Bovine Coronary Artery Research Articles

Endothelium-dependent vasodilatation in bovine coronary arteries: calcium dependence and inhibition by proadifen.

Vasodilator responses were examined in bovine coronary artery rings preconstricted with the thromboxane-mimetic, U46619. A23187 produced endothelium-dependent vasodilatation that was abolished in calcium-free solution. In contrast, endothelium-dependent vasodilatation produced by arachidonic acid was not altered in calcium-free solution. In calcium-free solution, indomethacin (10 mumol/l) did not affect arachidonate-induced relaxations whereas BW755C (100 mumol/l) reduced relaxations to low concentrations of arachidonate, and proadifen (SKF-525A, 1 mmol/l) abolished them at all concentrations. Endothelium-independent relaxations produced by glyceryl trinitrate were not affected by proadifen (1 mmol/l). It is clear that arachidonic acid bypasses a calcium-dependent step in the release of endothelium-derived relaxing factor, perhaps acting as an intermediate precursor. The data support the hypothesis that a cytochrome P-450 mono-oxygenase may also be involved.

Eicosonoid metabolism and beta-adrenergic mechanisms in coronary arterial smooth muscle: potential compartmentation of cAMP.

Beta-Adrenergic relaxation in bovine coronary arteries is enhanced by inhibition of eicosonoid metabolism and inhibited by its stimulation. We investigated the interaction between eicosonoid metabolism and beta-adrenergic mechanisms by studying the effect of perturbations of eicosonoid metabolism on vascular adenosine 3',5'-monophosphate (cAMP) content and the cAMP-dependent relaxation of isometric force and activation of glycogen phosphorylase. KCl (35 mM) elicited a contraction, activated phosphorylase, and slightly decreased cAMP content. Isoproterenol (10(-7) M) relaxed the KCl contraction, further increased phosphorylase activity, and increased cAMP. Neither indomethacin (5 X 10(-6) M) nor arachidonic acid (3 X 10(-5) M) affected the KCl contraction, but arachidonic acid increased both cAMP and phosphorylase activity and indomethacin decreased cAMP. Indomethacin potentiated the relaxation induced by isoproterenol but inhibited the activation of phosphorylase and had no effect on the isoproterenol-induced increase in cAMP. Arachidonic acid, on the other hand, inhibited the isoproterenol-induced relaxation but potentiated both the increases of phosphorylase activity and cAMP. Thus neither relaxation nor phosphorylase activity was related in a straightforward manner to the total cAMP content. A direct relation between cAMP, relaxation, and phosphorylase can be reconciled with the antiparallel effects of alterations of eicosonoid metabolism observed in this study by a proposed model in which the effects of cAMP are assumed to be functionally compartmentalized.

Stimulation of soluble coronary arterial guanylate cyclase by SIN-1

SIN-1, a metabolite of the vasodilating drug molsidomine, was found to stimulate dose dependently (0.01–1 mM) soluble guanylate cyclase from bovine coronary arteries up to 100-fold the control value. The stimulatory effect of SIN-1 increased with rising concentrations of MnCl 2 or MgCl 2 and was diminished in the presence of methylene blue or ferricyanide. The time course of SIN-1-induced guanylate cyclase stimulation was characterized by a lag phase which was not observed after preincubation of the enzyme with SIN-1. In contrast to nitroglycerin and sodium nitroprusside, SIN-1 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.

Multiple molecular forms of cyclic nucleotide phosphodiesterase in cardiac and smooth muscle and in platelets: Isolation, characterization, and effects of various reference phosphodiesterase inhibitors and cardiotonic agents

