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Stimulation of soluble coronary arterial guanylate cyclase by SIN-1
European Journal of Pharmacology, 122 (1986) 75-79
75
Elsevier
STIMULATION
OF SOLUBLE
CORONARY
ARTERIAL
GUANYLATE
CYCLASE
BY SIN-1
KURT SCHMIDT and WALTER R. KUKOVETZ * Institut fiir Pharmakodynamik und Toxikologie, Karl-Franzens-Universitiit, Universitiitsplatz 2, A-8010 Graz, Austria
Received 19 August 1985, revised MS received 4 November 1985, accepted 10 December 1985
K. SCHMIDT and W.R. KUKOVETZ, Stimulation of soluble coronary arterial guanylate cyclase by SIN-I, European J. Pharmacol. 122 (1986) 75-79. SIN-l, a metabolite of the vasodilating drug molsidomine, was found to stimulate dose dependently (0.01-l mM) soluble guanylate cyclase from bovine coronary arteries up to lOO-fold the control value. The stimulatory effect of SIN-l increased with rising concentrations of MnCl, or MgCl, and was diminished in the presence of methylene blue or ferricyanide. The time course of SIN-l-induced guanylate cyclase stimulation was characterized by a lag phase which was not observed after preincubation of the enzyme with SIN-l. In contrast to nitroglycerin and sodium nitroprusside, SIN-l did not require the presence of cysteine or other thiols to stimulate guanylate cyclase. The results presented in this study provide further evidence that SIN-1 exerts its dilating effect on coronary vessels via direct stimulation of guanylate cyclase. Guanylate cyclase
Coronary arteries
Nitrocompounds
1. Introduction Molsidomine (SIN-lo, N-ethoxycarbonyl-3morpholino-sydnonimine) is used as a vasodilating drug in the treatment of myocardial ischemia. Molsidomine itself is inactive but it is rapidly metabolized in the liver to SIN-l (3-morpholinosydnonimine) and further, nonenzymatically to SIN-1A (N-morpholino-N-nitrosaminoacetonitril) which has been found to be the active metabolite (BBhme et al., 1983). The vasodilator effects of molsidomine resemble those of nitrocompounds which are thought to be mediated by cyclic GMP (Katsuki et al., 1977; Schultz et al., 1977; Kukovetz et al., 1979; Gruetter et al., 1979). There is also evidence from our laboratory showing that SIN-l-induced relaxation of coronary arteries is closely associated with increases in cGMP levels (Kukovetz et al., 1982; Kukovetz and Holzmann, 1985a). Both relaxation and cGMP increase could be antagonized by methylene blue, an inhibitor of
* To whom all correspondence should be addressed. OO14-2999/86/%03.500 1986 Elsevier Science Publishers B.V.
Molsidomine
NO-induced guanylate cyclase stimulation (Arnold et al., 1977; Gruetter et al., 1979; Holzmann, 1983; Schmidt et al., 1985). It was therefore of interest to study the effects of SIN-l on soluble guanylate cyclase from coronary arteries in vitro and to investigate whether these effects could be correlated with the relaxant effects of SIN-l on isolated coronary strips.
2. Materials and methods 2. I. Materials SIN-l (3-morph lino-sydnonimine) was a gift of Dr. Nitz, Cas % lla AG, Frankfurt, FRG. [ 32P]GTP triethylammonium salt (0.37-1.86 TBq/mmol) and [8-3H]cyclic GMP (1.11-1.85 TBq/mmol were purchased from Amersham International, Amersham, UK or from NEN, Dreieich, FRG. Methylene blue, ferricyanide and L( + )cysteine were obtained from Merck, Darmstadt, FRG; all other chemicals were from Sigma, Munich, FRG.
