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     

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     

Anticholinergic action of quinidine sulfate in the rabbit atrioventricular node

Summary Anticholinergic action of quinidine sulfate was electrophysiologically studied by recording spontaneous action potentials and membrane currents of the rabbit atrioventricular node. In the presence of 0.1 mol/l carbachol, the spontaneous activity of the atrioventricular nodal preparations was markedly inhibited, whereas subsequent addition of 1, 5 and 20 mol/l quinidine restored automaticity in a concentration-dependent manner. In some preparations, quinidine at concentrations of 5 mol/l and higher slowed the spontaneous activity by its direct membrane action even in the presence of carbachol. The dose-response curve for acetylcholine action on the spontaneous firing frequency showed that one molecule of acetylcholine bound to one muscarinic receptor of the atrioventricular node cell (Hill coefficient = 1.2). A parallel shift of this curve towards higher acetylcholine concentrations was observed at 0.03, 0.1 and 0.3 mol/l but not at 1 and 3 mol/l quinidine, suggesting a noncompetitive antagonism of quinidine against acetylcholine. Voltage clamp experiments revealed that 5 mol/l quinidine reduced the slow inward current, hyperpolarization-activated inward current, and delayed rectifying K⁺ current, through its membrane actions. Quinidine at this concentration almost completely suppressed the acetylcholine-activated K⁺ current, which showed a relaxation phenomenon. Hence, the direct blockage of the acetylcholine-activated K⁺ current by quinidine was considered responsible for the anticholinergic action of this drug.We conclude that quinidine is a non-specific ionic channel blocker that inhibits all the membrane currents in the atrioventricular node including the acetylcholine-activated K⁺ current.Send offprint requests to Y. Watanabe 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     

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     

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 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.     

Selective modulation of the ATP-sensitive K+ channel by nicorandil in guinea-pig cardiac cell membrane

Summary Effects of a vasodilator, nicorandil (2-nicotinamidoethyl nitrate) on four kinds for cardiac K⁺ channels were investigated in guinea pig ventricular and atrial cells using inside-out patch recording combined with oilgate concentration jump method.Nicorandil of 300 mol/l failed to affect the inward-rectifier K⁺ channel and the Na⁺-activated K⁺ channel. The open probability of the muscarinic K⁺ channel, when activated by the application of GTP, was not changed by the drug. Nicorandil selectively increased the open probability of the ATP-sensitive K⁺ channel that was partly suppressed by intracellular ATP. The median effective concentration (EC50) of nicorandil was 74 mol/l and Hill coefficient was 1.32 in the concentration-open probability relationship. The closing rate of the K⁺ channel by ATP was markedly delayed by the drug, whereas the open rate on removal of ATP was scarcely affected. Nicorandil had only little effect on this channel after run-down. It was concluded that nicorandil selectively activates the ATP-sensitive K⁺ channel mainly by modulating the ATP-dependent gate.Send offprint requests to M. Takano 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     

Differential electrophysiologic and inotropic effects of phenylephrine in atrial and ventricular heart muscle preparations from rats

