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Nitroprusside enhances isoprenaline-induced increases in cAMP in rat aortic smooth muscle
471
European Journal of Pharmacology, 191 (1990) 471-475
Elsevier EJP 20747
Short communication
itroprusside enhances isopenaline-in smooth Donald
ncreases in c
H. Maurice 1 and Richard J. Haslam ‘,*
Departments of I Pathology and ’ Biochemistry, McMaster Uniuersiy, Hamilton. Ontario, Canada LBN 325
Received 20 September 1990, accepted 16 October 1990
Low concentrations phenylephrinc-induced
of sodium contraction
nitroprusside of rat aortic
(SNP) and of isoprenaline smooth
muscle.
acted synergistically to inhibit the In experiments with these concentrations, SNP
enhanced the increases in smooth muscle CAMP caused by isoprenaline by 4- to 5-fold, whereas the SNP-induced increases in tissue cGMP were unaffected by isoprenaline. We conclude that CAMP is likely to mediate the synergistic inhibition of the contraction of rat aortic smooth muscle by these compounds. Nitroprusside; Isoprenaline; cGMP; CAMP; Smooth muscle: Aorta (rat) 1. Introduction
We have recently demonstrated vasodilators,
that nitrosuch as sodium nitroprusside (SNP)
which stimulates soluble guanylyl cyclase, and isoprenaline which activates adenylyl cyclase, interact synergistically to inhibit contraction of rat aortic smooth muscle (Maurice et al., in press). These results parallel those obtained previously with blood platelets, in which SNP and endothelium-derived relaxing factor (EDRF) potentiate the ability of prostacyclin (PGI,) and of adenosine to inhibit platelet aggregation (Levin et al., 1982; Radomski et al., 1987; Maurice and Haslam, 1990). In the latter case, we demonstrated that this synergism was caused by an enhanced accumulation of cyclic AMP (CAMP) resulting from the inhibitory action of cyclic GMP (cGMP) on the platelet cGMP-inhibited CAMP phosphodiesterase (CGI-PDE) (Maurice and Haslam, 1990). A similar enzyme activity has recently been demonstrated in rat aorta (Lindgren et al., 1990). More-
Correspondence to: R.J. Haslam, Department of Pathology, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 325.
over, we have reported that rilostamide, a selective inhibitor of the CGI-PDE, p&entiates the effects of isoprenaline on rat aortic smooth muscle to the same extent as SNP (Maurice et al., in press). We have therefore studied the effects of SNP and isoprenaline on the intracellular cyclic nucleotide levels in rat aortic smooth muscle to determine whether the synergistic inhibition of contraction was also associated with an enhanced accumulation of CAMP in this tissue.
2. Materials and methods 2. I. Materials
Male Wistar-Kyoto (WKY) rats were supplied by Harlan Sprague-Dawley (Indianapolis, IN, U.S.A.). Polypropylene microtubes with pestles were from Mandel Scientific (Guelph, Ont., Canada). CAMP and cGMP ‘251-radioimmunoassay kits and r3H]cAMP (30-50 Ci/mmol) were obtained from Du Pont Canada (Mississauga, Ont., Canada). SNP, ( - )-isoprenaline-( + )-bitartrate, phenylephrine and protein standard solution were from Sigma (St. Louis, MO, U.S.A.).
0014-2999/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
er materials were obtained from sources listed reviously (Maurice and Haslam, 1990; Maurice ., in press). ?? p _.-.
