Strips Of Bovine Coronary Arteries Research Articles

Influence of several Styrian wines on bovine coronary artery contractions induced by endogenous agents

In the current study, seven Styrian white wine varieties, as well as their corresponding grape skin extracts (GSE), were tested for possible antagonistic effects on several endogenous vasoconstrictors known to be involved in the pathogenesis of cardiovascular diseases via tissue contraction experiments. Results were compared to those of Zweigelt, the most cultivated Styrian red wine. Bovine coronary artery strips were attached to a force transducer connected to a bridge amplifier, and isometric force was recorded on a multipen recorder. Preincubation of the vessels with the dealcoholized wines (DAW; 330 μl/ml) inhibited the constrictions to histamine and serotonin (5-HT) NO-dependently in the range of 5–80% and 30–90%, respectively (Zweigelt 47% and 90%, respectively). The corresponding GSE (20 mg/ml) inhibited the coronary constrictions to histamine in the range of 40% to 80% (Zweigelt 80%). Additionally, all DAW antagonized the contractile responses to the thromboxane-mimetic U46619 and the isoprostane 8- iso-PGF 2α, indicating a nonspecific inhibition. To characterize the vasoactive component(s), the Traminer GSE was separated representatively using ultrafiltration, solid phase extraction (SPE) on Isolute C18(EC), and Isolute SCX-2, as well as column chromatography (CC) on polyamide. The resulting fractions were subsequently bioassayed for vasorelaxing activity. Preliminary results refer to a very hydrophilic, nonpolyphenolic basic compound with a MW<1000 Da. Our findings demonstrate that certain Styrian wines have remarkable antagonistic effects on several endogenous agonists in vitro, and that also nonpolyphenolic components in grapes may contribute to cardiovascular protection. Further separation steps as well as phytochemical and pharmacological investigations are in progress.

Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells. 1985.

A cascade superfusion technique has been developed for the differential bioassay of prostacyclin and endothelium‐derived relaxing factor (EDRF) released from porcine aortic endothelial cells cultured on microcarriers, packed into a column and perfused. Bradykinin (Bk; 20–100 Nm) released prostacyclin (9.6 ± 1.5 Nm per 106 cells; mean ± s.e.mean, n = 9) and prostaglandin E2 (PGE2; 2.1 ± 0.6 Nm per 10° cells) from the column measured by relaxation of strips of bovine coronary artery (BCA) and rabbit mesenteric or coeliac artery, respectively. The presence of these prostanoids in the effluent was confirmed by specific radioimmunoassays. A23187 (500–2000 Nm) also released both prostacyclin and PGE2 from the cells. This release was long‐lasting and not reproducible. Bk (20–100 Nm) and A23187 (30–300 Nm) released EDRF from the column. This was detected in a cascade of four rabbit aortic strips (RbA), denuded of endothelium and contracted with U46619 or phenylephrine. The relaxation of the RbA strips caused by EDRF was progressively attenuated down the cascade (half‐life &lt; 7 s) and was not affected by indomethacin. EDRF and prostacyclin could be differentially bioassayed in a cascade of alternating RbAs and BCAs as prostacyclin did not relax RbAs and the time delay to the BCAs destroys EDRF. EDRF could be bioassayed on its own when the endothelial cells were treated with indomethacin. 5‐Hydroxytryptamine 0.2, noradrenaline 1.0, platelet‐activating factor (Paf‐acether) 1.0, formylmethionyl‐leucyl‐phenylalanine 1.0, acetylcholine 0.5, bethanecol 0.5, adenosine diphosphate 0.25 and angiotensin II 0.1 μm did not release either prostanoids or EDRF from the column.

Antagonistic affinity profile of imperialine on different subtypes of muscarinic receptors

