Differential inhibition by tedisamil (KC 8857) and glibenclamide of the responses to cromakalim and minoxidil sulphate in rat isolated aorta

Summary The effects of the K⁺ channel blockers tedisamil and glibenclamide on cromakalim- and minoxidil sulphate-induced ⁴²K⁺ and ⁸⁶Rb⁺ efflux and vasorelaxation in rat aorta, were investigated. In aortic strips preloaded with ⁴²K⁺ or ⁸⁶Rb⁺, cromakalim (1 mol/l) induced increases in tracer efflux, which were concentration-dependently inhibited by tedisamil with similar potencies (pD₂ 7.3) but different amplitudes (maximum inhibition of ⁸⁶Rb⁺ efflux to 0% of control, ⁴²K⁺ efflux to 10 ± 1%). The ⁴²K⁺ efflux elicited by a low concentration of cromakalim (100 nmol/l) was, however, fully inhibited by tedisamil. The tracer effluxes induced by minoxidil sulphate were fully inhibited by tedisamil and glibenclamide (300 nM).Cromakalim and minoxidil sulphate, produced a concentration-dependent inhibition of noradrenaline (100 nmol/l)-induced tone, with pD₂ values of 7.3. Tedisamil (300 nmol/1) and glibenclamide (300 nmol/l), which inhibited cromakalim- and minoxidil sulphate-induced ⁴²K⁺ and ⁸⁶Rb⁺ efflux by 80%, produced 2-fold and 40-fold shifts in the concentration-relaxation curve for cromakalim, and 3.5-fold and 2200-fold shifts in the concentration-relaxation curve for minoxidil sulphate, respectively. Similar shifts of the cromakalim concentration-relaxation curve in the presence of tedisamil and glibenclamide were also observed when the tissues were precontracted with potassium chloride (25 mmol/l).The results show that tedisamil and glibenclamide inhibit the cromakalim- and minoxidil sulphate-induced tracer effluxes with similar potencies whereas they differ greatly in their ability to inhibit the vasorelaxant effects of the two K⁺ channel openers. This suggests that the opening of ⁴²K⁺/⁸⁶Rb⁺ permeable K⁺ channels in the plasma membrane cannot fully explain the vasorelaxant effects of the two drugs. The mechanism(s) of vasorelaxation of cromakalim and minoxidil sulphate, which is not due to the opening of plasmalemmal K⁺ channels, is sensitive to inhibition by glibenclamide but comparatively insensitive to inhibition by tedisamil. Send offprint request to U. Quast 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.     

Comparison of cromakalim-induced relaxation of potassium precontracted rabbit,cat, and rat isolated cerebral arteries