ration of de-endothelialized rat aortic rings
ale WKY rats (225-250 g) were killed by cervical dislocation. The thoracic aortas were rapidly removed and placed in a physiological salt solution (PSS) containing (mM) 1.16 MgSO,, 2.5 CaCl,, 21.9 NaHCO,, 1.16 NaH2P0,, 4.6 KCl, 115.5 NaCl and 11.l dextrose, which was equilibrated with 95% O,-5 COZ. After removal of adherent connective tissue and fat, each aorta was cut transversely into four separate 4 mm rings. The endothelium was removed from the rings by gently rubbing the luminal surfaces with a wooden stick. 2.3. Measurement of the effects of SNP and isoprenaline on phenylephrine-induced aortic smooth muscle
contraction of rat
The methods used have been described in detail previously (Maurice et al., in press). Briefly, aortic rings at an initial tension of 2 g were equilibrated for 2 h at 37” C in PSS gassed with 95% O,-5% COZ. During this period the PSS was frequently changed and the rings were treated four times with 30 mM KC1 to recharge internal Ca2+ stores. Following equilibration, the aortic rings were incubated for 30 s with SNP and isoprenaline, either singly or in combination. Phenylephrine (100 nM) was then added and the increase in tension was measured after a further 2 min. Control contractions and the effects of all combinations of inhibitors were measured on each aortic ring (Maurice et al., in press). inhibitions of contraction were calculated as percentages of the control contraction in the same ring. Results were expressed as means + S.E.M. a;rd the significance of differences in the inhibition of contraction were evaluated by paired t-tests. 2.4. Cyclic nucleotide measurements
De-endothelialized rat aortic rings were equilibrated in PSS, as described for measurement of
the inhibition of contraction, except that the rings were not under tension. Rings were then incubated for 2.5 n-tin at 37” C in 1.0 ml of PSS containing appropriate concentrations of SNP and/or isoprenaline. Each incubation contained two rings. Aortic rings from different parts of the thoracic aorta were distributed identically in replicates of each incubation condition to prevent any effect attributable to possible differences in their responsiveness to additions. After incubation, the rings were rapidly transferred to polypropylene microtubes precooled in liquid nitrogen. The frozen tissue was immediately pulverized with a precooled polypropylene pestle and then homogenized in 1 ml of 5% (w/v) trichloroacetic acid, following which 6 000 dpm of [3H]cGMP and of [3H]cAMP were added to monitor the recoveries of the two cyclic nucleotides. cGMP and CAMP were then separated and purified by a dual column procedure and finally acetylated and quantitated by radioimmunoassay (Maurice and Haslam, 1990). Trichloroacetic acid-precipitated protein was dissolved in 1 M NaOH and assayed by the Lowry method, using protein standard solution from Sigma. Cyclic nucleotide levels were calculated as means +_S.E.M.; changes were evaluated by unpaired t-tests.
3. Results Although incubation of de-endothelialized rat aortic smooth muscle with isoprenaline caused a concentration-dependent inhibition of contraction, much more inhibition was observed when low concentrations of isoprenaline were added in combination with a concentration of SNP that had little effect alone (fig. 1A). Thus, when rings were incubated with 5 nM SNP which by itself inhibited contraction by only 12 + 2%, the inhibition caused by 100 nM isoprenaline was increased from a control value of 20 f 4 to 80 + 6% (mean + S.E.M., 10 aortic rings). Under these conditions, the effects of the two vasodilators were clearly supra-additive (P < O.OOl),as were the effects seen with 5 nM SNP and 500 nM isoprenaline (fig. 1A). Higher concentrations of isopre-
0
100
500
Isoprenaline
(nM)
Fig. 1. Effects of 5 nM SNP and various isoprenaline concentrations on the phenylephrine-induced contraction of rat aortk smooth muscle and on cyclic nuckotide levels in this tissue.(A) Rat aortic rings mounted in organ baths were in~bat~ for 30 s with the indicated concentrations of isoprenaiine in the absence (ail)or presence ( f 5 nM SNP. Phenylephrine (100 nM) was added and the tension developed was measured after a further 2 min. The inhibition o contraction was calculated from paired records obtained from the same aortic rings. Values are means* S.E.M. from 9-10 aortic rings. Also shown (inset) are representative recordings obtained from one aortic ring incubated for 30 s with (1) no addition, (2) 100 nM isoprenaiine (I), f3) 5 nM SNP (S) or (4) 100 nM isoprenaline and 5 nM SNP (I-t-S), before addition of phenyiepbrine (P). (B d C) Rat aortic rings were incubated for 2.5 tin with the indicated concentrations of isoprenaline in the absence (9 or presence ( of 5 nM SNP. Tissue CAMP ii%\and cGMP (C) were then determined. Values are means + S.E.M. from six to eight separate incubations, each containing two aortic rings. Significant effects of SNP relative to those obtained with isoprenaline alone are shown; * P c 0.005, * * P < 0.001.