In order to identify the receptor subtype responsible for acetylcholine (ACh)-induced relaxation of bovine coronary artery, we determined the affinity of six subtype-selective muscarinic antagonists and compared them with affinity estimates obtained for bovine left atria. At low concentrations, ACh potently relaxed circular strips of coronary artery with endothelium (EC50 0.15 μM), but contracted them at higher agonist concentrations with potencies that depended on the presence or absence of endothelium: EC50 1.8 μM (without endothelium); 4.6 JμM (with endothelium). The pA2 values obtained for antagonism of relaxant responses to ACh were: pirenzepine (M1-selective) 7.38 ± 0.12; AF-DX 116 (11-[2-(diethylamino-methyl)-1-piperidinylacetyl]-5, 11-dihydro-6H-pyrido(2,3-b)l,4-benzodiazepine-6-one; M2-selective) 5.79 ± 0.09; and 4-diphenylacetoxy-N-methylpiperidine-methobromide (4-DAMP; M3/M1-selective) 9.07 ± 0.12. The corresponding Schild slopes were 0.98 ± 0.07 for pirenzepine, 1.17 ± 0.09 for AF-DX 116 and 1.01 ± 0.04 for 4-DAMP. For the following three antagonists, pKg values were determined at two different antagonist concentrations: dicyclomine (M1-selective) 7.49 ± 0.10, cyclohexylphenyl-(2-piperidinoethyl)-silanol (CPPS; M3-selective) 8.0 ± 0.10, and parafluoro-hexahydrosila-difenidol (pFHHSiD; M3-selective) 7.87 ± 0.10. For comparison, the antagonism of methacholine-induced negative inotropy in left atria was determined for three antagonists, yielding the following pA2 values: pirenzepine 5.98 ± 0.14; AF-DX 116 6.81 ± 0.14 and 4-DAMP 7.99 ± 0.14. The slopes of the corresponding Schild plots were 1.05 ± 0.10,1.14 ± 0.12 and 1.08 ± 0.08, respectively. The results indicate that ACh relaxes precontracted circular strips of bovine coronary arteries with intact endothelium by activation of muscarinic receptors most closely corresponding to the M3 subtype.

Pharmacological interaction experiments differentiate between glibenclamide-sensitive K + channels and cyclic GMP as components of vasodilation by nicorandil

The relaxant effect of the vasodilator drug, nicorandil, was studied in circular strips of bovine coronary arteries. To differentiate between relaxation caused by cyclic GMP (cGMP) and by hyperpolarization, the influence of cGMP was blocked with methylene blue and that of hyperpolarization with the inhibitor of ATP-dependent K + channels, glibenclamide. Methylene blue and glibenclamide inhibited nicorandil-induced relaxation to similar extents. Cromakalim-induced relaxation but not that due to sodium nitroprusside (nitroprusside-Na) was inhibited by glibenclamide. Methylene blue inhibited the relaxation caused by nitroprusside-Na but not that due to cromakalim. The different modes of action of the two components of relaxation caused by nicorandil were studied in agonist-agonist interaction experiments. The interaction between nicorandil and nitroprusside-Na or 3-morpholino-sydnonimine (SIN-1) was overadditive in the absence of glibenclamide but additive, i.e. competitive, in the presence of glibenclamide. The interaction of nicorandil with cromakalim or pinacidil was overadditive in the absence of methylene blue but additive, i.e. competitive, in the presence of methylene blue. The results show that nicorandil relaxes smooth muscle through two independent mechanisms: ATP-dependent activation of K + channels and stimulation of guanylyl cyclase resulting in increases in cGMP.

Characterization of muscarinic receptors mediating endothelium-dependent relaxation of bovine coronary artery

In order to identify the receptor subtype responsible for acetylcholine (ACh)-induced relaxation of bovine coronary artery, we determined the affinity of six subtype-selective muscarinic antagonists and compared them with affinity estimates obtained for bovine left atria. At low concentrations, ACh potently relaxed circular strips of coronary artery with endothelium (EC 50 0.15 μM), but contracted them at higher agonist concentrations with potencies that depended on the presence or absence of endothelium: EC 50 1.8 μM (without endothelium); 4.6 JμM (with endothelium). The pA 2 values obtained for antagonism of relaxant responses to ACh were: pirenzepine (M 1-selective) 7.38 ± 0.12; AF-DX 116 (11-[2-(diethylamino-methyl)-1-piperidinylacetyl]-5, 11-dihydro-6H-pyrido(2,3-b)l,4-benzodiazepine-6-one; M 2-selective) 5.79 ± 0.09; and 4-diphenylacetoxy-N-methylpiperidine-methobromide (4-DAMP; M 3/M 1-selective) 9.07 ± 0.12. The corresponding Schild slopes were 0.98 ± 0.07 for pirenzepine, 1.17 ± 0.09 for AF-DX 116 and 1.01 ± 0.04 for 4-DAMP. For the following three antagonists, pKg values were determined at two different antagonist concentrations: dicyclomine (M 1-selective) 7.49 ± 0.10, cyclohexylphenyl-(2-piperidinoethyl)-silanol (CPPS; M 3-selective) 8.0 ± 0.10, and parafluoro-hexahydrosila-difenidol (pFHHSiD; M 3-selective) 7.87 ± 0.10. For comparison, the antagonism of methacholine-induced negative inotropy in left atria was determined for three antagonists, yielding the following pA 2 values: pirenzepine 5.98 ± 0.14; AF-DX 116 6.81 ± 0.14 and 4-DAMP 7.99 ± 0.14. The slopes of the corresponding Schild plots were 1.05 ± 0.10,1.14 ± 0.12 and 1.08 ± 0.08, respectively. The results indicate that ACh relaxes precontracted circular strips of bovine coronary arteries with intact endothelium by activation of muscarinic receptors most closely corresponding to the M 3 subtype.