Summary The effects of cromakalim were investigated in KCl-precontracted cat, rabbit, and rat isolated cerebral arteries with intact endothelium. Potassium induced contraction of all cerebral arteries studied, but exhibited marked vessel and species variation with no spasm to 20 or 30 mmol/l KCl in the rat basilar artery or 20 mmol/l KCl in the rabbit middle cerebral artery. On sustained tension to 20 mmol/l KCl, cromakalim induced concentration-related relaxation in the rabbit basilar artery and the cat basilar and middle cerebral arteries with Hill coefficients greater than unity. Cromakalim was more potent in the rabbit basilar artery precontracted with 20 or 30 mmol/KCl than in the rabbit middle cerebral artery or the cat basilar or middle cerebral artery. Elevation of the KCl concentration to 50 mmol/l inhibited cromakalim-induced relaxation and produced a decrease in the Hill coefficient. Preincubation of cerebral arteries with glibenclamide (100 nmol/l–1 mol/1) produced concentration-related inhibition of the cromakalim-induced relaxation in the rabbit basilar, cat basilar, and cat middle cerebral arteries precontracted with 20 mmol/l KCl. The degree of rightward shift of concentration-effect curves by glibenclamide was calculated at the EC₂₅, EC₅₀, and EC₇₅ levels. A good correlation was observed between the shifts at the EC₅₀ and EC₅₀ levels. However, the shift in concentration — effect curves for cromakalim produced at the EC₂₅ level was markedly less than the-EC₅₀ or EC₇₅ levels in the presence of 1 mol/1 glibenclamide. The pA ₂ values for glibenclamide calculated at the EC₅₀ level were 6.6 ± 0.09, 7.1 ± 0.1, and 6.5 ± 0.5 in the rabbit basilar, cat basilar, and cat middle cerebral artery, respectively. The slope of the Schild regression for the inhibitory effect of glibenclamide in the rabbit basilar artery was significantly greater than unity but did not differ from unity in cat cerebral arteries. Glibenclamide (1 mol/l) produced a similar degree of inhibition of the cromakalim-induced relaxation in the 30 mmol/l KCl precontracted rabbit middle cerebral artery and in the rabbit basilar artery exposed to 20 mmol/l KCl. In contrast, tolbutamide 10 mol/l was essentially inactive against the cromakalim-induced relaxation in all vessels studied. It is concluded that cromakalim produces concentration-dependent relaxations of rabbit and cat isolated cerebral arteries by a mechanism that is similar to that identified in peripheral vasculature and visceral smooth muscle. In this study we were unable to demonstrate effects of cromakalim on the KCl precontracted rat basilar artery, possibly due to the low sensitivity of this preparation to KCl. Send offprint requests to M. Wahl 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     

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     

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     

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     

Vasorelaxing effect in rat thoracic aorta caused by fraxinellone and dictamine isolated from the Chinese herb Dictamnus dasycarpus Turcz: comparison with cromakalim and Ca2+ channel blockers

Summary The components of Dictamnus dasycarpus Turcz were tested for their vasorelaxing effect on the rat aorta, and fraxinellone and dictamine were shown to be effective vasorelaxants. In high K⁺ (60 mmol/l) medium, Ca²⁺ (0.03 to 3 mmol/l)-induced vasoconstriction was inhibited concentration-dependently by both agents. The IC₅₀ for fraxinellone and dictamine were calculated to be about 25 mol/l and 15 mol/l (for Ca²⁺) concentration of (1 mmol/l), respectively. Cromakalim (0.2–10) mol/l relaxed aortic rings precontracted with 15 but not 60 mmol/l of K⁺. Fraxinellone and verapamil were more potent and effective in producing relaxation in 60 mmol/l than in 15 mmol/l K⁺-induced contraction. However, dictamine was more potent in producing relaxation in 5 mmol/l K⁺-induced contraction. Nifedipine (1 mol/l), dictamine (100 mol/l) and fraxinellone (100 mol/l) relaxed the aortic contraction caused by KCl or Bay K 8644. The tonic contraction elicited by nor adrenaline (NA, 3 mol/l) was also relaxed by dictamine (500 mol/l), but not by fraxinellone (500 mol/l) in the nifedipine (1 mol/l)-treated aorta. This relaxing effect of dictamine persisted in endothelium-denuded aorta. Glibenclamide (10 mol/l) shifted the concentration-relaxation curve of cromakalim, but not that of dictamine, to the right in rat aortic rings precontracted with NA. Dictamine (500 mol/l) did not affect tonic contraction of NA which are reduced by H-7 (1 mol/l) in Ca²⁺ depleted medium. In conclusion, fraxinellone is a selective blocker of voltage-dependent Ca²⁺ channel, while dictamine relaxed the rat aorta by suppressing the Ca²⁺ influx through both voltage-dependent and receptor-operated Ca²⁺ channels.This work was supported by a research grant from the Nationat Science Council of the Republic of China (NSC80-0420-B002-18) Send offprint requests to C. M. Teng, Pharmacological Institute, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Sect. 1, Taipei, 10018, Taiwan     

