Gerald Pöch

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.

Assay of phosphodiesterase with radioactively labeled cyclic 3',5'-AMP as substrate.

SummaryA highly sensitive method for the determination of cyclic 3′,5′-nucleotide phosphodiesterase (PDE) is described. 1.It is based on the use of radioactively labeled cyclic 3′,5′-AMP as substrate and the quantitative removal of the labeled product (3H-,14C-,32P-5′-AMP or32Pi).2.3H- or14C-labeled 3′,5′-AMP can be used without further additions if the labeled product remains unchanged, e.g. withpurified enzyme preparations.3.Whencrude PDE-preparations, which contain varying amounts of 5′-nucleotidase or phosphatase, are used either the labeled product of the PDE-reaction may be protected from further breakdown by the addition of high concentrations of unlabeled 5′-AMP, or the reaction may be carried out with32P-labeled substrate. The latter procedure is absolutely specific for measurements of PDE activity, since32Pi, formed by a subsequent step, is also quantitatively removed by precipitation.The effect ofaddition of 5′- AMP to the reaction mixture and the interference of 5′-nucleotidase activity was determined in various tissues.4.Examples are given for certain characteristics of crude tissue PDE, for inhibition by drugsin vitro, and for the influence of pH and Mg2+ on the inhibition of PDE. Ki-values and the type of inhibition by drugs are described for crude as well as for purified PDE, and the differences are discussed.5.Estimations of PDE in homogenized samples obtained fromisolated organs (heart, coronary arteries) are described.6.The use, advantages and limitations of the method are discussed as well as the possible significance of effects of drugs on this key enzyme for their pharmacodynamic effects.

Quantitative estimation of overadditive and underadditive drug effects by means of theoretical, additive dose-response curves

A graphical method is described that permits simple and adequate quantitative and statistical evaluation of combinations of synergistically acting drugs. Thereby, the dose-response (DR) of a drug A can be determined in the presence as well as in the absence of a certain concentration of a drug B. The effects of A + B combined are expressed as combined (total) effects related to the control value before addition of B. The DR curve obtained in this manner is compared with a theoretical DR curve of additive synergism, which can be constructed simply. This procedure allows differentiation of overadditive or underadditive from additive interactions of A + B and the quantitation of nonadditive drug effects. DR curves of additive interaction coincide with the respective theoretical additive DR curve. Overadditive DR curves are characterized by a left shift from the theoretical additive DR curve that can be expressed by a dose factor. Increased maximum effects of A + B over A can be treated statistically (t-test). Underadditive DR curves show a shift to the right from the theoretical additive DR curve that can likewise be expressed by a dose factor. Decreased effects of A + B below the maximum of A can likewise be treated by t-test. The quantitative expression of overadditive drug interactions further allows differentiation between sequential and functional synergism, the DR curve of the latter can also be constructed simply. The application of this method to experiments with isolated coronary arteries had yielded examples of additive, overadditive, and underadditive drug interactions. The method appears to be a superior alternative to the isoboles method of Loewe and Muischnek (1926).

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.

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 micorandil relayed vascular smooth muscle by increasing evelic GMP levels it was shown to activate K-channel, as well. an effect that also leads to relavation. In the present study. we attempted to differentiate quantitatively between these two elieels in isolated bovine coronary artery strips with simultaneous isotonic measurement of length and radioimmunoassay (RIA) determination of cyclie GMP. When the strips were contracted by the thromhovane A analogue U 46619 (1 μM) with 10 μM methylene blue added. nieorandil produced 30–50 relavation 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), nieorandil caused only 52 relaxation. whereas cyclic GMP increases were not significantly suppressed. Quantitative separation of both mechantsms f relaxation H nicorandil was further achicved through calculation of the cyclic GMP mediated component from a correlation between increases in cyclie GMP and percentage of relayation as, produced by mcorandil under condition, of inhibited hyperpolarization. i.e., in strips contracted with 1μM U 46619 or 26.8 mM K (10 fold) and exposed to either 30-fold K or 10 mM Ba Under both condition,. similar correlations between cylic GMP and relayation were ohtained. Because U 46619, in Addition to its contractile effect. partially antugonized the relaxation by nicorandil without changing cyclic GMP. the correlation was corrected for this effect and indicated a participation of cyclie GMP In the overall reluvunl response of 30–40 at low and 80–90 at high concentrations of nicorandil.

Molecular mechanism of action of nicorandil

Nicorandil relaxes coronary vascular smooth muscle by stimulating guanylyl cyclase and increasing cyclic GMP (cGMP) levels (as shown first in our laboratory) as well as by a second mechanism resulting in activation of K+ channels and hyperpolarization. Therefore, we studied the relative contributions of either mechanism to the overall response in bovine circular strips of coronary arteries by simultaneously measuring changes in length and in cGMP levels through radioimmunoassay. Blockade by 10 μM methylene blue of the cGMP increases in strips precontracted by 1 μM of the thromboxane A2 analogue U46619 reduced nicorandil-induced relaxation to 30–50%, and there were no significant changes in cGMP levels. Suppression of the hyperpolar-izing component of nicorandil by 80.4 mM K+ or 1 μM glibenclamide in precontracted strips reduced nicorandil relaxation to 50% (K+) or shifted the dose response to the right by a factor of two (glibenclamide) without alteration of increases in cGMP. A quantitative separation of both mechanisms of action was obtained by comparing the correlation between increases in cGMP and relaxation under conditions of inhibited versus noninhibited hyperpolarization. The results indicate that cGMP contributes to the total relaxing effect of nicorandil by 30–40% at low concentrations and 80–90% at high concentrations of nicorandil. From the experiments with glibenclamide, it can be concluded that the probable mechanism by which nicorandil hyperpolarizes is opening glibenclamide-sensi-tive K+ channels in coronary vascular smooth muscle and that this latter effect mimics those of other K+ channel openers such as cromakalim or pinacidil.

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.

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.

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.