Syntaxin-1A inhibition of P-1075, cromakalim, and diazoxide actions on mouse cardiac ATP-sensitive potassium channel.

AIMS: Syntaxin (Syn)-1A binds sulfonylurea receptor (SUR) nucleotide binding folds of cardiac myocyte (SUR2A) and islet beta-cells (SUR1) to inhibit ATP-sensitive potassium (K(ATP)) channels. We further reported that Syn-1A reduced the potency and efficacy of beta-cell-specific K(ATP) channel openers (KCOs). Here, we examined whether Syn-1A would influence non-specific (diazoxide) and SUR2-specific KCOs [N-cyano-N'-(1,1-dimethylpropyl)-N''-3-pyridylguanidine (P-1075) and cromakalim] on cardiac myocyte K(ATP) channels activation. METHODS AND RESULTS: Confocal microscopy and Western blotting verified the presence of both Syn-1A and -1B expressions on rodent cardiac ventricular myocytes. Inside-out patch-clamp electrophysiology was utilized to examine the effects of these syntaxins on K(ATP) macroscopic currents activated by various KCOs from a stable cell line expressing the potassium inward rectifier 6.2 (Kir6.2)/SUR2A and from C57BL/6 male mouse ventricular myocytes. Syn-1A inhibited the current amplitude activated by P-1075, cromakalim and diazoxide via its H3 but not Habc domain. Syn-1B exhibited similar inhibitory effects on P-1075 activation of K(ATP) currents. In examining for direct effects of Syn-1A on the KCO binding to cardiac SUR2 receptors, we found that Syn-1A did not directly affect [(3)H]-P-1075 binding to rat cardiac membrane SUR2A at maximum binding capacity, but was able to mildly reduce the affinity of cold P-1075 and cromakalim to displace [(3)H]-P-1075 binding. CONCLUSION: In conclusion, Syn-1A (and Syn-1B) could inhibit K(ATP) currents activated by SUR2A-acting KCOs. Potential fluctuations in the levels of these syntaxins in the myocardium may affect the therapeutic effectiveness of cardiac KCOs.

The endogenous cannabinoid anandamide inhibits cromakalim-activated K+ currents in follicle-enclosed Xenopus oocytes.

The effect of the endogenous cannabinoid anandamide on K(+) currents activated by the ATP-sensitive potassium (K(ATP)) channel opener cromakalim was investigated in follicle-enclosed Xenopus oocytes using the two-electrode voltage-clamp technique. Anandamide (1-90 microM) reversibly inhibited cromakalim-induced K(+) currents, with an IC(50) value of 8.1 +/- 2 microM. Inhibition was noncompetitive and independent of membrane potential. Coapplication of anandamide with the cannabinoid type 1 (CB(1)) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) (1 microM), the CB(2) receptor antagonist N-[(1S)endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (1 microM), or pertussis toxin (5 microg/ml) did not alter the inhibitory effect of anandamide, suggesting that known cannabinoid receptors are not involved in anandamide inhibition of K(+) currents. Similarly, neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor indomethacin (5 microM) affected anandamide inhibition of K(+) currents, suggesting that the effects of anandamide are not mediated by its metabolic products. In radioligand binding studies, anandamide inhibited the specific binding of the K(ATP) ligand [(3)H]glibenclamide in the oocyte microsomal fractions, with an IC(50) value of 6.3 +/- 0.4 microM. Gonadotropin-induced oocyte maturation and the cromakalim-acceleration of progesterone-induced oocyte maturation were significantly inhibited in the presence of 10 microM anandamide. Collectively, these results indicate that cromakalim-activated K(+) currents in follicular cells of Xenopus oocytes are modulated by anandamide via a cannabinoid receptor-independent mechanism and that the inhibition of these channels by anandamide alters the responsiveness of oocytes to gonadotropin and progesterone.

The role of potassium channels in the vasodilatory effect of levosimendan in human internal thoracic arteries.

