Dehydro-digitoxosides of digitoxigenin and digoxigenin: Binding to beef heart (Na++K+)-ATPase in relation to unchanged digitoxosides

Summary Dehydro-digitoxosides are metabolites of digitalis glycosides. In order to study their possible biological activity their affinity to (Na⁺+K⁺)-activated ATPase was determined and compared with unchanged glycosides. Based on the dissociation constants of glycoside-enzyme-complexes, the affinity of the dehydro-digitoxosides ranged in the same order of magnitude as that of the native glycosides. Comparing mono-, bis-, and tris-digitoxosides of digitoxigenin (dt-1, dt-2, dt-3) and of digoxin (dg-1, dg-2, dg-3) with the corresponding dehydrodigitoxosides (3-dehydro-dt-1, 9-dehydro-dt-2, 15-dehydro-dt-3, 3-dehydro-dg-1 and 9-dehydro-dg-2, respectively) the dehydro-digitoxosides had lower affinities to the enzyme. The highest dissociation constants (K _D)were found for 3-dehydro-dt-1 and 3-dehydro-dg-1. The half maximal inhibition of (Na⁺+K⁺)-ATPase activity (I₅₀) corresponded to affinity measurements in all but two cases: dehydro-dt-3 and dehydro-dt-2 showed very low I₅₀ values.     

Cromakalim (BRL 34915) restores in vitro the membrane potential of depolarized human skeletal muscle fibres

Summary The purpose of the present study was to analyze the effects of cromakalim (BRL 34915), a potent drug from a new class of drugs characterized as K⁺ channel openers, on the electrical activity of human skeletal muscle. Therefore, intracellular recordings were used to measure the effects of cromakalim on the membrane potential and input conductance of fibres from human skeletal muscle biopsies. Cromakalim in a concentration above 1 mol/l induced an increase in membrane K⁺ conductance. This effect resulted in a membrane hyperpolarization. The magnitude of this polarization depended on the difference between resting and K⁺ equilibrium potential. The effect had a rapid onset and was quickly reversible after washing. Fibres from two patients with hyperkalaemic periodic paralysis showed an excessive membrane depolarization during and also after exposure to an slightly elevated extracellular K⁺ concentration. In the latter situation, cromakalim repolarized the fibres to the normal resting potential. Tolbutamide (1 mmol/l) and Ba²⁺ (3 mmol/l) strongly antagonized the effect of cromakalim. The data show that cromakalim hyperpolarizes depolarized human skeletal muscle fibres maintained in vitro. The underlying mechanism is probably an activation of otherwise silent, ATP-regulated K⁺ channels. Such an effect may be of therapeutic benefit in a situation in which a membrane depolarization causes muscle paralysis. Send offprint requests to A. Spuler at the above address     

Differential effects of organotin compounds on voltage-gated potassium currents in lymphocytes and neuroblastoma cells

Effects of organotin compounds were studied on voltage-gated K⁺ current in whole-cell voltage clamped lymphocytes and in NlE-115 neuroblastoma cells. In human peripheral blood lymphocytes the immunotoxic compounds dibutyltinchloride (DBT, 2.5 M) and triphenyltinchloride (TPhT, 2.5 M) decrease the peak amplitude of the K⁺ current and prolong time to peak. Tributyltinchloride (TBT, 2.5 M) decreases the K⁺ current to a greater extent than DBT and TPhT, without affecting the time to peak. The neurotoxic organotin compound trimethyltinchloride (TMT, 2.5 M) does not affect the voltage-gated K⁺ current in lymphocytes. Similar effects of DBT were observed in freshly isolated and PHA-activated human lymphocytes and with rat thymocytes. On the other hand, in mouse NIE-115 neuroblastoma cells, none of the organotin compounds altered the voltage-dependent K⁺ current.In human lymphocytes DBT affects both the peak amplitude and the time to peak of the K⁺ current in a concentration-dependent manner. At the maximum concentration of 10 M tested, the peak amplitude of the K⁺ current was reduced to 22 ± 4% of the control current. The IC₅₀ and slope factor for block of the peak outward current by DBT amounts to 6.7 ± 0.4 M, and 2.7 ± 0.4, respectively. The delay in K⁺ current activation does not saturate. At 10 M DMT increases the time to peak to 332 ± 12% of the control value. The present results suggest that the effects by DBT originate from two separate interactions with the voltage-gated K⁺ channel at the extracellular site of the membrane: a direct effect on the closed K⁺ channel causing a delay in current activation and a membrane-related effect causing inhibition of the K⁺ current. The differential effects of the organotin compounds may relate to their differential toxicological action.     

