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Background and Purpose: ATP-sensitive K+ (KATP) channels are composed of pore-forming subunits (Kir6.x) and of sulphonylurea receptors (SUR). Both sulphonylureas and K(ATP) channel openers act by binding to SUR. Sulphonylureas reach their binding site from the cytosol but it remains unknown whether this holds for openers too., Experimental Approach: A poorly membrane-permeant sulphonic acid derivative of the benzopyran-type opener, bimakalim, was synthesized, descyano-bimakalim-6-sulphonic acid (BMSA). Binding of BMSA and bimakalim was compared in membranes and intact cells expressing the Kir6.2/SUR2B channel and channel opening was compared in inside-out patches and whole cells., Key Results: In membranes, bimakalim and BMSA bound to Kir6.2/SUR2B with Ki values of 61 nM and 4.3 microM, showing that the negative charge decreased affinity 69-fold. In intact cells, however, binding of BMSA was much weaker than in membranes (75-fold) whereas that of bimakalim was unchanged. The Ki value of BMSA decreased with increasing incubation time. In inside-out patches, bimakalim (1 microM) and BMSA (100 microM) opened the Kir6.2/SUR2B channel closed by MgATP to a similar degree whereas in whole-cell experiments, only bimakalim was effective., Conclusions and Implications: Despite its negative charge, BMSA is an effective channel opener. The fact that BMSA binds and acts more effectively when applied to the inner side of the cell membrane shows that benzopyran openers reach their binding site at SUR from the cytosol. This suggests that the binding pocket of SUR is only open on the cytoplasmic side., (Published online 14 August 2006.)

ATP-sensitive potassium (K(ATP)) channel openers can obviate experimental airways hyperreactivity (AHR) and have shown therapeutic benefit in asthma. However, the clinical potential of such compounds has been compromised by cardiovascular side-effects. We report here the pharmacological properties of (3 S,4 R)-3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-piperidinyl)- N-phenyl-2 H-1-benzopyran-6-sulphonamide (KCO912), a K(ATP) channel opener which suppresses AHR at doses devoid of cardiovascular effects.Specific interaction of KCO912 with the native vascular channel and the sulphonylurea receptor subunit (SUR2B) of the vascular K(ATP) channel was shown in radioligand binding assays. In rat aortic strips, KCO912 inhibited specific binding of [3H]P1075 and [3H]glibenclamide with up to 100% efficacy and with p Ki values of 8.28 and 7.96, respectively. In HEK cells transfected with the recombinant vascular K(ATP) channel (Kir6.1 + SUR2B), the compound elicited a concentration-dependent outward current (pEC50 6.8) and in preloaded rat aortic rings it induced a concentration-dependent glibenclamide-sensitive 86Rb+ efflux (pEC50 7.51). Following intratracheal (i.t.) administration of KCO912 to guinea pigs, AHR induced by immune complexes or ozone was rapidly (<5 min) reversed (ED50 values 1 microg/kg and 0.03 microg/kg, respectively). Changes in blood pressure were seen only at doses =100 microg/kg yielding 'therapeutic ratios' of 100 and 3333, respectively. In addition, KCO912 reversed AHR induced by lipopolysaccharide (LPS; ED50 0.5 microg/kg i.t.) and a dose of 1 microg/kg i.t. fully reversed AHR induced by subchronic treatment with salbutamol. At doses which suppressed AHR, KCO912 had no anti-bronchoconstrictor effects in normoreactive guinea pigs. In spontaneously hyperreactive rhesus monkeys, KCO912, given by inhalation, inhibited methacholine-induced bronchoconstriction (ED50 1.2 microg/kg) but had no significant effects on blood pressure or heart rate at all doses tested (therapeutic ratio >100). In rats given 3 mg/kg of KCO912 by inhalation, the ratio of the area under the concentration-time curve (AUC) for lung to the AUC in blood was 190 and the compound was rapidly cleared (initial t1/2 approximately 30 min). Thus, the wide therapeutic window following administration of KCO912 to the lung seems likely to reflect slow or incomplete passage of KCO912 from the lung into the systemic circulation coupled with rapid removal from the systemic circulation.Thus, when given locally to the airways in both guinea pigs and monkeys, KCO912 suppresses AHR at doses devoid of cardiovascular effects and has a significantly better therapeutic window than representative earlier generation K(ATP) channel openers defined in the same models. Given the pivotal role of AHR in the pathophysiology of asthma and the preclinical profile of KCO912, this compound was selected for clinical evaluation.

