W. R. Kukovetz

Evidence for cyclic GMP-mediated relaxant effects of nitro-compounds in coronary smooth muscle

SummaryThe effects of the four nitro-compounds nitroglycerin, nitroprusside-Na, NaNO2 and B 744-99 were studied simultaneously on length and on cGMP-levels in isolated circular strips of bovine coronary arteries. 1.All 4 nitro-compounds concentration dependently relaxed the strips in close association with pronounced increases in cGMP-levels which preceded the mechanical responses.2.The relaxant effects of all 4 nitro-compounds were significantly potentiated by the predominant inhibitor of cGMP-hydrolysis M & B 22,948, which also potentiated the increase in cGMP-levels of the two nitro-compounds in which it was studied (nitroglycerin and nitroprusside-Na).3.Non-substituted cGMP and — much stronger —its 8 bromo-derivative also relaxed the strips and these effects were likewise potentiated by M & B 22,948.4.When the log increase in cGMP produced by the 4 nitro-compounds were plotted against percent relaxation (probit scale) a linear and highly significant positive correlation was obtained.5.The results provide evidence that the increases in cGMP caused by the 4 nitro-compounds studied are responsible for the smooth muscle relaxing actions of these drugs.

Cardiostimulatory effects of cyclic 3′,5′-adenosine monophosphate and its acylated derivatives

SummaryThe effects of cyclic 3′,5′-AMP and of two acylated derivatives, dibutyryl (DBA) and dihexanoyl-3′,5′-AMP (DHA) were investigated in isolated perfused hearts of guinea pigs, rats and rabbits.In guinea pig hearts, DBA (Ca- and Na-salt) and DHA-Na in high doses (10 μmoles) produced strong and long lasting increases in the rate and amplitude of contractions, coronary flow, and moderate increases in phosphorylase activity in the majority of experiments. The positive ino- and chronotropic effects occured 3–5 min after injection of the drug, mostly in a fluctuating manner with several maxima. Theophylline augmented the effects of DBA-Na and revealed positive inotropic actions of non substituted 3′,5′-AMP.In rat hearts, similar, but more pronounced and dose-dependent effects were observed after 1, 5 and 10 μmoles DBA-Na. Propranolol (50 μg) did not block the action of 10 μmoles DBA-Na. Non substituted 3′,5′-AMP, 5′-AMP and ATP in doses of 10 μmoles had no significant positive inotropic effects.In rabbit hearts, DBA-Na (50 μmoles) produced moderate, non fluctuating rises in the amplitude of contraction.The results provide evidence that under certain conditions cyclic 3′, 5′-AMP itself, like its acylated derivatives DBA and DHA, may produce strong and direct positive inotropic and chronotropic effects in the heart. These findings support the view that cyclic 3′,5′-AMP is the cellular mediator of the cardiostimulant actions of substances that increase its rate of production in the myocardial cell.

Effect of catecholamines, histamine and oxyfedrine on isotonic contraction and cyclic AMP in the guinea-pig heart

SummaryExperiments in isolated, perfused guinea-pig hearts (Langendorff) have shown that1.Inotropic effects of catecholamines and histamine are potentiated by the PDE-inhibitor theophylline, but antagonized by the PDE-activators imidazole or N-methyl-imidazole.2.Maximum effective doses of isoprenaline (0.5 μg) or histamine (10 μg) increase cardiac cAMP 4–5-fold at 15 sec after injection whereas maximum effective doses of oxyfedrine (10 μg) produce a 2-fold increase in cAMP at 15 sec, along with a considerably weaker inotropic response than isoprenaline or histamine.3.Changes in cAMP precede the increase in isotonic contractions brought about by isoprenaline, histamine or oxyfedrine in maximum effective doses, and also clearly precede the decrease in the inotropic effect of isoprenaline or histamine.4.No dissociation between the increase in cAMP and the positive inotropic effect was observed after very small doses of isoprenaline or histamine which only increased the amplitude of contraction by about 12–14%. A significant correlation (r=0.94, p<0.01) between increases in isotonic contractions and increases in cAMP was found over the whole tested dose range of both compounds.5.Increases in cAMP as well as in contractions induced by 2 μg of histamine—in contrast to an equieffective dose of 0.1 μg of isoprenaline—were not blocked by the β-blocking compound Kö 592 (50 μg), demonstrating again a close relationship between cAMP and inotropic effect of histamine or isoprenaline.6.The data support the mediator role of cAMP for the inotropic effect of substances which activate the adenyl cyclase system in the heart.

Inhibition of a store‐operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908

1 The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2 LOE 908 inhibited store‐operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 μm and 4 μm, respectively. 3 LOE 908 did not affect thapsigargin‐ or ionomycin‐induced Ca2+ release from intracellular stores up to concentrations of 3 μm. 4 LOE 908 reversibly suppressed thapsigargin‐ as well as ionomycin‐induced whole‐cell membrane currents. 5 The LOE 908‐sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively. 6 Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store‐operated (capacitive) Ca2+ entry pathway in endothelial cells.