Tolbutamide-sensitivity of the adenosine 5′-triphosphate-dependent K+ channel in mouse pancreatic B-cells

Summary The patch-clamp technique was used to examine the tolbutamide-sensitivity of the adenosine 5-triphosphate (ATP)-dependent K⁺ channel in mouse pancreatic B-cells. When studied at 37°C in cell-attached membrane patches, this channel had a single-channel conductance of 88 pS and was half-maximally inhibited by 2.2 mol/l tolbutamide in the presence of 3 mmol/l d-glucose and 10 mol/l nifedipine. The tolbutamide-induced decrease in the amplitude of the single-channel currents indicated that the membrane potential was sufficiently depolarized for initiation of insulin release by 30 but not by 10 mol/l of tolbutamide. Using 300 mol/l diazoxide to open the ATP-dependent K⁺ channels already closed by 3 mmol/l d-glucose alone, it was demonstrated that initiation of insulin release requires closure of more than 98% of all ATP-dependent K⁺ channels. In excised inside-out membrane patches, the K⁺ channel-blocking potency of tolbutamide was maximally enhanced by 0.3 mmol/1 adenosine 5t'-diphosphate (ADP) at the cytoplasmic side. This ADP effect required the presence of Mg²⁺. Inhibition of K⁺ channel activity by ATP, ADP (Mg²⁺-free) or their non-hydrolyzable analogues adenylyl-imidodiphosphate (AMP-PNP) and , methylene adenosine 5-diphosphate (AMP-CP) was not accompanied by enhancement of tolbutamide-sensitivity. The results suggest that cytosolic MgADP controls tolbutamide-sensitivity by interaction with a receptor site not identical with the site mediating channel closure and that this control plays a role in the intact B-cell.Some of the results described here are part of the medical theses of F. Rosenberger and K. SchefferSend offprint requests to U. Panten at the above address     

Opposite modulation of ouabain cardiotoxicity by hexamethyleneamiloride and phenylephrine

Summary To see whether the Na/H antiporter plays a role in digitalis cardiotoxicity, we investigated the influence of modulators of Na/H exchange on the toxic effects of ouabain in isolated, paced (0.4 Hz) rat left atria. Ouabain (1 mmol/l) caused a transient positive inotropic effect followed by toxic events, including a complete loss of developed force and a gradual increase in resting force. In the presence of hexamethyleneamiloride (3 and 10 mo1/l), an inhibitor of Na/H exchange, ouabain (1 mmol/l) caused a sustained positive inotropic effect without toxicity. By contrast, phenylephrine (100 mol/ 1) an -adrenoceptor agonist reported to stimulate the antiporter, hastened the development of ouabain's toxicity. Neither ouabain, at a subtoxic concentration (650 ol/l), nor phenylephrine (100 mol/l) affected diastolic force, but in their combined presence, a substantial contracture developed and twitch contractions disappeared. Phenylephrine (30 or 100 mol/l) or adrenaline (30 mol/l), in the presence of a -adrenoceptor antagonist, increased the intracellular pH by up to 0.15 pH unit, as measured using ion-selective microelectrodes in quiescent preparations. This effect on pH₁ was prevented by hexamethyleneamiloride (10 mol/l). Consistent with phenylephrine's ability to stimulate Na⁺ influx via the Na/H antiporter, phenylephrine (100 mol/l) increased intracellular Na⁺ activity by about 3 mmol/l in ouabain (650 mol/l)-treated atria. These findings indicate that modulators of Na/H exchange affect the cardiotoxicity of digitalis glycosides and imply that the stimulation of myocardial -adrenoceptors may aggravate digitalis toxicity.This work was conducted in part under the auspices of the Association for US/French Biomedical Cooperation Send offprint requests to S. M. Vogel at the above address     

Comparison of the effluxes of 42K+ and 86Rb+ elicited by cromakalim (BRL 34915) in tonic and phasic vascular tissue

