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     

ATP-sensitive K+ channels in the kidney

ATP-sensitive K⁺ channels (K_ATP channels) form a link between the metabolic state of the cell and the permeability of the cell membrane for K⁺ which, in turn, is a major determinant of cell membrane potential. K_ATP channels are found in many different cell types. Their regulation by ATP and other nucleotides and their modulation by other cellular factors such as pH and kinase activity varies widely and is fine-tuned for the function that these channels have to fulfill. In most excitable tissues they are closed and open when cell metabolism is impaired; thereby the cell is clamped in the resting state which saves ATP and helps to preserve the structural integrity of the cell. There are, however, notable exceptions from this rule; in pancreatic -cells, certain neurons and some vascular beds, these channels are open during the normal functioning of the cell.In the renal tubular system, K_ATP channels are found in the proximal tubule, the thick ascending limb of Henle's loop and the cortical collecting duct. Under physiological conditions, these channels have a high open probability and play an important role in the reabsorption of electrolytes and solutes as well as in K⁺ homeostasis. The physiological role of their nucleotide sensitivity is not entirely clear; one consequence is the coupling of channel activity to the activity of the Na-K-ATPase (pump-leak coupling), resulting in coordinated vectorial transport. In ischemia, however, the reduced ATP/ADP ratio would increase the open probability of the K_ATP channels independently from pump activity; this is particularly dangerous in the proximal tubule, where 60 to 70% of the glomerular ultrafiltrate is reabsorbed.The pharmacology of K_ATP channels is well developed including the sulphonylureas as standard blockers and the structurally heterogeneous family of channel openers. Blockers and openers, exemplified by glibenclamide and levcromakalim, show a wide spectrum of affinities towards the different types of K_ATP channels. Recent cloning efforts have solved the mystery about the structure of the channel: the K_ATP channels in the pancreatic -cell and in the principal cell of the renal cortical collecting duct are heteromultimers, composed of an inwardly rectifying K⁺ channel and sulphonylurea binding subunit(s) with unknown stoichiometry. The proteins making up the K_ATP channel in these two cell types are different (though homologous), explaining the physiological and pharmacological differences between these channel subtypes.     

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     

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     

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     

Effects of the potassium channel openers KRN4884 and levcromakalim on the contraction of rat aorta induced by A23187, compared with nifedipine

We examined the different vasodilatory effects of the K⁺ channel openers levcromakalim and 5-amino-N2-[2-(2-chlorophenyl)ethyl]-N-cyano-3-pyridinecarboxamidine (KRN4884), and the Ca²⁺ channel blocker nifedipine in the rat aorta. KRN4884 (10^–10-10^–5 M) and nifedipine (10^–10–10^–5 M) produced concentration-dependent relaxation in the rat aorta precontracted by 25 mM KCl. The K⁺ channel blocker glibenclamide (1 M) inhibited the relaxation induced by KRN4884 but did not influence nifedipine-induced relaxation. KRN 4884 had almost no effect on contraction induced by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl. These results indicate that KRN4884 is a K+ channel opener. We investigated the relaxant effects of KRN4884 (10^–10-10^–5 M), levcromakalim (10^–9-10^–5 M) and nifedipine (10^–9-10^–5 M) on A23187 (1 M)-induced contraction. KRN4884 and levcromakalim had a potent relaxant effect but nifedipine only a weak effect on the smooth muscle contracted by A23187. Glibenclamide (1 M) inhibited the relaxation induced by KRN4884 and levcromakalim, but did not influence the nifedipine-induced relaxation. KRN 4884 (1 M) produced a larger relaxation of A23187-induced contraction but had little effect on the increase in intracellular [Ca²⁺] induced by A23187. These results suggest that KRN4884 is a specific K⁺ channel opener and its vasodilating mechanisms involve not only deactivation of Ca²⁺ channels but also a decrease in the Ca²⁺ sensitivity of contractile elements.     

Role of Na+-K+ ATPase enzyme in vascular response of goat ruminal artery

Objective:

To study the role of Na⁺, K⁺- ATPase enzyme in the vascular response of goat ruminal artery.

