Cyclic nucleotides and contractility in human and sheep umbilical arteries

European Journal of Pharmacology, 73 (1981) 283- 291 Elsevier/North-Holland Biomedical Press

283

CYCLIC NUCLEOTIDES AND CONTRACTILITY IN HUMAN AND SHEEP UMBILICAL ARTERIES RONALD R. FISCUS * and DONALD C. DYER **

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Iowa State University, Ames, 1,4 50011, U.S.A. Received 22 April 1981, accepted 21 May 1981

R.R. FISCUS and D.C. DYER, Cyclic nucleotides and contractility in human and sheep umbilical arteries, European J. Pharmacol. 73 (1981) 283-291. Strips of human umbilical artery (HUA) and sheep umbilical artery (SUA) were freeze-clamped at selected times during drug-induced contraction or relaxation. Tissue concentrations of cyclic AMP and cyclic GMP were measured. 5-Hydroxytryptamine (10 #M) contracted HUA and SUA, but had no detectable effect on cyclic GMP; cyclic AMP rose in SUA (but not in HUA) after contractions had begun. Histamine (4/~M) elevated cyclic GMP in HUA with a time course that lagged behind contraction. Prostaglandin Ej (PGE0, PGE 2 and PGF2,, (2.8 /~M with ,0.1% v / v ethanol) contracted HUA and decreased cyclic GMP. Nitroglycerin (3/~M) relaxed KCl-contracted HUA; this relaxation was preceded by an initial 29-fold elevation of cyclic GMP. Our data show no consistent correlation of cyclic nucleotide changes with contraction of umbilical arteries and suggest that such changes are not essential for contraction of vascular smooth muscle. In contrast, the temporal relationship between cyclic GMP elevation and relaxation induced by nitroglycerin suggests a possible role for cyclic GMP. Blood vessel

Cyclic AMP

Cyclic GMP

5-Hydroxytryptamine

1. Introduction

Relaxation of vascular smooth muscle, induced by fl-adrenergic agonists (Namm and Leader, 1976) or by E-type prostaglandins (PG) (Dunham et al., 1974; Kadowitz et al., 1975) has been associated with an increase in tissue concentrations of cyclic 3':5'-adenosine monophosphate (cyclic AMP). Vasoconstrictors, such as 5-hydroxytryptamine, bradykinin, histamine and K ÷ have been found to elevate cyclic Y:5'-guanosine monophosphate (cyclic GMP) concentrations in isolated segments of human umbilical artery (Clyman et al., 1975c). In bovine mesenteric artery, contractions induced by phenylephrine or histamine have been associated with an initial decrease in cyclic AMP

* Present address: Division of Pharmacology, M-013, Department of medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093, U.S.A. ** To whom reprint requests should be submitted.

Nitroglycerin

Prostaglandins

:(Andersson, 1973). Furthermore, an elevation in the ratio of cyclic GMP/cyclic AMP has been found in blood vessels of rats with experimentallyinduced hypertension and it has been proposed that these elevated ratios are involved in the increased tone and responsiveness of vascular smooth muscle in hypertensive animals (Amer, 1977). Based on these findings, it has been hypothesized that relaxation of vascular smooth muscle is mediated by an increase in cyclic AMP whereas contraction is mediated by an elevation of cyclic GMP and/or a decrease in cyclic AMP. However, more recent studies have found dissociations between changes in cyclic nucleotide concentrations and changes in the contractile state in a variety of smooth muscle preparations (Katsuki and Murad, 1977; Diamond, 1978; Janis and Diamond, 1979). At present, the role of cyclic nucleotides in contraction and relaxation of vascular smooth muscle remains unclear. Clyman et al. (1975a, b,c) reported that cyclic nucleotide concentrations in isolated segments of human umbilical artery (HUA) change following

0014-2999/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press

284 exposure of the tissue to a variety of vasoactive agents. However, contractile responses were not reported in their study and therefore no direct correlation could be made between changes in cyclic nucleotide concentrations and changes in the contractile state. In the present study, we measured tissue concentrations of cyclic AMP and cyclic GMP in isolated strips of HUA that had been freeze-clamped at various stages of isotonic contraction or relaxation induced by selected vasoactive agents. Changes in cyclic nucleotide concentrations were compared to changes in contractility. 5-Hydroxytryptamine (5-HT), histamine, and prostaglandins E t, E 2 and F2~ were used as vasoconstrictors and nitroglycerin was used as a vasodilator.