76
2.2. Guanylate cyclase assay
Soluble guanylate cyclase from bovine coronary arteries was prepared and assayed as previously described (Schmidt et al., 1985). Briefly, coronary arteries were homogenized and the 115 000 x g supernatant was used for measuring enzyme activity. The assay mixture contained, 0.1 mM GTP (3 X lo5 cpm [32P]GTP), 50 mM Tris-HCl (pH 1 mM cGMP, 0.3 mM 7.5), 3 mM MgCl,, 1-methyl-3-isobutylxanthine and 150-180 pg enzyme protein. Incubation was carried out for 10 min at 37°C and was stopped by boiling the samples for 5 min. [3H]Cyclic GMP was added to monitor recovery and labelled cGMP was separated by consecutive chromatography on Dowex50 and Al,O, columns. 2.3. Statistical analysis of data
Experiments were performed in triplicate and the results are expressed as the mean k S.E.M. The EC,, values were extrapolated from each experiment and are expressed as geometric means with 95% confidence limits calculated as the product of S.E.M. x Student’s t-values.
10-4
10-3
10-2 M
hb2+
Fig. 1. Influence of MnCl, and MgCl, on the stimulatory effect of SIN-l. Guanylate cyclase activity was measured at various MnCl, (0,O) or MgCI, (0, +) concentrations in the absence (0, 0) or presence (0, +) of 0.1 mM SIN-l. Data are mean values+ S.E.M. from 1 experiment representative of 2 separate experiments.
3.2. Effect of methylene blue and ferricyanide SIN-l stimulated guanylate cyclase dose dependently with an affinity (EC,,) of 25 PM (21-31 PM). As shown in fig. 2, methylene blue inhibited the stimulatory effect of SIN-l as evident from
3. Results 3.1. Effect of divalent cations
The influence of MnCl, and MgCl, on basal and SIN-1 stimulated guanylate cyclase activity is shown in fig. 1. While both cations stimulated basal guanylate cyclase in a linear manner, a typical sigmoid dose-response curve was obtained in the presence of 0.1 mM SIN-l, indicating a potentiation of the stimulatory effect of SIN-1 by these cations. Although MnCl, was found to be about two orders of magnitude more potent than the latter was used in the subsequent MgCl,, experiments in a concentration of 3 mM to provide more physiological assay conditions. cGMP production with both cations was linearly related to the protein concentration in the absence as well as in the presence of SIN-1 (data not shown).
SIN-l
Fig. 2. Influence of methylene blue on the stimulatory effect of SIN-l. Guanylate cyclase activity was measured at varying SIN-1 concentrations in the absence (0) and presence of 10 PM (0) or 100 pM (+) methylene blue. Data are mean values k S.E.M. from 1 experiment representative of 4 separate experiments.
SIN-l
Fig. 3. Influence of ferricyanide on the stimulatory effect of SIN-l. Guanylate cyclase activity was measured at varying SIN-l concentrations in the absence (0) and presence of 0.5 PM (0) or 1 pM (+) ferricyanide. Data are mean valuesf S.E.M. from 1 experiment representative of 3 separate experiments.
rightward shifts of the respective dose-response curves by factors of 2.0 (10 PM methylene blue) and 4.8 (100 I_IM methylene blue). The respective EC,, values and 95% confidence limits were 49 PM (40-59 PM) and 120 PM (74-194 PM). Basal activity was not affected by methylene blue. The influence of ferricyanide, also known to antagonize the stimulatory effect of nitrocompounds on guanylate cyclase is shown in fig. 3. In contrast to methylene blue, ferricyanide primarily reduced the maximal effect of SIN-l with
TABLE
Fig. 4. Time course of cGMP production. Guanylate cyclase activity was measured at the times indicated, in the absence (0) and presence of 0.1 mM SIN-1 (0). For preincubation experiments the enzyme preparation was incubated for 5 min at 37°C with 0.1 mM SIN-l then assayed in the presence of 0.1 mM SIN-l. Data are mean values + S.E.M. from 1 experiment representative of 2 experiments.
