Uwe Fricke

Nitric oxide (EDRF) enhances the vasorelaxing effect of nitrendipine in various isolated arteries

SummaryRecent studies suggest endothelium to be involved in the vasorelaxation of calcium antagonists of the 1,4-dihydropyridine type, which may at least in part be mediated by endothelium-derived relaxing factor (EDRF=NO). To study this effect further, the influence of L-NG-nitro arginine (L-NNA), a specific inhibitor of EDRF-synthesis, on nitrendipine-induced vasorelaxation was examined in different isolated porcine arteries. Coronary, basilary, and tail arteries were bathed in Krebs-Henseleit solution and endothelial function was verified by means of substance P, and EDRF releasing neuropeptide. Vasorelaxation of nitrendipine in PGF2α-precontracted arteries was studied in the presence and absence of L-NNA. Nitrendipine-induced vasorelaxation was markedly reduced by the addition of L-NNA in all vessels studied. Tachyphylactic effects of nitrendipine could be excluded. The obtained results may be explained by an enhancement of nitrendipine action by basally released EDRF, alternatively, by an increased EDRF-release induced by this calcium antagonist. Therefore, in a second series of experiments the release of EDRF was studied in isolated coronary arteries under cumulative application of nitrendipine. Using the nitric oxide scavenging properties of oxyhemoglobin, EDRF release was measured spectrophotometrically by means of methemoglobin formation. The application of nitrendipine resulted in a concentration-dependent increase in the extinction rate, indicating an increased release of NO which could be inhibited by preincubation with L-NNA. It may be concluded that, in functionally intact vessels, vasorelaxation induced by nitrendipine may additionally be mediated by an increased release of EDRF.

Evidence for two different Na+-dependent [3H]-ouabain binding sites of a Na+-K+-ATPase of guinea-pig hearts.

1 The influence of various Na+ concentrations on [3H]‐ouabain binding was studied in experiments on a microsomal Na+‐K+‐adenosine triphosphatase (ATPase) from guinea‐pig hearts. 2 The ATP‐independent cardiac glycoside binding was not influenced by increasing Na+ concentrations. However, a good correlation was found between the ATP‐dependent [3H]‐ouabain binding and Na+ concentration. 3 A more detailed analysis of these results according to Hofstee (1952) revealed two distinct processes involved in this interaction: one ouabain binding process was activated at rather low Na+ concentrations, (K0.5 = 4.5 mM); this type of [3H]‐ouabain binding was strongly correlated to the Na+ concentration necessary for half maximum phosphorylation (K0.5 = 1 mM). The other ouabain binding process was predominant at high Na+ concentrations (K0.5 = 69 mM). 4 On the basis of the commonly accepted ATPase reaction cycle a model for the interaction of cardiac glycosides with the Na+‐K+‐ATPase is proposed, assuming two different binding sites for cardiac glycosides (E2‐P and E1‐P) and involving a translocation of these drugs from an outer to an inner compartment of the cell membrane.

Influence of derivation on the lipophilicity and inhibitory actions of cardiac glycosides on myocardial Na+-K+-ATPase.

1 Lipophilicity and inhibitory actions on guinea‐pig heart Na+‐K+‐ATPase of twenty‐six digitalis and six strophanthus glycosides comprising the aglycones, mono‐, bis‐, tris‐sugar, alkylated (acylated) tris‐sugar, acyl steroid derivatives and three cardanolides were investigated. 2 Their octanol/water partition coefficients (P), reversed phase thin layer (r.t.l.c.) and reversed phase high performance liquid chromatography (r.h.p.l.c.) were determined and the viability of these methods as a measure of the lipophilicity of the cardiotonic steroids evaluated. 3 The influence of lipophilicity and so also structural changes on the inhibitory effects of the cardiac glycosides on myocardial Na+‐K+‐ATPase was then examined. 4 It is concluded that (a) r.t.l.c. and r.h.p.l.c. are just as effective as the conventional shake‐flask method for estimation of the lipophilicity of cardiac glycosides and (b) the inhibitory potencies of cardiotonic steroids on the myocardial Na+‐K+‐ATPase increase with growing lipophilicity. The relationship between these two parameters is, however, governed by the influence of substitution or derivation of structural components on their inhibitory potencies on the myocardial Na+‐K+‐ATPase.

