++ and Mg++ was used at room
Abbreviations: cGMP, guanosine 3':5'-monophosphate; cAMP,
adenosine 3':5'-monophosphate; PGE1, prostaglandin E1; EGTA,
ethylene glycol bis(,B-aminoethyl ether)-N,N'-tetraacetic acid.
temperature. Artery segments (300-400 mg wet weight)
were randomly assigned to 25 ml Erlenmeyer flasks contain-
ing 4 ml of medium as indicated in specific experiments.
The complete medium contained 127 mM NaCl, 5 mM KCl,
2.7 mM CaCl2, 1.27 mM KH2PO4, 1.27 mM MgSO4, glucose
(1 mg/ml), and 15 mM Tris-HCl buffer, pH 7.3. Artery segments to be incubated in Ca++-free medium were incubated
in Ca++-free medium with 4 mM ethylene glycol bis(fl-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) for 20 min before transfer to the experimental medium without EGTA.
Incubations were carried out in duplicate at 370 for a total
duration of 2 hr.
For incubation under atmospheres other than room air,
the flasks containing artery segments were sealed with rubber caps pierced with two 18 gauge needles. While the flasks
were shaking 100% N2 or 100% 02 was delivered to each
(above the surface of the medium) through one of the needles from a multiport glass manifold. Pressure and flow were
constant in each flask throughout the experiment. Preliminary experiments revealed that a steady state 02 tension was
achieved in the medium within 3 min following a change in
oxygen tension of the gas delivered to the flasks. Oxygen
tensions were determined using a Beckman Field Lab Oxygen Analyzer. The total duration of incubation was 2 hr. Artery segments to be incubated under 100% nitrogen for less
than 2 hr were incubated in room air until exposed to 100%
nitrogen for the indicated time to complete the 2 hr period.
Incubations were terminated by addition of 1 ml of cold
12% perchloric acid followed by homogenization of tissue
and medium with a Brinkman Polytron and centrifugation
at 3200 X g for 30 min. The precipitates, dissolved in 1 N
NaOH, were used for determination of protein (6) with bo-
c
0.
0)
o-.
E
0
1.0
Q.
0~
5
10
15
30
EXPOSURE TO 100% 02 (minutes)
FIG. 1. Effects of 02 on cGMP content of artery segments. Artery segments were incubated for 2 hr in an atmosphere of 100%
N2. The atmosphere was then changed to 100% 02 and the incubation was terminated at the indicated time. Values represent the
mean I SEM for the number of experiments indicated in parentheses.
3883
3884
Biochemistry: Clyman et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 1. Effect of 02 on cAMP content of umbilical artery
cAMP content,*
Atmosphere
A. Room air
PGEI pmol/mg of protein
0
+
100% N2
0
+
B. 100% N2
100% N2 then 100% 02
0
0
0.50
1.92
0.38
2.07
0.38
0.45
±
±
±
±
±
±
0.06
0.24
0.07
0.24
0.07
0.11
Artery segments were incubated for 1.5 hr in the atmosphere indicated. Theophylline, 1 mM, was then added to all samples. (A)
PGE1 (14 uM) was added after 30 min to some and incubations
were terminated 3 min later. (B) Atmosphere was changed from
100% N2 to 100% 02 in some (after 30 min of exposure to theophylline) and incubations were terminated 2 min later.
* Mean 4 SEM for data from seven experiments.
vine serum albumin as standard. The supernatants were purified and cGMP and cAMP was assayed as previously described (4, 5).
Sources of most materials have been noted (4, 5). Methylene blue (K&K) was kindly provided by Dr. Elise Ann
Brown. Sodium ascorbate was purchased from Nutritional
Biochemicals, oligomycin and 2,4-dinitrophenol from
Sigma. Stock solutions of oligomycin, 25 mM in ethanol, and
of 2,4-dinitrophenol, 1 M in acetone, were prepared on the
day of use. A freshly prepared stock solution of sodium ascorbate, 20 mM, was allowed to stand in room air for 30 min
before it was added to the incubation medium. Neither
methylene blue nor sodium ascorbate, in the concentrations
Table 2. Effect of inhibitors of oxidative phosphorylation
on 02-induced cGMP accumulation
Exp.
1.
2.