2. Materials and methods

2.1. Preparation of tissue Human umbilical cords were obtained at full term following normal vaginal delivery. Sheep umbilical cords were obtained by Cesarean section on day 120-125 of gestation (full gestation = 147 days) (Dyer, 1970). Umbilical cords were immediately placed in a modified Krebs-bicarbonate (Krebs) solution. The Krebs solution contained: 115.3 mM NaC1, 4.70 mM KC1, 1.17 mM KH2PO4, 1.17 mM MgSO4, 1.82 mM CaC12, 7.88 mM glucose, 1.00 mM pyruvic acid, 24.9 mM NaHCO 3, and 0.0269 mM Na2EDTA. Within 30 min of obtaining the umbilical cords, arteries from the placental halves of the cords were dissected free from other vessels and of the surrounding Wharton's jelly and were helically cut into strips approximately 2 cm long and 3 mm wide (Dyer, 1970). The strips were suspended under 1 g resting tension in 50 ml organ baths containing Krebs solution at 37°C aerated with O2:CO 2 (95:5). The strips were allowed to relax for 3-4 h before drug additions. During this time, the medium was replaced with fresh Krebs solution every 20-30 rain. This lengthy procedure for relaxation and washing of the strips was found to be necessary to assure that the strips were fully relaxed and quiescent. Prior to the addition of the test agonists, all strips were contracted with 5-HT

in cumulative doses (1 nM to 1/,M) and allowed to again relax (see fig. 1 as an example). This initial 5-HT-induced contraction, which represents approximately 95% of maximal contraction, was used as the standard contraction for normalizing contractile responses between different arterial preparations. 5-Hydroxytryptamine-creatinine sulfate (CalBiochem), histamine-HC1 (Cal-Biochem), and nitroglycerin (Eli Lilly) were dissolved in saline (0.9% NaC1) and were added to the 50-ml baths in a volume ranging from 10 to 500/,1. PGE1, PGE 2 and PGF2~ (a gift from the Upjohn Company) were dissolved in 100% ethanol and were added to the 50-ml baths in a volume of 50/,1. At selected times following the addition of the vasoactive agonists, the baths were rapidly lowered and the strips were frozen between large WoUenberger clamps precooled to -190°C; this was accomplished in less than 2 sec. The strips were stored in liquid nitrogen until they were assayed for their cyclic nucleotide content. Contractions were measured isotonically.

2.2. Determination of cyclic nucleotides Frozen strips of artery were homogenized in 4 ml of 0.2 M HC1-98.3% ethanol (Binder et al., 1975) with a Tekmar Tissumizer (Model SDT100EN). Prior to and during homogenization, tubes containing the extraction solution were partially submerged in an ethanol-dry ice bath. This technique maintained the temperature of the extraction solution below - 4 0 ° C throughout the homogenization procedure. The homogenate was split into two fractions: one for cyclic AMP determination and the other for cyclic GMP determination. Appropriate tracers were added to each fraction for the determination of fractional recovery. The homogenates were centrifuged at 10000 X g for 20 m i n at 4°C. The supernatant fractions were evaporated to dryness under a stream of air at room temperature; cyclic AMP and cyclic GMP were found to be stable under these conditions. The residues were taken up in 1.0 ml of acetate buffer (50 mM sodium acetate, pH 6.2) and 100-/,1 aliquots were used for RIA and for recovery determination. (Preliminary experiments showed

285

that purification of samples was unnecessary.) Contents of cyclic AMP and cyclic GMP were measured using RIA kits purchased from New England Nuclear or, in some experiments, using the RIA procedure of Harper and Brooker (1975) with antibody kindly provided by Dr. Gary Brooker. In order to increase the sensitivity of the assay, all samples were acetylated prior to RIA analysis (Harper and Brooker, 1975). The validity of this analysis of human and sheep umbilical arteries was verified by treating some samples with cyclic nucleotide phosphodiesterase or by spiking the samples with a known amount of cyclic nucleotide prior to RIA analysis. Further verification of the technique was obtained when measured cyclic nucleotide contents were found to be directly proportional to the amount of sample used. The protein pellets were dissolved in 1 N NaOH and assayed by the procedure of Lowry et al. (1951) using BSA (Sigma Chemical Company, A4378) as a standard.