OBJECTIVE: We investigated the role of potassium channels in vasodilatory effect of levosimendan in human internal thoracic arteries. METHODS: Samples of redundant internal thoracic arteries obtained from patients undergoing a coronary artery bypass graft surgery were cut into 3 mm wide rings and suspended in 20 ml organ baths. Isometric tension was continuously measured with an isometric force transducer connected to a computer-based data acquisition system. RESULTS: Levosimendan (10(-8)-10(-5) M) or cromakalim (10(-8)-10(-5) M) produced concentration-dependent relaxation responses in human internal thoracic arteries precontracted by 10(-6) M phenylephrine. The relaxant responses to levosimendan did not differ significantly between endothelium-intact and endothelium-denuded preparations. Incubation of human internal thoracic artery rings with adenosine 3',5'-triphosphate (ATP)-dependent potassium channel blocker glibenclamide (10(-6) M) for 30 min significantly inhibited the relaxant responses to both levosimendan and cromakalim. The Ca2+-activated potassium channel blocker iberiotoxin (10(-7) M) also caused a significant but smaller inhibition on relaxant responses to levosimendan. Incubation of the rings with the voltage-dependent potassium channel blocker 4-aminopyridine (5 mM) for 10 min did not cause significant alterations in relaxant responses to levosimendan. CONCLUSIONS: The findings of this study suggested that levosimendan-induced relaxation responses in human internal thoracic arteries were depended on the activation of ATP-dependent and Ca2+-activated potassium channels.

Alteration of cyclopiazonic acid-mediated contracture of mouse diaphragm after denervation.

As a major Ca(2+) source for muscle contraction, the sarcoplasmic reticulum (SR) of skeletal muscle maintains its Ca(2+) content by uptake of myoplasmic Ca(2+) and by replenishment with extracellular Ca(2+). Since transection of motor nerve alters the functions of SR Ca(2+) pump and sarcolemma ion channels, this study explored the effect of denervation on the contracture evoked by cyclopiazonic acid (CPA), an inhibitor of SR Ca(2+) pump. In innervated hemidiaphragm, CPA elicited a bimodal elevation of muscle tone, which was dependent on extracellular Ca(2+) and differentially inhibited by pretreatment with 2-aminoethoxydiphenylborane (APB) and U73122. Activation of muscle Na(+) channels to simulate denervation-induced membrane depolarization did not change the contracture profile. After denervation for 5-14 days when the contracture induced by caffeine was not yet depressed, CPA elicited only APB-sensitive monophasic contracture. Stimulation of ATP-regulated K(+) channels with lemakalim hyperpolarized muscle membrane and attenuated CPA contracture in denervated, but not innervated, hemidiaphragm. The effects of lemakalim were antagonized by glybenclamide. It is inferred that the bimodal CPA contracture is resulted from distinct recruitments of Ca(2+) entry and that denervation alters the voltage dependence and down-regulates CPA-mediated Ca(2+) influx.

Altered vascular reactivity and KATP channel currents in vascular smooth muscle cells from deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

This study was designed to evaluate the contribution of ATP-dependent potassium (KATP) channels to the changes in vascular reactivity and spontaneous tone observed in vessels isolated from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In phenylephrine preconstricted aortic rings, cromakalim induced concentration-dependent, glibenclamide-sensitive relaxation. The concentration response curve to cromakalim was shifted to the right in DOCA-salt hypertensive rats (EC50: 0.850 +/- 0.100 microM) compared with SHAM-normotensive rats (0.108 +/- 0.005 microM), and the maximum relaxation (Emax) evoked by cromakalim was significantly lower in aortic rings from the DOCA group (68 +/- 2%) compared with the SHAM group (108 +/- 5%). The results were similar in endothelium-denuded rings. Spontaneous tone was observed in aortic rings (5 g preload) from DOCA-salt but not SHAM rats. Cromakalim abolished spontaneous tone and the effect was blocked by glibencamide. In whole cell patch clamp studies, increasing extracellular K concentrations from 5.4 to 140 mM and the administration of cromakalim evoked dramatic increases in KATP channel currents in aortic cells isolated from SHAM rats. In contrast, in aortic cells from DOCA-salt hypertensive rats, KATP channel currents were either absent or weak in response to challenges by elevated extracellular K and by cromakalim. These findings suggest that the function of KATP channels is impaired in smooth muscle cells from aorta of DOCA-salt hypertensive rats, which may contribute to the impaired vasodilatation and spontaneous tone observed in these rats.

The synergistic effect of adenosine A2A receptors agonist, type IV phosphodiestease inhibitor and ATP-sensitive K channels activation on free radicals production and aggregation of human polymorphoneuclear leukocytes.