Dual Mechanism of the Relaxing Effect of Nicorandil by Stimulation of Cyclic GMP Formation and by Hyperpolarization

In addition to previous results from our laboratory showing that nicorandil relaxed vascular smooth muscle by increasing cyclic GMP levels, it was shown to activate K-channels as well, an effect that also leads to relaxation. In the present study, we attempted to differentiate quantitatively between these two effects in isolated bovine coronary artery strips with simultaneous isotonic measurement of length and radioimmunoassay (RIA) determination of cyclic GMP. When the strips were contracted by the thromboxane A2 analogue U 46619 (1 microM) with 10 microM methylene blue added, nicorandil produced 30-50% relaxation without significant changes in cyclic GMP. When in U 46619-contracted strips the hyperpolarizing effect of nicorandil was suppressed by increasing extracellular K+ to 80.4 mM (30-fold), nicorandil caused only 52% relaxation, whereas cyclic GMP increases were not significantly suppressed. Quantitative separation of both mechanisms of relaxation by nicorandil was further achieved through calculation of the cyclic GMP-mediated component from a correlation between increases in cyclic GMP and percentage of relaxation as produced by nicorandil under conditions of inhibited hyperpolarization, i.e., in strips contracted with 1 microM U 46619 or 26.8 mM K+ (10-fold) and exposed to either 30-fold K+ or 10 mM Ba2+. Under both conditions, similar correlations between cyclic GMP and relaxation were obtained. Because U 46619, in addition to its contractile effect, partially antagonized the relaxation by nicorandil without changing cyclic GMP, the correlation was corrected for this effect and indicated a participation of cyclic GMP in the overall relaxant response of approximately 30-40% at low and less than or equal to 80-90% at high concentrations of nicorandil.

Evidence for muscarinic receptors in endothelial cells from combined functional and binding studies

The aim of this study was to characterize muscarinic receptors of the bovine coronary artery by means of a combination of mechanical relaxation and contraction responses and radioligand binding data. Fresh helical strips of bovine coronary artery with intact endothelium relaxed in response to low concentrations (0.03−1 μM) of acetylcholine (ACh) and contracted at higher concentrations while endothelium-denuded strips only contracted. The ED 50 for relaxation was 0.13 μM and that for contraction 1.8 μM (without endothelium); in the presence of endothelium, contraction dose-response curves were shifted to the right and the maximum contraction was reduced. In order to determine the location of the receptors mediating vasorelaxation, apparent affinity constants (K A) of ACh for relaxant and contractile effects were determined by irreversible blockade of receptors with propyl benzilycholine mustard (PBCM). The affinity constants (K A) were 0.22 μM for relaxation and 13 μM (with endothelium) and 20 μM (without endothelium) for contraction. In competition binding experiments against the muscarinic antagonist, [ 3H]N-methylscopolamine ([ 3H]NMS), the apparent affinity (K 1) of ACh for binding sites in homogenates of endothelium-free coronary artery was 16 μM which was not different from the affinity constant determined in functional contraction experiments. Thus, the affinity constant of ACh determined for relaxation responses with endothelium-preserved vessels had no correlate in the binding affinity as determined with endothelium-free arteries. These findings indicate that bovine coronary arteries are relaxed by ACh through muscarinic receptors located on the endothelium whereas contractions are mediated by receptors on smooth muscle cells.