3.1. Effect of 5-HT in human umbilical artery Fig. 1 shows the levels of cyclic AMP and cyclic GMP in HUA strips frozen at the peak (times B and D) of contractions induced by cumulative doses or a single dose of 5-HT. Cyclic nucleotide levels are reported as a percentage of control concentrations (at time A). Comparisons were made between cyclic nucleotide concentrations in strips at: B versus A, C versus A, D versus A, and D versus C. No significant differences in cyclic nucleotide concentrations were found. In addition, some strips were frozen at 15 sec following the addition of 5-HT (10 #M). All strips had begun

B

g c o

o

2.3. Statistics

o

"E o

Data in the tables and graphs represent the mean__+ standard error of the mean (S.E.M.). Paired t-tests were used to compare cyclic nucleotide concentrations (in pmol/mg protein) in paired strips (one treated and another untreated) taken from the same umbilical artery. Arteries from 4 to 9 individual umbilical cords were used for each experiment.

Basal cyclic nucleotide concentrations irt HUA strips taken from male and female neonates were found to be essentially the same; an unpaired t-test was used to make this comparison. Cyclic AMP concentrations (pmol/mg protein) were 0.71 __+0.07 for female (n = 10) and 0 . 9 5 _ 0.20 for male (n = 6) and cyclic GMP concentrations (pmol/mg protein) were 0.20-+-.0.02 for female ( n = 10) and 0.22__+0.03 for male (n=6). The sex of the newborn was not considered in the following experiments.

101JM 5-HT

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3. Results

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B

C

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Fig. 1. Cyclic nucleotide concentrations in isolated strips of human umbilical artery during contractions induced by cumulative doses (1 nM-1 #M, added at half-log increments) or a single dose (10 #M) of 5-hydroxytryptamine (5-HT). The upper half of the graph is a typical recording of isotonic contraction induced by 5-HT. An upward deflection on the tracing represents a shortening of the arterial strip with a 10-fold amplification. Points A, B, C and D represent the times at which the strips were frozen. Shortly after point B, the recording paper was stopped and the strips were washed several times with fresh Krebs solution over a period of 40 to 60 rain. The cyclic nucleotide concentrations__+ S.E.M. of these strips are shown in the lower half of the graph. The number of arteries from individual umbilical cords that were analyzed in each treatment group are indicated within the bar graphs. Control cyclic AMP concentrations= 1.21 ___0.15 pmol/mg protein and cyclic GMP concentrations = 0.42__+0.10 pmol/mg protein.

286 contracting at 5 - 1 0 sec after 5-HT addition. Again, no change in the concentrations of either cyclic nucleotide was found at this early stage of contraction (data not shown).

o

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3.2. Effects of 5-HT in sheep umbilical artery In contrast to H U A strips, strips from sheep umbilical, artery (SUA) showed an elevation in cyclic A M P levels during 5-HT-induced contractions (fig. 2). Cyclic A M P concentrations were significantly elevated at 20, 40, 60 and 120 sec ( P < 0 . 0 5 ) and at 240 and 660 sec ( P < 0 . 0 1 ) after 5-HT addition. However, contractions of the strips began at 5 - 1 0 sec after addition of 5-HT and, therefore, the measurable elevation of cyclic A M P lagged behind the time course of contraction. Fig. 3 shows that the elevation of cyclic A M P in SUA was dependent on the dose of 5-HT; i.e., 10/~M 5-HT increased cyclic A M P concentrations more than 0.03 /~M 5-HT. Cyclic A M P concentrations were significantly ( P < 0 . 0 5 ) elevated by 5-HT at

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Fig. 3. Cyclic AMP levels in isolated strips of sheep umbilical artery during contractions induced by selected concentrations of 5-hydroxytryptamine(5-HT). The strips were frozen at 11 rain, the peak of contraction, after 5-HT addition. The lower half of the graph shows the cyclic AMP levels+ S.E.M. in these strips. The number of arteries from individual umbilical cords is indicated in the bar graphs. Control cyclic AMP concentrations= 1.24±0.09 pmol/mg protein.

all concentrations tested. No significant effect of 5-HT on cyclic G M P concentrations was observed in SUA (fig. 2).