Atmosphere
100% N2
Inhibitor
cGMP
content,*
pmol/mg
of protein
None
0.35
Oligomycin,
50 ,M
0.56
100% N2 then
100% 02 (2 min)
None
1.57
100% N2
Oligomycin,
50,uM
None
Dinitrophenol,
0.5 mM
2.30
0.76
None
3.19
100% N2 then
100% 02 (2 min)
2.25
Dinitropherqol,
0.5 mM
100% N2 then
100% 02 (6 min)
None
Dinitrophenol,
0.5 mM
10.25
1.31
3.60
Artery segments from a single umbilical cord were incubated for
1.5 hr in an atmosphere of 100% N2. Oligomycin or 2,4-dinitrophenol was then added to some samples. After 40 min the atmosphere was changed to 100% 02 where indicated and incubations
were terminated 2 or 6 min later. The final concentration of ethanol
in samples from Exp. 1 was 0.2% and of acetone in samples from
Exp. 2 was 0.05%. These concentrations of ethanol and acetone
were without effect on the cGMP content of the artery.
* Mean of values from duplicate incubations.
ZC
0.
05
E
0.5
0.
.~~~~~
100%N2
U
0
0.1
1
10
100
BRADYKI Nl N -jiM
FIG. 2. Influence of 02 on the response of the artery to bradykinin. Segments of umbilical artery from a single cord were incubated under 100% N2 atmosphere for 2 hr. Some segments were
then exposed to 100% 02 for an additional 30 min. Bradykinin was
added to the indicated concentration and incubation was terminated 2 min later. Each point represents the mean cGMP content
of duplicate samples of tissue plus medium.
used in tissue experiments, altered the binding of cGMP in
the radioimmunoassay.
RESULTS
Influence of 02 tension on cGMP and cAMP content of
umbilical arteries
In six experiments the cGMP content of artery segments incubated for 2 hr in room air was 0.71 4 0.10 pmol/mg of
protein and was significantly decreased (P < 0.01 for paired
differences) to 0.39 4 0.05 (mean :1 SEM) in arteries incubated for 2 hr in 100% N2. Within 1 min after changing the
atmosphere from 100% N2 to 100% 02, accumulation of
cGMP had occurred and maximal increases were observed
after 2 or 3 min (Fig. 1). In contrast, incubation of arteries
for 2 hr in 100% N2 did not alter their cAMP content or the
effects of prostaglandin E1 (PGE1) on it (Table 1). As shown
in Table 2, the 02-induced accumulation of cGMP in arteries previously deprived of 02 was not prevented by oligomycin or 2,4-dinitrophenol, inhibitors of oxidative phosphorylation, but was, in fact, somewhat enhanced.
Influence of 02 tension on the responses to other
agents that can cause accumulation of cGMP
Bradykinin, which caused significant increases in the cGMP
content of arteries incubated in room air, was without effect
on arteries that had been incubated for 2 hr in 100% N2.
When 02 was returned to the atmosphere for 30 min, bradykinin responsiveness was restored (Fig. 2). The cGMP
content of the 02-deprived arteries and the effects of bradykinin on it were unaffected by the phosphodiesterase inhibitor 3-isobutyl-l-methyl xanthine (Table 3). Like bradykinin, histamine and ionophore A23187 had no effect on
cGMP in arteries that had been incubated for 2 hr in 100%
N2 (Table 4). Responsiveness to these agents was restored
following a 15-min exposure to 100% 02 (data not shown).
The cGMP content of 02-deprived tissues was increased
by methylene blue and sodium ascorbate, both of which can
act intracellularly as electron acceptors and thereby alter the
redox state of the cell (Fig. 3 and 4). As shown in Table 4,
methylene blue, sodium ascorbate, and serotonin (unlike
bradykinin, histamine, and ionophore A23187) caused apparently greater accumulation of cGMP in arteries incubated in 100% N2 than they did in those incubated in room air.
Biochemistry: Clyman et al.