Analysis of cromakalim-, pinacidil-, and nicorandil-induced relaxation of the 5-hydroxytryptamine precontracted rat isolated basilar artery

Summary The effects of the K⁺ channel activators cromakalim, pinacidil, and nicorandil were investigated in endothelium intact, 5-hydroxytryptamine (5-HT) precontracted rat isolated basilar artery. Cromakalim, pinacidil, and nicorandil produced concentration-dependent relaxation of rat isolated basilar artery precontracted with 5-HT with a rank order of potency of cromakalim > pinacidil > nicorandil. All compounds produced full or nearly full relaxation. The calculated Hill coefficients for cromakalim-, pinacidil-, and nicorandil-induced relaxation of 5-HT-precontracted rat isolated basilar artery were 2.20 ± 0.36, 1.30 ± 0.07, and 1.00 ± 0.01, respectively. Under conditions of increased tone produced by 50 mmol/1 KCl (which inhibits cromakalim-induced relaxation) pinacidil and nicorandil produced marked reversal of spasm, with pinacidil being more potent than nicorandil. In arteries precontracted with 5-HT, preincubation with glibenclamide (0.1–1 mol/1) produced concentration-related inhibition of relaxation with calculated mean pA ₂ values (and slopes of Schild regression) ± SEM of 6.84 ± 0.20 (1.1 ± 0.20) against cromakalim, 6.60 ± 0.14 (0.95 ± 0.23) against nicorandil,and6.57 ± 0.26(1.04 ± 0.18) against pinacidil. For cromakalim, pinacidil, and nicorandil the slopes of Schild regression were not significantly different from unity. Tolbutamide 10 mol/l was without effect against the cromakalim-, pinacidil-, or nicorandil-induced relaxation. Tetraethylammonium (TEA; 1–10 mmol/l) produced noncompetitive inhibition of the cromakalim-induced relaxation, but appeared to produce competitive inhibition of the pinacidil- and nicorandil-induced relaxations. We conclude that cromakalim, pinacidil, and nicorandil produce relaxation of the 5-HT precontracted rat basilar artery by similar mechanisms to those identified in other peripheral vascular and visceral smooth muscle. Furthermore, pinacidil and nicorandil differ from cromakalim in possessing marked spasmolytic activity in 50 mmol/l KCl precontracted arteries. Send offprint requests to M. Wahl at the above address     

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

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

Large conductance Ca-activated K channel activation with NS1619 decreases myogenic and neurogenic contractions of rat detrusor smooth muscle

Large conductance voltage- and Ca²⁺-activated K⁺ (BK) channels are important in regulating detrusor smooth muscle (DSM) function. Here, we examined systematically how the BK channel pharmacological activation modulates DSM contractility. NS1619, a potent BK channel activator, was utilized as a pharmacological tool to investigate the effect of BK channel activation on rat DSM contractility. Isometric tension recordings of DSM strips isolated from rat urinary bladder were performed systematically under various experimental conditions. NS1619 (30 μM) substantially diminished DSM spontaneous contraction amplitude, muscle force integral, frequency, duration and muscle tone. This effect was blocked by iberiotoxin, a BK channel selective inhibitor. NS1619 inhibited the phasic and tonic contractions in DSM strips pre-contracted with either the cholinergic agonist, carbachol (0.1 μM), or the depolarizing agent, KCl (20 mM). In the presence of elevated KCl (60 mM KCl), the inhibitory effect of NS1619 was significantly reduced, indicating that BK channel activation is the underlying mechanism of NS1619 action. BK channel activation with NS1619 dramatically decreased the amplitude of electrical field stimulation (EFS)-induced contractions under a range of stimulation frequencies (0.5–50 Hz). In the presence of specific neurotransmitter inhibitors, BK channel activation with NS1619 significantly decreased both cholinergic and purinergic components of EFS-induced contractions. We conclude that BK channel activation with NS1619 significantly inhibited spontaneous, pharmacologically induced and nerve-evoked DSM contractions. Targeting the BK channel with selective openers may offer a unique opportunity to control DSM contractile activity, including pathophysiological conditions such as overactive bladder and detrusor overactivity, regardless of the underlying cause.     