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been identified previously in several tissues and cell types using a variety of different isolation methods. In the present study, the different molecular forms of phosphodiesterase (PDE) were isolated from cardiac muscle (guinea pig left ventricle), vascular smooth muscle (bovine coronary arteries) and human platelets using the same isolation procedure in each instance. These enzymes were then characterized kinetically, and the effects of various reference PDE inhibitors and cardiotonic agents on each form were examined. A low K m, low V max form of phosphodiesterase (PDE I) was found in all three tissue/cell types. PDE I activity was stimulated by calmodulin in cardiac and smooth muscle, but not in platelets. In smooth muscle and platelets, PDE I preferentially hydrolyzed cyclic GMP, whereas cardiac muscle PDE I hydrolyzed cyclic AMP and cyclic GMP equally. A high K m , high V max form of phosphodiesterase (PDE II) was found in cardiac muscle and platelets, but not in smooth muscle. PDE II activity was not stimulated by calmodulin and there was no substrate specificity. A low K m , low V max cyclic AMP-specific form of phosphodiesterase (PDE III) was found in all three tissue/cell types. The activity of PDE III was not stimulated by calmodulin. The reference inhibitors theophylline and papaverine exerted nonspecific inhibitory effects on all forms of phosphodiesterase. Other reference inhibitors (M & B 22,948 and dipyridamole) and several cardiotonic agents (AR-L 57, CI-914, CI-930, amrinone, and MDL 17,043) exerted selective inhibitory effects on only one molecular form of phosphodiesterase. The degree of selectivity was often dependent upon the tissue or cell from which the molecular form of phosphodiesterase was isolated. These studies demonstrate that (i) there is heterogeneity regarding the number of phosphodiesterases present in various tissue/cell types, as well as their substrate specificity and their ability to be stimulated by calmodulin, and (ii) these different molecular forms of phosphodiesterase can be selectively inhibited by different pharmacological agents. The possibility exists that such selective inhibitors may produce discrete changes in cyclic AMP or cyclic GMP levels, and that these changes may be produced in specific tissues and/or cells.

Cyclic GMP as the mediator of molsidomine-induced vasodilation

The mode of action of the in vitro active metabolites SIN-1 and SIN-1A of the vasodilator prodrug molsidomine was studied in bovine coronary artery strips. Both compounds increased cyclic GMP levels in close association with, but prior to their relaxing action. Relaxation and rises in cyclic GMP by SIN-1 were potentiated by M& B 22,948, an inhibitor of cyclic GMP phosphodiesterase and attenuated by methylene blue, a dye that inhibits activation of guanylate cyclase by SIN-1 and various nitrovasodilators. A single significant correlation between rises in cGMP and relaxation was obtained for both SIN compounds and various nitrovasodilators. Relaxation by SIN-1A was independent of the presence of endothelium and was not affected by various inhibitors of arachidonic acid metabolism. In contrast to nitroglycerin, SIN-1 did not induce substantial tolerance nor were its actions in artery strips that were tolerant to nitroglycerin. The results indicate that SIN-1A relaxes coronary smooth muscle by a direct stimulant effect on soluble guanylate cyclase in vascular smooth muscle cells.

Pharmacological evaluation of 5-(Z,E)-13,14-didehydro-20-methyl-carboprostacyclin (FCE 22509)

FCE 22509, 5-(Z,E)-13,14-didehydro-20-methyl-carboprostacyclin, a chemically stable prostacyclin (PGI 2) derivative, was 3.5 times more potent than PGI 2 in relaxing bovine coronary artery in vitro ; unlike PGI 2, it did not contract bovine coronary vein and it antagonized PGI 2-induced contractions of the coronary vein. In vitro smooth muscle(guinea pig ileum and trachea and rat stomach strip) was contracted to a lesser extent than with PGI 2. FCE 22509 was about five times less potent than PGI2 in deaggregating platelet clumps formed on rabbit Achilles tendon bathed with heparinized cat blood (ED 50 = 7.6 and 1.6 mcg/kg i.v. respectively). In the conscious normotensive and spontaneously hypertensive rat FCE 22509 lowered mean systemic arterial pressure (MSAP) (ED 25 = 11.5 and 4.8 mcg/kg i.v.) to a lesser extent than PGI2 (ED 25 = 2.1 and 2.34 mcg/kg i.v.) and increased heart rate but not dose-dependently. Orally administered, FCE 22509 likewise reduced MSAP though at high dosages (ED 30 = 1.33 and 1.02 mg/kg in normotensive and hypertensive rats). Heart rate (HR) was raised after i.v. and oral treatment but not dose-dependently. In the open-chest anaesthetized dog, FCE 22509, compared with PGI 2 at the same three doses, 0.2, 0.4 and 0.8 mcg/kg i.v., lowered MSAP but its hypotensive effect was less pronounced than that of PGI 2. Like PGI 2, FCE 22509 did not modify HR (though PGI 2 showed a tendency to a decrease and FCE 22509 seemed to increase this cardiovascular function) and mean pulmonary arterial pressure (MPAP) and likewise significantly reduced myocardial contractile force. After infusion of PGI 2 and FCE 22509 0.2 mcg/kg i.v. for 15 min in the open chest anaesthetized cat, the ex-vivo ADP-induced inhibition of platelet aggregation was the same for both compounds. Unlike PGI 2, which reduced MSAP and MPAP, FCE 22509 had no significant lowering effect on MSAP and MPAP.