3.3. Effect of histamine in human umbilical artery 02OO

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40 60 120 240 660 2O Seconds after 5-HT (10pM)

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Fig. 2. Cyclic nucleotide levels in isolated strips of sheep umbilical artery at selected times during contraction induced by

5-hydroxytryptamine (5-HT) (10 tLM). The lower half of the graph shows the cyclicnucleotide levels± S.E.M. in these strips. The number of arteries from individual umbilical cords that were analyzed is indicated in the bar graphs. Control cyclic AMP concentrations= 1.27±0.08 pmol/mg protein and cyclic GMP concentrations= 0.31 ~ 0.06 pmol/mg protein.

Histamine-induced contraction of isolated H U A strips was associated with an increase in cyclic G M P concentrations with no detectable change in cyclic A M P concentrations (fig. 4). Cyclic G M P elevations of 3.5- and 7.0-fold were observed at 60 sec and 240 sec, respectively. However, the concentrations of cyclic G M P were unchanged at 15 sec following the addition of histamine even though all strips had begun contracting at that time.

3.4. Effects of PGF~, PGE 2 and PGFea in human umbilical artery Since ethanol was used as the vehicle for the addition of the prostaglandins, strips exposed to ethanol alone were included as solvent controls

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Fig. 4. Cyclic nucleotide levels in isolated strips of human umbilical artery during contractions induced by ~stamine (4/~M). Contraction reported at 30 sec was from strips subsequently frozen at 60 sec. Contraction at 120 and 180 sec was from strips subsequently frozen at 240 sec. The lower half of the graph shows the cyclic nucleotide levels±S.E.M, in the strips at the times indicated. Control concentrations of cyclic A M P = 0 . 9 9 ~ 0 . 1 6 p m o l / m g protein and cyclic GMP=0.21 + 0.03 p m o l / m g protein. Arterial strips from 6 individual umbilical cords were used.

(table 1). Ethanol (0.1% v/v; 17.1 mM) by itself induced a slow, steady contraction of isolated HUA strips: the contraction began at approximately 30 sec following addition of ethanol and continued to increase for at least 60 min. Concentrations of cyclic AMP were unchanged throughout 60 min of exposure. Cyclic GMP concentrations, on the other hand, were significantly reduced at 4 min and 60 min, but not at 0.5 min, following exposure to ethanol. All three of the prostaglandins induced rapid concentrations of isolated HUA strips; contractions began at 0.5 min and reached their peak at approximately 4 min (table 1). A significant elevation of cychc AMP was observed following exposure for 0.5 min and 4 min to PGE~ (2.8 gM) or for 4 min to PGE 2 (2.8 gM). PGF2~ (2.8 #M) had no detectable effect on cyclic AMP concentrations. Following the addition of each prostaglandin, cyclic GMP concentrations were not significantly changed at 0.5 min but were significantly ( P < 0 . 0 1 ) lowered at 4 min (table 1). However, when cyclic GMP concentrations in prostaglandin-treated strips were compared to concentrations in ethanoltreated strips, no significant differences were found.