Summary Stimulation of ₁-adrenoceptors evokes a different pattern of inotropic responses in atrial and ventricular heart muscle preparations from rats. The inotropic effects are accompanied by different changes in membrane potential. In an attempt to clarify the question whether or to which extent these events are causally related, the effects of phenylephrine on force of contraction, transmembrane potential, Ca²⁺ current (I_Ca) and K⁺ currents were comparatively studied in either tissue.In atrial preparations, phenylephrine 10 mol/l caused an increase in force of contraction, a marked prolongation of the action potential duration and a depolarization of the membrane at rest. In the ventricle, however, the addition of phenylephrine 10 mol/l produced first a decline in force of contraction associated with a hyperpolarization of the membrane and a reduction in the action potential duration. These changes were followed by an increase in force,of contraction and a slight prolongation of the action potential, whereas the resting membrane potential remained increased. The hyperpolarization was eliminated in the presence of ouabain 100 mol/l.In enzymatically isolated atrial and ventricular myocytes, the whole-cell voltage clamp technique was used to study membrane currents on exposure to phenylephrine. Phenylephrine 30 mol/l did not affect the magnitude of I_Ca in either cell type. Transient and steady state K⁺ outward currents, however, were significantly diminished to a similar extent in atrial and in ventricular myocytes.It is concluded that the positive inotropic effect of ₁-adrenoceptor stimulation in the rat atrium is related to an increase in action potential duration and a decrease in resting membrane potential due to a decrease in K⁺ currents. In the ventricle, phenylephrine additionally activates the Na⁺/K⁺ pump thereby hyperpolarizing the membrane. The rapid onset of pump stimulation seems to overwhelm, in the beginning, the phenylephrine-induced decrease in K⁺ conductance and therefore to evoke a transient negative inotropic effect.It is assumed that phenylephrine can alter the intracellular Ca²⁺ concentration due to changes in the action potential duration. The way how Ca 2+ enters the cell remains speculative, since direct changes of Ica were not detected. The more complicated changes in membrane potential in the ventricle suggest that also other mechanisms for the positive inotropic response to phenylephrine must be considered. Send offprint requests to H. Nawrath at the above address     

Concentration-dependent effects of tolbutamide,meglitinide, glipizide,glibenclamide and diazoxide on ATP-regulated K+ currents in pancreatic B-cells

Summary The influence of the hypoglycemic drugs tolbutamide, meglitinide, glipizide and glibenclamide on ATP-dependent K⁺ currents of mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. In the absence of albumin, tolbutamide blocked the currents half maximally at 4.1 mol/l. In the presence of 2 mg/ml albumin half maximal inhibition of the currents was observed at 2.1 mol/l meglitinide, 6.4 nmol/l glipizide and 4.0 nmol/1 glibenclamide. The hyperglycemic sulfonamide diazoxide opened ATP-dependent K+channels. Half maximally effective concentrations of diazoxide were 20 mol/l with 0.3 mmol/1 ATPand102 mol/l with 1 mmol/1 ATP in the recording pipette. Thus, the action of diazoxide was dependent on the presence of ATP in the recording pipette. The free concentrations of the drugs which influenced ATP-dependent K⁺ currents were comparable with the free plasma concentrations in humans and the free concentrations which affected insulin secretion in vitro. The results support the view that the target for the actions of sulfonylureas and of diazoxide is the ATP-dependent K⁺ channel of the pancreatic B-cell or a structure closely related to this channel.Some of the results described here were obtained during medical thesis work by K. Männer Send offprint requests to B. J. Zünkler     

Inhibition by carbamazepine of various ion channels-mediated catecholamine secretion in cultured bovine adrenal medullary cells

The effects of carbamazepine (CBZ) on ²²Na⁺ influx, ⁴⁵Ca²⁺ influx, catecholamine secretion and cyclic GMP production were examined in cultured bovine adrenal medullary cells. 1 CBZ (40–120 mol/l) inhibited ²²Na⁺ influx evoked by carbachol in a concentration-dependent manner. CBZ inhibited carbachol-evoked ⁴⁵Ca²⁺ influx and catecholamine secretion at concentrations similar to those which suppressed ²²Na⁺ influx. 2 CBZ (4–120 mol/l) inhibited veratridine-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion. 3 CBZ (12 or 40–120 mol/l) suppressed 56 mmol/1 K⁺-evoked ⁴⁵Ca²⁺ influx and catecholamine secretion, respectively. 4 Combination of CBZ with nitrendipine or -agatoxin-IVA produced further inhibition of 56 mmol/l K⁺ - evoked ⁴⁵Ca²⁺ influx and catecholamine secretion, compared to the effect of CBZ alone, whereas CBZ plus -conotoxin-GVIA did not produce any further inhibition. 5 CBZ (40 mol/1) attenuated the production of cyclic GMP caused by muscarine. These results suggest that CBZ at therapeutic concentrations (16–48 mol/l: 4–12 g/ml) inhibits catecholamine secretion by interfering with nicotinic acetylcholine receptor-associated ion channels, voltage-dependent Na⁺ channels and N-type voltage-dependent Ca²⁺ channels, and may have an antimuscarinic effect in adrenal medullary cells.     