Bovine coronary artery endothelium: In vitro culture and production of plasminogen activator

A new method for isolation and culture of endothelial cells from bovine coronary artery (BCoAEC) is presented. This method involves in situ perfusion and digestion of main coronary arteries with a collagenase solution. The isolated cells were cultured and maintained through many cell passages in Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum derived from either whole blood or plasma. Confirmation of these cells' endothelial origin was obtained by demonstration of typical morphologic and growth characteristics of endothelium, immunofluorescent staining with antibodies to von Willebrand factor (Factor VIII: vWF), and measurement of plasminogen activator (PA). In addition, production of PA was inhibited by enzymatically active thrombin as has been previously described with bovine aortic endothelial cells in culture.

Effects of local anaesthetics on responses of human saphenous vein and bovine coronary artery to neurotransmitters, acetylcholine, noradrenaline and 5-hydroxytryptamine.

The effects of 5 different local anaesthetics, lignocaine, etidocaine, prilocaine, mepivacaine, bupivacaine, and 3 different neurotransmitters, acetylcholine (ACh), noradrenaline (NA), 5-hydroxytryptamine (5-HT) on tone and contractility of human saphenous vein and bovine coronary artery were studied and compared in vitro. The experiments were carried out to see if there were species or tissue differences in the action of local anaesthetics and neurotransmitters at vascular smooth muscle, i.e. saphenous vein and coronary artery. Another important aspect was to see if local anaesthetics modified cholinergic (ACh), adrenergic (NA) and serotoninergic (5-HT) responses in vascular smooth muscle. Among the local anaesthetics studied, lignocaine and etidocaine produced prolonged relaxations, whereas prilocaine, mepivacaine, bupivacaine produced contractions in the saphenous vein and coronary artery. High concentrations of the latter drugs produced relaxations in the blood vessels. Among the local anaesthetics, lignocaine produced differential effects on saphenous vein and coronary artery, its effect on the latter was 3 times larger than on the saphenous vein. ACh, NA and 5-HT contracted the saphenous vein. In the coronary artery, ACh and 5-HT contracted whereas NA relaxed the vessel. On a molar basis, ACh was less effective than NA in contracting the saphenous vein. 5-HT was equipotent on both the saphenous vein and coronary artery. Lignocaine reduced ACh, NA and 5-HT-induced contractions in the saphenous vein and those of ACh and 5-HT in the coronary artery, and shifted the control curves, non-competitively, to the right. The relaxation produced by NA in the coronary artery was enhanced by lignocaine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of calcium antagonists on vascular responses of bovine coronary artery to acetylcholine, noradrenaline, and 5-hydroxytryptamine.

Verapamil (0.05-5 microM), diltiazem (0.2-20 microM), and nifedipine (0.3-30 microM) produced concentration-dependent relaxation of bovine coronary artery. Based on the EC50 values (concentration to produce 50% maximum response), the calcium entry blocker verapamil (relative potency = 1) was 3.4 and 7 times as potent as diltiazem and nifedipine, respectively, in producing relaxation of bovine coronary artery. In addition, verapamil reduced the contractions produced by acetylcholine (0.01-10 microM), 5-hydroxytryptamine (5-HT) (0.01-10 microM) whereas it potentiated the relaxation produced by noradrenaline (0.01-10 microM). It was concluded that verapamil, diltiazem, and nifedipine (a) relax the bovine coronary artery, verapamil being more potent than diltiazem and nifedipine, and (b) the calcium entry blockers modified the contractile responses to neurotransmitter agents, acetylcholine, noradrenaline, and 5-HT, inhibiting the contractions produced by acetylcholine and 5-HT, and enhancing the relaxation produced by noradrenaline.