TABLE 1

Effects of PGE t , PGE 2, PGF2=, and ethanol on contractility and cyclic nucleotide concentrations of isolated human umbilical artery. Numbers in parentheses indicate the number of arteries from individual umbilical cords that were analyzed in each group. Data represents mean±standard error. * P<0.05; ** P<0.01. Additions

Contact time (rain)

Contraction (% of standard 5-HT [ I/~M])

pmol/mg protein Cyclic AMP

Cyclic G M P

None

-

-

0.72_+0.07 (7)

0.21 ±0.02 (8)

Ethanol (0.1% v/v)

0.5 4.0 60

0.8±0.5 4.8 ± 1.4 10 ± 2

0.55±0.05 (4) 0.53 ± 0.06 (6) 0.61±0.13 (6)

0.15 ___0.03 (4) 0.078 ~ 0.031 (5) ** 0.10 ___0.03 (4) **

PGE~ (2.8 gM)

0.5 4.0

1.2±0.9 29 ± 12

1.21 ± 0 . 2 0 (5) * 1.20±0.26 (6) *

0.22 ±0.06 (5) 0.076±0.023 (6) **

PGE 2 (2.8 gM)

0.5 4.0

1.7±0.8 44 ± 10

0.81 ±0.11 (5) 1.02__+0.17 (5) *

0.17 ± 0 . 0 4 (5) 0.062±0.011 (5) **

PGF2= (2.8/~M)

0.5 4.0

2.0±0.6 62 -+-7

0.91 -+-0.18 (6) 0.74±0.09 (6)

0.21 ± 0 . 0 6 (5) 0.058±0.009 (6) **

288 TABLE 2 Effect of nitroglycerin on contractile state and cyclic nucleotide concentrations of human umbilical arteries contracted with KC1. Relaxation began at 30-45 sec following addition of nitroglycerin. KCI (30 mM) produced a sustained contraction (94 ± 3% of the 5-HT standard contraction) which remained at a constant level for several hours. Numbers in parentheses indicate the number of arteries from individual umbilical cords that were analyzed in each group. Data represents mean +__standard error. * P < 0.05 versus KC1 (30 mM) alone; ** P<0.01 versus KC1 (30 mM) alone. Additions

Contact time (min)

None KC1 (30 mM)

30

KCI (30 mM) + nitroglycerin (3/~M)

30

KC1 (30 mM)+ nitroglycerin (3 #M)

30

0.5

4.0

Contractile state (% of KCl-induced contraction)

pmol/mg protein Cyclic AMP

Cyclic GMP

-

0.64±0.08 (5)

0.24"+'0.03 (5)

100

0.69~0.13 (5)

0.29±0.08 (4)

100

0.89-+-0.14 (5)

8.4 ±2.2 (5) **

85±3*

3.5. Effect of nitroglycerin in human umbilical artery Table 2 shows the concentrations of cyclic nucleotides during nitroglycerin-induced relaxation of KCl-contracted strips of HUA. No change in cyclic nucleotides was observed in HUA strips frozen after 30 min of continuous contraction induced by KC1 (30 mM). Exposure of the KC1contracted strips to nitroglycerin (3 ttM) for 30 sec caused a 29-fold increase in cyclic GMP concentrations with no detectable change in cyclic AMP. This initial elevation of cyclic GMP was followed by a partial relaxation of the contracted strips. A 130-fold increase in cyclic GMP and a 67% increase in cyclic AMP were noted at the time (4 min) strips had reached maximal relaxation to nitroglycerin.

4. Discussion

In a review of the involvement of cyclic nucleotides in regulating smooth muscle contractility, Diamond (1978) summarized findings which demonstrated a dissociation between changes in contractility and changes in cyclic nucleotide concentrations in a variety of smooth muscle preparations. In contrast, many other studies have pro-

1.14±0.24(5)*

38

±4(5)**

vided data which implicate cyclic nucleotides as mediators of contractile regulation in vascular smooth muscle (Andersson, 1973; Dunham et al., 1974; Clyman et al., 1975a,b,c; Kadowitz et al., 1975; Amer, 1977; Clyman, 1978). In many of these studies, however, changes in cyclic nucleotide concentrations were not directly compared to contractile changes. Our objective was to study the direct correlation between changes in contractility and changes in cyclic nucleotide concentrations in human vascular tissue. We selected the human umbilical artery (HUA), since it possesses many characteristics that make it particularly useful for these types of experiments. First, HUA has a high proportion of smooth muscle compared to other vascular components; HUA has very little elastin and essentially no collagen (Roach, 1973). Roach (1973) states that umbilical arteries are probably the most muscular arteries that occur in mammals. Second, the placental half (the part used in the present study) of HUA has been reported not to be innervated (Ellison, 1971). If HUA is noninnervated, then the release and subsequent action of endogenous neurotransmitters would not be a complicating factor. Third, HUA is a human blood vessel that is readily obtainable for research in a relatively healthy state. We found no change in the concentration of