only slight changes of EC,, values. At the highest SIN-l concentration used, ferricyanide (1 PM) diminished the maximal SIN-1 effect by about 30% while the ECS,, of 36 PM (25-50 PM) was only increased by a factor of 1.4. 3.3. Time course of cGMP production The time course of cGMP production was studied more closely in other series of experiments
1
Effect of SIN-I, sodium nitroprusside. nitroglycerin and sodium nitrite in the absence and presence of various thiols. Guanylate cyclase activity was measured in the presence of 1 mM nitrocompound or 5 mM thiol or combinations of both. A thiol concentration of 5 mM has been found to be optimal to potentiate the stimulatory effect of these nitrocompounds. Data are mean values of 3-5 separate experiments each performed in triplicate. The S.E.M. (not shown) were less than 5%. Guanylate None
None Cysteine Dithiothreitol Penicillamine
22 19 23 20
cyclase activity (pmol cGMP/mg
protein
per 10 min)
SIN-l
Nitroprusside
Nitroglycerin
NaNOz
(1 mM)
(1 mM)
(1 mM)
(1 mM)
4280 5138 5 249 5127
1943 4709 2 280 4432
17 1942 31 16
77 1941 166 4658
78
(fig. 4). In the presence of a maximal effective concentration of SIN-l (0.1 mM) cGMP production started rather slowly but was constant and much higher between 4 and 10 min. This lag phase could be avoided when the enzyme preparation was preincubated (5 min at 37°C) with SIN-l. 3.4. Effect of various thiols Thiols such as cysteine, dithiothreitol or penicillamine only slightly increased the stimulatory effect of SIN-l (table 1). This was in contrast to other nitrocompounds which often need the presence of certain thiols in the assay. As evident from table 1 the effect of sodium nitroprusside was potentiated by cysteine or penicillamine and was then comparable with the stimulatory effect of SIN-l obtained in the absence of any added thiol. Nitroglycerin stimulated the enzyme only in the presence of cysteine. Sodium nitrite was active in the presence of cysteine and to a higher extent also in the presence of penicillamine.
4. Discussion There is evidence from our laboratory indicating that the dilatation of coronary vessels is mediated by cyclic GMP (Kukovetz et al., 1982; Kukovetz and Holzmann, 1985a). In these studies SIN-1 was found to increase cGMP levels in isolated bovine coronary arteries in close relation to its relaxing effect. Further, the increases in cGMP preceded the mechanical response and both the relaxing effect and the rises in cGMP levels were potentiated by a specific inhibitor of cGMP phosphodiesterase (M & B 22,948) and antagonized by methylene blue. A mediator role of cGMP in coronary relaxation has already been described for various nitrocompounds such as nitroglycerin (Kukovetz et al., 1979; Gruetter et al., 1981), sodium nitroprusside (Kukovetz et al., 1979; Gruetter et al., 1979), sodium nitrite (Kukovetz et al., 1979; Gruetter et al., 1981), amyl nitrite (Gruetter et al., 1981) and nicorandil (Holzmann, 1983; Schmidt et al., 1985). The potency of SIN-l in stimulating coronary arterial guanylate cyclase (EC,, = 25 PM) was sim-
ilar to that described for nitroglycerin (EC,, = 100 PM; Gruetter et al., 1981) and sodium nitroprusside (EC,, = 45 PM; unpublished observations). It was, however, about lOO-fold lower than the EC,, for relaxation which was 0.3 PM (Kukovetz et al., 1982). Similar differences indicating that enzyme stimulation in vitro requires higher nitrite concentrations than does relaxation have also been reported for nitroglycerin and nicorandil (Schmidt et al., 1985). The reason for this phenomenon is not known. Both methylene blue and ferricyanide, which were shown to inhibit NO-induced stimulation of