The synthesis of a tritiated benzopyran-type opener of the ATP-dependent K+ channel (KATP channel), [3H]-PKF217 - 744 (3S,4R)-N-[3,4-dihydro-2,2-dimethyl-3-hydroxy-6-(2-methyl-4-pyridinyl)-2H-1-benzopyran-4-yl]-3-[2,6-3H]pyridinecarboxamide with a specific activity of 50 Ci mmol(-1) is described. Binding of the ligand was studied in membranes from human embryonic kidney cells transfected with the sulphonylurea receptor isoforms, SUR2B and SUR2A, respectively. PKF217 - 744 was confirmed as being a KATP channel opener by its ability to open the Kir6.1/SUR2B channel, the recombinant form of the vascular KATP channel, and to inhibit binding of the pinacidil analogue, [3H]-P1075, to SUR2B (Ki=26 nM). The kinetics of [3H]-PKF217 - 744 binding to SUR2B was described by rate constants of association and dissociation of 6.9x10(6) M(-1) min(-1) and 0.09 min(-1), respectively. Binding of [3H]-PKF217 - 744 to SUR2B/2A was activated by MgATP (EC50 approximately 3 microM) and inhibited (SUR2B) or enhanced (SUR2A) by MGADP: Binding of [3H]-PKF217 - 744 to SUR2B was inhibited by representatives of the different structural classes of openers and sulphonylureas. Ki values were identical with those obtained using the opener [3H]-P1075 as the radioligand. Glibenclamide accelerated dissociation of the SUR2B-[3H]-PKF217 - 744 complex. The data show that the affinity of [3H]-PKF217 - 744 binding to SUR2B is approximately 6 times lower than that of [3H]-P1075. This is due to a surprisingly slow association rate of the benzopyran-type ligand, suggesting a complex mechanism of opener binding to SUR. The other pharmacological properties of the two opener radioligands are identical.

The K+ channel openers activate ATP-sensitive K+ channels (KATP) 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 microM and a Hill-coefficient of 1.2. At concentrations >300 microM, 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 KATP 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 microM tedisamil blocked the relaxant effect of levcromakalim completely. With minoxidil sulfate as the KATP 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 (> or = 99.95%) the vasorelaxant effect of the openers is also abolished suggesting a link between both effects. This paradoxon remains to be solved.

Cromakalim (BRL 34915), a racemic trans-3,4-disubstituted benzopyran, is the prototype of a novel group of vasorelaxants which act by opening K+ channels in the cell membrane, the K+ channel openers. The enantiomers of cis-cromakalim were synthesized and their biological activity compared to that of the enantiomers of (trans-)cromakalim. Both the (+)-(3R,4R) enantiomer and its (-)-(3S,4S) antipode inhibited binding of the K+ channel opener [3H]P1075 in strips of rat aorta with pKi values of 5.4 and 5.2, respectively. They relaxed noradrenaline-induced contractions of rat isolated aorta under control conditions with pD2 values of 5.7 and 5.2; their vasorelaxant potency was greatly diminished under depolarized conditions (KCl = 55 mmol/l). Both compounds increased the permeability of the cell membrane for K+ as suggested by their ability to stimulate 86Rb+ efflux from rat aortic strips. The vasorelaxant and the 86Rb+ efflux-stimulating effects of the compounds were inhibited by the sulfonylurea, glibenclamide. These results show that the enantiomers of cis-cromakalim are genuine K+ channel openers. The (R,R) enantiomer is 50 times weaker than the (-)-(3S,4R) enantiomer of cromakalim (= levcromakalim, BRL 38227) but 3 times more potent than the (+)-(3R,4S) enantiomer. These data highlight the importance of the stereochemistry at both the 3 and 4 position of the benzopyran ring.