naline (2 and 10 PM) caused complete inhibition of contraction in the absence of SNP (not shown). At concentrations that i~bited phenylep~neinduced contraction, isoprenaline increased CAMP in rat aortic smooth muscle (fig. 1B). The basal value of CAMP (0.58 + 0.04 pmoI/mg of protein; mean rt: S.E.M.) was increased by 46, 68,196 and 263% after 2.5 min incubations with 100 nM, 500 nhi, 2 FM and 10 PM isoprenaline, respectively (from eight dete~nations~. With the exception of 100 nM isoprenaline, these increases in CAMP were significant (P < 0.02). Isoprenaline did not affect cGMP levels (fig.. 1C). However, as expected, SNP did cause concentration-dependent increases in aortic smooth muscle cGMP. The
basal value for cGMP (0.17 f 0.05 pma?l,!mg of protein; mean f S.E.M.) was increased by 126 and 358% after 2.5 min incubations with 5 and 50 nM SNP, respectively (from six to eight determinations); both values were significant (P < 0.05). AIthough 5 nM SNP did not increase cAMP significantly in the present study, incubation of rat aortic rings with 50 nM SNP, a concentration sufficient to inhibit contraction in response to 100 nM phenylep~ne completely (not shown}, did cause a significant increase CAMP (table 1). This increase in CAMP was similar to that seen with 500 nM isoprenaline under identical experimental conditions (table 1). Simultaneous incubation of rat aortic smooth
TABLE 1
Effects of 50 nM SNP and 500 nM isoprenaline on cyclic nuckotkk Ievels in rat aortic smo& muscle- Rat aortic rings were incubated for 2.5 min with SXP and isoprenaline, singly ot in conttktation. before determination of tissue cAMP and &HP. Values are means* SEM. from six to eight separate imcubations, each containing two aortic rings. Significant increases are ShoWI:9 P < 0.02; h P -z 0.001. Additions
cAMP @nWmg of protein)
cGMP (pmol/mg of protein)
NOM
0.58 f 0.04 1.01*0.14 it 0.98k0.13a
0.17+0.05 0.80 f 0.10 ’ 0.19+0.06
3.01*0.40 b
0.87kO.13 b
SNP (50 nM) lsoprcnaline (500 nM) SNP (50 nM) + isoprenaliie (500 nM)
have generally been assumed to be parallel and independent. However, we recently reported that low concentrations of either of two nitrovasodilators, SNP or 3-morpholinosydnonimine (SIN-l), act synergistically with isoprenaline to relax or inhibit the contraction of rat aortic smooth muscle (Maurice et al., in press). In the present study, we have examined the relationship between the synergistic effects of SNP and isoprenaline on the contraction of rat aortic smooth muscle and the associated changes in cyclic nucleotide levels addition of SNP or of isoprenaline alone caused concentration-dependent increases in rat aortic cGMP or CAMP, respectively. These effects were consistent with those reported by others using this tissue (Lincoln, 1983; Schoeffter and Stoclet, 1982). However, when low concentrations of both of these compounds were added simultaneously, 4- to 5-fold greater increases in CAMP were obtained than with isoprenaline alone. In contrast, the increases in cGMP caused by SNP were not affected by isoprenaline. These results indicate that CAMP rather than cGMP is likely to mediate the synergistic inhibition of rat aortic smooth muscle contraction seen when both SNP and isoprenaline are present. Although a synergistically effective concentration of SNP (5 nM) did not appear to affect tissue CAMP, a higher concentration of SNP (50 nM) did increase CAMP significantly. Comparison of the latter effect with the increases in CAMP caused by