Persistence of the response to SIN1 on isolated coronary arteries rendered tolerant to nitroglycerin in vitro or in vivo

Experiments were performed on isolated canine and human coronary arteries to provide more insight into the mechanisms responsible for the vascular tolerance to nitroglycerin that is induced under in vitro or in vivo conditions. In vitro tolerance was produced after an incubation of coronary ring segments with nitroglycerin (10 microM for 30 min at physiological pH). After elevation of tone with KCl (15 mM), dose-response curves were constructed for nitroglycerin or SIN1 (3-morpholino-syndnonimin) on control and tolerant rings. On canine tolerant rings the dose-response curve for nitroglycerin-induced relaxations was significantly (p less than 0.001) shifted to the right, and 50% of the maximal relaxation (ED50) increased from 55 +/- 9 nM to 1.2 +/- 0.2 microM. Pretreatment of tolerant rings with N-acetylcysteine (NAC, 10 microM 10 min before KCl-induced contraction) partially restored the responsiveness to nitroglycerin, with ED50 reducing to 0.56 +/- 0.03 microM (p less than 0.02). On the other hand, the dose-response curves to SIN1 were not significantly altered. Similar results were obtained on human preparations. On isolated canine coronary rings rendered tolerant in vivo by subcutaneous injections of 15 mg/kg nitroglycerin (two times daily for 4 consecutive days), ED50 for nitroglycerin was 0.67 +/- 0.08 microM (p less than 0.001 versus control rings), and NAC again partially restored the responsiveness to nitroglycerin. As for the in vitro tolerance, the relaxations to SIN1 were not significantly altered on these canine rings rendered nitrate tolerant in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of endogenous hydrogen peroxide in the development of nitrate tolerance

The present study was designed to test the hypothesis that hydrogen peroxide plays a role in the development of nitrate tolerance. Isolated rat aortic rings were suspended in organ chambers for isometric tension recording. The rings were incubated with (tolerant) and without (control) nitroglycerin (10(-4) M) for 90 min, followed by repeated rinsing for 1 h. Hydrogen peroxide release in control and tolerant tissues was measured fluorimetrically using amplex red. Nitroglycerin (10(-9)-10(-4) M) caused concentration-dependent relaxations in control (-logEC50=7.15+/-0.1) and tolerant rings (-logEC50=5.83+/-0.1) contracted with norepinephrine. Nitrate tolerance was evident by a >20-fold rightward shift in the nitroglycerin concentration-response curve in tissues exposed previously to nitroglycerin for 90 min. Incubation of the rings with the superoxide dismutase (SOD)-mimetic, tempol (10(-4) M), during the 90-min exposure period to nitroglycerin caused a leftward shift in the nitroglycerin concentration-response curve in tolerant rings (-logEC50=6.84+/-0.2), but had no effect on the response to nitroglycerin in control rings. Treatment of the rings with catalase (1200 U/ml) or ebselen (1.5x10(-5) M), a glutathione peroxidase-mimetic, during the 90-min exposure period to nitroglycerin resulted in a further rightward shift in the nitroglycerin concentration-response curve in tolerant rings (-logEC50=5.41+/-0.1 and 4.98+/-0.1; catalase and ebselen respectively), without altering the response to nitroglycerin in control rings. In the presence of catalase, the effect of tempol on nitrate tolerance was abolished (-logEC50=5.46+/-0.1). Hydrogen peroxide release was reduced by approximately 64% in nitrate tolerant tissues when compared to control. The decrease in hydrogen peroxide release was completely reversed by treatment with tempol, whereas treatment with ebselen caused a further decrease in hydrogen peroxide release in nitrate tolerant tissues. Addition of hydrogen peroxide (3x10(-5) M) to nitrate tolerant rings caused a leftward shift in the nitroglycerin concentration-response curve in tolerant rings (-logEC50=7.18+/-0.3), but had no effect on the response to nitroglycerin in control rings. These results suggest that nitrate tolerance is associated with decreased endogenous formation of hydrogen peroxide, which attenuates nitrate tolerance development. SOD-mimetics may reduce nitrate tolerance, in part, by increasing the formation of hydrogen peroxide.     

Reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril: a role of calcitonin gene-related peptide

Previous studies have shown that the development of tolerance to nitroglycerin is related to a decrease in the release of endogenous calcitonin gene-related peptide (CGRP). In the present study, we explored whether endogenous CGRP is involved in reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril in rats in vivo and vitro. Tolerance was induced by exposure to nitroglycerin (4.4 x 10(-6) M) for 10 min in vitro or by pretreatment with nitroglycerin (10 mg/kg, s.c.) three times a day for 8 days in vivo. Nitroglycerin (3 x 10(-9)-10(-6) M) caused a concentration-dependent relaxation in the isolated rat thoracic aorta, an effect that was reduced by CGRP-(8-37) (3 x 10(-7) M) or capsaicin (3 x 10(-7) M). Preincubation with nitroglycerin for 10 min significantly decreased its vasodilation, which was restored in the presence of N-acetylcysteine (10(-5) M) or captopril (10(-5) M). Nitroglycerin (150 microg/kg, i.v.) produced a depressor effect and an increase in concentrations of nitric oxide and CGRP, and the effects of nitroglycerin disappeared after pretreatment with nitroglycerin for 8 days. However, tolerance to nitroglycerin in vivo also was partially restored in the presence of N-acetylcysteine or captopril. The present results suggest that reversal of tolerance to nitroglycerin with N-acetylcysteine or captopril is related to the increased release of CGRP in the rat.     

Recovery of vascular smooth muscle relaxation from nitroglycerin-induced tolerance following a drug-free interval. A time-course in vitro study.

Hemodynamic tolerance occurs upon continuous exposure of vascular tissues to nitroglycerin (NTG). This phenomenon is believed to be due to the depletion of the tissue sulfhydryl (SH) group, which is essential for NTG-induced increase in tissue cyclic GMP and vasorelaxation. To determine the effect of an NTG-free interval on recovery of tissue cyclic GMP accumulation and vasorelaxation following development of NTG tolerance, isolated rat aortic rings were kept in Krebs physiologic buffer at 37 degrees, precontracted with epinephrine, and exposed to NTG. The mean concentration of NTG, which relaxed the rings by 50% (EC50) upon first exposure, was 1.1 x 10(-7) M (N = 20), and vascular cyclic GMP levels after NTG increased from 21 to 46 fmol/mg (P less than 0.02). A second exposure to NTG 15 min later increased the EC50 to 1.3 x 10(-4) M and cyclic GMP levels did not change (P less than 0.001 vs first NTG exposure), indicating tolerance to NTG. However, acetylcholine-mediated relaxation of aortic rings was preserved even in NTG-tolerant rings. A second exposure of tissues to NTG separated by 30, 60, and 120 min from the first exposure progressively decreased the EC50, such that at 120 min the EC50 of NTG was 0.4 x 10(-7) M (P = NS vs first NTG exposure). Tissue cyclic GMP levels increased from 14 to 71 fmol/mg (P = NS vs first NTG exposure). These data confirm development of tolerance to the vasorelaxant effects of NTG following initial exposure. An interval of 2 hr between multiple exposures of tissues to NTG results in preservation of the smooth muscle relaxation and an increase in tissue cyclic GMP in response to NTG.     

Nitroglycerin-induced release of calcitonin gene-related peptide from sensory nerves attenuates the development of nitrate tolerance

The present study was designed to determine if endogenous calcitonin gene-related peptide (CGRP) affects the process of nitrate tolerance development in blood vessels. Rat aortic rings were suspended in organ chambers and relaxations to nitroglycerin (10(-9) -10(-6) M) were obtained in nitrate tolerant and nontolerant rings contracted with norepinephrine (10(-7) M). Tolerance was induced by incubating the rings with (tolerant) or without (nontolerant) nitroglycerin (10(-4) M) for 90 minutes, followed by repeated rinsing for 1 hour. Some rings were treated with CGRP8-37 (10(-6) M), glyburide (10(-6) M), or iberiotoxin (10(-7) M) during the 90-minute desensitization period with nitroglycerin (10(-4) M), and were then washed out during the 1-hour rinsing period. Other rings were treated with capsaicin (10(-5) M) prior to the 90-minute desensitization period. Calcitonin gene-related peptide release was measured by radioimmunoassay. Relaxation to nitroglycerin was markedly reduced in tolerant rings, as compared with nontolerant. Incubation with CGRP8-37 (10(-6) M) specifically during the 90-minute desensitization period with nitroglycerin resulted in even greater impairment in the response to nitroglycerin in tolerant rings, even though the calcitonin gene-related peptide antagonist had been washed out before completion of the nitroglycerin dose-response curve. Similar results were obtained following depletion of calcitonin gene-related peptide stores in sensory nerves by treatment with capsaicin (10(-5) M) prior to the 90-minute desensitization period with nitroglycerin. Prior treatment with CGRP8-37 or capsaicin had no effect on the response to nitroglycerin in nontolerant rings. Incubation with glyburide (10(-6) M), but not iberiotoxin (10(-7) M), specifically during the 90-minute desensitization period, mimicked the effect of CGRP8-37 and capsaicin in tolerant rings, suggesting a role for KATP channels in the effect of calcitonin gene-related peptide. Nitroglycerin (10(-4) M) caused a greater than twofold increase over basal levels in calcitonin gene-related peptide release in nontolerant rings, which was abolished in rings treated with capsaicin and in nitrate tolerant rings. These results suggest that nitroglycerin releases calcitonin gene-related peptide from sensory nerves during the process of desensitization to nitrovasodilators, and that interference with either the release or action of endogenous calcitonin gene-related peptide during this period enhances the extent to which nitrate tolerance occurs. The finding that nitroglycerin-induced release of calcitonin gene-related peptide from sensory nerves attenuates the desensitizing effect of nitroglycerin represents a heretofore unknown event in the development of nitrate tolerance, and demonstrates a novel role for calcitonin gene-related peptide in the vasculature.     