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.     

Interaction between 5-HT uptake inhibition and activation of 5-HT autoreceptors by exogenous agonists in rat cerebral cortex slices and synaptosomes

Summary Rat cerebral cortex slices or synaptosomes were labelled with ³H-5-hydroxytryptamine (³H-5-HT) and subsequently superfused. They were depolarized by electrical stimulation (slices) or with high K⁺ (slices and synaptosomes). Continuous electrical stimulation (2 Hz, 24 mA, 2 ms) and continuous or discontinuous K⁺ depolarization (15–25 mM) were used. 1. Continuous electrical stimulation or continuous K⁺-depolarization of slices evoked a steady overflow of tritium that slowly decayed with time. 2. Exposure to increasing concentrations of 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole succinate (RU 24969) (0.001–0.1 M) during continuous electrical stimulation produced a concentration-dependent decrease in tritium overflow. Citalopram (1 M) counteracted the effect of RU 24969. 3. RU 24969 inhibited the evoked ³H-overflow and citalopram reduced the effect of RU 24969 also during continuous depolarization of slices with 20 mM K⁺. Similar results were obtained by using 5-methoxytryptamine or LSD. 4. In slices 1 M citalopram increased significantly the tritium overflow evoked by electrical stimulation or by 20 mM K⁺-depolarization. 5. Increasing the K⁺ concentration from 20 mM to 25 mM mimicked the effects of 1 M citalopram both on the RU 24969 activity and on the evoked tritium overflow. 6. RU 24969 (0.001–0.1 M) decreased in a concentration-dependent way the release of tritium from cortical synaptosomes depolarized with K⁺ (15–20 mM). The presence of 1 M citalopram did not modify significantly the effect of the agonist. Citalopram was ineffective also when the serotonin uptake carrier in superfused synaptosomes was activated by tryptamine. In conclusion, in slices of rat cerebral cortex, the action of exogenous 5-HT autoreceptor agonists is inhibited by 5-HT uptake blockers independently of the depolarizing agent (electrical stimulation or high-K⁺) used to elicit ³H-5-HT release. Increasing K⁺-concentration, which probably increases serotonin in the biophase, mimics the presence of the reuptake inhibitor. These data together with the finding that, in superfused synaptosomes, 5-HT uptake inhibition did not affect the potency of autoreceptor agonists, favours the idea that, in cerebral cortex slices, inhibitors of 5-HT reuptake prevent activation of autoreceptors by exogenous agonists by increasing the concentration of 5-HT in the autoreceptor biophase. Send offprint requests to M. Raiteri at the above address     

Tolbutamide- and diazoxide-sensitive K+ channel in neurons of substantia nigra pars reticulata

Summary Single-channel K⁺ currents were recorded in cell-attached patches from slices of rat substantia nigra. On the somata of neurons in the caudal half of the substantia nigra pars reticulata a K⁺ selective channel with a unitary conductance of 71 pS (154 mmol/l K⁺ in pipette filling solution) was identified. The channel was activated both by application of diazoxide (300 mol/l) and by energy-depleting conditions (200 mol/l cyanide) and was reversibly blocked by tolbutamide (0.1–1 mmol/l). It is concluded that neurons in the substantia nigra pars reticulata of the rat contain a typical ATP-sensitive K⁺ channel the activity of which can be modulated by diazoxide and sulfonylureas.Correspondence to: C. Schwanstecher 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     

Cocaine inhibits cromakalim-activated K<Superscript>+</Superscript> currents in follicle-enclosed<Emphasis Type="Italic"> Xenopus</Emphasis> oocytes