Materials and Methods:

Ruminal artery was obtained in chilled aerated modified Krebs-Henseleit solution (KHS) from a local slaughterhouse and transported in ice for further processing. The endothelium intact arterial ring was mounted in a thermostatically controlled (37 ± 0.5°C) organ bath containing 20 ml of modified KHS (pH 7.4) bubbled with oxygen (95%) and CO₂ (5%) under 2g tension. An equilibration of 90 min was allowed before addition of drugs into the bath. The responses were recorded isometrically in an automatic organ bath connected to PowerLab data acquisition system. In order to examine intact functional endothelium, ACh (10 μM) was added on the 5-HT (1.0 μM) - induced sustained contractile response. Similarly, functional characterization of Na⁺, K⁺-ATPase activity was done by K⁺-induced relaxation (10 μM-10 mM) in the absence and presence of ouabain (0.1 μM/ 0.1 mM), digoxin (0.1 μM) and barium (30 μM).

Results:

ACh (10⁻⁵ M) did not produce any relaxing effect on 5-HT-induced sustained contractile response suggesting that vascular endothelium has no significant influence on the activation of sodium pump by extracellular K⁺ in ruminal artery. Low concentration of Ba²⁺ (30 μM) (IC₅₀: 0.479 mM) inhibited K⁺-induced relaxation suggesting K_ir (inward rectifier) channel in part had role in K⁺-induced vasodilatation in ruminal artery. Vasorelaxant effect of KCl (10 μM-10 mM) in K⁺-free medium is also blocked by ouabain (0.1 μM and 0.1 mM) (IC₅₀:0.398 mM and IC₃₅: 1.36 mM), but not by digoxin (0.1 μM) (IC₅₀ 0.234 mM) suggesting that ouabain sensitive Na⁺, K⁺-ATPase isoform is present in the ruminal artery.

Conclusion:

In the goat ruminal artery functional regulation of sodium pump is partly mediated by K⁺ channel and ouabain sensitive Na⁺, K⁺ ATPase.     

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     

Evidence that BKCa channel activation contributes to K+ channel opener induced relaxation of the porcine coronary artery

The rank order of potency of a series of benzopyran and cyanoguanidine K⁺ channel openers (KCOs) for causing relaxation of the PGF₂-precontracted porcine coronary artery was determined. Glyburide, an inhibitor of K_ATP channels, showed an apparent competitive inhibition of the vasorelaxant activity of the KCOs. The pA₂ values of glyburide when cromakalim and CGP 14877 (P1060) were used as vasorelaxants were 7.66 and 7.83, respectively. Charybdotoxin (40 nM), an inhibitor of BK_Ca channels, also caused a significant inhibition of the cromakalim mediated relaxation of the porcine coronary artery. In order to clarify the site of action of these KCOs, we identified a K⁺ channel current in single porcine coronary arterial cells and measured channel activity in the presence of these compounds. The prominent K⁺ ion current in these cells had characteristics typical of the conventional large Ca²⁺-activated K⁺ channel BK_Ca present in other smooth muscle cells. Using symmetrical K⁺ concentrations, the channel had a conductance of 214 pS and was found to be sensitive to [Ca²⁺]_i and membrane potential. The KCOs were found to reversibly increase the open probability (P_o) of the channel without changing channel conductance. The potency of the KCOs to increase K⁺ channel opening was similar to the potency of these compounds to cause coronary artery relaxation. These results indicate that the porcine coronary artery contains the BK_Ca channel and that this channel, along with other types of K⁺ channels (K_ATP), mediate the vasorelaxant effects of K⁺ channel openers.     

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     

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     

Binding and effects of P1075, an opener of ATP-sensitive K+ channels, in the aorta from streptozotocin-treated diabetic rats