Cromakalim activates the KATP and enhances spontaneous transient outward potassium currents in rat saphenous arterial myocytes

Potassium channel openers, e.g. cromakalim are held to relax smooth muscle by hyperpolarizing the cell membrane via activation of ATP-sensitive K(+) (K(ATP)) channels. A recent report indicates that members of this group dilate cerebral arteries also by enhancing the K(Ca)-based spontaneous transient outward currents (STOCs) through the activation of mitochondrial K(ATP) channels. We extended the study to rat saphenous arterial myocytes, a model for peripheral resistance vessels, to investigate the effects of cromakalim on K(ATP) and STOCs, and the underlaying mechanisms. Smooth muscle myocytes were enzymatically dissociated from the saphenous branch of the femoral artery. Macroscopic currents were recorded from acutely isolated cells using the perforated-patch and whole-cell variants of the patch-clamp technique. Predictably metabolic inhibitors and cromakalim activated a background K(+) current blocked by glibenclamide, identified as the K(ATP) channel. However, in addition, cromakalim markedly increased the amplitude and frequency of STOCs. The latter action was not sensitive to the specific K(ATP) channel blocker glibenclamide, excluding the participation of mitocondrial K(ATP) channels in this action. In conclusion, this study suggests that, in addition to the opening of K(ATP) channels, the increased STOC activity may have an important role in the vasorelaxing action of cromakalim, but through a mechanism different from that reported on cerebral artery.     

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.     

Diclofenac, a nonsteroidal anti-inflammatory drug, activates the transient outward K+ current in rat cerebellar granule cells

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its direct effect on ion channels. Here, the effect of diclofenac on voltage-dependent transient outward K+ currents (I(A)) in cultured rat cerebellar granule cells was investigated using the whole-cell voltage-clamp technique. At concentrations of 10(-5)-10(-3) M, diclofenac reversibly increased the I(A) amplitude in a dose-dependent manner and significantly modulated the steady-state inactivation properties of the I(A) channels, but did not alter the steady-state activation properties. Furthermore, diclofenac treatment resulted in a slightly accelerated recovery from I(A) channel inactivation. Intracellular application of diclofenac could mimic the effects induced by extracellular application, although once the intracellular response reached a plateau, extracellular application of diclofenac could induce further increases in the current. These observations indicate that diclofenac might exert its effects on the channel protein at both the inner and outer sides of the cell membrane. Our data provide the first evidence that diclofenac is able to activate transient outward potassium channels in neurons. Although further work will be necessary to define the exact mechanism of diclofenac-induced I(A) channel activation, this study provides evidence that the nonsteroidal anti-inflammatory drug, diclofenac, may play a novel neuronal role that is worthy of future study.     

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.     

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     

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     

Bicuculline free base blocks voltage-activated K+ currents in rat medial preoptic neurons

The effects of the well-known GABA(A)-receptor blocker bicuculline on voltage-gated K(+) currents were studied in neurons from the medial preoptic nucleus (MPN) of rat. Whole-cell currents were recorded using the perforated-patch technique. Voltage steps from -54 to +6 mV resulted in tetraethylammonium-sensitive K(+) currents of delayed rectifier type. The total K(+) current (at 300 ms), including Ca(2+)-dependent and Ca(2+)-independent components, was reversibly reduced (17 +/- 4%) by 100 microM bicuculline methiodide and (37 +/- 5%) by 100 microM bicuculline as free base. The Ca(2+)-independent fraction (77 +/- 2%) of K(+) current evoked by a voltage step was, however, reduced (54 +/- 6%) only by bicuculline free base, but was not affected by bicuculline methiodide. The half-saturating concentration of bicuculline free base for blocking this purely voltage-gated K(+) current was 113 microM, whereas for blocking a steady Ca(2+)-dependent K(+) current it was 36 microM. The bicuculline-sensitive voltage-gated K(+) current was composed of 4-AP-sensitive and 4-AP-resistant components with different kinetic properties. No component of the purely voltage-gated K(+) current was affected neither by 100 nM alpha-dendrotoxin nor by 100 nM I-dendrotoxin. The possible K(+)-channel subtypes mediating the bicuculline-sensitive current in MPN neurons are discussed.     