isoprenaline suggest that CAMP could have contributed to the inhibition of contraction by this concentration of SNP. Most workers have not reported increases in CAMP in vascular smooth muscle preparations incubated with nitrovasodilators. In platelets, in which increases in CAMP can be measured more accurately, we have previously shown that SNP causes small increases in cAMP by itself, in addition to potentiating the increases in CAMP seen in the presence of activators of adenylyl cyclase; we have attributed these effects to inhibition of the major low K, CAMP phosphodiesterase of platelets by cGMP (Maurice and Haslam, 1990). This enzyme (CGI-EDE) has recently been shown to be partly responsible for CAMP breakdown in rat aortic smooth muscle mechanisms
muscle with SNP and low concentrations of isoprenahne greatly increased the accumulation of CAMP caused by the latter (fig. 1B). Thus, on average, 5 nM SNP enhanced the increases in CAMP observed in the presence of 100 and 500 isoprenaline by 4.8- and 4.3-fold, respectively. This additional CAMP accumulation was highly significant (P < 0.001; fig. 1B). With higher isoprenahne concentrations (2 and 10 PM), 5 nM SNP caused similar increases in the amount of cAMP found in the tissue though, relative to the effect of isoprenaline alone, these increases were smaller (l-8- and 19-fold, respectively). Substitution of 50 nM SNP for 5 nM caused a somewhat larger increase in the amount of CAMP accumulating during simultaneous incubation with 500 nM isoprenaline (table 1); although 50 nM SNP itself increased CAMP, the effects of the two compounds in combination were clearly supra-additive (P < 0.001). Isoprenaline (100 or 500 nM) did not affect the increases in cGMP caused by 5 or 50 nM SNP (fig. 1C and tabie 1).
4. Discussion It is generally accepted that nitrovasodilators exert their effects on vascular smooth muscle by activating guanylyl cyclase and increasing cGMP, and that $adrenoceptor agonists act by stimulating adenylyl cyclase and increasing CAMP. These
475
(Lindgren et al., 1990). We therefore propose that inhibition of CGI-PDE by cGMP mediates the synergistic effects of SNP and isoprenaline on CAMP accumulation that we have observed in the this tissue, as well as the smaller increase in CAMP seen with 50 nM SNP alone. Our results suggest that CAMP formation in vascular smooth muscle could play a previously unsuspected role in the actions of nitrovasodilators and EDRF, which under physiological conditions are likely to function in the presence of endogenous activators of adenylyl cyclase. In this context, Shimokawa et al. (1988) noted an apparent synergism between EDRF and PG12 in pig coronary arteries. It may also be relevant that both cGMP and CAMP levels are lower in de-endothelialized than intact arteries (Rapoport and IMurad, 1983). Thus, our results may offer a partial explanation of observations showing that vasodilators, such as isoprenaline, adenosine and PGI,, that potentially exert direct effects on vascular smooth muscle through CAMP formation, are much more effective when the endothelium is present (Rubanyi and Vanhoutte, 1985; Shimokawa et al., 1988). Finally, a role for CAMP in the actions of nitrovasodilators may help to explain some of the quantitative discrepancies between cGMP formation and relaxation in smooth muscle noted in a recent review (Nakatsu and Diamond, 1989).
Acknowledgement This work was supported by a Grant-in-Aid the Heart and Stroke Foundation of Ontario.
