Some degree of overlap exists between the K+-channels opened by cromakalim and those opened by minoxidil sulphate in rat isolated aorta

Summary The effects of the K⁺ channel opening drugs minoxidil sulphate and cromakalim, on ⁴²K⁺ and ⁸⁶Rb⁺ efflux and on vasorelaxation in rat isolated aorta, were compared. In rat aortic rings precontracted with noradrenaline (100 nmol/l), minoxidil sulphate and cromakalim concentration-dependently inhibited induced tension by up to 90%, with pD₂ values of 7.35±0.1 and 7.17±0.1, respectively. Glibenclamide (300 nmol/l), produced 2200- and 19-fold rightward shifts in the concentration-relaxation curves to minoxidil sulphate and cromakalim, respectively, without an effect on the maximum relaxation.Both minoxidil sulphate and cromakalim increased the efflux of ⁴²K⁺ and ⁸⁶Rb⁺ from aorta in a concentration-dependent manner, with midpoints in the µmol/l range; the maximum efflux induced by minoxidil sulphate being approximately one tenth of that induced by cromakalim. The ratio of stimulated ⁸⁶Rb⁺/⁴²K⁺ efflux increased from 0.22 to 0.48 with increasing cromakalim concentrations, but was approximately constant (0.39) when the minoxidil sulphate concentration was varied. In the presence of minoxidil sulphate, the effects of cromakalim on ⁴²K⁺ and ⁸⁶Rb⁺ efflux were inhibited in a concentration-dependent manner, by up to 60%. In the continuing presence of cromakalim (300 nmol/l), minoxidil sulphate (10 µmol/l)-induced increases in ⁴²K⁺ and ⁸⁶Rb⁺ efflux were inhibited by 45%, whereas conditioning with cromakalim (1 µmol/l) inhibited the ⁸⁶Rb⁺ efflux stimulated by additional superfusion of cromakalim (1 µmol/l) by 85%. Glibenclamide inhibited minoxidil sulphate (10 µmol/l)- and cromakalim (1 µmol/l)-induced increases in ⁴²K⁺ and ⁸⁶Rb⁺ efflux in a concentration-dependent manner with IC₅₀ values of approximately 80 nmol/l.In conclusion, the efflux data suggest that considerable overlap exists between the channels opened by minoxidil sulphate and those opened by cromakalim in rat aorta. Minoxidil sulphate has a weak efficacy as a K⁺ channel opener, and may act to open a homogeneous population of K⁺ channels. In contrast, the actions of cromakalim (1 µmol/l) are associated with large increases in tracer efflux, which are probably mediated via a heterogeneous population of K⁺ channels. However, only a small proprtion of this induced efflux appears to be required for relaxation. The differential inhibition by glibenclamide of the vasorelaxant effects of minoxidil sulphate and cromakalim may result from (a) the partial agonist properties of minoxidil sulphate in opening K⁺ channels and/or (b) additional mechanisms of vasorelaxation, which differ in their sensitivity to glibenclamide. Send offprint requests to U. Quasi 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     

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     

Ba2+ differentially inhibits the Rb+ efflux promoting and the vasorelaxant effects of levcromakalim and minoxidil sulfate in rat isolated aorta

The K⁺ channel openers activate ATP-sensitive K⁺ channels (K_ATP) in vascular smooth muscle and induce relaxation. In this study, the relationship between these two effects was examined in rings of rat aorta using levcromakalim and minoxidil sulfate as the openers and Ba²⁺ as the K⁺ channel blocker; K⁺ channel opening was assessed by determining the rate constant of ⁸⁶Rb⁺ efflux from the preparation.Ba²⁺ inhibited the ⁸⁶Rb⁺ efflux stimulated by levcromakalim in a noncompetitive manner with an IC₅₀ value of 29 M and a Hill-coefficient of 1.2. At concentrations > 300 M, Ba²⁺ increased the tension of rat aortic rings concentration-dependently. Levcromakalim relaxed contractions to Ba²⁺ (0.5 and 1 mM) with potencies similar to those determined against KCl (25 mM) or noradrenaline as spasmogens (EC₅₀ values 15–40 nM). The vasorelaxant effect against Ba²⁺ was inhibited by the K_ATP channel blockers, glibenclamide and tedisamil, and abolished in depolarizing medium (55 mM KCl). At 3 mM Ba²⁺, levcromakalim was still able to transiently induce complete relaxation; however, within 1 h oscillations in tension developed, leading to a stable level of only 15% relaxation. A similar level of relaxation was achieved against 10 mM Ba²⁺ whereas the combination of 0.5 mM Ba²⁺ and 3 M tedisamil blocked the relaxant effect of levcromakalim completely. With minoxidil sulfate as the K_ATP channel opener the results of the ⁸⁶Rb⁺ efflux and tension experiments were similar to those obtained with levcromakalim.It is concluded that Ba²⁺ is more potent in inhibiting the K⁺ channel opening than the vasorelaxant effects of the openers. On the basis of the ⁸⁶Rb⁺ efflux experiments it is estimated that at least 97% of the channels opened by the activators can be blocked without major effects on vasorelaxation suggesting a dissociation between the two effects. However, if the block is pushed to extremes ( 99.95%) the vasorelaxant effect of the openers is also abolished suggesting a link between both effects. This paradoxon remains to be solved.     