Catecholamine release and potentiation of thromboxane A2 production by nicotine in the greyhound.

Thromboxane A2 was generated by infusing arachidonic acid (2.5 micrograms ml-1) into an extra-corporeal circuit of blood withdrawn from anaesthetized dogs, and assayed on a blood-bathed bioassay cascade of porcine and bovine coronary artery strips, chick rectum and rat stomach strip. All tissues except chick rectum were treated with phentolamine and propranolol to abolish direct effects of catecholamines. The arachidonate-induced contractions of artery strips were abolished by a thromboxane synthetase inhibitor UK-38485 (3 mg kg-1, i.v.), but were not altered by the 5-hydroxytryptamine antagonist ketanserin (10 microM) administered over the tissues. Intravenous infusion of adrenaline (0.2 and 0.4 micrograms kg-1 min-1) reversibly potentiated the coronary contractions produced by arachidonate, but did not alter contractions when applied directly over the bioassay tissues. Intra-aortic infusion of nicotine (5 or 10 micrograms kg-1 min-1) also increased the arachidonate-induced contractions of the bioassay tissues but only on those experiments where nicotine caused appreciable adrenaline release, as indicated by relaxation of chick rectum. Phenoxybenzamine (2 mg kg-1, i.v.) blocked the potentiation effect of adrenaline and nicotine on coronary contractions. The specific alpha 2-adrenoceptor antagonist, idazoxan (1 mg kg-1, i.v.), also blocked nicotine-induced potentiation of the contractions. These findings suggest that the ability of nicotine to potentiate thromboxane release from circulating platelets and blood cells is dependent upon the release of adrenaline, and probably involves an action on alpha-adrenoceptors of the circulating blood elements.

Effects of dipyridamole on the glyceryl-trinitrate-induced inhibition of coronary artery muscle tone and platelet aggregation in relation to cyclic nucleotide metabolism.

The effects of glyceryl-trinitrate (GTN) and dipyridamole (DIP) on relaxation of bovine coronary arteries and on inhibition of aggregation of human platelets have been studied in vitro with special reference to the cyclic GMP (cGMP) system. GTN had a dose-dependent relaxant effect on bovine coronary arteries, and at a high concentration (10(-5) M) it had an inhibiting effect on platelet aggregation. The effects were associated with an increase in the cGMP levels of the tissues. DIP (5 X 10(-7) M respectively 5 X 10(-6) M) potentiated the coronary artery relaxation induced by GTN (10(-8) M) and the inhibition of platelet aggregation caused by GTN in the concentrations 10(-7)-10(-4) M. The potentiation was associated with higher levels of cGMP than those produced by GTN alone, at least in bovine coronary arteries. However, at a concentration of 10(-4) M, GTN, in combination with DIP, caused a significant fall in the cGMP level compared to GTN alone. GTN and DIP were not found to significantly increase the cAMP levels in the concentrations tested. DIP was shown to inhibit phosphodiesterase (PDE) from both platelets and bovine coronary arteries. This might be one of the possible mechanisms that can explain the above mentioned potentiation. It is suggested that the combination of DIP + GTN may be of some clinical importance since the potentiating effects were seen at concentrations comparable to the therapeutic plasma concentration for the respective drugs.