Specific inhibition by burimamide of histamine effects on myocardial contraction and cyclic AMP

Summary1.In the isolated perfused guinea-pig heart, the increases in isotonic contraction and in cyclic AMP, produced by 2 μg of histamine were significantly and almost completely inhibited in the presence of 2 mg of burimamide, whereas the adrenergic β-receptor blocking drug Kö 592 (50 μg) was ineffective.2.Under the same conditions similar effects of 0.1 μg of isoprenaline, which was equieffective with 2 μg of histamine, were not changed by 2 mg of burimamide, but prevented by 50 μg of Kö 592.3.The results support the hypothesis, that histamine exerts its cardiac effects by stimulation of a receptor (H2) that can be clearly distinguished from the adrenergic (β1) receptor. The close parallelism between changes in contractile amplitude and in cyclic AMP produced by histamine in the absence and in the presence of burimamide is in accordance with the concept that cyclic AMP mediates the mechanical response of the heart to histamide.

Function of cyclic GMP in acetylcholine-induced contraction of coronary smooth muscle

SummaryIncreasing evidence indicates that cyclic GMP is involved in smooth muscle relaxation by various nitrocompounds which stimulate guanylate cyclase. Since, however, rises in cGMP were also observed in association with contractile effects, the role of cGMP in acetylcholine-induced contraction was studied in isolated bovine coronary artery strips.Concentration response experiments were performed with acetylcholine (ACh) in the absence and in the presence of a) the cGMP-phosphodiesterase inhibitor M & B 22,948, and of b) methylene blue which was found to inhibit NO-, and azide-induced stimulation of guanylate cyclase (Katsuki et al. 1977b), and changes in cGMP-levels (RIA) and in smooth muscle tone were monitored.1.ACh (55 nM to 55 μM) concentration dependently raised cGMP up to the 4.4-fold control value concomitantly with, but slightly prior to its contractile effects.2.In the presence of 370 μM M & B 22,948, cGMP-levels as well as their ACh-induced increases were 2–3 times higher than in its absence, whereas the contractile responses to ACh were diminished at normal (2.68 mM) K+ (DR=8.7) and —to a lesser extent — also at high (26.8 mM) K+-concentration (DR=2.2).3.Methylene blue (50 μM) at normal K+ (2.68 mM) attenuated the ACh-induced rises in cGMP-levels (DR=4.4; lower maximum response) but potentiated the contractile effects (DR=4.0; higher maximum response). At high (26.8 mM) K+ the changes in dose ratios were less pronounced but the lower maximum rise in cGMP and the higher maximum contractile response were even more pronounced than at normal K+.4.These results demonstrate that the rises in cyclic GMP-levels under the different conditions studied are inversely correlated with the magnitude of the contractile responses, suggesting that cGMP is likely to function as a physiologic negative feedback signal to limit and/or to reverse the contractile effects of ACh in smooth muscle.

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 (EC50 0.15 microM), but contracted them at higher agonist concentrations with potencies that depended on the presence or absence of endothelium: EC50 1.8 microM (without endothelium); 4.6 microM (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-piperidinyl-acetyl]-5,11- dihydro-6H-pyrido(2,3-b)1,4-benzodiazepine-6-one; M2-selective) 5.79 +/- 0.09; and 4-diphenylacetoxy-N-methyl-piperidine-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, pKB 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-adrenerge Effekte und ihr zeitlicher Verlauf unter Tetrahydropapaverolin und Isoprenalin am Langendorff-Herzen

SummaryThe β-adrenergic effects of tetrahydropapaveroline (THP) and their timecourse were studied in comparison with the effects of isoprenaline in the isolated perfused guinea pig heart. In extension of the results of Holtz et al. (1964) it was found that 50 nM THP not only raised the isotonic contractions, rate and coronary flow but also the percentage of active phosphorylase (%-a) of the heart in a similar manner as, and equieffective with 0.4 nM isoprenaline. 200 nM pronethalol prevented all effects of THP as well as isoprenaline. No clear dissociation could be observed in the time course of the positive inotropic action and the raise in %-a after THP-administration. %-a reached its climax at the same time or a few seconds before the contraction. A similar correlation between these parameters was found after isoprenaline, when given in small doses of 0.02 nM, except that the climax for both events was reached faster than after THP and that %-a in 4 of 5 experiments returned earlier toward control level than contractility. A permissive role of phosphorylase activation for the positive inotropic action of catecholamines is discussed.ZusammenfassungDie β-adrenergen Wirkungen von Tetrahydropapaverolin (THP) und ihr zeitlicher Verlauf wurden vergleichend mit Isoprenalin an isoliert durchströmten Meerschweinchenherzen untersucht. Als Erweiterung zu den Ergebnissen von Holtz u. Mitarb. (1964) wurde gefunden, daß 50 nM THP nicht nur die isotonischen Kontraktionen, die Frequenz und den Coronardurchfluß, sondern auch den Prozentgehalt an aktiver Phosphorylase steigern. Diese Wirkungen sind mit den Effekten einer Dosis von 0,4 nM Isoprenalin vergleichbar und wie diese durch 200nM Pronethalol aufzuheben. Zwischen dem zeitlichen Verlauf der positiv inotropen und der %-a-steigernden Wirkung von THP konnte keine eindeutige Dissoziation beobachtet werden. %-a erreichte gleichzeitig oder einige Sekunden vor der Kontraktion das Maximum. Eine ähnliche Korrelation zwischen diesen Parametern wurde nach kleinen Dosen von 0,02 nM Isoprenalin gefunden, wobei das Maximum beider Effekte unter Isoprenalin rascher als unter THP erreicht wurde und %-a in vier von fünf Versuchen rascher wieder abfiel als die Kontraktion. Eine permissive Rolle der Phosphorylase-Aktivierung für die positiv inotrope Katecholaminwirkung wird diskutiert.