Activators of protein kinase A induce a glibenclamide-sensitive 86Rb+ efflux in rat isolated aorta

The effect of activators of protein kinase A on membrane K⁺ permeability and the interaction of these compounds with cromakalim, an opener of ATP-sensitive K⁺ channels (K_ATP channels), were investigated. Membrane K⁺ permeability was assessed by measuring ⁸⁶Rb⁺ efflux from rings of rat aorta. Forskolin, an activator of adenylate cyclase, and isobutylmethylxanthine (IBMX), a nonselective phosphodiesterase inhibitor, induced small, concentration-dependent increases in tracer efflux up to 20-40% over the basal level. The effect of forskolin was abolished by the K⁺ channel blocker tedisamil (1 μM) and partially inhibited by glibenclamide (1 μM), a relatively selective blocker of K_ATP channels. Further studies were conducted in the presence of 35mM KCl in the bath in order to increase the size of the ⁸⁶Rb⁺ efflux stimulated by forskolin and IBMX. At high concentrations, these compounds produced a biphasic effect with a peak increase being followed by a lower plateau value. Glibenclamide inhibited the ⁸⁶Rb⁺ efflux response to forskolin and IBMX by 50-80%. The K⁺ channel blockers tedisamil (1 μM), Ba²⁺ (1mM) and tetraethylammonium (10mM) also reduced the peak response to forskolin by about 50% and abolished or greatly inhibited the plateau response. In addition to the small effect on basal ⁸⁶Rb⁺ efflux, forskolin (0.3 μM) increased cromakalim-induced ⁸⁶Rb⁺ efflux 3.4 times. At higher concentrations, however, a concentration-dependent inhibition was observed with an IC₅₀ value of 7.6 ±0.4 μM. 1,9-dideoxyforskolin, which does not increase cAMP, increased neither basal nor cromakalim-induced ⁸⁶Rb⁺ efflux; however, it inhibited cromakalim-stimulated tracer efflux with an IC₅₀ value of 22 ±2 μM. It is concluded that forskolin and IBMX, probably by increasing intracellular cAMP levels, induce a ⁸⁶Rb⁺ efflux from rat aorta, the major part of which is glibenclamide-sensitive and may pass through K_ATP channels. In addition, low concentrations of forskolin greatly facilitate the K_ATP channel opening effect of cromakalim whereas high concentrations block the channel; this blocking effect of forskolin is unrelated to the cAMP elevating action. Received: 25 September 1996 / Accepted: 20 December 1996     

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.     

The dualistic mode of action of the vasodilator drug,nicorandil, differentiated by glibenclamide in 86Rb flux studies in rabbit isolated vascular smooth muscle