(T.1265) from
References Levin, R.L, B.B. Weksler and E.A. Jaffe, 1982, The interactjon of sodium nitroprusside with human endothelial cells and platelets: nitroprusside and prostacyclin synergi.sticaBy inhibit platelet function, Circulation 66. 1299. Lincoln. T.M., 1983. Effects of nitroprusside and %bromo_ cyclic GMP on the contractile activity of the rat aorta, J. Pharmacol. Exp. Ther. 224,100. Lindgren, S., A. Rasccn, E. Degerman. P. Be&age, V. Manganiello and K.-E. Anderson, 1990, Identification of the cGMP-inhibited low K, CAMP phosphodiesterase in rat aortic smooth muscle, European J. Pharmacol. 183,806. Maurice, D.H.. D. Crankshaw and R.J. Haslam, Synergistic actions of nitrovasodilators and isoprenaline on rat aortic smooth muscle, European J. Pharmacol. (in press). Maurice, D.H. and R.J. Haslam, 1990, Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase: inhibition of cyclic AMP breakdown by cyclic GMP, Mol. Pharmacol. 37,671. Nakatsu, K. and J. Diamond, 1989, Role of cGMP in relaxation of vascular and other smooth muscle, Can. J. Physiol. Pharmacol. 67, 251. Radomski. M.W.. R.M.J. Palmer and S. Moncada. 1987, The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide, Br. J. Pharmacol. 92. 639. Rapoport, R.M. and F. Murad, 1983, Agonist-induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cGMP, Circ. Res. 52. 352. Rubanyi, G. and P.M. Vanhoutte. 1985, Endothelium-removal decreases relaxations of canine coronary arteries caused by /3-adrenergic agonists and adenosine. J. Cardiovasc. Pharmacol. 7, 139. Schoeffter, P. and J.-C. Stoclet, 1982. Age-related decrease of in vitro isoproterenol-induced cyclic AMP accumulation in rat aorta, European J. Pharmacol. 77, 183. Shimolcawa. H., N.A. Flavahan, R.R. Lorenz and P.M. Vanhoutte. 1988, Prostacyclin releases endothelium-derived relaxing factor and potentiates its action in coronary arteries of the pig, Br. J. Pharmacol. 95. 1197.
European Journal of Pharmacology, 191 (1990) 471-475
Elsevier EJP 20747
Short communication
itroprusside enhances isopenaline-in smooth Donald
ncreases in c
H. Maurice 1 and Richard J. Haslam ‘,*
Departments of I Pathology and ’ Biochemistry, McMaster Uniuersiy, Hamilton. Ontario, Canada LBN 325
Received 20 September 1990, accepted 16 October 1990
Low concentrations phenylephrinc-induced
of sodium contraction
nitroprusside of rat aortic
(SNP) and of isoprenaline smooth
muscle.
acted synergistically to inhibit the In experiments with these concentrations, SNP
enhanced the increases in smooth muscle CAMP caused by isoprenaline by 4- to 5-fold, whereas the SNP-induced increases in tissue cGMP were unaffected by isoprenaline. We conclude that CAMP is likely to mediate the synergistic inhibition of the contraction of rat aortic smooth muscle by these compounds. Nitroprusside; Isoprenaline; cGMP; CAMP; Smooth muscle: Aorta (rat) 1. Introduction
We have recently demonstrated vasodilators,
that nitrosuch as sodium nitroprusside (SNP)
which stimulates soluble guanylyl cyclase, and isoprenaline which activates adenylyl cyclase, interact synergistically to inhibit contraction of rat aortic smooth muscle (Maurice et al., in press). These results parallel those obtained previously with blood platelets, in which SNP and endothelium-derived relaxing factor (EDRF) potentiate the ability of prostacyclin (PGI,) and of adenosine to inhibit platelet aggregation (Levin et al., 1982; Radomski et al., 1987; Maurice and Haslam, 1990). In the latter case, we demonstrated that this synergism was caused by an enhanced accumulation of cyclic AMP (CAMP) resulting from the inhibitory action of cyclic GMP (cGMP) on the platelet cGMP-inhibited CAMP phosphodiesterase (CGI-PDE) (Maurice and Haslam, 1990). A similar enzyme activity has recently been demonstrated in rat aorta (Lindgren et al., 1990). More-
Correspondence to: R.J. Haslam, Department of Pathology, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 325.
over, we have reported that rilostamide, a selective inhibitor of the CGI-PDE, p&entiates the effects of isoprenaline on rat aortic smooth muscle to the same extent as SNP (Maurice et al., in press). We have therefore studied the effects of SNP and isoprenaline on the intracellular cyclic nucleotide levels in rat aortic smooth muscle to determine whether the synergistic inhibition of contraction was also associated with an enhanced accumulation of CAMP in this tissue.
2. Materials and methods 2. I. Materials
Male Wistar-Kyoto (WKY) rats were supplied by Harlan Sprague-Dawley (Indianapolis, IN, U.S.A.). Polypropylene microtubes with pestles were from Mandel Scientific (Guelph, Ont., Canada). CAMP and cGMP ‘251-radioimmunoassay kits and r3H]cAMP (30-50 Ci/mmol) were obtained from Du Pont Canada (Mississauga, Ont., Canada). SNP, ( - )-isoprenaline-( + )-bitartrate, phenylephrine and protein standard solution were from Sigma (St. Louis, MO, U.S.A.).