Melatonin inhibits nitrate tolerance in isolated coronary arteries

(1) The present study was designed to test the hypothesis that melatonin inhibits nitrate tolerance in coronary arteries. (2) Rings of porcine coronary arteries were suspended in organ chambers for isometric tension recording. Nitrate tolerance was induced by incubating the tissues with nitroglycerin (10(-4) M) for 90 min, followed by repeated rinsing for 1 h. Control rings that had not been exposed previously to nitroglycerin, but were otherwise treated identically, were studied simultaneously. The rings were contracted with U46619 (1-3 x 10(-9) M) and concentration-response curves to nitroglycerin (10(-9)-10(-4) M) were obtained. (3) Nitrate tolerance was evident by a 15- to 20-fold rightward shift in the concentration-response curve to nitroglycerin in rings with and without endothelium exposed previously to the drug for 90 min. Addition of melatonin (10(-9)-10(-7) M) to the organ chamber during the 90-min incubation period with nitroglycerin partially inhibited nitrate tolerance in coronary arteries with intact endothelium; however, melatonin had no effect on nitrate tolerance in coronary arteries without endothelium. (4) The effect of melatonin on nitrate tolerance in coronary arteries with endothelium was abolished by the melatonin receptor antagonist, S20928 (10(-6) M). In contrast to melatonin, the selective MT(3)-melatonin receptor agonist, 5-MCA-NAT (10(-8)-10(-7) M), had no effect on nitrate tolerance in coronary arteries. (5) The results demonstrate that melatonin, acting via specific melatonin receptors, inhibits nitrate tolerance in coronary arteries and that this effect is dependent on the presence of the vascular endothelium.     

Effect of resveratrol on nitrate tolerance in isolated human internal mammary artery

The present study aims to examine whether resveratrol, a natural antioxidant present in red wine, restores the tolerance to nitroglycerin (GTN) on isolated human internal mammary artery (IMA), using an in vitro model of nitrate tolerance. IMA rings were obtained from 53 male patients undergoing coronary bypass operation. Nitrate tolerance was induced by incubating the artery ring with 100 microM GTN for 90 minutes. Concentration-response curves to GTN (10(-9) to 10(-4) M) were obtained on IMA rings precontracted with noradrenaline. A low concentration (5 microM) of lucigenin was used as a tool to measure superoxide production in IMA segments. GTN produced concentration-dependent relaxation in isolated human IMA rings. Preexposure of artery rings to GTN reduced the relaxations to GTN [E(max) values: 105 +/- 2% and 76 +/- 3%, n = 10 to 12, P < 0.05; EC(50) values (-log M): 6.72 +/- 0.05 and 4.95 +/- 0.06, P < 0.05, respectively]. Relaxation to sodium nitroprusside remained unchanged. Diminished relaxation to GTN is partially restored after removing endothelium or L(G)-nitro-L-arginine (L-NOARG, 10 M) or superoxide dismutase (20 and 200 U/mL) or catalase (200 U/mL) pretreatments. Pretreatments with resveratrol (1, 10, and 20 microM) for 20 minutes relatively improved the reduced relaxation to GTN in tolerant IMA rings. Coadministration of L-NOARG with resveratrol did not abolish the beneficial effect of resveratrol on nitrate tolerance. The inhibitory effect of resveratrol on GTN-induced tolerance was not abolished in arterial rings without endothelium. Exposure to GTN increased superoxide production in IMA segments with endothelium. Endothelium denudation, L-NOARG, or superoxide dismutase pretreatments markedly inhibited the increased superoxide production in tolerant arteries. Resveratrol (1 and 10 microM) almost completely abolished basal or NAD(P)H-stimulated superoxide production in tolerant and nontolerant arteries. Vascular tolerance to GTN, in in vitro tolerant human IMA rings, can be induced by endothelial superoxide anions. Resveratrol partially restored the reduced relaxation to GTN by inhibiting NAD(P)-derived superoxide production in endothelium.     