Summary (1) In rabbit isolated aorta, the effects of the antianginal drug, nicorandil, of the K⁺ channel opener, cromakalim, and of the nitrovasodilator, sodium nitroprusside, on ⁸⁶Rb efflux and on contractile force were compared. (2) In ion flux studies using ⁸⁶Rb as a marker of K⁺ ions, both nicorandil and cromakalim, but not sodium nitroprusside, increased the efflux of ⁸⁶Rb in non-stimulated preparations. The increase of membrane K⁺ conductance induced by nicorandil and cromakalim was totally suppressed by 10^–5 mol/l of the sulfonylurea derivative, glibenclamide. (3) The vasoconstrictor drug, noradrenaline (3 × 10^–7 mol/l), effectively increased the rate of ⁸⁶Rb efflux. This stimulatory response was unaffected by glibenclamide, but was totally inhibited by Ca²⁺ depletion suggesting that the activation of Ca²⁺-sensitive K⁺ channels was responsible for this action of noradrenaline. (4) Sodium nitroprusside and nicorandil, the latter in the presence of glibenclamide to suppress the glibenclamide-sensitive stimulatory component on ⁸⁶Rb efflux, antagonized the noradrenaline-induced increase in ⁸⁶Rb efflux, while cromakalim in the presence of glibenclamide had no effect. (5) All of the vasodilators relaxed rabbit aortic strips contracted by 10^–7 mol/l noradrenaline in a concentration-dependent manner. (6) The vasodilatory response to cromakalim was antagonized by glibenclamide, whereas the relaxant action of nicorandil and of sodium nitroprusside remained unaffected. (7) These results demonstrate that under particular experimental circumstances, nicorandil can behave in vascular smooth muscle both as an opener of glibenclamide-sensitive K⁺ channels, and as a directly acting nitrovasodilator which acts via reduction of cellular calcium levels. Nevertheless, its vasorelaxant action seems to depend primarily on its nitrovasodilator-like properties. (8) ⁸⁶Rb efflux studies are a suitable technique to differentiate between the cellular actions of vasodilators involving a reduction of intracellular calcium such as the nitrovasodilators, or acting similarly as cromakalim, i. e. presumably via opening of K⁺ channels. Supported by The Deutsche Forschungsgemeinschaft Offprint requests to V. A. W. Kreye at the above address     

The action of diazoxide and minoxidil sulphate on rat blood vessels: a comparison with cromakalim.

1. The actions of diazoxide and minoxidil sulphate have been compared with those of cromakalim in rat aorta and portal vein. 2. Diazoxide and minoxidil sulphate hyperpolarized the rat portal vein in a similar manner to cromakalim. 3. Cromakalim, diazoxide and minoxidil sulphate increased 42K and 86Rb efflux from rat portal vein, although minoxidil sulphate had only a small effect on 86Rb efflux. 4. Cromakalim, diazoxide and minoxidil sulphate increased 42K efflux from rat aorta but only cromakalim and diazoxide increased 86Rb efflux from this tissue. 5. Glibenclamide inhibited the relaxant actions of cromakalim, diazoxide and minoxidil sulphate on rat aorta and the increase in 42K efflux produced by these agents in this tissue. 6. Diazoxide relaxed an 80 mM KCl-induced contraction of rat aorta, whilst cromakalim and minoxidil sulphate were without effect. 7. Cromakalim, diazoxide and minoxidil sulphate had no effect on cyclic AMP or cyclic GMP concentrations in rat aorta. 8. It is concluded that diazoxide and minoxidil sulphate like cromakalim exhibit K+ channel opening properties in vascular smooth muscle. Diazoxide exerts an additional inhibitory action not related to the production of cyclic AMP or cyclic GMP. The action of minoxidil sulphate may be primarily located at a K+ channel which is relatively impermeable to 86Rb.     

The effects of (−)-cromakalim and glibenclamide on uptake2 in guinea-pig trachealis muscle

Summary In a recent study, we have shown that hyperpolarization of cells by -adrenoceptor agonists results in stimulation of the uptake₂ process for catecholamines. The aim of the present study was to further explore the hypothesis that uptake₂ is dependent on membrane potential by examining the effects of the K⁺-channel opening drug, (–)-cromakalim, and the K⁺-channel blocking drug, glibenclamide, on uptake₂ of isoprenaline. The effects of these drugs were examined in guinea-pig trachealis muscle, in which isoprenaline and cromakalim cause hyperpolarization, and in rat heart, in which isoprenaline and cromakalim have little effect on membrane potential.In guinea-pig trachealis muscle segments, 1 mol/l glibenclamide reduced uptake₂ (as measured by the steady-state rate of corticosterone-sensitive formation of ³H-3-O-methylisoprenaline normalized for the isoprenaline concentration) in tissues incubated in concentrations of ³H-(±)-isoprenaline that hyperpolarize the muscle (25 and 250 nmol/l) but not at an isoprenaline concentration that did not hyperpolarize the muscle (1 nmol/l). (–)-Cromakalim (10 mol/l), which hyperpolarizes the trachealis muscle, increased uptake₂ of isoprenaline (1 or 25 nmol/l) and this effect of (–)-cromakalim was inhibited by glibenclamide. In rat hearts perfused with 1 or 25 nmol/l ³H-(±)-isoprenaline and 10 mol/l U-0521 to inhibit catechol-O-methyltransferase, the rate of uptake₂ of isoprenaline was unaffected by cromakalim or glibenclamide.The results show that hyperpolarization of cells by various mechanisms can result in stimulation of uptake₂ of catecholamines and provide further evidence to support the hypothesis that the uptake₂ transport process is driven by the membrane potential of cells.Preliminary results of part of this study were presented to the German Society for Pharmacology and Toxicology (Bryan-Lluka and Vuocolo 1991)Correspondence to L. J. Bryan-Lluka at the above address     