SDZ PCO 400 (30 nM-100 microM) suppressed the spontaneous tone of guinea-pig isolated trachealis. Glibenclamide (1-10 microM), phentolamine (100 microM), guanethidine (50 microM) and bretylium (50 microM) each antagonized SDZ PCO 400 without antagonizing isoprenaline or theophylline. Charybdotoxin (100 nM) failed to antagonize SDZ PCO 400 but antagonized theophylline. The relaxant action of SDZ PCO 400 was ablated when spasm was induced by a K(+)-rich (120 mM) medium. In bovine and guinea-pig trachea, SDZ PCO 400 (10 microM) suppressed spasm evoked by lower (less than 40 mM) but not higher (greater than 40 mM) concentrations of KCl. In guinea-pig trachea the relaxant action of SDZ PCO 400 was associated with suppression of electrical slow waves and with marked cellular hyperpolarisation. SDZ PCO 400 (0.5 and 10 microM) promoted the efflux of 86Rb+ from bovine trachealis, an effect inhibited by glibenclamide (1 microM). It is concluded that the tracheal relaxant action of SDZ PCO 400 is associated with the opening of a plasmalemmal K(+)-channel analogous to the ATP-sensitive K(+)-channel observed in insulin-secreting cells.

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 mumol/l) induced increases in tracer efflux, which were concentration-dependently inhibited by tedisamil with similar potencies (pD2 approximately 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 approximately 7.3. Tedisamil (300 nmol/l) and glibenclamide (300 nmol/l), which inhibited cromakalim- and minoxidil sulphate-induced 42K+ and 86Rb+ efflux by greater than or equal to 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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 mumol/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 (approximately 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 mumol/l)-induced increases in 42K+ and 86Rb+ efflux were inhibited by 45%, whereas conditioning with cromakalim (1 mumol/l) inhibited the 86Rb+ efflux stimulated by additional superfusion of cromakalim (1 mumol/l) by 85%. Glibenclamide inhibited minoxidil sulphate (10 mumol/l)- and cromakalim (1 mumol/l)-induced increases in 42K+ and 86Rb+ efflux in a concentration-dependent manner with IC50 values of approximately 80 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of the K+ channel opener cromakalim on phasic contractions induced by noradrenaline and caffeine were studied in the rat isolated mesenteric bed. In the presence of 1.4 mM Ca2+, 1-s pulses of noradrenaline increased the perfusion pressure of the preparation concentration dependently (midpoint at 92 +/- 10 microM noradrenaline). Cromakalim (0.3 and 1 microM) inhibited these contractions in a non-competitive manner. Contractions elicited by 1-s pulses of noradrenaline (100 microM) were inhibited by the dihydropyridine Ca2+ antagonist isradipine by maximally 24 +/- 1%, indicating that only a minor component of this contraction depended on Ca2+ entry via dihydropyridine-sensitive Ca2+ channels. Cromakalim was a much more effective inhibitor of these contractions (maximum inhibition by 80%, midpoint of the inhibition curve at 171 +/- 15 nM). The effect of cromakalim was stereoselective, inhibited by the sulphonylurea glibenclamide, and abolished in partially depolarizing media (KCl = 35 and 50 mM). In Ca(2+)-free medium, cromakalim inhibited the contraction induced by noradrenaline (100 microM) by maximally 69 +/- 4%, with a midpoint at 58 +/- 14 nM. The effect of cromakalim was again stereoselective, inhibited by glibenclamide, and abolished in the presence of 50 mM KCl. Contractions induced by caffeine (10 and 100 microM) were not affected by cromakalim (1 microM). The results indicate that, in rat mesenteric resistance vessels, cromakalim interferes with the ability of noradrenaline, but not caffeine, to mobilize Ca2+ from intracellular stores. The antivasoconstrictor effect of cromakalim against noradrenaline is inhibited by glibenclamide and appears to be linked to the ability of cromakalim to hyperpolarize the cell membrane.

Tedisamil, a blocker of cardiac K+ channels, potently inhibited cromakalim-induced 86Rb+ efflux from rat aorta with a pIC50 = 7.3, a value similar to that obtained with the sulphonylurea glibenclamide. However, tedisamil was approximately 30 times less potent than glibenclamide in inhibiting the vasorelaxant effects of cromakalim. The data suggest that tedisamil can dissociate between the efflux-inducing and vasorelaxant effects of cromakalim and may therefore prove to be an important tool in elucidating the mechanism of action of this vasorelaxant.