Microsomal calcium-accumulating ability of bovine coronary artery and aorta

Calcium uptake and binding activities of microsomal fractions from bovine coronary artery and aorta were examined. The isolated microsomal fraction of the coronary artery and aorta showed 7- to 8-fold higher glucose-6-phosphatase activity and 4- to 6-fold higher NADPH-cytochrome c reductase activity as compared with the corresponding values for the homogenate fraction. Coronary artery and aorta microsomal calcium uptake activities were 118 and 159 nmoles Ca 2+/mg protein/10 min in the presence of 100 μM CaCl 2, respectively. These activities for bovine vascular smooth muscle microsomes are higher than those of other species investigated. The calcium uptake activities were dependent on calcium concentrations ranging from 5 to 50 μM in the assay medium. The onset of the reaction for aorta microsomal calcium uptake was faster than that for the coronary artery. The calcium uptake activity was also dependent on ATP, but it was practically independent of oxalate ions in the assay medium. Microsomal calcium binding activities of the coronary artery and aorta were maximal at 20 min of incubation under the present experimental conditions. A lower K m value of the aortic calcium binding for ATP was obtained as compared with that for the coronary artery. The present experiment explored several characteristics of the microsomal calcium-accumulating ability of vascular smooth muscle, which provides meaningful information for further study on cellular calcium movements in vascular smooth muscle.

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.

Studies on the mode of action of isosorbide dinitrate: A physiologic and biochemical approach

The action of isosorbide dinitrate (ISDN) and diltiazem on coronary artery diameter, vascular resistance, and coronary blood flow was determined in instrumented postoperative conscious dogs. Low doses of ISDN were found to increase the diameter of large arteries without affecting coronary blood flow. Higher doses of ISDN produced an increase in both coronary diameter and blood flow. Diltiazem, on the other hand, increased coronary artery diameter and blood flow at all doses tested. Coronary vascular resistance was more sensitive to diltiazem than to ISDN. In isolated canine cardiac Purkinje strands, ISDN produced a concentration-dependent decrease in force development and action-potential duration measured at 50% of repolarization (APD 50). ISDN did not significantly affect action potentials recorded in Purkinje strands depolarized by potassium (22 mmol) and treated with isoproterenol (10 −6M). Diltiazem also decreased Purkinje strand force development and APD 50 in a concentration-dependent manner. Diltiazem, however, was several orders of magnitude more potent than ISDN and completely abolished action-potential genesis in potassium-depolarized, isoproterenol-restored Purkinje strands. Both diltiazem and ISDN were found to relax porcine coronary artery strips contracted by KCl or histamine. Studies on 45Ca flux, in isolated coronary artery rings, indicate that ISDN inhibited both histamine-induced Ca ++ influx and efflux from intracellular sources. The inhibition of Ca ++ efflux and intracellular Ca ++-dependent contraction occurred over a similar ISDN concentration range. ISDN also relaxed bovine coronary artery strips contracted with KCl. No change in cyclic adenosine monophosphate levels occurred during ISDN-induced relaxation of bovine coronary arterial strips. In contrast, cyclic guanosine monophosphate (cGMP) increased markedly in a dose- and time-dependent manner. Moreover, the extent of ISDN-induced relaxation was directly correlated with the increase in cGMP. Calcium transport in mitochondria, sarcoplasmic reticulum, and sarcolemmal membranes of cardiac and vascular smooth muscles was not affected by ISDN. It is postulated that increases in cGMP may be causally linked to relaxation of vascular smooth muscle due to changes in either Ca ++ flux or phosphorylation of proteins or both. In ischemic myocardium of dogs, ISDN appears to prevent damage of mitochondrial phosphorylative respiration.

Inhibition of thromboxane and 12-HPETE formation by dazoxiben and its two thiophenic acid-substituted derivatives

LG 82-4-00 (5-(1-(1-imidazolyl)-ethoxy)-thiophene-2-carboxylate) and LG 82-4-01 (4-chloro-thiophenic-substituted derivative) were examined for specific inhibition of thromboxane (TX) synthetase. Thromboxane formation was measured by a radioimmunoassay specific for TBX2. In thrombin (0.6 IU/ml)-stimulated, washed human platelet suspensions (WPS) the IC50 (μM) for inhibition of TX formation were 1.1 (LG 82-4-00), 1.3 (LG 82-4-01) and 0.7 (dazoxiben). LG 82-4-00, LG 82-4-01 and dazoxiben also inhibited collagen (0.6–2.5 μg/ml)-induced TXB2 formation and platelet aggregation in human platelet-rich plasma. Neither LG 82-4-00 nor LG 82-4-01 had vasoconstrictor, proaggregatory or TX antagonistic activity or affected primary wave ADP aggregation. There was less than 10% inhibition of PGI2 formation from bovine coronary artery slices with concentrations up to 100 μM. At 100 μM, dazoxiben inhibited thrombin-induced 12-HPETE formation in WPS by 81 ± 10% whereas LG 82-4-00 and LG 82-4-01 were much less active. These data indicate that LG 82-4-01 are specific inhibitors of thromboxane synthetase in human platelets.