Summary The cromakalim-induced effluxes of ⁴²K⁺ and ⁸⁶Rb⁺ were compared in rat aortic segments and in guinea-pig portal vein. In both vessels, low concentrations of cromakalim (0.1 M) increased the permeability to ⁸⁶Rb⁺ 3–4 times less than that to ⁴²K⁺; at 10 M the difference was about a factor of 1.3–2. In rat aorta, the threshold concentration of cromakalim for ⁴²K⁺ efflux was 0.03 M; with ⁸⁶Rb⁺ as the tracer ion it was 0.1 M. At similar concentrations, cromakalim relaxed the tension of aortic segments precontracted with 23 mM KCl (IC₅₀ = 0.06 ± 0.01 M). However, no concomitant increase in ⁴²K⁺ or ⁸⁶Rb⁺ efflux could be detected from this stimulated preparation at these concentrations. In guinea-pig portal vein, ⁴²K⁺ efflux measurements were performed in the presence and absence of the dihydropyridine Ca²⁺ entry blocker PN 200-110 (isradipine) yielding comparable results. In the presence of PN 200-110, where spontaneous activity and the K⁺ efflux associated with it were abolished, the threshold concentration of cromakalim for ⁴²K⁺ efflux was 0.02 M as compared to 0.06 M for ⁸⁶Rb⁺ efflux. In the absence of PN 200-110, spontaneous activity of the portal vein was inhibited by 70% and 90% at these concentrations. In double isotope experiments, the K⁺ channel inhibitor tetraethylammonium did not discriminate between the effluxes of ⁴²K⁺ and ⁸⁶Rb⁺ stimulated by cromakalim.It is concluded that in the two vascular tissues examined, cromakalim increased the permeability to ⁴²K⁺ more than to ⁸⁶Rb⁺, the difference being more marked at low cromakalim concentrations. The use of ⁴²K⁺ as the tracer ion narrows the apparent gap between the concentrations of cromakalim which elicit vasorelaxant effects and those which induce an observable increase in K⁺ permeability; however a significant difference persists.Part of the data was presented at the Winter Meeting of the British Pharmacological Society London 1988 [Br J Pharmacol 93 (1988) p 19] Send offprint requests to U. Quasi at the above address     

Inhibition of minK protein induced K+ channels in Xenopus oocytes by estrogens

Previously it was shown that minK protein expression in uterus is regulated by estrogen. In the present study, we were interested in putative direct effects of estrogen on minK protein induced K⁺ currents (I_minK) in Xenopus oocytes. Superfusion with 17--estradiol (1 M) resulted in an inhibition of minK-induced currents, but had no appreciable effects on the delayed rectifier and inward rectifier K⁺ channels Kv1.1 and Kir2.1, respectively. The inhibition of I_minK by 17--estradiol was concentration-dependent, with an IC₅₀ of approximately 0.5 M. In the presence of 17--estradiol, the conductance-voltage relationship was shifted to more depolarized potentials. I_minK inhibition occurred also in the presence of the estrogen-receptor antagonist tamoxifen, suggesting that a mechanism independent of estrogen receptors is involved. The synthetic estrogen diethylstilbestrol (DES) also inhibited I_minK but with a lower affinity (IC₅₀ of 4.5 M), while cortisol and progesterone had only weak effects on I_minK. In summary, the results indicate that estrogens directly inhibit I_minK.     

Effects of ouabain on human bronchial muscle in vitro

The effects of ouabain, an inhibitor of the plasmalemmal Na⁺/K⁺-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with –logEC₅₀=7.16±0.11 and maximal effect of 67±4% of the response to acetylcholine (1 mM). Ouabain (10 M)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H₁-receptors and -adrenoceptors, or neuronal Na⁺ channel blockade. The inhibition of ouabain contraction in tissues bathed in K⁺-free medium, and the inhibition by ouabain of the K⁺-induced relaxation confirm that the contractile action of ouabain is mediated by inhibition of Na⁺/K⁺-ATPase. Furthermore, depolarization (16.4±0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 M)-induced contractions were abolished by a Ca²⁺-free solution but not by blockers of L-type Ca²⁺ channels. In human cultured bronchial smooth muscle cells, ouabain (10 M) produced a sustained increase in [Ca²⁺]_i (116±26 nM) abolished in Ca²⁺-free medium. Incubation with a Na⁺-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of ouabain thus suggesting the role of Na⁺/Ca²⁺ exchange in ouabain contraction while selective inhibitors of Na⁺/H⁺-antiport, Na⁺/K⁺/Cl^–-antiport, or protein kinase C had no effect. Ouabain (10 M) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 M) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca²⁺ entry unrelated to L-type channels and involving the Na⁺/Ca²⁺-antiporter.     