1. The effects of acetylcholine and substance P on the efflux of 86Rb+ and 42K+ from rat aorta and pig coronary artery, respectively, were compared with those of the K+ channel opening agent, cromakalim. 2. In rat aorta preloaded with 86Rb+ and/or 42K+, acetylcholine produced transient, concentration-dependent increases in the efflux rate coefficients of these tracers (maximum approximately 35%). These effects were abolished by endothelial cell removal. 3. Donor/acceptor experiments with rat aorta suggested that at least some of the efflux of 86Rb+ seen in the presence of acetylcholine was not derived from the endothelium, but came from the smooth muscle itself. 4. Acetylcholine (10 microM)-induced 86Rb+ efflux was reduced by tetraethylammonium (TEA, 10 mM) to 33% and ouabain (300 microM) to 54% of control. Preincubation with Ba2+ (100 microM) did not significantly inhibit acetylcholine-induced efflux. 5. Acetylcholine-induced 42K+/86Rb+ efflux was unaffected by preincubation with glibenclamide (10 microM). In contrast, the 42K+/86Rb+ efflux induced by cromakalim was inhibited by glibenclamide (50 nM) by 50%. 6. Acetylcholine (0.3-10 microM)-induced inhibition of phenylephrine (1 microM)-induced tone was abolished by endothelial cell removal but unaffected by glibenclamide. Cromakalim-induced relaxations were endothelium-independent and were inhibited by glibenclamide in a concentration-dependent manner. 7. LG-monomethyl L-arginine (L-NMMA, 250 microM) produced a significant (37 +/- 14%) inhibition of acetylcholine-induced 86Rb+ efflux whereas DG-monomethyl L-arginine was without effect. In the tissue bath L-NMMA inhibited relaxations produced by acetylcholine (0.3-10 microM), but was without effect on responses to cromakalim. 8. In the pig coronary artery, substance P induced an endothelium-dependent efflux of 86Rb+ and 42K+, which was unaffected by preincubation with glibenclamide (10 microM) or L-NMMA (250 microM). 9. The present study shows that acetylcholine and substance P each open K(+)-channels in arterial smooth muscle. However, the insensitivity of the stimulated 86Rb/42K+ efflux to inhibition by glibenclamide suggests that the K(+)-channel opened by these agents is different from the K(+)-channel opened by cromakalim. In addition, the inability of L-NMMA to inhibit fully the acetylcholine- and substance P-stimulated 86Rb+ efflux suggests that in rat aorta and pig coronary artery the endothelium-derived hyperpolarizing factor(s) (EDHF) is different from endothelium-derived relaxing factor (EDRF).

The present study compares the in vitro and in vivo activities of pinacidil with another new vasodilator drug, BRL 34915, claimed to act via the opening of K+ channels in vascular smooth muscle. In the rabbit aorta, BRL 34915 and pinacidil caused rightward shifts of the KCl concentration-response curve and noncompetitively antagonized angiotensin II contractions, yielding an IC50 of 5 and 10 microM, respectively. In 86Rb-loaded guinea pig portal veins, both BRL 34915 and pinacidil stimulated 86Rb+ efflux over the concentration range 0.3-30 microM. At saturating concentrations, the maximum efflux elicited by pinacidil was only one-third that of BRL 34915. Spontaneous activity measured simultaneously from the same vessels was inhibited by BRL 34915 and pinacidil with an IC50 of 12 and 32 nM, respectively. Effects on mechanical activity were thus observed at drug concentrations 100-fold lower than those required to stimulate 86Rb+ efflux. In anesthetized rats, both compounds rapidly lowered blood pressure, with BRL 34915 being threefold more potent than pinacidil in this respect. Tachycardia was more pronounced after BRL 34915 than after pinacidil. Angiotensin II pressor responses were poorly antagonized by these two vasodilators in rats, in marked contrast to the potent effects of Ca2+ antagonists on these responses at equieffective hypotensive doses. We conclude the pinacidil, like BRL 34915, is a potent antihypertensive that is able to enhance the K+ permeability of vascular smooth muscle. The similarities between these two drugs suggest they have a common mechanism of action. However, the discrepancy between the concentrations of these drugs necessary to stimulate 86Rb efflux, and those at which mechanical effects are seen in the portal vein, do not rule out the possibility that other actions may contribute to their vasodilator activities.