Effects of a thromboxane synthetase inhibitor and a thromboaxane antagonist on release and activity of thromboxane A 2 and prostaglandin in vitro

The TxA 2 synthetase inhibitor, dazoxiben, and the TxA 2 antagonist, ±SQ 29, 548, were examined for effects on release and vasoactivity of TxA 2 and prostacyclin. Isolated perfused guinea pig lungs were used as the enzyme source from which TxA 2 and prostacyclin were released in response to injections of arachidonic acid or bradykinin. Both dazoxiben and ±SQ 29, 548 inhibited contraction of the superfused rat aorta and bovine coronary artery after arachidonic acid injection through the lung. ±SQ 29, 548 abolished contractions of the rat aorta, but significant aorta contracting activity persisted during dazoxiben treatment. Dazoxiben significantly inhibited arachidonate-induced release of TxA 2 (immunoreactive TxB 2)iinto the superfusate, but TxA 2 release was significantly potentiated by ±SQ 29, 548. Thus, in the presence of enhanced TxA 2 concentrations, ±SQ 29, 548 effectively antagonized the vasospastic effect of TxA 2. Dazoxiben diverted a significantly greater amount of arachidonic acid into prostacyclin synthesis (immunoreactive 6-keto-PGF 1α), changing original coronary vasoconstriction into relaxation. ±SQ 29, 548 did not significantly modify lung prostacyclin synthesis. Moreover, with ±SQ 29, 548, the absence of TxA 2-mediated coronary contraction unmasked active relaxation of the superfused bovine coronary artery, coincident with thromboxane and prostacyclin release. Dazoxiben consistently inhibited TxA 2 synthesis and enhanced prostacyclin synthesis. ±SQ 29, 548 augmented TxB 2 release in response to arachidonate, but not bradykinin, and did not significantly alter 6-keto-PGF 1α release in response to either arachidonate or bradykinin. In terms of vasoactivity measured in vitro , ±SQ 29, 548 and dazoxiben produced similar anti-vasospastic effects, although this was accomplished by completely different mechanisms.

Dissociation of cysteine and glutathione levels from nitroglycerin-induced relaxation

The present study investigated possible involvement of cysteine (CSH) and reduced glutathione (GSH) as critical cellular sulfhydryls which mediate nitroglycerin (GTN)-induced cyclic GMP accumulation and relaxation in bovine coronary artery (BCA). Tolerance to the relaxant effects of GTN was induced in BCA in vitro by preincubation with 1 mM GTN for 2 h. GTN-tolerant BCA were at least 100-fold less sensitive than non-tolerant BCA to the relaxant effects of GTN. Consistent with a relationship between tolerance to both GTN-induced cyclic GMP accumulation and relaxation, cyclic GMP accumulation induced by 1 μM GTN was markedly reduced in GTN-tolerant BCA when compared with non-tolerant BCA. Incubation with 1 mM CSH for 1 h did not significantly alter GTN-induced cyclic GMP accumulation or relaxation in either GTN-tolerant or non-tolerant BCA. Levels of CSH, GSH and glutathione-disulfide (GSSG) were measured in non-tolerant BCA, GTN-tolerant BCA and GTN-tolerant BCA incubated with 1 mM CSH for 1 h. Levels of CSH and GSH were lower in GTN-tolerant BCA that in non-tolerant BCA, whereas GSSG levels were similar in both. In GTN-tolerant BCA incubated with 1 mM CSH, CSH levels were more than 10-fold above, and GSH levels were similar to corresponding values obtained in non-tolerant BCA. These data indicate that although incubation with CSH did not significantly reverse tolerance to GTN-induced cyclic GMP accumulation and relaxation in BCA, it did effectively raise the level of CSH and GSH in GTN-tolerant BCA, at least to corresponding levels found in non-tolerant BCA. These results indicate that the relaxant effects of GTN in BCA do not correlate with tissue levels of CSH and GSH. The findings do not support the hypothesis that CSH and GSH are the cellular sulfhydryls involved in mediating GTN-induced guanylate cyclase activation, cyclic GMP accumulation and relaxation in intact BCA.