Pharmacological analysis of the inhibitory effects of KRN2391 on endothelin-1-induced contraction in isolated large coronary artery of the pig

1. The relaxant effect of KRN2391, N-cyano-N′-(2-nitroxyethyl)-3-pyridine-carboximidamide-monomethanesulfonate (with both K⁺ channel opener and nitrate actions), nifedipine (Ca²⁺ channel blocker), nitroglycerin (nitrate) and cromakalim (K⁺ channel opener) were investigated in isolated porcine large coronary arteries contracted by endothelin-1. These drugs inhibited endothelin-1-induced contraction in a concentration-dependent manner.2. The relaxation induced by KRN2391 was nearly complete at their maximum effects, but nifedipine and cromakalim could not produce complete relaxation.3. The concentration-relaxation curves for KRN2391 underwent a rightward shift in the presence of methylene blue or glibenclamide. The concentration ratios of KRN2391 calculated based on EC₅₀ values were 2.8 and 3.7 in the presence of methylene blue and glibenclamide, respectively.4. The concentration-relaxation curves for nitroglycerin and cromakalin underwent a rightward shift in the presence of methylene blue and glibenclamide, respectively, and the concentration ratios of nitroglycerin and cromakalim were 12.0 and 6.3.5. These relaxant effects of KRN2391 and nitroglycerin on endothelin-1-induced contraction of porcine coronary artery were greater than those of cromakalim and nifedipine. This potent relaxant action of KRN2391 on endothelin-induced contraction is thought to be based on both a nitrate action and a K⁺ channel opening action.     

Pertussis toxin treatment does not inhibit the effects of the potassium channel opener BRL 34915 on rat isolated vascular and cardiac tissues

Summary The experiments were undertaken to determine whether the effects of the K⁺ channel opener BRL 34915 on rat isolated vascular smooth muscle and atria were sensitive to pertussis toxin (PTx). PTx treatment of rats (100 g/kg, infused over 15 min) affected some baseline parameters of the isolated tissues: in the atria, heart rate was increased, contractile force was decreased and the basal efflux of ⁸⁶Rb⁺ was increased; in portal veins, the spontaneous activity was decreased but the contractility of aortic rings was unaffected. In the isolated atria removed from saline-treated rats, carbamylcholine decreased heart rate and contractile force, shortened the action potential duration by increasing the maximum rate of repolarization and increased ⁸⁶Rb⁺ efflux. These effects of carbamylcholine were completely abolished in the atria from PTx-treated rats, demonstrating the efficacy of the toxin. The ability of 300 M BRL 34915 and of 55 mM KCl to increase atrial ⁸⁶Rb⁺ permeability was, however, only slightly affected by PTx treatment. In portal veins from PTx-treated rats, the efficacy of BRL 34915 to inhibit spontaneous activity and to increase ⁸⁶Rb⁺ efflux was the same as in control organs. Similarly, in aortic rings, the ability of BRL 34915 to inhibit contractions to low concentrations of KCl or to noradrenaline was unaffected by PTx treatment as was the ⁸⁶Rb⁺ efflux response to BRL 34915 in this tissue. It is concluded that PTx treatment does not inhibit the effects of BRL 34915 in the tissues investigated. The results are compatible with the notion that BRL 34915 does not open K⁺ channels by acting through a PTx-sensititive G-protein. Send offprint requests to U. Quast     

Differential antagonism by glibenclamide of the relaxant effects of cromakalim,pinacidil and nicorandil on canine large coronary arteries