0014-2999/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
er materials were obtained from sources listed reviously (Maurice and Haslam, 1990; Maurice ., in press). ?? p _.-.
ration of de-endothelialized rat aortic rings
ale WKY rats (225-250 g) were killed by cervical dislocation. The thoracic aortas were rapidly removed and placed in a physiological salt solution (PSS) containing (mM) 1.16 MgSO,, 2.5 CaCl,, 21.9 NaHCO,, 1.16 NaH2P0,, 4.6 KCl, 115.5 NaCl and 11.l dextrose, which was equilibrated with 95% O,-5 COZ. After removal of adherent connective tissue and fat, each aorta was cut transversely into four separate 4 mm rings. The endothelium was removed from the rings by gently rubbing the luminal surfaces with a wooden stick. 2.3. Measurement of the effects of SNP and isoprenaline on phenylephrine-induced aortic smooth muscle
contraction of rat
The methods used have been described in detail previously (Maurice et al., in press). Briefly, aortic rings at an initial tension of 2 g were equilibrated for 2 h at 37” C in PSS gassed with 95% O,-5% COZ. During this period the PSS was frequently changed and the rings were treated four times with 30 mM KC1 to recharge internal Ca2+ stores. Following equilibration, the aortic rings were incubated for 30 s with SNP and isoprenaline, either singly or in combination. Phenylephrine (100 nM) was then added and the increase in tension was measured after a further 2 min. Control contractions and the effects of all combinations of inhibitors were measured on each aortic ring (Maurice et al., in press). inhibitions of contraction were calculated as percentages of the control contraction in the same ring. Results were expressed as means + S.E.M. a;rd the significance of differences in the inhibition of contraction were evaluated by paired t-tests. 2.4. Cyclic nucleotide measurements
De-endothelialized rat aortic rings were equilibrated in PSS, as described for measurement of
the inhibition of contraction, except that the rings were not under tension. Rings were then incubated for 2.5 n-tin at 37” C in 1.0 ml of PSS containing appropriate concentrations of SNP and/or isoprenaline. Each incubation contained two rings. Aortic rings from different parts of the thoracic aorta were distributed identically in replicates of each incubation condition to prevent any effect attributable to possible differences in their responsiveness to additions. After incubation, the rings were rapidly transferred to polypropylene microtubes precooled in liquid nitrogen. The frozen tissue was immediately pulverized with a precooled polypropylene pestle and then homogenized in 1 ml of 5% (w/v) trichloroacetic acid, following which 6 000 dpm of [3H]cGMP and of [3H]cAMP were added to monitor the recoveries of the two cyclic nucleotides. cGMP and CAMP were then separated and purified by a dual column procedure and finally acetylated and quantitated by radioimmunoassay (Maurice and Haslam, 1990). Trichloroacetic acid-precipitated protein was dissolved in 1 M NaOH and assayed by the Lowry method, using protein standard solution from Sigma. Cyclic nucleotide levels were calculated as means +_S.E.M.; changes were evaluated by unpaired t-tests.