Characterization of contractile prostanoid receptors on human airway smooth muscle

In human bronchial rings the thromboxane A2 (TxA2) mimetic, U46619, produced cumulative concentration-related contractions up to a maximum of 141 +/- 23% of the response induced by carbachol or acetylcholine. The geometric mean EC50 value was 3.2 X 10(-8) M (95% confidence interval: 1.2, 8.9 X 10(-8) M) (n = 5). Contractions to U46619 were unaffected by atropine (10(-6) M) or verapamil (10(-5) M), but were competitively antagonized by the TxA2 antagonist GR32191 with a pA2 value of 8.40 +/- 0.41. The maximum contractile response to prostaglandin (PG) F2 alpha was smaller (90 +/- 9%, n = 13) and the potency was less (EC50 = 2 X 10(-6) M) than that of U46619. Contractions to PGF2 alpha were also competitively antagonized by GR32191 with a pA2 value of 8.18 +/- 0.08. Concentration-response curves to PGE2 were biphasic, relaxation at concentrations from 10(-9) to 10(-6) M and contraction from 10(-6) to 3 X 10(-5) M. GR32191 10(-7) M inhibited the contractile portion of the response curve in 8 of 11 tissues. Based on these results we conclude that U46619, PGF2 alpha and PGE2 all contract human airways by stimulation of the TxA2 (TP) receptor.     

Contribution of cyclic GMP formation to KRN2391-induced relaxation in coronary artery of the pig.

1. In the present study, we investigated the relationship between relaxation and guanosine 3':5'-cyclic monophosphate (cyclic GMP) formation induced by KRN2391, compared with those induced by nicorandil and nitroglycerin, in the coronary artery of the pig. 2. KRN2391 (10(-8)-3 X 10(-5) M), nicorandil (10(-8)-3 X 10(-4) M) and nitroglycerin (10(-9)-10(-5) M) antagonized the contraction caused by 25 mM KCl in a concentration-dependent manner. 3. The concentration-relaxation curves for KRN2391, nicorandil and nitroglycerin shifted rightward in the presence of methylene blue (10(-5) M). 4. KRN2391 (10(-6) M), nicorandil (10(-4) M) and nitroglycerin (10(-6) M) induced an increased in cyclic GMP. 5. The magnitude of the shift of the concentration-relaxation curve caused by methylene blue and the increase in cyclic GMP with KRN2391 were lower than those with nicorandil and nitroglycerin. 6. The adenosine 3':5'-cyclic monophosphate (cyclic AMP) level was not increased by KRN2391 even at a concentration that produced full relaxation. 7. The present results suggest that KRN2391-induced relaxation in the coronary artery of the pig is partly due to the increase in cyclic GMP formation through the stimulation of guanylate cyclase.     

N-acetylcysteine modifies the acute effects of isosorbide-5-mononitrate in angina pectoris patients evaluated by exercise testing

Nitrates are well established in the treatment of angina pectoris and the presence of sulfhydryl groups seems to be fundamental to nitrate-induced vasodilatation. The present study was performed to elucidate if large oral doses of N-acetylcysteine (NAC, 2,400 mg X 2), a donor of sulfhydryl groups, given together with a single oral dose of the long-acting nitrate, isosorbide-5-mononitrate (5-ISMN, 60 mg), would modify the nitrate effect evaluated by exercise testing before and after additional sublingual doses of nitroglycerin (NTG). Ten patients with angina pectoris and angiographically proven significant coronary artery disease were included. All patients received a baseline therapy with beta blockers. None of the patients had developed nitrate tolerance at inclusion. NAC/5-ISMN treatment significantly prolonged the total exercise time as compared with placebo/5-ISMN (7.7 +/- 2.1 min vs. 6.8 +/- 1.7 min, p less than 0.05). This increase was of such magnitude that no further effect was obtained after additional NTG doses. This study demonstrated that increased availability of sulfhydryl groups can increase the exercise capacity in angina pectoris patients treated with 5-ISMN without nitrate tolerance.     