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 4. Effect of 02 deprivation on agonist-induced
accumulation of cGMP
Table 3. Influence of 02 on effects of bradykinin and
3-isobutyl-1-methyl xanthine on cGMP content
100% N2
0
0
0
+
0
0.23
0.34
0.37
0.33
0.85
1.25
4.62
7.89
+
+
+
Room air
A cGMP
cGMP content,*
pmol/mg
of protein
Bradykinin
IBMX
Atmosphere
0
0
0
0
+
+
+
+
Artery segments from a single umbilical cord were incubated for
2 hr in the atmosphere indicated. 3-isobutyl-1-methyl xanthine
(IBMX), 0.1 mM, was added to some samples. After 30 min bradykinin, 1.1 ztM, was added to some and the incubations were terminated 2 min later.
* Mean of values from duplicate incubations.
Influence of medium Ca++ concentration on the
responses to oxygen and other agents that can cause
accumulation of cGMP
In 02-deprived arteries incubated in medium without Ca
the introduction of 02 failed to cause accumulation of
cGMP (Table 5). Bradykinin, histamine, and ionophore
A23187, which failed to increase cGMP in 02-deprived arteries, were also ineffective in tissue incubated in medium
without Ca++ (in room air) as previously reported (5). As
shown in Table 6, however, the effects of methylene blue
and sodium ascorbate on cGMP, like those of serotonin (5),
were somewhat enhanced in arteries incubated in medium
without Ca++, just as they were in the 02-deprived arteries
(see Table 4).
DISCUSSION
In the umbilical artery the concentrations of cAMP and
cGMP appear to be independently controlled in a manner
that mirrors the contractile behavior of this tissue (4). PGE1,
which causes relaxation of the artery (17), induces accumulation of cAMP (4) without affecting the cGMP content.
This effect of PGE1 is not dependent on the concentration of
3885
due to
Exp.
Atmosphere
1
Room air
100% N2
Room air
100% N2
Room air
100% N2
2
3
4
Room air
100%N2
Room air
100% N2
Room air
100% N2
5
6
Agonist
agonist, *
pmol/mg
of protein
Bradykinin,
6.14
0.16
2.23
0.31
6.54
1.1 ,uM
Histamine,
1 ,uM
Ionophore A23187,
20 MM
Serotonin,
0liM
Sodium ascorbate,
0.5 mM
Methylene blue,
27 ,uM
-0.17
2.03
3.25
2.34
6.95
0.97
1.89
For each experiment, artery segments from a single umbilical
cord were incubated in the indicated atmosphere for 30 min.
Agonists were added and incubations were terminated after 2 min
(bradykinin, histamine, ionophore A23187, serotonin, and methylene blue) or 6 min (sodium ascorbate).
*Difference between mean cGMP content of duplicate samples
treated with and without agonist.
Ca++ in the medium (5) and was unimpaired in the 02-deprived artery, as reported above. In addition, changes in the
02 tension did not alter the basal cAMP content of the artery.
We have reported (4) that a number of humoral agents
previously known to cause contraction of the human umbilical artery in vitro also induce accumulation of CGMP (without altering cAMP concentration). The increase in 02 tension to which the umbilical artery is exposed at birth acts in
at least two separate but related ways to initiate closure of
the vessel. 02, itself, apparently directly induces constriction
(1, 2, 3, 9, 12). It plays further a "permissive" role in the action of other chemical agents that cause contraction (3, 1012). A similar dual role for 02 has been demonstrated in
other vascular tissues (12-15).
C
METHYLENE
BLUE
.
0 0.
Cr
SODIUM
ASCORBATE
0
E
E
1
Q
E
/
METHYLENE
BLUE
1.5
27 pM
SODIUM
ASCORBATE
500WM
-2
Ec0.4~
EXP. 1
5
10 15
EXP. 1
EXP. 2
30
5 10 15
TIME (minutes)
30
FIG. 3. Effects of methylene blue and sodium ascorbate on
cGMP content of artery segments. In each experiment segments of
artery from a single umbilical cord were incubated for 2 hr, in an
atmosphere of 100% N2, before addition of agonist. Incubations
were terminated at the indicated times. Each point represents the
mean cGMP content of duplicate samples of tissue plus medium.
2.7
27
270
5
50
CONCENTRATION (,UM)
EXP. 2
500
FIG. 4. Effects of methylene blue and sodium ascorbate on
cGMP content of artery segments. In each experiment segments of
artery from a single umbilical cord were incubated for 2 hr in an
atmosphere of 100% N2, after which the agonist was added. The incubations with methylene blue were terminated 2 min later while
those with sodium ascorbate were terminated 5 min later. Each
point represents the difference between cGMP content of samples
treated with and without agonist.