guanylate cyclase (Arnold et al., 1977; Gruetter et al., 1979; Schmidt et al., 1985) also diminished the stimulatory effect of SIN-1 on this enzyme. It is noteworthy that the relaxing effect of SIN-1 was also inhibited by methylene blue (Kukovetz et al., 1982; Kukovetz and Holzmann, 1985a) and ferricyanide (unpublished observations). As evident from the dose-response curves, methylene blue and ferricyanide probably act by two different mechanisms. We had already observed this different pattern of inhibition when we studied the effect of nicorandil (N-2-hydroxyethyl-nicotinamide nitrate) on guanylate cyclase (Schmidt et al., 1985) but it has been not yet described for other nitrocompounds. Bohme and coworkers (1983) recently showed that the active metabolite of molsidomine is SIN1A which probably interacts directly with guanylate cyclase through its free -N-NO group. The degradation of SIN-l is pH-dependent, i.e. OH--catalyzed. At pH 8, e.g. 50% of SIN-l is metabolized to SIN-1A within 6 min (Bohme et al., 1982). This is in good agreement with our results which show that the stimulatory effect of SIN-l did not occur immediately upon addition of SIN-l to the assay but started 3-4 min later. Further, SIN-l stimulated guanylate cyclase even in the absence of thiols and addition of cysteine, dithiothreitol or penicillamine increased the stimulatory effect only slightly. This is in contrast to other nitrocompounds (e.g. sodium nitroprusside, sodium nitrite) which stimulate nitroglycerin, guanylate cyclase only in the presence of (added) thiols, probably by formation of S-nitrosothiols as active intermediates (Ignarro and Gruetter, 1980;
19
Ignarro et al., 1981). This thiol-independent stimulation of guanylate cyclase by SIN-l has been also described for other tissues such as human platelets (Bohme et al., 1982; Nishikawa et al., 1982; Yamakado et al., 1982) as well as with the purified enzyme from bovine lung (Bohme et al., 1982). It could explain why SIN-l, in contrast to other nitrovasodilators, does not induce tolerance (Kukovetz and Holzmann, 1985b). In conclusion, the data presented in this study indicate that SIN-1 stimulates guanylate cyclase in coronary arteries by a direct, thiol-independent interaction of SIN-IA with the enzyme and provide further evidence that the relaxation of coronary vessels is mediated by cyclic GMP.
Acknowledgement The authors wish to thank competent help in the preparation
Dr. A. Buchmann of the manuscript.
for
her
References Arnold, W.P., C.K. Mittal, S. Katsuki and F. Murad, 1977, Nitric oxide activates guanylate cyclase and increases guanosine 3’ : 5’-cyclic monophosphate levels in various tissue preparations, Proc. Natl. Acad. Sci. U.S.A. 74, 3203. Bohme, E., G. Grossmann and C. Spies, 1983, Effects of molsidomine and -NO containing vasodilators on cyclic GMP formation, European Heart J. 4, 19. Bohme, E., C. Spies and G. Grossmann, 1982, Wirksamer Metabolit von Molsidomin und Stimulation der cGMPBildung durch Sydnonimine, in: Molsidomin, Neue Aspekte zur Therapie der Ischamischen Herzerkrankung, eds. E. Bassenge and H. Schmutzler (Urban & Schwarzenberg, Munchen, Wien, Baltimore) p. 37. Gruetter, C.A., B.K. Barry, D.B. McNamara, D.Y. Gruetter, P.J. Kadowitz and L.J. Ignarro, 1979, Relaxation of bovine coronary artery and activation of coronary arterial guanylate cyclase by nitric oxide, nitroprusside and a carcinogenic nitrosamine, J. Cycl. Nucl. Res. 5, 211. Gruetter, C.A., P.J. Kadowitz and L.J. Ignarro, 1981, Methylene blue inhibits coronary arterial relaxation and guanylate cyclase activation by nitroglycerin, sodium nitrite and amylnitrite, Can. J. Physiol. Pharmacol. 59, 150.