3. Results Although incubation of de-endothelialized rat aortic smooth muscle with isoprenaline caused a concentration-dependent inhibition of contraction, much more inhibition was observed when low concentrations of isoprenaline were added in combination with a concentration of SNP that had little effect alone (fig. 1A). Thus, when rings were incubated with 5 nM SNP which by itself inhibited contraction by only 12 + 2%, the inhibition caused by 100 nM isoprenaline was increased from a control value of 20 f 4 to 80 + 6% (mean + S.E.M., 10 aortic rings). Under these conditions, the effects of the two vasodilators were clearly supra-additive (P < O.OOl),as were the effects seen with 5 nM SNP and 500 nM isoprenaline (fig. 1A). Higher concentrations of isopre-
0
100
500
Isoprenaline
(nM)
Fig. 1. Effects of 5 nM SNP and various isoprenaline concentrations on the phenylephrine-induced contraction of rat aortk smooth muscle and on cyclic nuckotide levels in this tissue.(A) Rat aortic rings mounted in organ baths were in~bat~ for 30 s with the indicated concentrations of isoprenaiine in the absence (ail)or presence ( f 5 nM SNP. Phenylephrine (100 nM) was added and the tension developed was measured after a further 2 min. The inhibition o contraction was calculated from paired records obtained from the same aortic rings. Values are means* S.E.M. from 9-10 aortic rings. Also shown (inset) are representative recordings obtained from one aortic ring incubated for 30 s with (1) no addition, (2) 100 nM isoprenaiine (I), f3) 5 nM SNP (S) or (4) 100 nM isoprenaline and 5 nM SNP (I-t-S), before addition of phenyiepbrine (P). (B d C) Rat aortic rings were incubated for 2.5 tin with the indicated concentrations of isoprenaline in the absence (9 or presence ( of 5 nM SNP. Tissue CAMP ii%\and cGMP (C) were then determined. Values are means + S.E.M. from six to eight separate incubations, each containing two aortic rings. Significant effects of SNP relative to those obtained with isoprenaline alone are shown; * P c 0.005, * * P < 0.001.
naline (2 and 10 PM) caused complete inhibition of contraction in the absence of SNP (not shown). At concentrations that i~bited phenylep~neinduced contraction, isoprenaline increased CAMP in rat aortic smooth muscle (fig. 1B). The basal value of CAMP (0.58 + 0.04 pmoI/mg of protein; mean rt: S.E.M.) was increased by 46, 68,196 and 263% after 2.5 min incubations with 100 nM, 500 nhi, 2 FM and 10 PM isoprenaline, respectively (from eight dete~nations~. With the exception of 100 nM isoprenaline, these increases in CAMP were significant (P < 0.02). Isoprenaline did not affect cGMP levels (fig.. 1C). However, as expected, SNP did cause concentration-dependent increases in aortic smooth muscle cGMP. The
basal value for cGMP (0.17 f 0.05 pma?l,!mg of protein; mean f S.E.M.) was increased by 126 and 358% after 2.5 min incubations with 5 and 50 nM SNP, respectively (from six to eight determinations); both values were significant (P < 0.05). AIthough 5 nM SNP did not increase cAMP significantly in the present study, incubation of rat aortic rings with 50 nM SNP, a concentration sufficient to inhibit contraction in response to 100 nM phenylep~ne completely (not shown}, did cause a significant increase CAMP (table 1). This increase in CAMP was similar to that seen with 500 nM isoprenaline under identical experimental conditions (table 1). Simultaneous incubation of rat aortic smooth
TABLE 1
Effects of 50 nM SNP and 500 nM isoprenaline on cyclic nuckotkk Ievels in rat aortic smo& muscle- Rat aortic rings were incubated for 2.5 min with SXP and isoprenaline, singly ot in conttktation. before determination of tissue cAMP and &HP. Values are means* SEM. from six to eight separate imcubations, each containing two aortic rings. Significant increases are ShoWI:9 P < 0.02; h P -z 0.001. Additions
cAMP @nWmg of protein)
cGMP (pmol/mg of protein)
NOM
0.58 f 0.04 1.01*0.14 it 0.98k0.13a
0.17+0.05 0.80 f 0.10 ’ 0.19+0.06
3.01*0.40 b
0.87kO.13 b
SNP (50 nM) lsoprcnaline (500 nM) SNP (50 nM) + isoprenaliie (500 nM)