Captopril-induced reversal of nitroglycerin tolerance: role of sulfhydryl group vs. ACE-inhibitory activity.

The angiotensin-converting enzyme (ACE) inhibitor captopril has been shown to reverse vascular tolerance to nitroglycerin (NTG). Whether captopril reverses NTG tolerance by providing sulfhydryl (SH) groups or by inhibiting ACE is not clear. To examine this issue, we treated rat aortic rings with buffer, captopril (SH +, ACE inhibitory activity +), enalaprilat (SH-, ACE inhibitory activity +), or N-acetylcysteine (NAC, SH+, ACE inhibitory activity-) prior to their contraction with epinephrine and subsequent relaxation with NTG. Previous exposure of NTG-treated rings resulted in marked resistance to the vasorelaxant effect of a subsequent exposure to NTG in buffer-treated rings. Both NAC and captopril, but not enalaprilat, potentiated the vasorelaxant effects of NTG during the first exposure of vascular rings to NTG and also prevented the development of tolerance to NTG during a second exposure. Buffer-treated rings showed an inability to accumulate cyclic guanosine monophosphate (GMP) in response to a second exposure to NTG. In contrast, both NAC and captopril-pretreated rings demonstrated a persistence of cyclic GMP accumulation during the second NTG exposure. The endothelium-dependent vasodilator acetylcholine (ACh) caused relaxation of the NTG-tolerant rings and also induced cyclic GMP accumulation in these rings. In other experiments, we found that prior exposure of vascular rings to ACh did not cause resistance to the subsequent vasorelaxant effects of ACh. NAC, captopril, and enalaprilat did not modulate the effects of ACh during either the first or subsequent exposures to ACh. In addition, indomethacin did not influence the "protective" effects of NAC or captopril against NTG tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in vivo interactions of nitrovasodilators and zaprinast, a cGMP-selective phosphodiesterase inhibitor.

We have examined the interaction of zaprinast, a selective inhibitor of cGMP phosphodiesterase, with guanylate cyclase activators on vascular smooth muscle relaxation in vitro and in vivo. Isolated porcine coronary arterial rings precontracted with prostaglandin F2 alpha (PGF2 alpha) were relaxed dose dependently by the guanylate cyclase activators nitroglycerin and nitroprusside, the cGMP phosphodiesterase inhibitor zaprinast and the endothelium-dependent agent bradykinin. A 1 h pretreatment with 0.5 mM nitroglycerin shifted the dose-response curve to nitroglycerin to the right by a factor of 90, reflecting the development of tolerance. The dose-response curve to sodium nitroprusside was also affected, albeit to a much lesser degree (9-fold increase in IC50). Both zaprinast and bradykinin remained unaffected by nitroglycerin pretreatment. A 30 min pretreatment of rings with zaprinast (1 microM) had no effect on nitroglycerin- or nitroprusside-induced relaxation in control rings, but enhanced vasorelaxation to both nitrovasodilators 7- and 2-fold, respectively, in tolerant rings. Similarly, a 30 min pretreatment of rings with 0.1 microM nitroprusside enhanced zaprinast-induced vasorelaxation 4- and 8-fold, respectively, in control and tolerant rings. Similar observations were made in vivo in anesthetized spontaneously hypertensive rats where zaprinast (0.1-3.0 mg/kg i.v.), caused dose-dependent reductions in mean arterial pressure. This effect was enhanced when rats had been pretreated with nitroprusside (1 micrograms/kg per min). In comparison, in zaprinast-pretreated rats the magnitude of depressor responses to nitroprusside (0.5-5.0 micrograms/kg) was not altered, but the duration of hypotensive response to the highest dose of nitroprusside was enhanced by zaprinast. These data demonstrate an enhanced vasodilatory response of nitrocompounds in combination with peak I-selective phosphodiesterase inhibitors.     

Increased nitroglycerin-induced relaxation by genistein in rat aortic rings.