289

cyclic GMP or cyclic AMP in isolated HUA strips frozen at the beginning or at the peak of contraction induced by 5-HT. These results conflict with those of Clyman et al. (1975c), who found that 5-HT increased cyclic GMP in incubated segments of HUA. This difference in 5-HT response may be due to differences in experimental technique. Nevertheless, our results agree with the findings from studies of other smooth muscle preparations (Diamond, 1978). Our data are inconsistent with the hypothesis that increases in cyclic GMP or decreases in cyclic AMP mediate contraction in vascular smooth muscle. For comparative purposes, we also determined the effect of 5-HT on cyclic nucleotide concentrations in isolated strips of sheep umbilical artery (SUA). 5-HT induced a time-dependent and dosedependent increase" in cyclic AMP concentrations without changing the concentrations of cyclic GMP. The elevation of cyclic AMP in SUA appeared to lag behind the time course of contraction. The data with umbilical arteries from sheep further demonstrate a dissociation between increases in cyclic GMP or decrease in cyclic AMP and contraction in vascular smooth muscle. The functional significance of the cyclic AMP elevation in 5-HT contracted SUA strips is presently unclear. The fact that cyclic AMP rose in SUA but not in HUA following exposure to 5-HT may be related to species differences or differences in gestational age. Histamine has been reported to induce a large, Ca2+-dependent elevation of cyclic GMP in isolated segments of HUA (Clyman et al., 1975b). We also found that histamine induced an increase in cyclic GMP with no detectable change in cyclic AMP in isolated HUA strips. However, the increase in cyclic GMP appeared to lag behind the time course of contraction. Therefore, our data are inconsistent with the hypothesis that increases in cyclic GMP or decreases in cyclic AMP mediate histamine-induced contraction in vascular smooth muscle. Similar dissociations between the time course of cyclic GMP elevation and contraction were observed in guinea pig myometrium and taenia coli (Diamond, 1978) and in bovine tracheal smooth muscle (Katsuki and Murad, 1977). PGFE~-induced contraction of vascular smooth

muscle has been reported to be associated with an increase in cyclic GMP concentrations, whereas relaxation induced by PGE l or PGE 2 has been associated with an increase in cyclic AMP concentrations (Dunham et al., 1974; Kadowitz et al., 1975). These observations support the hypothetical involvement of cyclic nucleotides in the regulation of vascular contractility as stated in the Introduction. However, discrepancies in this hypothesis are apparent. For example, PGF2~, at a concentration that is known to contract HUA strips, had no effect on cyclic nucleotides in HUA segments (Clyman et al., 1975c). Furthermore, PGE 1 caused an elevation of cyclic AMP in HUA segments (Clyman et al., 1975c); yet PGE~ has been shown to contract isolated HUA strips (Park et al., 1972). In the present study, HUA strips were contracted by PGEI, PGE2, or PGF2~ (each at 2.8/~M). This concentration is approximately the ECs0 for PGE 2 and PGF2~ in causing HUA contraction (Park et al., 1972). PGE I is somewhat less potent as a vasoconstrictor of HUA (Park et al., 1972) and this is reflected in the smaller contractile response elicited by PGE~ as compared to the response to the other prostaglandins (table 1). The prostaglandins in the present study were dissolved in 100% ethanol and were added to the organ baths to give a final ethanol concentration of 0.1% v / v (17.1 mM). This procedure is in accordance with that of Clyman et al. (1975c) and Kadowitz et al. (1975). However, in contrast to their findings, we found that ethanol by itself induced a significant reduction in the cyclic GMP concentrations when measured at 4 min and 60 min after ethanol addition. No change in cyclic GMP concentrations was noted at 0.5 min or in cyclic AMP concentrations at 0.5, 4, and 60 min. Reduction in cyclic GMP following exposure to ethanol is not unique to vascular tissue, since such responses have been reported to occur in brain (Redos et al., 1976) and heart (Vesely et al., 1978). Furthermore, ethanol has been reported to inhibit guanylate cyclase activity in heart (Vesely et al., 1978) and mammary gland (Rillema, 1978). A similar mechanism may be involved in ethanolinduced decrease of cyclic GMP in HUA strips. PGE 1 and PGE2, but not PGF2~, elevated cyclic AMP concentrations in HUA. These findings agree