BRL34915 (BRL) is a vasodilator with a novel structure. Its mechanism of action, its effects on depolarization-induced and receptor-mediated blood vessel contraction, and its hemodynamic effects were investigated. In the rat portal vein, BRL inhibited spontaneous mechanical activity [IC50 0.013 +/- 0.001 microM (mean +/- SEM) for (-)-BRL], the initial effect being a reduced frequency of contraction. At higher concentrations, the spontaneous contractions were abolished and 86Rb+ efflux was increased. These results suggest that BRL preferentially acts on the pacemaker cells, the K+ channels in other cells being activated only at higher BRL concentrations in this vessel. In experiments on the rabbit aorta, (-)-BRL shifted the KCl concentration-response curve to the right and noncompetitively inhibited responses to angiotensin II. A concentration of 3 microM (-)-BRL reduced maximal angiotensin II contractions by around 50%, higher concentrations having little further effect. This inhibition of angiotensin II contractions is notably greater than that seen with Ca2+ antagonists in this vessel. In anesthetized rabbits, (-)-BRL was a peripheral vasodilator at doses of 3-30 micrograms/kg, but it had no relevant effects on heart rate and myocardial contractile force. This suggests tissue selectivity of this compound or this mechanism of action. BRL preferentially dilated the coronary, gastrointestinal, and cerebral vessels but not those of the kidneys or skeletal muscle as measured with tracer microspheres. This profile of activity is different from that of calcium antagonists or nonspecific vasodilators like dihydralazine. All effects were stereoselective, the (-)-enantiomer being 100 to 200 times more active than the (+)-enantiomer.

Diazoxide caused an increase in 86Rb+ efflux from the rat aorta and portal vein and inhibited spontaneous activity of the latter at concentrations 100 times higher than the K+ channel opener cromakalim. In the rabbit aorta both drugs inhibited vasoconstrictor responses to angiotensin II, noradrenaline and low concentrations (less than or equal to 30 mM) of KCl in a similar manner, the antivasoconstrictor activities being abolished in vessels depolarized with greater than or equal to 35 mM K+. In vivo cromakalim was about 100 times more potent than diazoxide at lowering blood pressure in rats. Diazoxide (30 mg/kg) caused a more than 2-fold increase in plasma glucose in rats and prevented any return toward base line within 1.5 hr after a glucose load. Cromakalim had minimal effects upon glucose homeostasis at equihypotensive doses. Glibenclamide, a potent blocker of ATP-dependent K+ channels, inhibited the stimulation by cromakalim and diazoxide of 86Rb+ efflux from the portal vein and aorta (IC50 approximately 0.1 microM), antagonized their vasorelaxant effects in vitro and in vivo (20-30 mg/kg i.v.) and reversed the diazoxide-induced changes in plasma glucose and insulin levels. These results provide evidence that diazoxide, like cromakalim, is able to open 86Rb+-permeable K+ channels in vascular smooth muscle. This action is likely to be responsible for the in vitro and in vivo vasodilator activity of these two drugs. However, there would seem to be pharmacological differences between the K+ channels affected by these drugs in vascular smooth muscle and the (ATP-sensitive) K+ channels of pancreatic beta-cells, which are thought to be responsible for the effects of diazoxide on plasma glucose.

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 micrograms/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 microM 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-sensitive G-protein.