Die Hemmung mechanischer und metabolischer Katecholaminwirkungen durch Isopropylmethoxamin und K 592 am Herzen

SummaryIn isolated perfused guinea pig hearts the blocking effects of isopropylmethoxamine (d,l- and l-IMA) on the mechanic as well as the metabolic stimulation, caused by about equieffective doses of 1 nM noradrenaline (N), 0.4 nM isoprenaline (I) and 50 nM tetrahydropapaveroline (THP) were studied and compared with the effects of the β-adrenergic blocking agent 1-(3-methylphenoxy)-2-hydroxy-3-isopropylaminopropan (Kö 592). In doses of 100 nM, which already depressed contractility, d,l-IMA did not reduce the positive inotropic and the phosphorylase (% a) activating actions of I. Higher doses of 500 nM d,l-IMA, which also caused more cardiac depression, significantly but transiently reduced the stimulant effects of N on contractility and % a. The l-isomer of IMA, which depressed contractility and rate to the same extent as the racemate, exerted a higher degree of adrenergic blockade. It completely inhibited the positive ino- and chronotropic effects as well as the increase in % a, caused by N. Similar actions of I, and, to a lesser extent, of THP were reduced significantly. In no case IMA inhibited the increase in % a, as caused by N, I and THP to a higher-, but rather to a lower degree than their mechanic actions. When compared with Kö 592, which in doses of 50 nM completely abolished all the actions of N, I and THP, IMA may be classified as a weak and short acting β-blocker, with its activity bound to the l-isomer. The present results do not support the assumption that different β-receptors for mechanic and metabolic adrenergic effects exist in the heart.ZusammenfassungAn Meerschweinchen-Langendorffherzen wurde die Hemmwirkung von Isopropylmethoxamin (d,l- und l-IMA) auf die mechanische und die metabolische Erregung durch gleich- und annähernd maximal wirksame Dosen von 1 nM Noradrenalin (N), 0,4 nM Isoprenalin (I) und 50 nM Tetrahydropapaverolin (THP) untersucht und mit den Wirkungen des adrenergen β-Blociers 1-(3-Methylphenoxy)-2-hydroxy-3-isopropylaminopropan (Kö 592) verglichen. d,l-IMA zeigte bereits in Dosen von 100 nM, welche die Wirkungen von I noch nicht deutlich beeinflußten, einen negativ inotropen Eigeneffekt. Nach 500 nM d,l-IMA, die auch stärker negativ inotrop wirkten, war die Kontraktions- und auch die % a-Steigerung durch N für einige Minuten signifikant vermindert. l-IMA hatte in Dosen von 500 nM die gleiche negativ ino- und chronotrope Eigenwirkung wie das Racemat, hemmte jedoch die adrenergen Effekte deutlicher. Nach l-IMA war sowohl die Kontraktions-und Frequenzzunahme, als auch die % a-Steigerung durch N vollständig blockiert. Auch die entsprechenden Wirkungen von I und in geringerem Ausmaß jene von THP waren signifikant reduziert. IMA hemmte die % a-Steigerung durch N, I und THP niemals ausschließlich bzw. stärker, sondern eher etwas schwächer als ihre mechanischen Wirkungen. Im Vergleich zu Kö 592, das in Dosen von 50 nM sämtliche Effekte von N, I und THP vollständig blockierte, ist IMA seiner Herzwirkung nach als schwacher adrenerger β-Blocker einzustufen, dessen kurze Wirksamkeit an das l-Isomer gebunden ist. Die vorliegenden Ergebnisse bieten keinen Anhaltspunkt für die Existenz verschiedener β-Receptoren für mechanische und metabolische Effekte im Herzen.

Die Wirkung von g-Strophanthin auf den Papillarmuskel der Katze bei Hypothermie, Normaltemperatur und Hyperthermie

Summary1.Isometric systolic tension (I.S.T.) was measured in the papillary muscle of the cat-heart at temperatures of 37° C, 27° C and 41° C, before and after administration of g-Strophanthine.2.In the majority of experiments an increase in temperature was associated with a decrease of I.S.T.3.g-Strophanthine in concentrations of 0.05–0.2 μ/ml increased the I.S.T. of the failing papillary muscle at 37° C, but did not change the I.S.T. at 27° C and 41° C.4.It is assumed that induction of hypothermia as such compensates for the failure of the papillary muscle which is necessary for the action of cardiac glycosides.