Diabetes mellitus is associated with major vascular complications. It was the aim of this study to examine the function of the ATP-sensitive K⁺ channel (K_ATP channel) in aortic rings prepared from diabetic rats and from age-matched controls. Diabetes was induced by injection of streptozotocin (60mg/kg i.p.) and the animals were sacrificed 10 weeks after treatment. The binding of the K_ATP channel opener, P1075 (N-cyano-N’-(1,1-dimethylpropyl)-N’’-3-pyridylguanidine), as well as the vasorelaxant and ⁸⁶Rb⁺ efflux stimulating effects of the drug were measured. ATP channel opener P1075 against noradrenaline was shifted rightwards by a factor of 1.3 and the maximum relaxation was reduced from 81 to 71% of initial tension (P<0.01). However, specific binding of ³H-P1075 was increased by 20% without a change in affinity, indicating that the number of binding sites for the opener was increased as a consequence of diabetes. In addition, P1075-induced ⁸⁶Rb⁺ efflux, a qualitative measure of K_ATP channel opening, was augmented by 50%. + channel opening response to P1075 is markedly increased; however, the vasorelaxant effect to the K_ATP channel opener is slightly impaired. A possible explanation of these findings is that the vasorelaxant mechanisms (which are in part independent of plasmalemmal K_ATP channel opening) may be altered; alternatively, the link between membrane potential and smooth muscle tone may be changed in this model of insulin-dependent diabetes mellitus. Received: 11 February 1997 / Accepted: 9 May 1997     

Effects of monovalent cations on cardiac Na+, K+-ATPase activity and on contractile force

Summary The relationship between Na⁺, K⁺-ATPase inhibition by monovalent cations and their inotropic effect was studied in guinea pig hearts. The activity of partially purified cardiac enzyme was assayed in the presence of 5.8 mM KCl and either 20 or 150 mM NaCl. Rb⁺ and Tl⁺ inhibited Na⁺, K⁺-ATPase activity, the magnitude of the inhibition by these cations being greater in the assay media containing lower Na⁺ concentrations. Tl⁺ produced a dose-dependent inhibition of Na⁺, K⁺-ATPase activity in the presence of 20 mM Na⁺ and 75 mM K⁺, a cationic condition similar to that of intracellular fluid. Other monovalent cations such as K⁺, Cs⁺, NH₄ ⁺, Na⁺ or Li⁺ produced essentially no effect on the Na⁺, K⁺-ATPase activity or slightly stimulated it. In left atrial strips stimulated with field electrodes and bathed in Krebs-Henseleit solution (5.8 mM K⁺ and 145 mM Na⁺), addition of Cs⁺ failed to alter the isometric contractile force significantly. NH₄ ⁺ and K⁺ caused a transient positive inotropic effect which was partially blocked by propranolol. The positive inotropic response to K⁺ was followed by a negative inotropic response. Rb⁺ produced a sustained, dose-dependent inotropic response reaching a plateau at 1–2 min, whereas Tl⁺ produced a dose-dependent positive inotropic effect which developed slowly over a 30-min period. The positive inotropic effects produced by Rb⁺ and Tl⁺ were insensitive to propranolol pretreatment. Concentrations of Tl⁺ and cardiac glycosides which produce similar inotropic effects appear to cause the same degree of Na⁺-pump inhibition. The onset of the positive inotropic response to Rb⁺ or Tl⁺ was not dependent on the number of contractions which is in contrast to the cardiac glycoside-induced inotropic response. Substitution of 20 mM LiCl for an equimolar amount of NaCl in Krebs-Henseleit solution produced a significantly greater inotropic response than that observed when sucrose was substituted for NaCl. It appears that, among monovalent cations, only sodium pump inhibitors produce a sustained positive inotropic response.     

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.     

Modulation of intracellular calcium by potassium channel openers in vascular muscle