Holzmann, S., 1983, Cyclic GMP as possible mediator of coronary arterial relaxation by nicorandil (SG-75). J. Cardiovasc. Pharmacol. 5, 364. Ignarro, L.J. and CA. Gruetter, 1980, Requirement of thiols for activation of coronary arterial guanylate cyclase by glyceryl trinitrate and sodium nitrite, B&him. Biophys. Acta 631, 221. Ignarro, L.J., H. Lippton, J.C. Edwards, W.H., Baricos, A.L. Hyman, P.J. Kadowitz and C.A. Gruetter, 1981, Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxid: evidence for the involvement of S-nitrosothiols as active intermediates, J. Pharmacol. Exp. Ther. 220, 183. Katsuki, S., W.P. Arnold, C. Mittal and F. Murad, 1977, Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine, J. Cycl. Nucl. Res. 3. 23. Kukovetz, W.R. and S. Holzmann. 1985a, Mode of action of the vasodilator drug molsidomine, in: Vascular Neuroeffector Mechanisms, eds. J.A. Bevan, T. Godfraind, R.A. Maxwell, J.C. Stoclet and M. Worcel (Elsevier, Amsterdam, New York, Oxford) p. 139. Kukovetz, W.R. and S. Holzmann, 1985b. Mechanism of vasodilation by molsidomine, Am. Heart J. 109, 637. Kukovetz, W.R., S. Holzmann, M. Straka and K. Schmidt. 1982, Mechanismus der gefasserweiternden Wirkung von Molsidomin, in: Molsidomin, Neue Aspekte zur Therapie der Ischamischen Herzerkrankung, eds. E. Bassenge and H. Schmutzler (Urban & Schwarzenberg, Miinchen, Wien, Baltimore) p. 32. Kukovetz, W.R., S. Holzmann, A. Wurm and G. Piich, 1979, Evidence for cyclic GMP-mediated relaxant effects of nitro-compounds in coronary smooth muscle, NaunynSchmiedeb. Arch. Pharmacol. 310, 129. Nishikawa, M., M. Kanamori and H. Hidaka, 1982, Inhibition of platelet aggregation and stimulation of guanylate cyclase by an antianginal agent molsidomine and its metabolites, J. Pharmacol. Exp. Ther. 220, 183. Schmidt, K., R. Reich and W.R. Kukovetz, 1985, Stimulation of coronary guanylate cyclase by nicorandil (SG-75) as a mechanism of its vasodilating action, J. Cycl. Nucl. Prot. Phosphor. Res. 10, 43. Schultz, K.D., K. Schultz and G. Schultz, 1977. Sodium nitroprusside and other smooth muscle-relaxants increase cyclic GMP levels in rat ductus deferens, Nature 265, 750. Yamakado, T., M. Nishikawa and M. Hidaka, 1982, Stimulation of human platelet guanylate cyclase by nitroso compounds, Thromb. Res. 26, 135.
75
Elsevier
STIMULATION
OF SOLUBLE
CORONARY
ARTERIAL
GUANYLATE
CYCLASE
BY SIN-1
KURT SCHMIDT and WALTER R. KUKOVETZ * Institut fiir Pharmakodynamik und Toxikologie, Karl-Franzens-Universitiit, Universitiitsplatz 2, A-8010 Graz, Austria
Received 19 August 1985, revised MS received 4 November 1985, accepted 10 December 1985
K. SCHMIDT and W.R. KUKOVETZ, Stimulation of soluble coronary arterial guanylate cyclase by SIN-I, European J. Pharmacol. 122 (1986) 75-79. SIN-l, a metabolite of the vasodilating drug molsidomine, was found to stimulate dose dependently (0.01-l mM) soluble guanylate cyclase from bovine coronary arteries up to lOO-fold the control value. The stimulatory effect of SIN-l increased with rising concentrations of MnCl, or MgCl, and was diminished in the presence of methylene blue or ferricyanide. The time course of SIN-l-induced guanylate cyclase stimulation was characterized by a lag phase which was not observed after preincubation of the enzyme with SIN-l. In contrast to nitroglycerin and sodium nitroprusside, SIN-l did not require the presence of cysteine or other thiols to stimulate guanylate cyclase. The results presented in this study provide further evidence that SIN-1 exerts its dilating effect on coronary vessels via direct stimulation of guanylate cyclase. Guanylate cyclase
Coronary arteries
Nitrocompounds
1. Introduction Molsidomine (SIN-lo, N-ethoxycarbonyl-3morpholino-sydnonimine) is used as a vasodilating drug in the treatment of myocardial ischemia. Molsidomine itself is inactive but it is rapidly metabolized in the liver to SIN-l (3-morpholinosydnonimine) and further, nonenzymatically to SIN-1A (N-morpholino-N-nitrosaminoacetonitril) which has been found to be the active metabolite (BBhme et al., 1983). The vasodilator effects of molsidomine resemble those of nitrocompounds which are thought to be mediated by cyclic GMP (Katsuki et al., 1977; Schultz et al., 1977; Kukovetz et al., 1979; Gruetter et al., 1979). There is also evidence from our laboratory showing that SIN-l-induced relaxation of coronary arteries is closely associated with increases in cGMP levels (Kukovetz et al., 1982; Kukovetz and Holzmann, 1985a). Both relaxation and cGMP increase could be antagonized by methylene blue, an inhibitor of
* To whom all correspondence should be addressed. OO14-2999/86/%03.500 1986 Elsevier Science Publishers B.V.