290 with those reported by Clyman et al. (1975c), who suggested that cyclic AMP may be involved in prostaglandin-induced relaxation of HUA. However, we found that H U A was contracted by a l l three prostaglandins. In addition, it has been reported that isoproterenol fails to elevate cyclic AMP concentrations in H U A (Clyman et al. 1975c). Therefore, we feel that there is presently no strong evidence linking cyclic AMP to contractile changes in HUA. All prostaglandins (with ethanol as the vehicle) caused depression of cyclic G M P concentrations in H U A after 4 min of exposure. Since this response is the same as that observed with ethanol alone, the cyclic G M P decrease is attributed to the action of the vehicle. Although no significant changes in cyclic G M P concentrations were observed after 0.5 min of exposure, it is possible that ethanol may have interfered with the ability of the prostaglandins to activate guanylate cyclase at this early time. We emphasize, therefore, that previously reported data from experiments in which ethanol was used as a vehicle for drug delivery may need to be re-evaluated. Our data clearly show that contractions of H U A induced by PGE~, PGE 2, or PGF2~ (in the presence of ethanol) are not associated with increases in cyclic G M P concentrations or decreases in cyclic AMP concentrations. Sodium azide, sodium nitrite, organic nitrates, and a number of nitroso-containing compounds have been shown to elevate cyclic G M P concentrations in a variety of tissues (Katsuki and Murad, 1977, Katsuki et al., 1977, Schultz et al., 1977, Diamond, 1978, Janis and Diamond, 1979). Diamond (1978) reported that nitroglycerin caused a 16-fold elevation of cyclic G M P concentrations in canine femoral arteries. However, the temporal relationship between the cyclic G M P elevation and relaxation was not established. Holzmann et al. (1978) reported that sodium nitroprusside elevated cyclic G M P concentrations in strips of bovine coronary artery; this elevation was found to have a dose-dependency which correlated with relaxation. In the present study, we found that nitroglycerin (3 #M) caused a 29-fold elevation of cyclic G M P which clearly preceded relaxation. Although cyclic AMP concentrations were also slightly elevated at

4 min, this response occurred well after the beginning of relaxation. Our data show that cyclic G M P concentrations can be greatly elevated in H U A strips without the occurrence of smooth muscle contraction. In addition, our data and the data of others suggest that cyclic G M P may be involved in the relaxation of vascular smooth muscle induced by nitroglycerin and related vasodilators. There is evidence, owever, that this latter relationship may not hold for all forms of smooth muscle (Diamond, 1978; Janis and Diamond, 1979). We conclude that changes in cyclic nucleotide concentrations do not correlate with contractions of umbilical arteries. At this time, however, we can not rule out the possibility that cyclic nucleotides may regulate vascular contractility in a way undetected by measurements of whole tissue concentrations of cyclic nucleotides. Diamond (1978) has pointed out that smooth muscle contractility may be regulated by cyclic nucleotides in specific subcellular pools or by turnover of cyclic nucleotides rather than by whole tissue concentrations. Therefore, future experiments will need to test these possibilities. At present, however, the experimental evidence argues against the proposed involvement of cyclic nucleotides in vascular contractions. Relaxation induced by nitroglycerin, on the other hand, appears to be temporally linked to cyclic G M P elevation.

Acknowledgements This project was supported in part by the Iowa Heart Association, Inc. and the Iowa State UniversityGraduate College. We are grateful to Ann Schmitt and the obstetrical nursing staff at Mary Greeley Hospital, Ames, Iowa, for their cooperation in obtaining the umbilical cords.

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