Anti-cholinergic effect of verapamil on the muscarinic acetylcholine receptor-gated K+ channel in isolated guinea-pig atrial myocytes

Summary Effects of verapamil on the acetylcholine (ACh)-induced K⁺ current were examined in single atrial cells, using the tight-seal whole-cell clamp technique. The pipette solution contained guanosine-5-triphosphate (GTP) or guanosine-5-O-(3-thiotriphosphate) (GTP-S, a non-hydrolysable GTP analogue). In GTP-loaded cells, ACh induced a specific K⁺ current, which is known to be mediated by pertussis toxin-sensitive GTP-binding (G) proteins. Verapamil (0.1–100 M) depressed the ACh-induced K⁺ current in a concentration-dependent fashion. In GTP-S-loaded cells, the K⁺ current remained persistently after wash-out of ACh, probably due to irreversible activation of G proteins by GTP-S. Verapamil (0.1–100 M) also depressed the intracellular GTP-S-induced K⁺ current. However, the magnitude of verapamil-depression of the K⁺ current in GTP-S-loaded cells was significantly smaller than that in GTP-loaded cells at concentrations between 1 and 10 M of the drug. From these results, it is suggested that verapamil may block not only the function of muscarinic ACh receptors but also of G proteins and/or the K⁺ channel itself and thereby depress the ACh-induced K⁺ current in isolated atrial myocytes.Supported by grants from the Ministry of Education, Science and Culture of Japan and the Research Program on Ca Signal Control Send offprint requests to Y. Kurachi at the above address     

Differentiation between isoforms of Na+/K+-transporting ATPase from human and guinea-pig cardiac muscle through use of digitalis derivatives as analytical probes

The aims of the study included: to explore the protein structure basis for the differences in digitalis sensitivity between isoforms of Na/K-ATPase from human and guinea-pig cardiac muscle; to determine the relative significance of the constituents of tripartite digitalis compounds in their inhibitory action on these Na/K-ATPase isoforms; to evaluate the potential significance of the receptor kinetics for pharmacological characteristics. The analytical method has been the recording of the inhibitory interaction of various digitalis derivatives with the Na/K-ATPase isoforms.The protein structure basis for the isoform differences in digitalis susceptibility has been explored by analysing in free-energy plots the kinetics of their inhibitory interaction with 53 digitalis derivatives of grossly different structure. The slope of the regression line and the parameters of the regression equation proved to be similar for the two isoforms in spite of the great difference in their digitalis susceptibilities. This surprising uniformity indicates that a uniform "macroscopic" mechanism underlies the inhibitory effect of the various derivatives on the two isoforms. On the other hand, the differences in the positions of G _infon ^sup* and G _infoff ^sup* values for particular inhibitors relative to the regression line reveal differences in the microscopic interaction energy surfaces of the two isoforms. In conclusion, the origin of the isoform distinctions in their susceptibility towards inhibition by various digitalis derivatives is essentially confined to differences in the chemotopology of the digitalis recognition matrix and binding cleft.Specific observations allowed to disentangle the impact of various steroid derivatizations at carbon atoms 3, 17, and diverse other positions on the kinetics of their interaction with the enzyme isoforms. The steroid nucleus of the cardiac glycosides, 5,14-androstane, proves to be the basal structural element for discrimination of Na/K-ATPase isoforms. This discrimination becomes much enlarged by steroid glycosidation at C3-OH and/or by steroid substitution of C17-H by a lactone ring. The higher inhibitory sensitivity of the human isoform is based either on an increased association rate or a decreased dissociation rate, depending on the nature of derivatization.-The significance of specific findings is discussed as to the following aspects: the types of intermolecular forces and the characteristics of the digitalis binding matrix; the nature of the rate-determining step in the formation and dissociation of the digitalis-enzyme complex; the receptor kinetics as a potential basis of the therapeutic range of cardiac glycosides; the potentiality of the use of 5,14-androstane as the newly discovered lead structure for the design of novel inotropic steroids possibly analogous in structure to the putative endogenous digitalis compounds. Abbreviations and definitions Na/K-ATPase, Na⁺/K⁺-transporting ATPase (EC 3.6.1.37) · Isoform, generic name for Na/K-ATPase preparations from different species which may or may not consist of more than one isoenzyme · digitalis, generic name for steroids with C/D-cis ring junction including cardenolides, bufadienolides, and their glycosides     