3886
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Clyman et al.
Table 5. Effect of Ca++ omission on 02-induced
cGMP accumulation
Atmosphere
100% N2
100% N2 then 100% 02
100% N2
100% N2 then 100% 02
Incubation
medium
Complete
Complete
Ca++-free
Ca++-free
Table 6. Effect of Ca++ omission on the agonist-induced
accumulation of cGMP
cGMP
content,*
pmol/mg of
A cGMP
due to
agonist,*
protein
pmol/mg
0.51 + 0.12
1.34 t 0.30t
0.51 ± 0.11
0.47 ± 0.06
Artery segments were incubated for 2 hr in an atmosphere of
100% N2 in medium containing 2.7 mM Ca+ + or no Ca+ +. The gas
entering some flasks was changed to 100% 02 and the incubations
were terminated 2 or 3 min later.
* Mean 4 SEM for data from six experiments.
t cGMP content was significantly greater than 100% N2 control
(P < 0.02, paired t-test).
02 deprivation decreased the cGMP content of the umbilical artery and, on exposure to 02, accumulation of cGMP
occurred as rapidly as it did following the addition of other
contractile agents (4). This direct effect of 02 required the
presence of Ca++ in the medium. Similarly, cGMP accumulation in response to acetylcholine, bradykinin, histamine,
K+-ion or ionophore A23187 (all of which cause contraction)
is dependent on exogenous Ca++ (5). Thus 02 appears to
mimic the Ca++-dependent agonists in their effects on cyclic nucleotide concentrations in the umbilical artery.
It has been assumed that the dependence of contractile
tension on 02 is a result of its function as the final electron
acceptor for oxidative phosphorylation in the mitochondria
of smooth muscle cells (14, 15). The effects of 02 on cGMP
in the umbilical artery, however, were not inhibited by oligomycin or 2,4-dinitrophenol. It would appear that 02 has
significant actions in the cell other than those secondary to
the production of ATP.
In addition to itself increasing cGMP, 02 was necessary to
permit demonstration of accumulation of cGMP in response
to the Ca++-dependent agonists (bradykinin, histamine, and
ionophore A23187). Whether these two effects of 02 are a
result of increasing the rate of cGMP synthesis or decreasing
the rate of degradation remains to be proven. The failure of
the phosphodiesterase inhibitor 3-isobutyl-l-methyl xanthine to increase cGMP in arteries incubated in an 02-deprived' atmosphere (with or without bradykinin) is consistent
with the view that guanylate cyclase activity is decreased
and its responsiveness to bradykinin impaired in the 02-depleted artery (assuming that isobutyl methyl xanthine effectively inhibited cGMP degradation under these conditions).
Methylene blue and sodium ascorbate can alter the intracellular "redox" state (7, 8) and sodium ascorbate has been
reported to cause contraction of ileal muscle strips (16). Both
sodium ascorbate and methylene blue caused accumulation
of cGMP in 02-deprived umbilical arteries. Neither agent
affected the cAMP content of the arteries (data not shown).
Unlike 02, however, sodium ascorbate and methylene blue
did not restore responsiveness of the 02-deprived artery to
the 02- and Ca++-dependent agonists, bradykinin, histamine, and ionophore A23187 (data not shown). In addition,
although the effect of 02 alone on cGMP was dependent on
Ca++, the effects of sodium ascorbate and methylene blue
did not require exogenous Ca++ and were perhaps somewhat diminished when 2.7 mM Ca++ was present in the medium. Serotonin-induced accumulation of cGMP was also
Exp.
Medium
Agonist
1
Complete
No Ca++
Complete
NoCa++
Complete
NoCa++
Complete
No Ca+
Complete
No Ca++
Complete
No Ca+
Serotonin,
10 ,M
Sodium ascorbate,
0.5 mM
Methylene blue,
27,uM
Bradykinin,
1.1 AM
Histamine,
2
3
4
5
6
1 gM
lonophore A23187,
20,uM
of
protein
2.40
3.17
2.64
7.51
0.26
0.51
5.30
0.05
7.16
0.15
3.42
0.14
For each experiment, artery segments from a single umbilical
cord were incubated for 2 hr in the indicated medium in room air.