Mechanism of vasodilation by molsidomine

Molsidomine is enzymatically metabolized in the liver to SIN-1 and readily converted into the active metabolite SIN-1A, which carries a free nitroso group. Evidence obtained in isolated circular strips from bovine coronary arteries indicates that SIN-1 increases cyclic guanosine monophosphate in close association with its relaxant effects in coronary strips under various pharmacologic conditions, suggesting that cyclic guanosine monophosphate mediates relaxation. Various nitrovasodilators act by the same mechanism, which is stimulation of guanylate cyclase. In this study the effect of nitroglycerin depended on the presence of a special thiol, cysteine, whereas SIN-1 was active also in the absence of cysteine. Cysteine deficiency was found to be associated with tolerance. After prolonged exposure to the drug, tolerance toward nitroglycerin developed in coronary strips that was antagonized by cysteine. SIN-1 produced no significant tolerance and was also fully active in nitroglycerin-tolerant strips. We conclude that SIN-1 relaxes vascular smooth muscle by direct stimulation of guanylate cyclase, whereas nitroglycerin probably must be converted into a cyclase stimulator by a cysteine-dependent reaction.

Alterations of arachidonic acid metabolism modulate adenosine relaxation in isolated coronary arteries.

The possible modulation of adenosine-induced relaxation by endogenous prostaglandins was studied in isolated porcine, bovine and canine coronary arteries. Artery rings were mounted for isometric tension recording in organ bath filled with Krebs-Ringer-bicarbonate solution (37 degrees C). Increasing concentrations (10(-6)-10(-4) M) of adenosine relaxed contractions induced by 35 mM KCl. The cyclo-oxygenase inhibitor indomethacin (5 X 10(-6) M) significantly augmented the relaxations in response to adenosine in coronary arteries isolated from all three species. In the porcine coronary artery (which was most sensitive to exogenous adenosine) the mixed lipoxygenase/cyclo-oxygenase inhibitor phenidone augmented, while exogenous arachidonic acid (3 X 10(-5) M) or a decrease in bath O2-level from 95 to 20% depressed adenosine-induced relaxations. Indomethacin completely prevented the inhibitory effect of decreasing bath O2, but only partially antagonized the suppression caused by arachidonic acid. The data demonstrate that arachidonic acid and its cyclo-oxygenase metabolites modulate (depress) adenosine responsiveness in isolated coronary arteries.

Effects of isoproterenol and forskolin on tension, cyclic AMP levels, and cyclic AMP dependent protein kinase activity in bovine coronary artery

The effects of isoproterenol and forskolin on tension, cyclic AMP levels, and cyclic AMP dependent protein kinase activity were compared in helical strips of bovine coronary artery. Elevation of cyclic AMP and activation of the protein kinase appeared to be well correlated with relaxation of potassium-contracted arteries by isoproterenol. Forskolin, at 1 microM or higher concentrations, also markedly elevated cyclic AMP levels, activated the kinase, and relaxed the arteries. However, a lower concentration of forskolin (0.1 microM) caused significant increases in both cyclic AMP levels and cyclic AMP dependent protein kinase activity, but did not relax the muscles. Relaxation caused by isoproterenol was accompanied by an apparent translocation of cyclic AMP dependent protein kinase activity from the soluble to the particulate fraction in these preparations. A similar shift in the distribution of the kinase was caused by various concentrations of forskolin, irrespective of whether the arteries were relaxed or not. In contrast to previous results in other tissues, low concentrations of forskolin (less than or equal to 1 microM), which themselves markedly elevated cyclic AMP levels in the arteries, did not potentiate the effects of isoproterenol on cyclic AMP levels or tension in these preparations. These results suggest that either cyclic AMP is not solely responsible for the relaxation caused by these agents, or some form of functional compartmentalization of cyclic AMP and cyclic AMP dependent protein kinase exists in this tissue.