Effects of glibenclamide and tetracaine on 86Rb+ fluxes in mouse pancreatic β-cells

Summary Potassium transport was measured in -cell-rich islets from ob/ob-mice using the K⁺-analogue ⁸⁶Rb⁺. Both tetracaine (0.1 mM) and glibenclamide (0.1 M) reduced the oubain-resistant ⁸⁶Rb⁺ influx but did not significantly affect the oubain-sensitive portion (Na⁺/K⁺ pump). Tetracaine (0.5–1 mM) or glibenclamide (0.2 mM) decreased the ⁸⁶Rb⁺ equilibrium content and glibenclamide (1 M) transiently reduced the ⁸⁶Rb⁺ efflux rate but 0.1 mM tetracaine had only a slight effect on this flux rate. The results suggest that a change in ouabain-resistant (passive) K⁺ fluxes, but not the Na⁺/K⁺ pump, is involved in stimulation of insulin secretion by glibenclamide and tetracaine. Both drugs may exert similar effects on the -cell plasma membrane.     

On the mechanism of action of phenylephrine in rat atrial heart muscle

Both in rat left atrial heart and in aortic smooth muscle preparations, phenylephrine (PE) caused a concentration-dependent increase in force of contraction (F_c) in the presence of atenolol (10 mol/l), which was antagonized by phentolamine, prazosin and WB 4101 in a competitive manner. The pA₂ values of the antagonists in the cardiac tissue were 10–20fold lower than those in the rat thoracic aorta. In the spontaneously beating right atrium, PE exerted a positive chronotropic action, which was not significantly antagonized by phentolamine or prazosin. It is therefore assumed that the effects of phenylephrine in the left atrium and in the aorta are mediated by different subtypes of ₁-adrenoceptors, whereas the effects in the sino-atrial node are probably unrelated to ₁-adrenoceptors. To further elucidate the mechanisms of the positive inotropic effect of PE, action potential configuration and ⁴⁵Ca²⁺ fluxes were monitored in the rat left atrium. The increase in F_c by PE was associated with an increase in action potential duration (APD) and a reduction in resting membrane potential (RP). In the presence of (–)-devapamil (13888), the effects of PE on APD and RP persisted, whereas the increase in F_c was antagonized in a non-competitive manner. Forskolin (300 nmol/l) enhanced the positive inotropic effect of PE. PE exerted a significant increase in ⁴⁵CA²⁺ uptake in beating preparations, which was abolished in the presence of (–)13888 (1 mol/l). In addition to the PE-induced increase in ⁴⁵Ca²⁺ uptake, a decrease in ⁴⁵Ca²⁺ efflux was observed. Similarly, depolarization of the membrane by raising [K⁺]_o to 85 mmol/l revealed an increase in ⁴⁵Ca²⁺ uptake and a decrease in ⁴⁵Ca²⁺ efflux. The latter observations support the view that the membrane potential strongly determines the movement of ⁴⁵Ca²⁺ across the membrane. It is assumed that the ₁-adrenoceptor-mediated changes in APD and RP may enhance F_c, first, by increasing net Ca²⁺ entry from the extracellular space through voltage-dependent Ca²⁺ channels and, second, by decreasing Ca²⁺ efflux possibly via the Na ⁺/Ca²⁺ exchange mechanism.     

Distribution of cardiac glycosides in heart and brain of dogs and their affinity to the (Na++K+)-ATPase

Summary The tissue distribution after repeated intravenous administration of tritium-labelled digoxin, -methyldigoxin and ouabain was examined in heart and brain of 6 beagle dogs. In addition, the (Na⁺+K⁺)-ATPase activity was measured in various heart and brain areas, and its affinity to the cardiac glycosides was determined. The glycoside concentrations in the atria are lower than in the ventricles, and the left heart areas show higher concentrations than the right areas. Significant differences in the (Na⁺+K⁺)-ATPase activity or its binding capacity in the various heart areas, which could be responsible for this characteristic distribution pattern, were not found. In agreement with its greater lipid-solubility, -methyldigoxin shows a higher accumulation in the brain than digoxin and ouabain. However, while -methyldigoxin is evenly distributed throughout all brain areas, concentration differences are found for digoxin and ouabain in the telencephalon, cerebellum and brain stem. This characteristic distribution of the more polar glycosides may be partly determined by the different structure of the capillaries in the central nervous system. In addition, the binding affinities for digoxin and ouabain also differ in the various crude brain preparations. In the diencephalon, pons, cerebellum and medulla the dissociation constants as a reciprocal measure of the binding affinity were lower for digoxin with 7.5 to 9.9×10^–9 than in the telencephalon, mesencephalon and spinal cord with dissociation constants of 1.1 to 1.45×10^–8 M. Since, in these brain areas higher glycoside concentrations per g wet weight were also measured, the glycoside accumulation in the various brain areas could be dependent on the higher receptor affinity of these brain areas. On the other hand, the binding affinities for -methyldigoxin were the same in all brain areas, with a mean dissociation constant of 1.45×10^–8 M.     