The effect of genistein, a tyrosine kinase inhibitor, on nitroglycerin-induced relaxation was examined in rat aortic rings contracted by phenylephrine. In rat aortic rings, genistein (10(-5) M and 3x10(-5) M), a tyrosine kinase inhibitor, but not daidzein, an analogue of genistein, increased relaxation induced by nitroglycerin in a concentration-dependent manner. Iberiotoxin, an inhibitor of Ca2+ -activated K+ channels, inhibited the relaxation induced by nitroglycerin, but it did not affect the effect of genistein. Glibenclamide, an inhibitor of ATP-sensitive K+ channels, did not affect the relaxation induced by nitroglycerin. Theophylline, an inhibitor of cyclic AMP-dependent phosphodiesterase, increased the relaxation induced by nitroglycerin, and genistein (10(-5) M) failed to affect the relaxation induced by nitroglycerin in the presence of theophylline. Genistein also inhibited the activity of cyclic AMP-dependent phosphodiesterase. In addition, 6-[4-(4'-pyridyl)amino phenyl]-4,5-dihydro-3(2H)-pyridazinone hydrochloride, an inhibitor of cyclic GMP-inhibitable cyclic AMP phosphodiesterase, inhibited the relaxation induced by nitroglycerin. These results suggest that, in the rat aortic rings, genistein inhibits cyclic AMP-dependent phosphodiesterase activities, resulting in the increase of the relaxation induced by nitroglycerin.     

Studies on the effects of viprostol in isolated small blood vessels and thoracic aorta of the rat.

1. The effects of viprostol, prostaglandin E2 (PGE2) and nitroglycerin were studied in basilar artery, small mesenteric artery and the vein parallel to it as well as thoracic aorta of the rat. 2. In KCl-contracted basilar artery, viprostol produced a concentration-related biphasic response, contraction at concentrations less than 3 X 10(-6) M and relaxation at concentrations greater than 3 X 10(-6) M. PGE2 produced a concentration-related contraction while nitroglycerin produced a concentration-related relaxation. 3. In KCl-contracted small mesenteric artery, viprostol produced a biphasic response which was similar to that in the basilar artery. PGE2 produced a contraction and nitroglycerin produced relaxation in a concentration-dependent manner. 4. In KCl-contracted small mesenteric vein, in contrast to basilar and mesenteric artery, viprostol produced only a concentration-related relaxation in the range of 1 X 10(-6) to 1 X 10(-4) M. PGE2 produced a contraction and nitroglycerin produced a concentration-related relaxation. 5. In KCl-contracted thoracic aorta, PGE2 produced a biphasic response, relaxation at concentrations less than 3 X 10(-7) M and a concentration-related contraction at concentrations greater than 3 X 10(-7) M. Viprostol only produced a concentration-related contraction at concentrations greater than 1 X 10(-6) M, which was significantly less in magnitude than the contraction produced by PGE2. Nitroglycerin produced a concentration-related relaxation as seen in the small vessels. 6. In conclusion, the present data demonstrate that viprostol is a vasorelaxant agent which effectively dilates KCl-contracted basilar, small mesenteric artery and vein, but not the thoracic aorta of rat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasodilator effects of organotransition-metal nitrosyl complexes, novel nitric oxide donors

Nitrovasodilators cause endothelium-independent relaxation of blood vessels by generating nitric oxide (NO). We examined the relaxation and depressor effects of two organotransition-metal nitrosyl complexes, CpCr(NO)2Cl and CpMo(NO)2Cl, relative to those of the prototypal nitrovasodilators, nitroglycerin, and sodium nitroprusside (SNP), in phenylephrine-preconstricted aortic rings and conscious, unrestrained rats. CpCr(NO)2Cl, CpMo(NO)2Cl, nitroglycerin and SNP caused dose-dependent relaxation of aortic rings at maximal responses (Emax) of -118+/-4, -113+/-4, -104+/-1, and -128+/-5% and EC50 of 0.14+/-0.04, 22+/-4, 1.23+/-0.65, and 0.063+/-0.013 microM, respectively. The dose-response curve of CpCr(NO)2Cl was displaced to the right by hemoglobin, as well as methylene blue, showing involvement of the NO/cGMP pathway. Unlike nitroglycerin, preexposure for 1 h to CpCr(NO)2Cl did not alter subsequent relaxation response to the compound. Intravenous bolus injections of CpCr(NO)2Cl, CpMo(NO)2Cl, nitroglycerin, and SNP caused dose-dependent decreases in MAP with Emax of -42+/-2, -51+/-8, -56+/-6, and -58+/-2 mm Hg and EC50 of 0.041+/-0.010, 13+/-4, 1.6+/-0.4, and 0.037+/-0.004 micromol/kg, respectively. These results show that CpCr(NO)2Cl and CpMo(NO)2Cl are efficacious nitrovasodilators in vitro and in vivo.     