Summary The relaxant mechanisms of action of cromakalim, pinacidil and nicorandil, potassium channel openers, on large epicardial coronary arteries were investigated in isolated canine left circumflex arteries contracted by 10^–7 mol/l U46619, a thromboxane A₂ analogue, or addition of 25 mmol/l KCl in comparison with nitroglycerin.Cromakalim (3 × 10^–8–3 × 10^–5 mol/l), pinacidil (10^–6–10^–4 mol/l), nicorandil (3 × 10^–6–10^–3 mol/l) and nitroglycerin (3 × 10^–8–10^–5 mol/l) all produced a concentration-dependent relaxation in both U46619- or KCl-contracted arteries. At their maximum effects pinacidil, nicorandil and nitroglycerin produced full relaxation in arteries contracted by either means. In contrast, cromakalim produced about a 73% relaxation in KCl-contracted arteries, although it caused full relaxation in U46619-contracted ones. In the presence of glibenclamide the concentration-relaxation curves for cromakalim in U46619- or KCl-contracted arteries underwent rightward parallel shifts. Schild regression had a slope of 1.00 and yielded a pA₂ of 7.47 for glibenclamide in U46619-contracted arteries, and corresponding values obtained in KCl-contracted arteries were 0.86 (not significantly different from unity) and 7.28. The concentration-relaxation curves for pinacidil in U46619-contracted arteries also underwent rightward parallel shifts in the presence of glibenclamide, however, Schild regression had a slope of 0.60. The concentration-relaxation curves for pinacidil in KCl-contracted arteries underwent rightward parallel shifts only to a limited extent in the presence of glibenclamide. The concentration-relaxation curves for nicorandil and nitroglycerin in U46619- or KCl-contracted arteries were not affected by glibenclamide in concentrations which antagonized cromakalim. The concentration-relaxation curves for nicorandil or nitroglycerin in U46619-contracted arteries were shifted by methylene blue (10^–5 mol/l) to the right without suppression of the their maximum effects. Similar curves for cromakalim were not affected at all by this concentration of methylene blue. The concentration-relaxation curves for nicorandil in U46619-contracted arteries determined in the presence of methylene blue (10^–5 mol/l) and glibenclamide (3 × 10^–7, 10^–6 and 3 × 10^–6 mol/l) were not significantly different from those in the presence of methylene blue alone.These results indicate the following: In canine large epicardial coronary arteries (1) cromakalim produced relaxation by the mechanism antagonized by glibenclamide, probably opening ATP-sensitive potassium channels, (2) pinacidil did so by the mechanism shared with cromakalim and by one not antagonized by glibenclamide as well, and (3) nicorandil did so exclusively by the mechanism of action as a nitrate. Send offprint requests to N. Taira at the above address     

Analysis of the hyperpolarizing effects of forskolin in guinea-pig atrial heart muscle

Summary The effects of forskolin on action potential configuration and on both uptake and efflux of ⁸⁶Rb⁺ were studied in guinea-pig left atria. The action potential was prolonged by forskolin in the plateau range but shortened at the end of repolarization; maximal upstroke velocity and amplitude of slow response potentials were enhanced. In partially depolarized preparations, the resting potential was increased by forskolin; this effect was not prevented by atropine 1 mol/1. Forskolin augmented the rate constant of ⁸⁶Rb⁺ efflux in beating and in resting preparations. The uptake of ⁸⁶Rb^s+ was enhanced by forskolin in resting preparations. It is concluded that forskolin stimulates the Na⁺, K⁺ -pump and activates a background potassium conductance. Both effects may account for the shortening effect of the drug on the action potential and the increase in resting potential seen in partially depolarized preparations. Send offprint requests to H. Nawrath     

Inhibition by protein kinase C of the 86Rb+ efflux and vasorelaxation induced by P1075, a KATP channel opener, in rat isolated aorta