have generally been assumed to be parallel and independent. However, we recently reported that low concentrations of either of two nitrovasodilators, SNP or 3-morpholinosydnonimine (SIN-l), act synergistically with isoprenaline to relax or inhibit the contraction of rat aortic smooth muscle (Maurice et al., in press). In the present study, we have examined the relationship between the synergistic effects of SNP and isoprenaline on the contraction of rat aortic smooth muscle and the associated changes in cyclic nucleotide levels addition of SNP or of isoprenaline alone caused concentration-dependent increases in rat aortic cGMP or CAMP, respectively. These effects were consistent with those reported by others using this tissue (Lincoln, 1983; Schoeffter and Stoclet, 1982). However, when low concentrations of both of these compounds were added simultaneously, 4- to 5-fold greater increases in CAMP were obtained than with isoprenaline alone. In contrast, the increases in cGMP caused by SNP were not affected by isoprenaline. These results indicate that CAMP rather than cGMP is likely to mediate the synergistic inhibition of rat aortic smooth muscle contraction seen when both SNP and isoprenaline are present. Although a synergistically effective concentration of SNP (5 nM) did not appear to affect tissue CAMP, a higher concentration of SNP (50 nM) did increase CAMP significantly. Comparison of the latter effect with the increases in CAMP caused by isoprenaline suggest that CAMP could have contributed to the inhibition of contraction by this concentration of SNP. Most workers have not reported increases in CAMP in vascular smooth muscle preparations incubated with nitrovasodilators. In platelets, in which increases in CAMP can be measured more accurately, we have previously shown that SNP causes small increases in cAMP by itself, in addition to potentiating the increases in CAMP seen in the presence of activators of adenylyl cyclase; we have attributed these effects to inhibition of the major low K, CAMP phosphodiesterase of platelets by cGMP (Maurice and Haslam, 1990). This enzyme (CGI-EDE) has recently been shown to be partly responsible for CAMP breakdown in rat aortic smooth muscle mechanisms
muscle with SNP and low concentrations of isoprenahne greatly increased the accumulation of CAMP caused by the latter (fig. 1B). Thus, on average, 5 nM SNP enhanced the increases in CAMP observed in the presence of 100 and 500 isoprenaline by 4.8- and 4.3-fold, respectively. This additional CAMP accumulation was highly significant (P < 0.001; fig. 1B). With higher isoprenahne concentrations (2 and 10 PM), 5 nM SNP caused similar increases in the amount of cAMP found in the tissue though, relative to the effect of isoprenaline alone, these increases were smaller (l-8- and 19-fold, respectively). Substitution of 50 nM SNP for 5 nM caused a somewhat larger increase in the amount of CAMP accumulating during simultaneous incubation with 500 nM isoprenaline (table 1); although 50 nM SNP itself increased CAMP, the effects of the two compounds in combination were clearly supra-additive (P < 0.001). Isoprenaline (100 or 500 nM) did not affect the increases in cGMP caused by 5 or 50 nM SNP (fig. 1C and tabie 1).
4. Discussion It is generally accepted that nitrovasodilators exert their effects on vascular smooth muscle by activating guanylyl cyclase and increasing cGMP, and that $adrenoceptor agonists act by stimulating adenylyl cyclase and increasing CAMP. These
475
(Lindgren et al., 1990). We therefore propose that inhibition of CGI-PDE by cGMP mediates the synergistic effects of SNP and isoprenaline on CAMP accumulation that we have observed in the this tissue, as well as the smaller increase in CAMP seen with 50 nM SNP alone. Our results suggest that CAMP formation in vascular smooth muscle could play a previously unsuspected role in the actions of nitrovasodilators and EDRF, which under physiological conditions are likely to function in the presence of endogenous activators of adenylyl cyclase. In this context, Shimokawa et al. (1988) noted an apparent synergism between EDRF and PG12 in pig coronary arteries. It may also be relevant that both cGMP and CAMP levels are lower in de-endothelialized than intact arteries (Rapoport and IMurad, 1983). Thus, our results may offer a partial explanation of observations showing that vasodilators, such as isoprenaline, adenosine and PGI,, that potentially exert direct effects on vascular smooth muscle through CAMP formation, are much more effective when the endothelium is present (Rubanyi and Vanhoutte, 1985; Shimokawa et al., 1988). Finally, a role for CAMP in the actions of nitrovasodilators may help to explain some of the quantitative discrepancies between cGMP formation and relaxation in smooth muscle noted in a recent review (Nakatsu and Diamond, 1989).
Acknowledgement This work was supported by a Grant-in-Aid the Heart and Stroke Foundation of Ontario.
(T.1265) from
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