Prevention and reversal of tolerance to nitroglycerine with N-acetylcysteine

A recent study demonstrated that the sulfhydryl donor N-acetylcysteine (NAC) potentiated hemodynamic responsiveness to nitroglycerine (NTG) in patients with ischaemic heart disease. The interaction between NTG and NAC in rings of bovine coronary artery was examined. Vasodilator responses to NTG were determined after elevation of tone with the thromboxane mimetic U46619 [(15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano) prosta-5Z, 13E-dienoic acid]. NAC (1 microM-3 mM) induced no changes in tone of the preparation, but 10 microM NAC significantly potentiated responses to NTG (EC50 reduced from 0.69 +/- 0.19 microM to 0.22 +/- 0.06 microM; p less than 0.01). Increasing degrees of tolerance to NTG were produced at pH 7.4 by preincubating coronary rings with NTG in concentrations of 4.4 and 44 microM, and 0.22 mM. With 0.22 mM NTG, EC50 for subsequently administered NTG was increased to 11.0 +/- 1.8 microM (p less than 0.001 vs. control vessels). The degree of tolerance produced with this concentration of NTG was markedly attenuated by simultaneous (EC50 = 0.50 +/- 0.30 microM; p less than 0.001 vs. tolerant vessels) or subsequent (EC50 = 1.17 +/- 0.59 microM, p less than 0.001 vs. control vessels) incubation with 10 microM NAC. These data confirm that responses to NTG are modulated by sulfhydryl (or specifically cysteine) availability and suggest that in vitro tolerance to NTG is related to sulfhydryl (or cysteine) depletion. It is therefore possible that in vivo potentiation of NTG responses by NAC will be of clinical benefit in preventing or reversing loss of hemodynamic responsiveness to NTG.     

Relative importance of calcium-activated potassium channels in nipradilol-induced aortic relaxation in rats.

Nipradilol (CAS 81486-22-8), a vasodilatory beta-blocker, has been shown to dilate smaller vessels than nitroglycerin does, and the vasodilative effects of nipradilol have been reported to be less mediated by cyclic GMP (guanosine monophosphate) than those of nitroglycerin. To test the hypothesis that cyclic GMP-independent potassium channels have a larger role in nipradilol-induced aortic relaxation than cyclic GMP-dependent mechanisms, the effects of a potassium channel blocker, tetraethylammonium (TEA, CAS 56-34-8), and of a guanylate cyclase inhibitor, methylene blue (MB, CAS 61-73-4), on nipradilol-induced aortic relaxation were investigated and compared with those on nitroglycerin-induced aortic relaxation in isolated rat aortic rings. Relaxation response was expressed as percent relaxation, which is a percentage of the tension developed by 10(-7) mol/l norepinephrine. Nitroglycerin and nipradilol similarly relaxed the aortic ring in a concentration-dependent manner (10(-9)-10(-4) mol/l). In contrast, desnitronipradilol, a nipradilol analogue which has no nitroxy group, induced almost no aortic relaxation. TEA at 10(-3) mol/l, which is selective for calcium-activated potassium channels, inhibited the aortic relaxation induced by nipradilol (10(-5) mol/l) to a significantly greater extent than that induced by nitroglycerin (10(-5) mol/l) (% relaxation: 30.0 +/- 6.8 vs. 51.1 +/- 6.1%, p < 0.05). MB (10(-5) mol/l) suppressed the relaxation by nitroglycerin slightly but not significantly more than that by nipradilol. (% relaxation: 54.7 +/- 9.9 vs. 64.6 +/- 5.7%). The combination of TEA and MB almost completely eliminated the relaxation induced by nipradilol as well as by nitroglycerin. Thus, cyclic GMP-independent calcium activated potassium channels may be more involved in the aortic relaxation by nipradilol than that by nitroglycerin in rats.     