Agonists were added and incubations were terminated after 2 min
(serotonin, ionophore A23187, bradykinin, and histamine) or 6 min
(sodium ascorbate, methylene blue).
*Difference between mean cGMP content of duplicate samples
treated with and without agonist.
somewhat reduced by exogenous Ca++ (5). The effects of
serotonin, like those of sodium ascorbate and methylene
blue, tended to be enhanced in 02-deprived arteries.
Interrelationships between cellular Ca++ movement and
oxidation have been observed (20, 21). In the umbilical artery the effects of 02 and agonists dependent on it (bradykinin, histamine, and ionophore A23187) appear to be exerted
through Ca++-dependent processes, whereas serotonin, sodium ascorbate, and methylene blue cause accumulation of
cGMP through mechanisms that do not require and may be
inhibited by exogenous Ca++ and 02. Recently, two evidently separable guanylate cyclases have been reported in several tissues (18, 19): a soluble enzyme that is stimulated by
Ca++ and a particulate enzyme that is inhibited by Ca
The demonstration in umbilical artery of two seemingly distinct mechanisms, influenced differently by Ca++ and 02,
for control of cGMP content may reflect the existence of two
separate guanylate cyclases in this tissue also.
We thank Mrs. Betty Hom for excellent technical assistance. We
are grateful to the obstetrical staffs and especially the obstetrical
nursing staffs of Sibley Memorial Hospital in the District of Columbia, Suburban Hospital in Maryland, and National Naval Medical
Center, whose cooperation and assistance made possible this study.
1. Panigel, M. (1962) Am. J. Obstet. Gynecol. 84, 1664-1683.
2. Bor, I. & Guntheroth, W. G. (1970) Can. J. Physiol. Pharmacol. 48, 500-502.
3. Eltherington, L. G., Stoff, J., Hughes, T. & Melmon, K. L.
(1968) Circ. Res. 22, 747-752.
4. Clyman, R. I., Sandler, J. A., Manganiello, V. C. & Vaughan,
M. (1975) J. Clin. Invest. 55, 1020-1025.
5. Clyman, R. I., Blacksin, A. S., Sandler, J. A., Manganiello, V.
C. & Vaughan, M. (1975) J. Biol. Chem., 250,4718-4721.
6. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.
(1951) J. Biol. Chem. 193,265-275.
7. Sass, M. D., Caruso, C. J. & Axelrod, D. R. (1967) J. Lab. Clin.
Med. 69,447-455.
Proc. Nat. Acad. Sct. USA 72(1975)
Biochemistry: Clymantei al.
8. Bigley, R. H. & Stankova, L. (1974) 1. ETp. Med. 139, 10841092.
9. Jones, J. K., Guevara, A., Engleman, E. & Melmon, K. L.
(1970) PharMacologist 12,263.
10. Nair, X. & Dyer, D. C. (1973) Am. J. Phystol. 225, 11181122.
_
11. Nair, X. & Dyer, D. C. (1974) Res. Commun. Chem. Pathol.
Pharmacol. 7,31-388.
12. Oberhansli-Weiss, I., Heymann, M. A., Rudolph, A. M. &
Melmon, K. L. (1972) Pediat. Res. 6, 693-700.
13. Kovalcik, V. (1963) J. Physiol. 169, 185-197.
14.
15.
16.
17.
18.
19.
20.
21.
3887
Fay, F. (1971) Am. J. Phystol. 221,470-479.
Detar, R. & Bohr, D. F. (1968) Am. J. Physiol. 214, 241-244.
Joiner, P. D. (1973) J. Pharmacol. Exp. Ther. 185,84-93.
Karim, S. M. M. (1967) Br. J. Pharmacol. Chemother. 29,
230-237.
Kimura, H. & Murad, F. (1974) J. Biol. Chem. 249, 69106916.
Chrisman, T. D., Garbers, D., Parks, M. A. & Hardman J. G.
(1975) j. Biol. Chem. 250, 374-381.
Lehninger, A. (1970) Btochem. J. 119,129-138.
Otto, D. A. & Ontko, J. A. (1974) Biochem. Biophys. Res.
Commun. 61, 743-750.