Amiloride analogues induce responses in isolated rat cardiovascular tissues by inhibition of Na+/Ca2+ exchange

Summary The role of inhibition of Na⁺/Ca²⁺ exchange in the positive inotropic, negative chronotropic and vasorelaxant responses to amiloride and some of its analogues was investigated in isolated cardiovascular tissues from female Wistar rats. The compounds tested were amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, a potent inhibitor of Na⁺/H⁺ exchange), phenamil and 2,4-dimethylbenzamil (DMB), both potent Na⁺ channel inhibitors with activity against Na⁺/Ca²⁺ exchange, and 5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil (CBDMB), a potent inhibitor of Na⁺/Ca²⁺ exchange with reduced activity against Na⁺ channels compared with its parent compound DMB.Phenamil, DMB and CBDMB increased the force of contraction of right ventricular papillary muscles with similar potencies (-log EC₅₀ values: 4.77 ± 0.06, 5.09 ± 0.09, 4.97 ± 0.17 respectively), while amiloride and EIPA gave small negative inotropic responses. All compounds gave negative chronotropic responses at similar concentrations to those which exerted inotropic effects. Inhibition of KCl contraction of endothelium-free aortic rings was observed with all compounds tested. Phenamil, DMB and CBDMB but not amiloride or EIPA showed a shift to the left of the concentration-response curves in the presence of intact endothelium.These results provide further evidence for positive inotropic and endothelium-dependent vasorelaxant effects of amiloride analogues mediated by inhibition of Na⁺/Ca²⁺ exchange. Send offprint requests to J. R. Bourke at the above address     

Quinidine inhibition of the muscarine receptor-activated K+ channel current in atrial cells of guinea pig

Summary Postsynaptic mechanisms underlying the anticholinergic effects of quinidine were examined in single atrial cells, using the tight-seal whole-cell recording technique. The solution in the glass pipettes contained guanosine-5triphosphate (GTP) or guanosine-5-O-(3-thiotriphosphate) (GTP-S, a non-hydrolyzable GTP analogue). In both cases, acetylcholine (ACh), applied to the bath, induced a specific K⁺ current. In GTP-loaded cells, quinidine in the bath solution depressed the ACh-induced K⁺ current concentration-dependently. Atropine also blocked the K⁺ current. On the other hand, in GTP-S-loaded cells, the ACh-induced current was not blocked by atropine and persisted even when ACh was washed out from the bath, indicating that GTP-S causes uncoupling of the K⁺ channels from the muscarine receptors. Quinidine, however, did depress the increased K⁺ current concentration-dependently. The percent inhibition curves for quinidine to depress the K⁺ current were very similar between GTP-loaded and GTP-S-loaded cells. From these observations, we suggest that direct inhibition of the muscarine receptor-activated K⁺ channel current by quinidine, and not blockade of the muscarine receptor itself, is mainly responsible for the anticholinergic effects of the drug in atrial myocytes. Send offprint requests to Y. Kurachi at the above address     

Effects of the K+ channel blocker tedisamil on 86Rb efflux induced by cromakalim,high potassium and noradrenaline,and on mechanical tension in rabbit isolated vascular smooth muscle