Molsidomine
NO-induced guanylate cyclase stimulation (Arnold et al., 1977; Gruetter et al., 1979; Holzmann, 1983; Schmidt et al., 1985). It was therefore of interest to study the effects of SIN-l on soluble guanylate cyclase from coronary arteries in vitro and to investigate whether these effects could be correlated with the relaxant effects of SIN-l on isolated coronary strips.
2. Materials and methods 2. I. Materials SIN-l (3-morph lino-sydnonimine) was a gift of Dr. Nitz, Cas % lla AG, Frankfurt, FRG. [ 32P]GTP triethylammonium salt (0.37-1.86 TBq/mmol) and [8-3H]cyclic GMP (1.11-1.85 TBq/mmol were purchased from Amersham International, Amersham, UK or from NEN, Dreieich, FRG. Methylene blue, ferricyanide and L( + )cysteine were obtained from Merck, Darmstadt, FRG; all other chemicals were from Sigma, Munich, FRG.
76
2.2. Guanylate cyclase assay
Soluble guanylate cyclase from bovine coronary arteries was prepared and assayed as previously described (Schmidt et al., 1985). Briefly, coronary arteries were homogenized and the 115 000 x g supernatant was used for measuring enzyme activity. The assay mixture contained, 0.1 mM GTP (3 X lo5 cpm [32P]GTP), 50 mM Tris-HCl (pH 1 mM cGMP, 0.3 mM 7.5), 3 mM MgCl,, 1-methyl-3-isobutylxanthine and 150-180 pg enzyme protein. Incubation was carried out for 10 min at 37°C and was stopped by boiling the samples for 5 min. [3H]Cyclic GMP was added to monitor recovery and labelled cGMP was separated by consecutive chromatography on Dowex50 and Al,O, columns. 2.3. Statistical analysis of data
Experiments were performed in triplicate and the results are expressed as the mean k S.E.M. The EC,, values were extrapolated from each experiment and are expressed as geometric means with 95% confidence limits calculated as the product of S.E.M. x Student’s t-values.
10-4
10-3
10-2 M
hb2+
Fig. 1. Influence of MnCl, and MgCl, on the stimulatory effect of SIN-l. Guanylate cyclase activity was measured at various MnCl, (0,O) or MgCI, (0, +) concentrations in the absence (0, 0) or presence (0, +) of 0.1 mM SIN-l. Data are mean values+ S.E.M. from 1 experiment representative of 2 separate experiments.
3.2. Effect of methylene blue and ferricyanide SIN-l stimulated guanylate cyclase dose dependently with an affinity (EC,,) of 25 PM (21-31 PM). As shown in fig. 2, methylene blue inhibited the stimulatory effect of SIN-l as evident from
3. Results 3.1. Effect of divalent cations
The influence of MnCl, and MgCl, on basal and SIN-1 stimulated guanylate cyclase activity is shown in fig. 1. While both cations stimulated basal guanylate cyclase in a linear manner, a typical sigmoid dose-response curve was obtained in the presence of 0.1 mM SIN-l, indicating a potentiation of the stimulatory effect of SIN-1 by these cations. Although MnCl, was found to be about two orders of magnitude more potent than the latter was used in the subsequent MgCl,, experiments in a concentration of 3 mM to provide more physiological assay conditions. cGMP production with both cations was linearly related to the protein concentration in the absence as well as in the presence of SIN-1 (data not shown).