The effect of cocaine on K⁺ currents activated by the K_ATP channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3–500 M reversibly inhibits cromakalim-induced K⁺ currents. The IC₅₀ value for cocaine was 96 M. Inhibition of the cromakalim-activated K⁺ current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca²⁺ chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K⁺ currents, suggesting that Ca²⁺-activated second messenger pathways are not involved in the actions of cocaine. Outward K⁺ currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC₅₀ and slope values for the activation of K⁺ currents by cromakalim were 184±19 M and 1.14 in the absence of cocaine as compared to 191±23 M and 1.03 in the presence of cocaine (300 M). Cocaine also blocked K⁺ currents mediated through C-terminally deleted form of Kir6.2 (KirC26) in the absence of sulfonylurea receptor with an IC₅₀ value of 87 M, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 M) did not affect the binding characteristics of the K_ATP ligand, [³H]glibenclamide. These results demonstrate that cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes are modulated by cocaine.     

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     

Gastric acid secretion in the dog: a mechanism-based pharmacodynamic model for histamine stimulation and irreversible inhibition by omeprazole

A mechanism-based pharmacodynamic model was used to describe the inhibitory effect by omeprazole on gastric acid secretion measured after histamine stimulation in the dog. The model identifies parameters that are related to the physiological system, the histamine stimulation, and the irreversible effect of omeprazole on the H⁺, K⁺-ATPase enzyme. Four different experiments with omeprazole (Exps. 1–4) and two placebo experiments were performed in each of the four Heidenhain pouch dogs used. For placebo and experiments 1–3, saline or omeprazole 0.81 mol/kg was infused during 3 hr with measurements of histamine-stimulated gastric acid secretion in two periods of 3.5–6.5 hr, one period starting just before the omeprazole infusion and a second later period up to 29 hr post infusion. In experiment 4, 0.18 mol/kg of omeprazole was infused for 22.5 min and gastric juice was collected for 5 hr post infusion. The response data was well described by the model. Similar parameter estimates were obtained by three different analysis methods; naïve pooling, two-stage method and nonlinear mixed effects modeling. The elimination rate constant for the H⁺, K⁺-ATPase enzyme, k _out, was estimated to be 0.040 hr^-1, corresponding to a half-life of about 17 hr. This rate constant determines the duration of omeprazole inhibition after long-term exposure. For short-term omeprazole exposure the duration is determined by the rate constant for transfer of enzymes from active to resting state, estimated to be 1.88 hr^-1. The second-order rate constant for histamine stimulation was estimated to be 0.064 hr^-1 per histamine concentration unit and the maximum acid secretion was estimated to be 5.0 mmol H⁺/30 min. The second-order rate constant for the irreversible binding of omeprazole to H⁺, K⁺-ATPase, k _ome, was estimated to be 2.39 L/mol hr. By modeling the histamine-induced baseline response simultaneously with active treatment, predictions of the response are possible not only following different dosing regimens of omeprazole, but also following different degrees of histamine stimulation.     

K+-evoked release of met-enkephalin from rat striatum in vitro: Effect of putative neurotransmitters and morphine

Summary The effects of drugs on the K⁺-evoked release of met-enkephalin from superfused rat striatal slices were investigated using a specific radioimmunoassay. GABA, at concentrations of 50M and 100 M, and the GABA agonist muscimol (50 M), significantly inhibited the release. The inhibitory effect of GABA was reversed by picrotoxin suggesting that GABA inhibition is mediated by GABA receptors. Selected concentrations of the dopamine agonists apomorphine and ergonovine, as well as of haloperidol, acetylcholine, carbachol, noradrenaline, glutamic acid and substance P, had no effect on the release of metenkephalin. Increases in the evoked release (80%) and striatal enkephalin content (60%) were found in rats after chronic haloperidol administration, pointing to an increase in the synthesis and utilization of striatal enkephalin. No differences were found between the release from slices from morphine-tolerant/dependent and naive rats or after addition of naloxone to slices derived from tolerant/dependent animals. Selected concentrations of morphine and naloxone had no effect on release suggesting the absence of a mechanism for the regulation of enkephalin release involving autoreceptors.