In rat aortic rings, P1075, an opener of ATP-dependent potassium channels (K_ATP channels), produces relaxation and ⁸⁶Rb⁺ efflux from preloaded tissues; the increase in ⁸⁶Rb⁺ efflux qualitatively reflects K_ATP channel opening. In this study we have investigated the effects of protein kinase C modulation on the ⁸⁶Rb⁺ efflux stimulating, the vasorelaxant and the binding properties of P1075. Phorbol 12,13-dibutyrate (PDBu), a direct activator of protein kinase C, inhibited the ⁸⁶Rb⁺ efflux produced by P1075 with an IC₅₀ value of 20±2nM. Phorbol 12-myristate 13-acetate (PMA), another stimulator of protein kinase C, was 150 times weaker in this respect whereas 4α-PDBu, the inactive stereoisomer of PDBu, was ineffective. Staurosporine (300nM), an inhibitor of protein kinase C, induced a small but significant increase of P1075-induced tracer efflux and partially reversed the inhibitory effect of PDBu on P1075-stimulated tracer efflux. The vasorelaxant effect of P1075 was inhibited only to a moderate degree by PDBu at concentrations which inhibited P1075-induced ⁸⁶Rb⁺ efflux to >90%; however, in the presence of PDBu, the relaxation kinetics of P1075 were increasingly slowed. The vasorelaxant effect of P1075 in the presence of PDBu was still sensitive to inhibition by glibenclamide (100nM), the standard inhibitor of the K_ATP channel openers. Specific binding of [³H]-P1075 to rat aortic rings was unaffected by PDBu and PMA even in the micromolar concentration range. The data show that stimulation of protein kinase C inhibits the K⁺ channel opening effect of P1075 in rat aorta and suggest that protein kinase C may exert a weak tonic inhibition on the K_ATP channels in this vessel under quasiphysiological conditions. At concentrations of PDBu which essentially abolished P1075-induced tracer efflux, the glibenclamide-sensitive vasorelaxant effect of P1075 was slowed down but not prevented; this supports earlier suggestions that K⁺ channel openers are also able to relax smooth muscle cells by a mechanism independent of K_ATP channel opening. Received: 11 March 1997 / Accepted: 12 May 1997     

Interaction between thiol-modifying agents and P1075, a KATP channel opener, in rat isolated aorta

In vascular smooth muscle, openers of ATP-dependent potassium channels (K _ATP channels), such as P1075 (N-cyano-N’-(1,1-dimethylpropyl)-N’’-3-pyridylguanidine), produce relaxation. In this study we have investigated the effects of thiol-modifying agents on the binding of P1075 and on the ⁸⁶Rb⁺ efflux stimulating and vasorelaxant effects of the opener in rat aortic rings. The increase in ⁸⁶Rb⁺ efflux induced by P1075 was taken as a qualitative measure of K⁺ channel opening. The hydrophilic SH-group-oxidizing substance, thimerosal (1 to 100μM), abolished specific binding of [³H]-P1075 with an IC₅₀ value of 7.6±1.2μM; at 30μM, the half time for inhibition was 38min. Two other thiol-oxidizing agents, PMB (4-hydroxy-mercuribenzoic acid) and DTBNP (2,2’-dithio-bis(5-nitropyridine)), inhibited binding up to 86% and 44%, respectively. The disulphide bond reducing substance, DTT (1,4-dithiothreitol, 0.1 to 1mM), reduced [³H]-P1075 binding by up to 20% and partially reversed the inhibitory effect of thimerosal. In ⁸⁶Rb⁺ efflux experiments, thimerosal (3 to 100μM) concentration-dependently increased basal efflux but inhibited P1075-stimulated tracer efflux with an IC₅₀ value of 7±1μM. The inhibitory effect occurred with a half-time of approximately 8min and was essentially reversed by DTT. In rings precontracted with noradrenaline, thimerosal inhibited the vasorelaxant effect in a noncompetitive manner, shifting the concentration-relaxation curves to the right and reducing maximum relaxation.The data show that oxidation of thiol groups interferes with the binding of the K _ATP channel opener, P1075; concomitantly, the ⁸⁶Rb⁺ efflux stimulating and the vasorelaxant effects are inhibited. Reduction of disulphide bonds by DTT has only minor effects on the action of P1075. Collectively, the results suggest that intact thiol groups are essential for the functioning of the K_ATP channel in rat aorta. The different kinetics governing the inhibition of opener binding and of opener-stimulated ⁸⁶Rb⁺ efflux suggest that the SH-groups involved in the two processes differ in their accessibility to thimerosal and/or in their reactivity. Received: 7 April / Accepted: 9 July 1997     

Concentration-Response Relationship of α1-Adrenoceptor-Stimulated Increase of 86Rb+ Efflux in Rat Heart*