Stereospecific antagonism by d-butaclamol of dopamine-induced relaxation of the isolated rabbit mesenteric artery

We characterized the properties of vascular dopamine receptors on isolated rabbit mesenteric arteries preincubated with phenoxybenzamine (10(-5) M) and contracted with prostaglandin F2 alpha (PGF2 alpha). The dose-response curve for dopamine-induced relaxation was shifted to the right by the dopamine receptor antagonist d-butaclamol (10(-7)--3 X 10(-6) M) in a concentration-dependent manner. The pA2 value for d-butaclamol was calculated as 6.77. In contrast, even a very high concentration (3 X 10(-6) M) of l-butaclamol had no effect, indicating that vascular dopamine receptors require stereospecificity of antagonists. In the same preparation the mechanism of relaxation by 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (A-6,7-DTN; 3 X 10(-7)--10(-4) M) and bromocriptine (10(-6)--3 X 10(-4) M) was found to be dopaminomimetic, since only the dopamine receptor antagonists droperidol (10(-5) M) and metoclopramide (5 X 10(-5) M) could inhibit relaxations, whereas the beta-adrenoceptor antagonists pindolol (10(-7) M) and propranolol (10(-6) M) were without effect. It is concluded that receptors specific for dopamine exist on the rabbit mesenteric artery, which may tentatively be classified as belonging to the D1-type.     

Comparative vasorelaxing profiles of nicorandil, isosorbide dinitrate and nitroglycerin in isolated coronary arteries of the dog

The vasorelaxing effects of nicorandil (NCR), isosorbide dinitrate (ISDN) and nitroglycerin (NTG) were studied in isolated canine coronary arteries. In rings of coronary arteries precontracted with prostaglandin F2 alpha (3 x 10(-6) M) or KCl (30 mM), removal of the endothelium significantly augmented the relaxing effects of NCR, while it did not affect those of ISDN and NTG. In unrubbed rings precontracted with KCl (30 mM), methylene blue (5 x 10(-6) M) significantly inhibited vasorelaxing responses to the three drugs. The order of the inhibition was as follows: NTG greater than ISDN greater than NCR. When the unrubbed tissue was incubated with NTG (10(-5) M) or ISDN (10(-4) M) for 10 min, it developed acute tolerance in relaxing response to NTG or ISDN. Unlike NTG and ISDN, NCR did not develop any tolerance. The treatment with N-acetylcysteine (5 x 10(-5) M) tended to potentiate relaxant effects of NTG and to reduce the degree of acute tolerance to NTG. The results suggest that cGMP plays a role in the relaxation of the coronary artery induced by the drugs and furthermore that the mode of the vasorelaxing action of NCR may be somewhat different from that of NTG or ISDN.     

Biochemical and pharmacological interactions between nitroglycerin and thiols. Effects of thiol structure on nitric oxide generation and tolerance reversal

Co-administration of N-acetylcysteine (NAC) with nitroglycerin (NTG) has been shown to partially reverse nitrate tolerance and to potentiate the hypotensive effect of NTG in humans. However, a high clinical dose of NAC was required for this pharmacologic interaction resulting in the production of unwanted side-effects. Therefore, sulfhydryl compounds more active than NAC need to be identified if this interaction is to be exploited clinically. We previously suggested that the effect of sulfhydryl compounds on NTG may be mediated by the formation of S-nitrosothiol or nitric oxide (NO) extracellularly to the vascular smooth muscle cell (e.g. in plasma) (Fung et al., J. Pharmacol Exp Ther 245: 524-530, 1988). In an attempt to understand the structural features which govern this thiol-catalyzed NO generation from NTG, nineteen different aliphatic and ten aromatic sulfhydryl compounds were examined with respect to their catalytic activity to generate NO from NTG in plasma. Significantly enhanced production of NO was observed with most sulfhydryl compounds examined when compared to buffer control. Among the aliphatic thiols, only mercaptosuccinic acid was more potent than NAC (2x), whereas among the aromatic thiols, both thiosalicylic acid (TSA, 10x) and TSA-methyl ester (3x) were more potent than NAC. Comparative in vitro relaxation studies were carried out using isolated (and nitrate-tolerant) rat aortic rings with NTG/TSA and NTG/NAC, in the presence of 0.5% (v/v) plasma. Under these conditions, partial reversal of NTG tolerance could be achieved with TSA, but not with NAC. These data are consistent with the view that extracellular production of NO or S-nitrosothiol serves as a tolerance-reversing mechanism of thiols on NTG. TSA appears to be a more potent sulfhydryl compound than NAC in this biochemical and pharmacologic interaction.