Sigrid Holzmann

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.

Anandamide‐induced mobilization of cytosolic Ca2+ in endothelial cells

Experiments were designed to determine whether anandamide affects cytosolic Ca2+ concentrations in endothelial cells and, if so, whether CB1 cannabinoid receptors are involved. To this effect, human umbilical vein‐derived EA.hy926 endothelial cells were loaded with fura‐2 to monitor changes in cytosolic Ca2+ using conventional fluorescence spectrometry methods. Anandamide induced an increase in Ca2+ in endothelial cells which, in contrast to histamine, developed slowly and was transient. Anandamide caused a concentration‐dependent release of Ca2+ from intracellular stores without triggering capacitative Ca2+ entry, contrary to histamine or the endoplasmic reticulum Ca2+‐ATPase inhibitor thapsigargin. Anandamide pretreatment slightly reduced the mobilization of Ca2+ from intracellular stores that was evoked by histamine. The mobilization of Ca2+ from intracellular stores evoked by anandamide was impaired by 10 mM caffeine. Anandamide and histamine each significantly increased NO synthase activity in EA.hy926 cells, as determined by the enhanced conversion of L‐[3H]‐arginine to L‐[3H]‐citruline. The CB1 cannabinoid receptor antagonist SR141716A (1 μM) only produced a marginal reduction of the mobilization of Ca2+ produced by 5 μM anandamide. However, at 5 μM SR141716A elicited the release of Ca2+ from intracellular stores. This concentration strongly impaired the mobilization of cytosolic Ca2+ evoked by either anandamide, histamine or thapsigargin. Pretreatment of the cells with either 200 μM phenylmethylsulphonyl fluoride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml−1 pertussis toxin (to uncouple CB1 cannabinoid receptors from Gi/o proteins) had no significant effect on the mobilization of cytosolic Ca2+ evoked by either anandamide, or histamine. Taken together the results demonstrate that anandamide mobilizes Ca2+ from a caffeine‐sensitive intracellular Ca2+ store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classical CB1 cannabinoid receptor‐mediated and pertussis toxin‐sensitive mechanism does not mediate this novel effect of anandamide in endothelial cells. The mobilization of cytosolic Ca2+ in endothelial cells may account for the endothelium‐dependent and NO‐mediated vasodilator actions of anandamide. Due to its non‐specific inhibition of Ca2+ signalling in endothelial cells, SR141716A may not be used to assess the physiological involvement of endogenous cannabinoids to endothelium‐dependent control of vascular smooth muscle tone.

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).

Pharmacologic differentiation between endothelium-dependent relaxations sensitive and resistant to nitro-L-arginine in coronary arteries.

Summary: We investigated whether formation of endothelium-derived relaxing factor (EDRF) and endothelium-derived hyperpolarizing factor (EDHF) in porcine and bovine endothelial cells (PAECs) was stimulated by different kinin receptors and studied pharmacologic differences and similarities between the two types of bradykinin-induced relaxation of bovine or porcine coronary arteries. Cultured PAECs were used for [3H]bradykinin binding assay and for measurement of the endothelial free [Ca2+]i by the fura-2/AM method. In organ bath studies with strips of bovine and porcine coronary arteries (endothelium intact), changes in length were recorded and cyclic GMP was measured by radioimmunoassay (RIA). Two bradykinin binding sites were detected, suggesting the presence of two subtypes of B2 kinin receptors. Bradykinin increased [Ca2+]i, and this action was antagonized by the B2 kinin receptor antagonist Hoe 140 and the K channel inhibitor tetrabutylammonium (TBA). Hoe 140 competitively antagonized the relaxing effects of bradykinin, whereas a B1 antagonist was inactive. L-ωN-nitro-arginine (L-NNA) diminished one part of bradykinin-induced relaxation and abolished the increases in cyclic GMP; TBA inhibited another part of the relaxing effect and attenuated (but not significantly) increases in cyclic GMP, and Hoe 140 completely inhibited relaxation and increases in cyclic GMP. The results indicate that the bradykinin response is mediated by biosynthesis of EDRF, which is sensitive to L-NNA, and of EDHF, which is sensitive to TBA.

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.

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.

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.

Mechanisms of L‐NG nitroarginine/indomethacin‐resistant relaxation in bovine and porcine coronary arteries

1 Coronary arteries from bovines (BCA) and pigs (PCA) were used for measuring endothelium‐dependent relaxation in the presence of L‐NG nitroarginine and indomethacin. As some compounds tested have been found to have an inhibitory effect on autacoid‐activated endothelial Ca2+ signalling, endothelium‐dependent relaxation was initiated with the Ca2+ ionophore A23187. 2 The common compounds for modulating arachidonic acid release/pathway, mepacrine and econazole only inhibited L‐NG nitroarginine‐resistant relaxation in BCA not in PCA. In contrast, proadifen (SKF 525A) diminished relaxation in BCA and PCA. Mepacrine and proadifen inhibited Hoe‐234‐initiated relaxation in BCA and PCA, while econazole only inhibited Hoe 234‐induced relaxation in PCA. Due to the multiple effects of these compounds, caution is necessary in the interpretation of results obtained with these compounds. 3 The inhibitor of Ca2+‐activated K+ channels, apamin, strongly attenuated A23187‐induced L‐NG nitroarginine‐resistant relaxation in BCA while apamin did not affect L‐NG nitroarginine‐resistant relaxation in PCA. 4 Pertussis toxin blunted L‐NG nitroarginine‐resistant relaxation in BCA, while relaxation of PCA was not affected by pertussis toxin. 5 Thiopentone sodium inhibited endothelial cytochrome P450 epoxygenase (EPO) in PCA but not in BCA, while L‐NG nitroarginine‐resistant relaxation of BCA and PCA were unchanged. Protoporphyrine IX inhibited EPO in BCA and PCA and abolished L‐NG nitroarginine‐resistant relaxation of BCA not PCA. 6 An EPO‐derived compound, 11,12‐epoxy‐eicosatrienoic acid (11,12‐EET) yielded significant relaxation in BCA and PCA in three out of six experiments. 7 These findings suggest that L‐NG nitroarginine‐resistant relaxation in BCA and PCA constitutes two distinct pathways. In BCA, activation of Ca2+‐activated K+ channels via a pertussis‐toxin‐sensitive G protein and EPO‐derived compounds might be involved. In PCA, no selective inhibition of L‐NG nitroarginine‐resistant relaxation was found.

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.