The influence of 3-ester side chain variation on the cardiovascular profile of nitrendipine in porcine isolated trabeculae and coronary arteries

SummaryIt has been reported that the lipophilicity of dihydropyridine-type calcium entry blockers may influence both their negative inotropic and their vasodilator activities. The action of nitrendipine and six related 3-ester side-chain derivatives with increasing alkyl and aryl substituents have been investigated in isolated porcine trabecular muscles and coronary artery rings. The lipophilicity of the drugs was determined by high-pressure liquid chromatography. In addition, some sterical parameters of the ester derivatives were considered. For the drugs tested, an increase in 3-ester side chain volume correlated well with increasing lipophilicity. Compared to nitrendipine, vascular selectivity of the ester side-chain derivatives, as expressed by the ratio of their negative inotropic and vasodilator activities, was much reduced. Neither vasodilator nor negative inotropic activity was directly related to the corresponding lipophilicity. Based on these results, earlier suggestions about the influence of the ester side-chain in dihydropyridines on their cardiovascular profile are extended.

Reduced responses of nitrendipine in PGF2α-precontracted porcine isolated arteries after pretreatment with methylene blue

SummaryVascular activity of nitrendipine (NTD) in different depolarized and prostaglandin F2α (PGF2α)-precontracted isolated porcine arteries was examined in the presence and absence of methylene blue (MB). Presence of MB potentiated the PGF2α-induced contractions in all vessels studied and reduced the response of NTD in coronary and basilar arteries seven- to 23-fold. Only in ulnar arteries was the affinity of NTD slightly increased. In contrast, MB did not modify the affinity of papaverine under these conditions. Furthermore, MB had no influence on KCl-induced contractions and subsequent vasorelaxation by NTD. It is proposed that MB impaired endothelium derived relaxing factor, thereby stimulating and/or increasing the calcium influx through receptor-operated calcium channels.

Sodium-dependent cardiac glycoside binding: experimental evidence and hypothesis.

1 The influence of increasing Na+ concentrations on the binding of digitoxin, digoxin and ouabain was examined in a Na+‐K+‐ATPase preparation of guinea‐pig hearts 2 Two distinct processes seem to be involved in this interaction: one binding process was activated at low Na+ concentrations. The maximum binding capacities were different and the K0.5 values were nearly identical for the cardiac glycosides studied. 3 In contrast, the second binding process was activated at appreciably higher Na+ concentrations, the maximum binding capacities were almost identical and the K0.5 values were different for the cardiac glycosides studied. 4 On the basis of these results attempts are made to explain the well known differences in the myocardial accumulation of cardiac glycosides.

Erythrosin B inhibits high affinity ouabain binding in guinea‐pig heart Na+‐K+‐ATPase without influence on cardiac glyoside induced contractility

1 Binding of [3H]‐ouabain to guinea‐pig heart membranes enriched in Na+‐K+‐ATPase revealed two different cardiac glycoside binding sites. High affinity binding was obtained at a KD = 2.2 × 10−7 mol 1−1 (Bmax = 16.8 pmol ouabain mg−1 protein) whereas low affinity ouabain binding occurred at a KD > 10−6 mol 1−1. 2 To discover whether the two ouabain binding sites are functional in guinea‐pig heart muscle, erythrosin B, an inhibitor of the high affinity ouabain binding in rat brain tissue, was tested in guinea‐pig isolated heart muscle preparations. Erythrosin B proved to be a potent inhibitor of the Mg2+(Na+)‐dependent‐, as well as Na+‐K+‐activated ATPase (ID50 = 9 × 10−6 mol 1−1). Contractility of guinea‐pig isolated papillary muscles, however, was not influenced by erythrosin B in concentrations up to 1 × 10−5 mol 1−1. Only very high concentrations (4 × 10−4 mol 1−1) resulted in a slightly negative inotropic effect (about 20%). 3 Erythrosin B dose‐dependently inhibited [3H]‐ouabain binding to the Na+‐K+‐ATPase (KD = − 3.6 × 10−6 mol 1−1). In a concentration of 1 × 10−5 mol 1−1 the dye abolished high affinity [3H]‐ouabain binding without affecting the low affinity binding sites. 4 In contrast, in guinea‐pig isolated atria, no functional antagonism between erythrosin B (5 × 10−5 mol 1−1) and ouabain was observed. 5 As there is a coincidence between the high affinity binding (KD = 2.2 × 10−7 mol 1−1) and the concentration for half maximum inotropic effects of ouabain (ED50 = 1.6 × 10−7 mol 1−1), the lack of effect of erythrosin B on ouabain‐induced inotropy may be caused by an inaccessibility of the dye to the (internal) ATP‐site of the Na+‐K+‐ATPase.