Differential vasorelaxant effects of KR-30075, a new cyclic AMP-phosphodiesterase inhibitor, on guinea-pig pulmonary, bovine coronary and renal arteries

The vasorelaxant effects of KR-30075 in guinea-pig pulmonary, bovine coronary and renal arterial strips contracted by either K+-depolarization, phenylephrine, or prostaglandin F2α (FGF2α) were evaluated. KR-30075 was more potent than imazodan as a vasorelaxant against PGF2α-induced contractions in bovine coronary and renal arteries, whereas against K+-induced contractions KR-30075 was less potent than imazodan in guineapig pulmonary arteries and more potent in bovine coronary arteries. KR-30075 was more potent against contractions induced by phenylephrine or PGF2α than the contractions induced by K+. This profile of activity for KR-30075 was similar to that of imazodan and dissimilar from the calcium entry blocking agent nifedipine. There was no vascular selectivity of KR-30075 between bovine coronary and renal arterial strip preparations. In conclusion, this study shows that KR-30075 represents the vasorelaxant effects on guinea-pig pulmonary, bovine coronary and renal arteries without specific vascular selectivity. The vasorelaxant profile of KR-30075, with different sources of vascular smooth muscle, is unlike that of calcium entry blocking agent and more similar to the profile of the agent that inhibit cyclic nucleotide phosphodiesterase.

Mechanism of Vasodilation by Nitrates: Role of Cyclic GMP

Nitrovasodilators relax vascular smooth muscle by stimulating soluble guanylate cyclase (GC). The resulting rise in cGMP probably initiates Ca extrusion from the smooth muscle cell which causes relaxation. Since repeated administration of organic nitrates, particularly nitroglycerin, leads to tolerance, i.e. a decrease in the vasodilator effect, it was studied whether (a) tolerance was a peripheral phenomenon occurring in the vascular smooth muscle, and (b) was due to an impairment of GC activation. In isolated circular strips of bovine coronary arteries, 90 min pretreatment with nitroglycerin greatly lowered the relaxing as well as the cGMP increasing response to nitroglycerin, indicating tolerance induction. Tolerance, although to a lesser extent, was also obtained with other organic nitrates under similar conditions, including IS 5-MN. Little (nitroprusside Na) to negligible tolerance was obtained with sodium nitrate and SIN-1, the active metabolite of molsidomine. The latter group of drugs stimulated soluble GC in vitro in the absence of cysteine whereas organic nitrates required the presence of this thiol. Preincubation with nitroglycerin almost completely inactivated GC whereas other organic nitrates had little effect. The results indicate that tolerance is caused by an impairment of GC function in the smooth muscle cell, particularly when elicited by nitroglycerin, and that differences in the degree of tolerance development by various nitrovasodilators are possibly due to different mechanisms of activation and inactivation of GC as well as differences in cysteine requirement.

Cyclic GMP in Nicorandil-Induced Vasodilatation and Tolerance Development

Nicorandil (SG-75) is a new organic nitrate with pronounced vasodilator properties. We studied whether nicorandil, in analogy to other nitrovasodilatators, exerted its relaxing effects on vascular smooth muscle by stimulating guanylate cyclase, and whether this effect was susceptible to tolerance development. Dose-response curves for the relaxing and cyclic guanosine monophosphate (cGMP) increasing effects of nicorandil were obtained in isolated strips of bovine coronary arteries and compared with those of other nitrovasodilatators. It was found that nicorandil dose-dependently relaxed the strips precontracted with 26.7 mM K+ and that this effect was closely associated with increases in cGMP levels (measured by RIA under various conditions). The correlation between relaxation and rises in cGMP was steeper than with other nitrovasodilatators, suggesting that nicorandil, in addition to its cGMP-mediated effect, also relaxed vascular smooth muscle by a cGMP independent mechanism. In contrast to nitroglycerin (NG), nicorandil caused little development of tolerance or cross-tolerance toward ISDN or IS-5-MN when tested after preincubation of the strips toward the respective substance. Pretreatment with N-acetylcysteine during the preincubation period prevented tolerance towards nicorandil. The results indicate that the relaxant effects of nicorandil consist of a larger cGMP-mediated component and a smaller one which is independent of this nucleotide.

Effects of sulfhydryl reagents on nitroglycerin-induced relaxation of bovine coronary artery.