Abstract: The aim of the present study was to establish a concentration-response relationship for the α₁-adrenoceptor mediated increase of ⁸⁶Rb⁺ efflux, and to characterize the sensitivity of this response to the selective α₁-adrenoceptor antagonist prazosin. Isolated rat hearts were perfused retrogradely at constant flow and at 31°. Timolol (10^?6 mol/l) was used to block β-adrenoceptors. After a loading period with ⁸⁶Rb⁺ and 55 min. washout, the hearts were exposed to phenylephrine in a concentration range from 3×10^?8 mol/l to 10^?4 mol/l. Control experiments comparing the effects of α₁-adrenoceptor stimulation on ⁸⁶Rb⁺ efflux and ⁴²K⁺ efflux were performed. α₁-Adrenoceptor stimulation increased the ⁸⁶Rb⁺ efflux with a pD₂=6.35±0.20 (mean±S.E.M.). The maximal response to phenylephrine was 22.5±2.0% (mean±S.E.M.) of the control values. The concentration-response curve was shifted to higher concentration of agonist in the presence of the α₁-adrenoceptor antagonist prazosin (3× 10^?10 mol/l). The calculated inhibition constant for prazosin was 6.1×10^?11 mol/l. ⁸⁶Rb⁺ was found to be a suitable K⁺ analogue in the study of relative changes in K⁺ efflux although the basal efflux kinetics were different for the two isotopes. Conclusion: Phenylephrine increased the ⁸⁶Rb⁺ efflux concentration-dependently. A high sensitivity to prazosin confirmed the involvement of the α₁-adrenoceptor population.     

Activators of potassium channels enhance calcium influx into endothelial cells as a consequence of potassium currents

Summary Ca²⁺ influx into stimulated endothelial cells is attenuated by depolarization. We hypothesized that Ca²⁺ influx is driven by the membrane potential and may be enhanced by hyperpolarizing drugs like activators of K⁺ channels. Therefore we studied the effects of pinacidil, cromakalim, and cicletanine on membrane currents and on the intracellular free calcium concentration ([Ca²⁺]i) in cultured endothelial cells from porcine aorta. In patch-clamped cells, pinacidil (1 mol/l) and cromakalim (1 mol/l) elicited outward currents carried by K⁺ and significantly prolonged the Ca²⁺-dependent K⁺ currents induced by bradykinin and ATP. Peak currents in response to bradykinin were not affected. In cells loaded with the fluorescent Ca²⁺ indicator indo-1 and prestimulated with thimerosal, pinacidil (0.1–1 mol/l elicited long-lasting increases in [Ca²⁺)_i from 100 ± 10 to 550 ± 110 nmol/l. These effects were completely abolished in a medium containing 90 mmol/l K⁺. Similar results were obtained with cromakalim. Likewise, in cells stimulated with bradykinin, pinacidil raised [Ca²⁺]_i when applied during the decline of [Ca²⁺]_i after the initial peak. Cicletanine elicited K⁺ currents in resting and attenuated K⁺ currents in bradykinin-stimulated cells. It elevated [Ca²⁺]_i even in the absence of extracellular Ca²⁺ and in K⁺-rich medium. Hence, the effects of cicletanine cannot be explained by direct actions on K⁺ channels. However, our studies demonstrate that pinacidil and cromakalim elevate [Ca²⁺]_i secondary to their activation of K⁺ channels by inducing hyperpolarization and augmenting the driving force for potential-dependent Ca²⁺ influx. In this way, the two drugs may promote Ca²⁺-dependent formation of endothelium-derived relaxing factor. Send offprint requests to A. Lückhoff at the above address     

Interaction of the diuretics torasemide and U-37883A with the KATP channel in rat isolated aorta