SIN-l
Fig. 2. Influence of methylene blue on the stimulatory effect of SIN-l. Guanylate cyclase activity was measured at varying SIN-1 concentrations in the absence (0) and presence of 10 PM (0) or 100 pM (+) methylene blue. Data are mean values k S.E.M. from 1 experiment representative of 4 separate experiments.
SIN-l
Fig. 3. Influence of ferricyanide on the stimulatory effect of SIN-l. Guanylate cyclase activity was measured at varying SIN-l concentrations in the absence (0) and presence of 0.5 PM (0) or 1 pM (+) ferricyanide. Data are mean valuesf S.E.M. from 1 experiment representative of 3 separate experiments.
rightward shifts of the respective dose-response curves by factors of 2.0 (10 PM methylene blue) and 4.8 (100 I_IM methylene blue). The respective EC,, values and 95% confidence limits were 49 PM (40-59 PM) and 120 PM (74-194 PM). Basal activity was not affected by methylene blue. The influence of ferricyanide, also known to antagonize the stimulatory effect of nitrocompounds on guanylate cyclase is shown in fig. 3. In contrast to methylene blue, ferricyanide primarily reduced the maximal effect of SIN-l with
TABLE
Fig. 4. Time course of cGMP production. Guanylate cyclase activity was measured at the times indicated, in the absence (0) and presence of 0.1 mM SIN-1 (0). For preincubation experiments the enzyme preparation was incubated for 5 min at 37°C with 0.1 mM SIN-l then assayed in the presence of 0.1 mM SIN-l. Data are mean values + S.E.M. from 1 experiment representative of 2 experiments.
only slight changes of EC,, values. At the highest SIN-l concentration used, ferricyanide (1 PM) diminished the maximal SIN-1 effect by about 30% while the ECS,, of 36 PM (25-50 PM) was only increased by a factor of 1.4. 3.3. Time course of cGMP production The time course of cGMP production was studied more closely in other series of experiments
1
Effect of SIN-I, sodium nitroprusside. nitroglycerin and sodium nitrite in the absence and presence of various thiols. Guanylate cyclase activity was measured in the presence of 1 mM nitrocompound or 5 mM thiol or combinations of both. A thiol concentration of 5 mM has been found to be optimal to potentiate the stimulatory effect of these nitrocompounds. Data are mean values of 3-5 separate experiments each performed in triplicate. The S.E.M. (not shown) were less than 5%. Guanylate None
None Cysteine Dithiothreitol Penicillamine
22 19 23 20
cyclase activity (pmol cGMP/mg
protein
per 10 min)
SIN-l
Nitroprusside
Nitroglycerin
NaNOz
(1 mM)
(1 mM)
(1 mM)
(1 mM)
4280 5138 5 249 5127
1943 4709 2 280 4432
17 1942 31 16
77 1941 166 4658
78
(fig. 4). In the presence of a maximal effective concentration of SIN-l (0.1 mM) cGMP production started rather slowly but was constant and much higher between 4 and 10 min. This lag phase could be avoided when the enzyme preparation was preincubated (5 min at 37°C) with SIN-l. 3.4. Effect of various thiols Thiols such as cysteine, dithiothreitol or penicillamine only slightly increased the stimulatory effect of SIN-l (table 1). This was in contrast to other nitrocompounds which often need the presence of certain thiols in the assay. As evident from table 1 the effect of sodium nitroprusside was potentiated by cysteine or penicillamine and was then comparable with the stimulatory effect of SIN-l obtained in the absence of any added thiol. Nitroglycerin stimulated the enzyme only in the presence of cysteine. Sodium nitrite was active in the presence of cysteine and to a higher extent also in the presence of penicillamine.