Summary Tedisamil, a new bradycardic agent with an inhibitory action on K⁺ channels in cardiac muscle, was found to inhibit in a non-competitive manner the relaxation induced by the K⁺ channel opener cromakalim in noradrenaline-stimulated helical strips from rabbit aortae. Tedisamil tended to be more potent in this respect than glibenclamide; the latter however competitively antagonized the cromakalim-induced relaxation. In rabbit aorta preloaded with ⁸⁶Rb as a marker of K⁺, 10 mol/l tedisamil inhibited the ⁸⁶Rb efflux induced by 10 mol/l cromakalim. — While the ⁸⁶Rb efflux evoked by depolarization with 100 mmol/l K⁺ aspartate was inhibited by tedisamil, too, the rise of ⁸⁶Rb efflux induced by noradrenaline was unaffected by the drug.In non-stimulated rabbit aorta, tedisamil increased mechanical tension in a concentration-dependent manner (EC₅₀ for peak contractions: 32 mol/l; for maintained tension: 24 mol/l), and enhanced ⁸⁶Rb efflux. Both stimulant actions were antagonized by the calcium antagonist diltiazem.In conclusion, tedisamil affects different K⁺ channels in vascular smooth muscle. Its stimulant effects are assumed to be secondary to membrane depolarization and subsequent activation of voltage-dependent Ca²⁺ channels.Supported by the Deutsche Forschungsgemeinschaft Send offprint requests to V. A. W. Kreye at the above address     

Characterization of the K+-channel-coupled adenosine receptor in guinea pig atria

Summary In the present work we studied the pharmacological profile of adenosine receptors in guinea pig atria by investigating the effect of different adenosine analogues on⁸⁶Rb⁺-efflux from isolated left atria and on binding of the antagonist radioligand 8-cyclopentyl-1,3-[³H]dipropylxanthine ([³H]DPCPX) to atrial membrane preparations. The rate of⁸⁶Rb⁺-efflux was increased twofold by the maximally effective concentrations of adenosine receptor agonists. The EC₅₀-values for 2-chloro-N⁶-cyclopentyladenosine (CCPA), R-N⁶-phenylisopropyladenosine (R-PIA), 5-N-ethylcarboxamidoadenosine (NECA), and S-N⁶-phenylisopropyladenosine (S-PIA) were 0.10, 0.14, 0.24 and 12.9 M, respectively. DPCPX shifted the R-PIA concentration-response curve to the right in a concentration-dependent manner with a K_B-value of 8.1 nM, indicating competitive antagonism. [³H]DPCPX showed a saturable binding to atrial membranes with a B_max-value of 227 fmol/mg protein and a K_D-value of 1.3 nM. Competition experiments showed a similar potency for the three agonists CCPA, R-PIA and NECA. S-PIA is 200 times less potent than R-PIA. Our results suggest that the K⁺ channel-coupled adenosine receptor in guinea pig atria is of an A₁ subtype.Abbreviations CCPA 2-chloro-N⁶-cyclopentyladenosine - DPCPX 8-cyclopentyl-1,3-dipropylxanthine - NECA 5-N-ethylcarboxami-doadenosine - PIA N⁶-phenylisopropyladenosine Send offprint requests to H. Tawfik-Schlieper at the above address     

A target K+ channel for the LP-805-induced hyperpolarization in smooth muscle cells of the rabbit portal vein

Summary The resting membrane potential of smooth muscle cells of the rabbit portal vein was –51.2 mV. LP-805 (8-tert-butyl-6,7-dihydropyrrolo[3,2-e] 5-methylpyrazolo [1,5-a] pyrimidine-3-carbonitrile) hyperpolarized the membrane to –62.3 mV (10 M) and inhibited the burst spike discharges as measured using the microelectrode method. In dispersed smooth muscle cells, LP-805 (10 M) generated an outward-current with a maximum amplitude of 68 pA at a holding potential of –40 mV in experiments using the voltage-clamp procedure. The reversal potential of the outward current evoked by LP-805 was –82 mV and this value was close to the equilibrium potential for K⁺ (–80 mV) in the present ionic conditions, suggesting that LP-805 activated the K⁺ channel. Generation of both the hyperpolarization and the outward c urrent by LP-805 was inhibited by glibenclamide ( 1 M). Using the cell-attached and cell-free patch-clamp (in the presence of GDP) procedures, the maxi-K⁺ channel current (150 pS) could be recorded in the absence of LP-805; application of LP-805 additionally opened a small conductance K⁺ channel current (15 pS) without change in the activity of the maxi-K⁺ channel. The maxi-K⁺ channel was sensitive to charybdotoxin (0.1 M) and to intracellular Ca²⁺ ([Ca²⁺]_i) concentration. The 15 pS channel was insensitive to [Ca²⁺]_i and charybdotoxin, but sensitive to intracellular ATP concentration. Glibenclamide (> 1 M) inhibited the 15 pS K⁺ channel activated by LP-805. These actions of LP-805 on the maxi-K⁺ and 15 pS K⁺ channels are the same as those previously observed for nicorandil and pinacidil. Thus, LP-805 is a K⁺ channel opener with a chemical structure different from those of the known openers. Correspondence to M. Kamouchi at the above address     