Summary We investigated two putative K⁺ channel openers, pinacidil and BRL34915 (cromakalim), and demonstrated their vasorelaxant effectiveness on rat artery contractions induced by K⁺, tetraethylammonium (TEA), or norepinephrine. The K⁺ channel opener-induced decrease in tension was rapid, even when tension was stimulated by 100 mmol/l K⁺. Measurements of intracellular free Ca⁺⁺ (activity) by ultra-high sensitivity digital imaging microscopy was carried out by briefly loaded fura2 (fluorescence ratio) quantitation in isolated, contracting cells of rat azygos vein. Submicron resolution was achieved by measuring cytoplasmic Ca⁺⁺-sensitive fluorescence at each pixel, and size and intensity of areas with high Ca⁺⁺ concentrations, called hot spots, were determined by a computer-generated, 3 algorithm. Hot spots, which most likely represent the sites of Ca⁺⁺ release and re-uptake by Ca⁺⁺-regulatory organelles, increased in size and intensity upon addition of K⁺ or norepinephrine, reaching an early peak prior to the whole cell average peak in cytoplasmic Ca⁺⁺ activity. Both norepinephrine and K⁺-induced stimulation resulted in Ca⁺⁺ activity increases that were primarily due to Ca⁺⁺ release from storage sites. Reduction of free Ca⁺⁺ activity to resting or lower levels occurred upon addition of pinacidil or cromakalim. Intracellular Ca⁺⁺ decreases due to K⁺ channel openers appeared abruptly beginning at the central portions of the cells, resulting in a pronounced early drop in central Ca⁺⁺ activity while elevated Ca⁺⁺ levels persisted at the periphery. While this late stage residual of peripheral Ca⁺⁺ appears to be a significant step in the vascular muscle relaxant action of both K⁺ channel opener drugs, the level of Ca⁺⁺ at peripheral sites was greater in response to pinacidil than to cromakalim. The results of this study suggest that in addition to increasing K⁺ conductance, pinacidil and cromakalim cause 1) decreased Ca⁺⁺ activity in central regions of the myocytes, and 2) a shift in Ca⁺⁺ distribution to primarily subsarcolemmal sites. These observations lead us to hypothesize separate control of peripheral and central Ca⁺⁺ activity within a vascular muscle cell, with Ca⁺⁺ redistribution that can be altered by vasorelaxants. We suggest that intracellular Ca⁺⁺ redistribution may contribute the membrane potential-independent part of the vasorelaxant action of the K⁺ channel openers.This study was supported by NIH grants HL38537 and HL38645, and Eli Lilly Co. P.E. was supported by the Swiss Foundation of Cardiology and by the SNF 32-029 975.90     

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.     

K+ channel openers,cromakalim and Ki4032, inhibit agonist-induced Ca2+ release in canine coronary artery

Summary The effects of K⁺ channel openers, cromakalim and an acetoxyl derivative of KRN 2391 (Ki 4032), were studied on force of contraction, increases in intracellular calcium concentration ([Ca²⁺]_i) measured by fura-2 and inositol 1,4,5-trisphosphate (IP₃) production induced by the thromboxane A₂ analogue, U46619, in canine coronary arteries. Upon single dose applications of U46619 at 300 nmol/l, phasic and tonic increases in [Ca²⁺]_i and force were seen, which were almost abolished by cromakalim (10 mol/l) and Ki4032 (100 mol/l).In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction. Cromakalim (0.01–10 mol/l) and Ki4032 (0.1–100 mol/l) concentration-dependently inhibited the increases in [Ca²⁺]_i and contraction. The inhibitory effects of cromakalim and Ki4032 were blocked by the K⁺ channel blocker tetrabutylammonium (TBA) and counteracted by 20 mmol/l KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production significantly and TBA blocked this inhibitory effect. These results suggest that the hyperpolarization of the plasma membrane by K⁺ channel openers inhibits the production of IP₃ and Ca ²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.Correspondence to T. Yanagisawa at the above address     

Role of ATP-sensitive K+ channels in antinociception induced by R-PIA,an adenosine A1 receptor agonist

The influence of several K⁺ channel-acting drugs on antinociception induced by the adenosine A₁ receptor agonist (–)-N⁶-(2-phenylisopropyl)-adenosine (R-PIA) was evaluated with a tail flick test in mice. The subcutaneous administration of R-PIA (0.5–8 mg/kg) induced a dose-dependent antinociceptive effect. The ATP-sensitive K⁺ (K_ATP) channel blocker gliquidone (2–8 g/mouse, i.c.v.) produced a dose-dependent displacement to the right of the R-PIA dose-response line, whereas the K_ATP channel opener cromakalim (32 g/mouse, i.c.v.) shifted it to the left. Several K_ATP channel blockers dose-dependently antagonized the antinociceptive effect of R-PIA, the order of potency being gliquidone > glipizide > glibenclamide (i.e., the same order of potency shown by these drugs in blocking K_ATP channels in neurons). In contrast, the K⁺ channel blockers 4-aminopyridine and tetraethylammonium did not antagonize the effect of R-PIA. These data suggest that antinociception produced by adenosine A₁ receptor agonists is mediated by the opening of ATP-sensitive K⁺ channels. The present results, together with those of previous studies, further support a role for K⁺ channel opening in the antinociceptive effect of agonists of receptors coupled to G_i/G_o proteins. Correspondence to: José M. Baeyens at the above address     

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