In this study we have investigated the interaction of the loop diuretics torasemide and furosemide and of the eukalemic diuretic U-37883A (4-morpholinocarboximidine-N–1-adamantyl-N’-cyclohexylhydrochloride) with the ATP-sensitive K⁺ channel (K_ATP channel) in rat aortic rings. Torasemide contains a sulphonylurea group which might enable the compound to interfere with K_ATP channels; this group is lacking in furosemide. U-37883A blocks several types of K_ATP channels. The interaction with the vascular K_ATP channel was probed in binding studies, ⁸⁶Rb⁺ efflux experiments and vasorelaxation assays. Torasemide inhibited the binding of the K_ATP channel inhibitor [³H]glibenclamide and of the opener [³H]P1075 with IC₅₀ values of 19 and 45 μM, respectively; furosemide and U-37883A were inactive or interfered with binding in a nonspecific way. In ⁸⁶Rb⁺ efflux experiments, the loop diuretics, at μM concentrations, inhibited basal tracer efflux to 50% whereas U-37883A had no effect. P1075-stimulated ⁸⁶Rb⁺ efflux, a qualitative measure of K_ATP channel opening, was inhibited by U-37883A and torasemide with IC₅₀ values of 0.06 and 130 μM, respectively; furosemide induced only a small (23%) inhibition. In experiments measuring isometric force, torasemide and furosemide partially relaxed endothelium-denuded aortic rings precontracted with noradrenaline or KCl with EC₅₀ values between 6 and 10 μM. The vasorelaxant effect of P1075 was inhibited in a noncompetitive manner by torasemide (300 μM) but unaffected by furosemide. U-37883A increased noradrenaline-induced force and inhibited the vasorelaxant effect of P1075 in an apparently competitive manner with an inhibition constant of 0.4 μM. The data show that torasemide interferes specifically with the binding of the K_ATP channel modulators [³H]glibenclamide and [³H]P1075 and with the K_ATP channel opening and vasorelaxant effects of P1075 whereas furosemide is inactive. This suggests that the interaction of torasemide with the vascular K_ATP channel is due to the sulphonylurea group present in torasemide. U-37883A, which does not inhibit P1075 binding, is one of the most potent blockers of P1075-induced ⁸⁶Rb⁺ efflux yet described but is relatively weak as an inhibitor of P1075-mediated vasorelaxation. The opposite vascular actions of torasemide and U-37883A are expected to contribute to the renal effects of these drugs. Received: 28 January 1998 / Accepted: 20 April 1998     

Activation of central ATP-sensitive potassium channels produces the antinociception and spinal noradrenaline turnover-enhancing effect in mice

ICV cromakalim, a K⁺ channel opener, produced antinociception. This effect was completely antagonized by ICV glibenclamide, a selective adenosine triphosphate-sensitive K⁺ channel (K_ATP channel) blocker. Furthermore, direct opening of central K_ATP channels by ICV cromakalim increased the spinal noradrenaline (NA) turnover. On the other hand, the antinociception induced by ICV morphine ( opioid agonist), but not ICV U-50,488H ( opioid agonist) was markedly potentiated by cromakalim. These findings suggest that the opening of central K_ATP channels may elicit the antinociceptive effect and activate the descending NAergic pathway, and central K_ATP channels play an important role as a modulator of the antinociception induced by agonists but not agonists.     

Comparison of the cromakalim antagonism and bradycardic actions of a series of novel alinidine analogues in the rat

Alinidine, and eight derivatives, were synthesized and tested for their ability to antagonise the actions of the K⁺ channel opener cromakalim in rat thoracic aorta, and for their ability to induce bradycardia in rat isolated spontaneously beating right atria. Ring segments of rat thoracic aorta were suspended in organ baths to record isometric tension. Tissues were precontracted with K⁺(20 mM), and full concentration-relaxation curves constructed to cromakalim (0.01–30 M) in the absence and presence of increasing concentrations of alinidine/derivative. The majority of the compounds tested caused rightward shifts in the cromakalim concentration-effect curves. Rat spontaneously beating right atria were suspended in organ baths to record rate of contraction. Addition of alinidine/derivative caused a concentration-dependent negative chronotropic response. In terms of structure-activity relationships, increasing the length of the N-allyl side-chain on the alinidine molecule (from 3 carbon (3C), to 5 C) resulted in a significant increase in the activity of the compounds as both bradycardic agents and cromakalim antagonists. The most potent compounds in both cases (bradycardic agent and cromakalim antagonist) had no double bond in the side chain. The results suggest that the carbon side-chain influences the activity of alinidine-related compounds both as cromakalim antagonists and as bradycardic agents. However, while similar structure-activity relationships appear to apply for both effects in some instances, there was no significant correlation between the two actions of the alinidine analogues. The results suggest that the ability of alinidine-derivatives to induce bradycardia or to block K⁺ channels opened by cromakalim can be differentiated on the basis of structure. Correspondence to: G. A. McPherson at the above address