The adenosine A2A receptor agonist CGS21680 (50, 100 and 200 microg/ml), the phosphodiserease type IV (PDE IV) inhibitor Rolipram (50, 100 and 200 microg/ml) and, ATP-sensitive K+ channels activator Cromakalim (30 and 40 microg/ml), when added separately, inhibit oxygen free radicals production from isolated human polymorphoneuclear leukocytes (PMNLs), stimulated with phorbol myristate acetate (PMA), in a dose dependent manner. When both CGS21680 and Rolipram were combined, in vitro, the inhibitory effect on PMNLs free radicals production was synergistic. On the other hand, when both the ATP-sensitive K+ channels opener (KATP) Cromakalim and the type IV PDE inhibitor Rolipram were combined, produced negative synergism (the inhibitory effect of both drugs disappeared). Furthermore, CGS21680, Rolipram, Cromakalim and Forskolin produced no significant inhibitory effect on PMNLs aggregation when added separately. But when various combinations of the above drugs were used, produced significant inhibition of aggregation. Only CGS21680 exhibited a scavenging effect on free radicals production. From the above results, combination of adenosine A2A agonists and type IV PDE inhibitors could serve as potentially novel anti-inflammatory drugs. Furthermore, ATP-sensitive K+ channels activators should be considered for further investigation as anti-inflammatory drug.

Blocking actions of glibenclamide on ATP-sensitive K+ channels in pig urethral myocytes.

The inhibitory effects of glibenclamide on the levcromakalim-induced ATP-sensitive K+ (K(ATP)) channels were investigated with cell-attached configuration in pig proximal urethra. Application of 10 microM glibenclamide reversibly inhibited the activity of the 100 microM levcromakalim-induced K(ATP) channel, decreasing not only the channel open probability but also the amplitude of unitary current. The inhibitory concentration-response curve of the glibenclamide-induced sublevel conductance of K(ATP) channel was shifted to the right (IC50 = 4.7 microM), compared with the levcromakalim-induced K(ATP) channel (full conductance, IC50 = 0.5 microM). Glibenclamide is the first reported sulphonylurea to selectively block K(ATP) channel, not only by decreasing the channel activity but also by reducing the unitary amplitude in smooth muscle.

Differential effects of sulphonylureas on the vasodilatory response evoked by K(ATP) channel openers.

The potency of three sulphonylureas, glibenclamide, glimepiride and gliclazide in antagonizing the vasorelaxant action of openers of adenosine triphosphate (ATP)-regulated K+ channel (KATP) was studied in vivo and in vitro in micro- and macrovessels, respectively. In the hamster cheek pouch, the vasodilatation and the increase in vascular diameter and blood flow induced by diazoxide were markedly reduced by the addition of either glibenclamide or glimepiride (0.8 microm) while they were not affected by gliclazide up to 12 microm. Similarly, in rat and guinea-pig isolated aortic rings, glibenclamide, glimepiride and gliclazide reduced the vasodilator activity of cromakalim. However, the inhibitory effect of gliclazide was considerably less when compared with either glimepiride or glibenclamide. These results suggest that, in contrast to glibenclamide and glimepiride, therapeutically relevant concentrations of gliclazide do not block the vascular effects produced by KATP channel openers in various in vitro and in vivo animal models.

Dimethylsulfoxide and ethanol, commonly used diluents, prevent dilation of pial arterioles by openers of K(ATP) ion channels.

Ethanol and dimethylsulfoxide are commonly used as diluents for water-insoluble drugs. Both are antioxidants. An earlier study of cats presented pharmacological evidence indicating that oxidants could open the K(ATP) ion channel in cerebral surface arterioles [pial arterioles] and that antioxidants including dimethylsulfoxide and L-cysteine prevented opening of these channels. Ethanol was not tested. The present study extends the older observations to a second species, the rat, and examines ethanol as well as dimethylsulfoxide and L-cysteine. A microscope and image splitter were used to measure arteriolar diameters under a closed cranial window in pentobarbital-anesthetized, paralyzed rats. Drugs were topically applied. Dose-dependent dilations produced by two well-established openers of the K(ATP) ion channel were inhibited in dose-dependent manner by ethanol at doses from 0.01% to 0.075%. Above this dose, the effect disappeared. Dilation by sodium nitroprusside was not affected. Dimethylsulfoxide and L-cysteine inhibited dilation produced by pinacidil. Dimethylsulfoxide inhibited pinacidil in a dose-dependent manner at doses from 0.01% to 0.2%. L-Cysteine inhibited pinacidil. Since all the inhibitory drugs have antioxidant properties, their effect may be a reflection of that property as suggested in an earlier paper. Ethanol and dimethylsulfoxide inhibited in doses frequently present when these agents are used as solvents. When investigators use these solvents to dissolve water-insoluble, topically applied drugs, we suggest that they first test the possibility that their observations are being made under conditions in which opening of the K(ATP) ion channel is inhibited.