The mechanism whereby nitroglycerin relaxes vascular smooth muscle remains uncertain. A current hypothesis suggests that nitroglycerin reacts with critical cellular sulfhydryl groups to form an intermediate, which activates guanylate cyclase, resulting in cGMP accumulation and relaxation. This study investigated further the potential involvement of sulfhydryls in nitroglycerin-induced vascular smooth muscle relaxation by evaluating effects of a variety of sulfhydryl alkylating and reducing agents on responses to nitroglycerin and other relaxants in bovine coronary arterial strips submaximally contracted using 30 mM K. Whereas 10(-4) M 5,5'-dithiobis-(2-nitrobenzoic acid), 10(-5) MN-ethylmaleimide, and 10(-4) MN-naphthylmaleimide did not affect nitroglycerin-induced relaxation, 10(-4) MN-ethylmaleimide and 10(-4) M ethacrynic acid significantly inhibited relaxation induced by nitroglycerin. Both ethacrynic acid and N-ethylmaleimide at 10(-4) M also inhibited relaxation induced by sodium nitroprusside. N-ethylmaleimide, but not ethacrynic acid, inhibited relaxation induced by isoproterenol and forskolin. Ethacrynic acid significantly reduced both relaxation and cGMP elevation induced by both 10(-7) M nitroglycerin and 10(-7) M sodium nitroprusside. Ethacrynic acid, but not N-ethylmaleimide, significantly reduced relaxation induced by 8-Br-cGMP. Pretreatment with the sulfhydryl-containing agents N-acetylcysteine, 2-mercaptoethanol, or dithiothreitol, at 10(-3) M did not affect nitroglycerin-induced relaxation in nontolerant arteries. Similarly, N-acetylcysteine and dithiothreitol did not alter the depressed responses to nitroglycerin in arteries in which tolerance to nitroglycerin was induced in vitro. A slight but statistically significant reversal of nitroglycerin-tolerance occurred after treatment of tolerant arteries with 2-mercaptoethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevation of cyclic AMP by prostacyclin is accompanied by relaxation of bovine coronary arteries and contraction of rabbit aortic rings

The role of cyclic AMP (cAMP) in the control of vascular smooth muscle tension was examined by comparing the effects of prostacyclin (PGI 2) on tension and cAMP levels in helical strips of bovine coronary arteries and in rabbit aortic rings, both denuded of endothelium. In bovine coronary arteries, PGI 2 elevated cAMP levels and relaxed the muscles. The PGI 2-induced cAMP elevation preceded the relaxation and both parameters were altered in a dose-dependent manner by increasing concentrations of PGI 2 (0.3, 3 and 30 μM). These results are consistent with a role for cAMP as a mediator of vascular smooth muscle relaxation. Cyclic AMP levels were also elevated by PGI 2 in a concentration- and time-dependent manner in rabbit aortic rings. However, in direct contrast to the results in the bovine coronary arteries, PGI 2-induced elevation of cAMP in the aortic rings was accompanied by contraction rather than relaxation. Isoproterenol, a drug which is generally believed to relax smooth muscles by virtue of its ability to increase tissue levels of cAMP, relaxed PGI 2-contracted aortic rings with no further elevation of cAMP beyond that caused by the PGI 2 alone. These results demonstrate that cAMP elevation and relaxation of vascular smooth muscle are not always well correlated. It is possible that some form of functional compartmentalization of cAMP or cAMP-dependent protein kinase exists in these tissues.

Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells.

A cascade superfusion technique has been developed for the differential bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) released from porcine aortic endothelial cells cultured on microcarriers, packed into a column and perfused. Bradykinin (Bk; 20-100 nM) released prostacyclin (9.6 +/- 1.5 nM per 10(6) cells; mean +/- s.e.mean, n = 9) and prostaglandin E2 (PGE2; 2.1 +/- 0.6 nM per 10(6) cells) from the column measured by relaxation of strips of bovine coronary artery (BCA) and rabbit mesenteric or coeliac artery, respectively. The presence of these prostanoids in the effluent was confirmed by specific radioimmunoassays. A23187 (500-2000 nM) also released both prostacyclin and PGE2 from the cells. This release was long-lasting and not reproducible. Bk (20-100 nM) and A23187 (30-300 nM) released EDRF from the column. This was detected in a cascade of four rabbit aortic strips (RbA), denuded of endothelium and contracted with U46619 or phenylephrine. The relaxation of the RbA strips caused by EDRF was progressively attenuated down the cascade (half-life less than 7s) and was not affected by indomethacin. EDRF and prostacyclin could be differentially bioassayed in a cascade of alternating RbAs and BCAs as prostacyclin did not relax RbAs and the time delay to the BCAs destroys EDRF. EDRF could be bioassayed on its own when the endothelial cells were treated with indomethacin. 5-Hydroxytryptamine 0.2, noradrenaline 1.0, platelet-activating factor (Paf-acether) 1.0, formylmethionyl-leucyl-phenylalanine 1.0, acetylcholine 0.5, bethanecol 0.5, adenosine diphosphate 0.25 and angiotensin II 0.1 microM did not release either prostanoids or EDRF from the column.