4. Discussion There is evidence from our laboratory indicating that the dilatation of coronary vessels is mediated by cyclic GMP (Kukovetz et al., 1982; Kukovetz and Holzmann, 1985a). In these studies SIN-1 was found to increase cGMP levels in isolated bovine coronary arteries in close relation to its relaxing effect. Further, the increases in cGMP preceded the mechanical response and both the relaxing effect and the rises in cGMP levels were potentiated by a specific inhibitor of cGMP phosphodiesterase (M & B 22,948) and antagonized by methylene blue. A mediator role of cGMP in coronary relaxation has already been described for various nitrocompounds such as nitroglycerin (Kukovetz et al., 1979; Gruetter et al., 1981), sodium nitroprusside (Kukovetz et al., 1979; Gruetter et al., 1979), sodium nitrite (Kukovetz et al., 1979; Gruetter et al., 1981), amyl nitrite (Gruetter et al., 1981) and nicorandil (Holzmann, 1983; Schmidt et al., 1985). The potency of SIN-l in stimulating coronary arterial guanylate cyclase (EC,, = 25 PM) was sim-
ilar to that described for nitroglycerin (EC,, = 100 PM; Gruetter et al., 1981) and sodium nitroprusside (EC,, = 45 PM; unpublished observations). It was, however, about lOO-fold lower than the EC,, for relaxation which was 0.3 PM (Kukovetz et al., 1982). Similar differences indicating that enzyme stimulation in vitro requires higher nitrite concentrations than does relaxation have also been reported for nitroglycerin and nicorandil (Schmidt et al., 1985). The reason for this phenomenon is not known. Both methylene blue and ferricyanide, which were shown to inhibit NO-induced stimulation of guanylate cyclase (Arnold et al., 1977; Gruetter et al., 1979; Schmidt et al., 1985) also diminished the stimulatory effect of SIN-1 on this enzyme. It is noteworthy that the relaxing effect of SIN-1 was also inhibited by methylene blue (Kukovetz et al., 1982; Kukovetz and Holzmann, 1985a) and ferricyanide (unpublished observations). As evident from the dose-response curves, methylene blue and ferricyanide probably act by two different mechanisms. We had already observed this different pattern of inhibition when we studied the effect of nicorandil (N-2-hydroxyethyl-nicotinamide nitrate) on guanylate cyclase (Schmidt et al., 1985) but it has been not yet described for other nitrocompounds. Bohme and coworkers (1983) recently showed that the active metabolite of molsidomine is SIN1A which probably interacts directly with guanylate cyclase through its free -N-NO group. The degradation of SIN-l is pH-dependent, i.e. OH--catalyzed. At pH 8, e.g. 50% of SIN-l is metabolized to SIN-1A within 6 min (Bohme et al., 1982). This is in good agreement with our results which show that the stimulatory effect of SIN-l did not occur immediately upon addition of SIN-l to the assay but started 3-4 min later. Further, SIN-l stimulated guanylate cyclase even in the absence of thiols and addition of cysteine, dithiothreitol or penicillamine increased the stimulatory effect only slightly. This is in contrast to other nitrocompounds (e.g. sodium nitroprusside, sodium nitrite) which stimulate nitroglycerin, guanylate cyclase only in the presence of (added) thiols, probably by formation of S-nitrosothiols as active intermediates (Ignarro and Gruetter, 1980;
19
Ignarro et al., 1981). This thiol-independent stimulation of guanylate cyclase by SIN-l has been also described for other tissues such as human platelets (Bohme et al., 1982; Nishikawa et al., 1982; Yamakado et al., 1982) as well as with the purified enzyme from bovine lung (Bohme et al., 1982). It could explain why SIN-l, in contrast to other nitrovasodilators, does not induce tolerance (Kukovetz and Holzmann, 1985b). In conclusion, the data presented in this study indicate that SIN-1 stimulates guanylate cyclase in coronary arteries by a direct, thiol-independent interaction of SIN-IA with the enzyme and provide further evidence that the relaxation of coronary vessels is mediated by cyclic GMP.
Acknowledgement The authors wish to thank competent help in the preparation
Dr. A. Buchmann of the manuscript.
for
her
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