Effects of Ca2+ channel antagonists and ryanodine on H1-receptor mediated electromechanical response to histamine in guinea-pig left atria

Summary Effects of organic Ca²⁺ channel antagonists, Ni²⁺ and ryanodine on the electrophysiological and positive inotropic responses to histamine were examined in isolated guinea-pig left atria. Histamine increased force of contraction, prolonged action potential duration (APD) and hyperpolarized the membrane in a concentration-dependent manner. Histamine at a concentration of 1 mol/l produced a dual-component positive inotropic response composed of an initial increasing phase (initial component) and a second an late developing, greater positive inotropic phase (second component), whereas causing monophasic changes in APD and resting potential. The electrophysiological and dual-component positive inotropic effects induced by histamine were antagonized by chlorpheniramine (1 mol/l) but not by cimetidine (10 mol/l), indicating that both effects are exclusively mediated by H₁-receptors. The positive inotropic response to 1 mol/l histamine was changed by the pretreatment with nifedipine (1 mol/l) and nisoldipine (1 mol/l). In the presence of these dihydropyridines, the second component was almost completely abolished, while the initial component was hardly affected. On the other hand, verapamil (3 mol/l) and diltiazem (10 mol/l) failed to modify the multiphasic inotropic response to histamine. None of the Ca²⁺ channel antagonists affected the histamine-induced APD prolongation. In the presence of Ni²⁺ at a concentration of 0.3 mmol/l, at which it produced no negative inotropic action, the second component of the positive inotropic effect of histamine was specifically suppressed whereas the histamine-induced APD prolongation was unaffected. Preferential attenuation of the second component was also observed in the presence of 30 nmol/l ryanodine. However, the electrophysiological alterations caused by histamine remained unchanged. These results suggest that in guinea-pig left atria the H₁-receptor-mediated prolongation of APD seems unlikely to be due to enhancement of the slow inward Ca²⁺ current. Conversely, the increased Ca²⁺ influx as a result of the APD prolongation may contribute to the second component of the positive inotropic effect of histamine. Dihydropyridine Ca²⁺ channel antagonists, Ni²⁺ and ryanodine are all capable of inhibiting the second component, but do so possibly via different mechanisms, implying the complicated mechanisms underlying the H₁-receptor-mediated positive inotropic effect.Send offprint requests to Y. Hattori     

Heparin specifically inhibits the inositol 1,4,5-trisphosphate-induced Ca2+ release from skinned rat aortic smooth muscle cells in primary culture

Summary By measuring the ⁴⁵Ca²⁺ movement in saponin-skinned primary cultured rat aortic smooth muscle cells, we examined the specificity of the inhibitory effect of heparin on the IP₃-induced Ca²⁺ release. IP₃ (100 mol/l) markedly (98%) decreased the MgATP-dependent ⁴⁵Ca²⁺ content in the non-mitochondrial Ca²⁺ stores in the presence of 1 mol/l free Ca²⁺. Heparin (1–100 g/ml) dose-dependently inhibited this Ca²⁺ release by IP₃. In Ca²⁺-free solution, heparin (100 g/ml) inhibited the increases in ⁴⁵Ca²⁺ efflux rate evoked by 10 mol/l IP₃. De-N-sulfated heparin did not inhibit the IP₃-induced Ca²⁺ release. Hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C and 2,6-disulfated d-glucosamine had no inhibitory effects on the IP₃-induced Ca²⁺ release. High concentrations (over 1 mg/ml) of heparin inhibited the ⁴⁵Ca²⁺ influx and decreased the Ca²⁺ content in skinned cells. These results suggest that heparin (1–100 g/ml) specifically inhibits the IP₃-induced increase in Ca²⁺ permeability of Ca²⁺ stores and that three sulfate groups at different locations on the molecule of heparin, two at the d-glucosamine and one at the iduronic acid, may be important for this action, in skinned vascular smooth muscle cells, in culture. Send offprint requests to H. Kanaide at the above address