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.

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.

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.

Temporal relationships between isometric force, phosphorylase, and protein kinase activities in vascular smooth muscle.

Vascular smooth muscle contractility is tightly coupled to ATP production by intermediary metabolism. To elucidate mechanisms underlying coordination of metabolism and contractility we studied the time course of isometric force, and the activation of phosphorylase and cAMP-dependent protein kinases during stimulation of bovine coronary arterial strips with KCl. Isometric force reached a maximum after 10 min of exposure to 30 mM KCl (ED90) and was sustained throughout the subsequent 20-min period of contraction. In contrast, activation of phosphorylase was biphasic: enzymic activity reached a maximum (176 +/- 10% of control) after 3 min of contraction and then, though remaining above control, activity declined to a lower level (135 +/- 7% of control). However, no change occurred in the activity ratios for cAMP-dependent protein kinase assessed in either the presence (type II isozyme) or absence (type I isozyme) of 0.5 M NaCl. These data suggest that the activation of phosphorylase during K+-induced contraction is independent of the cAMP system. The biphasic activation of phosphorylase may reflect transient changes in the intracellular concentration of Ca2+ or the activation of a phosphatase(s) during the response.

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.

Interaction of ketanserin and its metabolite ketanserinol with 5HT2 receptors in pulmonary and coronary arteries of calf

A comparison of 5-hydroxytryptamine (5HT)-induced contractions was made on cocaine-treated strips of bovine pulmonary arteries and large coronary arteries. The affinities of the 5HT2 antagonist ketanserin, its metabolite ketanserinol, yohimbine and rauwolscine were estimated for both arteries. Ketanserin was a competitive antagonist of the effects of 5HT in both arteries. The KB values (-log mol/l) were 9.5 for large coronary arteries and 9.4 for pulmonary arteries. Ketanserinol was also a competitive antagonist of the effects of 5HT in both arteries. The KB values (-log mol/l) were 6.5 for large coronary arteries and 6.4 for pulmonary arteries. A combination of ketanserin with ketanserinol antagonized the 5HT effects as expected from competition of the 3 drugs for a single class of receptor in both arteries. Yohimbine and rauwolscine were competitive antagonists of the effects of 5HT in pulmonary artery. The KB-values (-log mol/l) were 6.8 for yohimbine and 6.5 for rauwolscine. A combination of ketanserin with either yohimbine or rauwolscine antagonized the 5HT effects as expected from competition of all 4 drugs for a single class of receptors in pulmonary arteries. The evidence is consistent with the assumption that smooth muscle 5HT receptors of both pulmonary artery and large coronary artery are of the 5HT2 class. The reduction of the ketone of ketanserin to alcohol (i.e. ketanserinol) causes an approximately 1000-fold decrease in affinity for arterial 5HT2 receptors. Reported peak plasma levels of ketanserinol are too low (less than or equal to 10(-7) mol/l) in humans to account for a contribution of the metabolite to the blockade of 5HT effects by ketanserin.

Comparison of the effects of forskolin and isoproterenol on cyclic AMP levels and tension in bovine coronary artery.

The effects of isoproterenol and forskolin on cyclic AMP levels and tension were compared in helical strips of bovine coronary artery. Both compounds produced time- and dose-dependent increases in cyclic AMP levels and both compounds relaxed potassium-contracted arteries. However, in contrast to the results with isoproterenol, cyclic AMP elevation and relaxation were not well correlated at lower concentrations of forskolin. For example, 0.1 microM forskolin increased cyclic AMP levels in the arteries by approximately 5.5-fold but did not relax the muscles. A smaller elevation of cyclic AMP produced by 1.0 microM isoproterenol, on the other hand, was accompanied by an almost complete relaxation of the arteries. These results suggest that either cyclic AMP is not responsible for the relaxation caused by these agents, or some form of functional compartmentalization of cyclic AMP exists in this tissue.