Intervascular and stimulus selectivity of nitrendipine and related derivatives in KCl and prostaglandin F2α precontracted porcine arteries

1 Dihydropyridine‐type calcium entry blockers exhibit a different vasodilator potency depending on the arterial tissue (intervascular selectivity) as well as on the precontracting stimulus used (stimulus selectivity). In addition, the structure of their ester side chains seems to influence their activity. 2 Vascular activity of nitrendipine and six related 3‐ester side chain derivatives was investigated in isolated coronary, ulnar and basilar arteries of the pig following precontraction with KCl or prostaglandin F2α (PGF2α). 3 After depolarization, all dihydropyridines exhibited a weak preferential action on coronary arteries. Bay E 6927 produced the strongest effect in all vessel types. By contrast, precontraction with PGF2α resulted in a marked preferential action in basilar arteries, although higher concentrations of the dihydropyridines were required for half maximal vasorelaxation. In each case, ulnar arteries were less sensitive. 4 Except with Bay O 5572, the most bulky substituted and least active derivative, only moderate differences were observed within the dihydropyridines studied. On the other hand, there was a pronounced increase in the ratios of the half maximal active concentrations required after precontraction of the vessels with PGF2α compared to KCl (stimulus selectivity) following a limited prolongation of the 3‐ester side chain up to an isopropyl‐group. 5 It is suggested that the observed shift in the intervascular selectivity after precontraction with PGF2α is a consequence of different contractile mechanisms in the three vessel types studied. The degree of the stimulus selectivity may also depend on the structure of the dihydropyridines.

Novel halogenated dihydropyridine derivatives with high vascular selectivity

SummaryCalcium channel antagonists of the dihydropyridine type exhibit preferential vasodilator properties. To study whether this vascular selectivity is due to distinct steric modifications or may be influenced by the physicochemical nature of these drugs, contractility in guinea pig heart isolated papillary muscles, vasodilator properties in isolated rabbit femoral arteries and the lipophilicity of some novel halogenated dihydropyridines have been examined. All newly synthesized derivatives exhibited dose-dependent negative inotropic and vasodilator effects. The negative inotropic potency of all the halogenated derivatives was weaker than that of the parent compound nitrendipine. In contrast, compared to nitrendipine the vasodilator potency of the ester substituted derivatives was slightly increased, while halogen substitution in position 2 and 6 of the dihydropyridine nucleus decreased the vasodilator potency. As a result of the different influence on cardiac and vascular smooth muscle an improved vascular selectivity of the drugs was attained. The ester-substituted dihydropyridine derivatives showed a 9 times (3-bromoethyl-nitrendipine) or 11 times (3-chloroethyl-nitrendipine) higher vascular selectivity with respect to nitrendipine. Correlation of the lipophilicity with the physiological properties showed an increase in biological activity with decreasing lipophilicity. Within the ester-halogenated dihydropyridine derivatives an inverse trend was observed (increasing vasodilation with increasing lipophilicity), indicating a different influence of lipophilicity with the ester-substituted compounds on the different tissues examined. The improved vascular selectivity of the novel halogenated dihydropyridines may be at least in part a consequence of the different lipophilicity of the drugs. In addition, differences in the binding affinities of the dihydropyridines subordinate to distinct voltage dependent conformation states of the calcium channel may contribute.

Lipophilicity and pharmacodynamics of cardiotonic steroids in guinea-pig isolated heart muscle preparations.

1 The inhibitory potency of cardiotonic steroids on myocardial Na+‐K+‐ATPase increases with increasing lipophilicity. Employing the inotropic action on guinea‐pig isolated left atria, the relationship between lipophilicity, chemical structure and the pharmacodynamic properties of cardiac glycosides was further examined. Nineteen digitoxigenin‐and digoxigenin‐derivatives, whose lipophilic nature and inhibitory effects on the myocardial Na+‐K+‐ATPase have been previously investigated, were tested. 2 All steroids exhibited positive inotropic effects which varied with the lipophilicity of these drugs. The dependence of the relationship between these two parameters on structural transformations of the steroids showed qualitatively very close parallelism to that between lipophilicity and their inhibitory effects on myocardial Na+‐K+‐ATPase. 3 The positive inotropic effects and the inhibitory effects on myocardial Na+‐K+‐ATPase also correlated very well, exhibiting similar patterns in their respective correlations with the individual lipophilicity parameters. 4 It is inferred that (a) the positive inotropic effects of the cardiac glycosides vary with their lipophilicity, exhibiting trends similar to those shown for their inhibitory effects on myocardial Na+‐K+‐ATPase, (b) this interdependence is secondary to the influence of structural changes, particularly on the steroid nucleus, and (c) both the lipophilic nature and pharmacodynamic behaviour of the cardiac glycosides almost exclusively depend on the steroid nucleus itself.