Sustained K+ Outward Currents are Sensitive to Intracellular Heteropodatoxin2 in CA1 Neurons of Organotypic Cultured Hippocampi of Rats

Blocking or regulating K⁺ channels is important for investigating neuronal functions in mammalian brains, because voltage-dependent K⁺ channels (Kv channels) play roles to regulate membrane excitabilities for synaptic and somatic processings in neurons. Although a number of toxins and chemicals are useful to change gating properties of Kv channels, specific effects of each toxin on a particular Kv subunit have not been sufficiently demonstrated in neurons yet. In this study, we tested electrophysiologically if heteropodatoxin2 (HpTX₂), known as one of Kv4-specific toxins, might be effective on various K⁺ outward currents in CA1 neurons of organotypic hippocampal slices of rats. Using a nucleated-patch technique and a pre-pulse protocol in voltage-clamp mode, total K⁺ outward currents recorded in the soma of CA1 neurons were separated into two components, transient and sustained currents. The extracellular application of HpTX₂ weakly but significantly reduced transient currents. However, when HpTX₂ was added to internal solution, the significant reduction of amplitudes were observed in sustained currents but not in transient currents. This indicates the non-specificity of HpTX₂ effects on Kv4 family. Compared with the effect of cytosolic 4-AP to block transient currents, it is possible that cytosolic HpTX₂ is pharmacologically specific to sustained currents in CA1 neurons. These results suggest that distinctive actions of HpTX₂ inside and outside of neurons are very efficient to selectively reduce specific K⁺ outward currents.     

An approach to differentiate between noradrenaline-elicited contractile processes in the rat isolated aorta

Summary The aim of the present study was to assess the different processes contributing to the contraction induced by noradrenaline (NA, 1 gmol/l) in the rat isolated aorta. Pretreatment with maximally effective concentrations of nifedipine or cromakalim reduced the NA-induced contraction to 80 ± 3.5% or 63 ± 2.0%, respectively, without alteration of the shape of the response. After pretreatment with Mn²⁺, NA caused a transient phasic contraction followed by a sustained tonic component, comparable to the response obtained in Ca²⁺-free medium. Ryanodine — in the presence of extracellular Ca²⁺ — caused a slight increase of resting tension, but did not modify the NA-induced contraction. In Ca²⁺-free medium the contraction elicited by NA consisted of a transient phasic and a sustained tonic component. The amplitude of the phasic contraction decreased exponentially with the time of exposure to Ca²⁺-free medium. The phasic component was identified as elicited by Ca²⁺ released from the sarcoplasmic reticulum (SR) by means of ryanodine. If Ca²⁺ depleted tissues (80 min in Ca²⁺-free solution) were exposed to Ca²⁺ in the presence of Mn²⁺ or cromakalim, the NA-induced phasic response was inhibited, suggesting that Mn²⁺ and cromakalim blocked the refilling of the store. It can be concluded that activation of ₁-adrenoceptors in the rat aorta by NA elicits Ca²⁺-entry processes which have a different sensitivity to nifedipine, cromakalim and Mn²⁺. The Ca²⁺ released from SR contributes about 20% to the overall contractile response. Our data suggest that the depleted SR can be refilled from the extracellular space via a direct cromakalim- and Mn²⁺-sensitive pathway. Send offprint requests to: B. Wilffert at the above address