The effect of BRL 34915 on 86Rb+ efflux and myogenic activity was studied simultaneously in guinea-pig portal vein. 86Rb+ was used as a tracer ion for K+. BRL 34915 inhibited myogenic activity with an IC50 value of 12 +/- 2 nM by reducing primarily the frequency of spontaneous contractions. Washout of the substance was followed by hyperreactivity of the vessel. 86Rb+ efflux was slightly reduced by concentrations of BRL 34915 below 100 nM; above 300 nM efflux was increased in a concentration-dependent manner. Above 10 microM BRL 34915, a slow desensitization of the effect on flux was observed during the 10 min application period of the agonist. The Ca2+ entry blocker, isradipine (PN 200-110, 200-500 nM) did not modify BRL 34915-stimulated 86Rb+ efflux at any BRL 34915 concentration tested, indicating that the influx of extracellular Ca2+ through dihydropyridine-sensitive Ca2+ channels is not necessary for this effect. However, by abolishing spontaneous activity, it allowed the 86Rb+ efflux promoting effect of BRL 34915 to be observed at a concentration of 60 nM. The K+ channel blockers tetraethylammonium and 3,4 diaminopyridine inhibited the BRL 34915-induced 86Rb+ efflux with IC50 values of 13 and 3 mM, respectively. Cell permeable derivatives of cyclic AMP and cyclic GMP had no major effect on BRL 34915-induced 86Rb+ flux, indicating that cyclic nucleotide-induced phosphorylation does not play an important modulatory role here. In conclusion, there is an at least 5 fold difference between the concentrations of BRL 34915 necessary to inhibit myogenic activity and those needed to stimulate 86Rb+ efflux. This may be explained by a primary effect of BRL 34915 on the pacemaker cells of the portal vein. explained by a primary effect of BRL 34915 on the pacemaker cells of the portal efflux. This may be

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 microM) increased the permeability to 86Rb+ 3-4 times less than that to 42K+; at 10 microM the difference was about a factor of 1.3-2. In rat aorta, the threshold concentration of cromakalim for 42K+ efflux was greater than or equal to 0.03 microM; with 86Rb+ as the tracer ion it was 0.1 microM. At similar concentrations, cromakalim relaxed the tension of aortic segments precontracted with 23 mM KCl (IC50 = 0.06 +/- 0.01 microM). 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 microM as compared to 0.06 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of the crude venom of the Israeli scorpion Leiurus quinquestriatus hebraeus on the 86Rb+ efflux stimulated by the K+ channel opener BRL 34915 in the rat portal vein was examined. Applied alone, the venom greatly increased the spontaneous mechanical activity of and the concomitant 86Rb+ efflux from the vessel. When the excitability of the vein was suppressed by the dihydropyridine calcium antagonist, PN 200-110, the 86Rb+ efflux stimulated by BRL 34915 could be shown to be inhibited by the venom. From the concentration dependence of this inhibition an IC50 value of 0.17 +/- 0.01 mg/ml was estimated. This venom is thus the most potent blocker of BRL 34915-evoked 86Rb+ efflux reported so far.

Background and purpose:ATP-sensitive K+ (KATP) channels are composed of pore-forming subunits (Kir6.x) and of sulphonylurea receptors (SUR). Both sulphonylureas and KATP channel openers act by binding to SUR. Sulphonylureas reach their binding site from the cytosol but it remains unknown whether this holds for openers too.Experimental approach:A poorly membrane-permeant sulphonic acid derivative of the benzopyran-type opener, bimakalim, was synthesized, descyano-bimakalim-6-sulphonic acid (BMSA). Binding of BMSA and bimakalim was compared in membranes and intact cells expressing the Kir6.2/SUR2B channel and channel opening was compared in inside-out patches and whole cells.Key results:In membranes, bimakalim and BMSA bound to Kir6.2/SUR2B with Ki values of 61 nM and 4.3 μM, showing that the negative charge decreased affinity 69-fold. In intact cells, however, binding of BMSA was much weaker than in membranes (75-fold) whereas that of bimakalim was unchanged. The Ki value of BMSA decreased with increasing incubation time. In inside-out patches, bimakalim (1 μM) and BMSA (100 μM) opened the Kir6.2/SUR2B channel closed by MgATP to a similar degree whereas in whole-cell experiments, only bimakalim was effective.Conclusions and implications:Despite its negative charge, BMSA is an effective channel opener. The fact that BMSA binds and acts more effectively when applied to the inner side of the cell membrane shows that benzopyran openers reach their binding site at SUR from the cytosol. This suggests that the binding pocket of SUR is only open on the cytoplasmic side.British Journal of Pharmacology (2006) 149, 199–205. doi:10.1038/sj.bjp.0706858; published online 14 August 2006 [ABSTRACT FROM AUTHOR]