Cyclic GMP regulates cromakalim-induced relaxation in the rat aortic smooth muscle: role of cyclic GMP in K(ATP)-channels.

Recent studies have shown that nitric oxide (NO) modulates K+-channel activity which play an important role in controlling vascular tone. The formation of cyclic guanosine 3',5'-monophosphate (cyclic GMP) has also been recognized to be associated with the vasodilatory effect of NO. Both cyclic GMP and NO increase whole-cell K+-current by activating Ca2+-activated K+-channels (K(Ca)-channels). Here, we show evidence that activators of soluble guanylyl cyclase sodium nitroprusside or 3-morpholino-sydnonimine (SIN-1), and an analogue of cyclic GMP 8-bromo-cyclic GMP enhance the relaxation induced by cromakalim which is blocked by glibenclamide (a specific inhibitor of ATP-sensitive K+-channels [K(ATP)-channels]), and partially attenuated by methylene blue (an inhibitor of cyclic GMP formation). However, this is not due to the increase of cyclic GMP level by cromakalim itself because the relaxation induced by cromakalim is not associated with the changes of cyclic GMP level formed in the aortic smooth muscle. Thus, it is most likely that cyclic GMP also modulates activity of K(ATP)-channels, in addition to K(Ca)-channels, in the rat aorta.

The role of endothelium-derived nitric oxide in relaxations to levcromakalim in the rat aorta.

The present study was designed to examine the role of basally released nitric oxide in relaxations to an ATP-sensitive K+ channel opener. Whether relaxations to levcromakalim are modulated by endothelial removal or the inhibitors of vasodilator effects of endothelium-derived nitric oxide, were investigated in the rat aorta. During contractions to phenylephrine (3 x 10(-7) to 10(-6) M), levcromakalim (10(-8) to 10(-5) M) or a nitric oxide donor, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7, 10(-9) to 10(-5) M), was added in a cumulative fashion. Relaxations to levcromakalim (10(-8) to 10(-5) M) were significantly reduced by the endothelium-removal. In aortas with endothelium, relaxations in response to levcromakalim were decreased by selective inhibitors of nitric oxide synthase (N(G)-nitro-L-arginine methyl ester, 10(-4) M) and soluble guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one; ODQ, 10(-5) M) and a scavenger of nitric oxide (carboxy-PTIO, 10(-3) M). Relaxations to levcromakalim in aortas treated with these inhibitors are comparable to those seen in aortas without endothelium. KCl (30 mM) and an ATP-sensitive K+ channel inhibitor, glibenclamide (10(-5) M), abolished relaxations to levcromakalim in aortas with or without endothelium, whereas glibenclamide did not alter relaxations to NOC-7 (10(-9) to 10(-5) M) in aortas without endothelium. These results suggest that in rat aortas, inhibition of vasodilator effects of basally released nitric oxide can reduce relaxations via ATP-sensitive K+ channels, although these channels do not mediate relaxations to exogenously applied nitric oxide.

Coordinate interaction between ATP-sensitive K+ channel and Na+, K+-ATPase modulates ischemic preconditioning.

BACKGROUND: We reported that digoxin abolishes the infarct size (IS)-limiting effect of ischemic preconditioning (IPC). Because ATP-sensitive K+ (KATP) channels are involved in IPC, we studied whether Na+,K+-ATPase and KATP channels functionally interact, thereby modulating IPC. METHODS AND RESULTS: Rabbits received 30 minutes of coronary artery occlusion followed by 3 hours of reperfusion. IPC was elicited by 5 minutes of occlusion followed by 10 minutes of reperfusion. The IS, expressed as a percentage of the area at risk, was 40.2+/-2.8% in control and 39.8+/-5.0% in digoxin pretreatment rabbits. Both IPC and pretreatment with cromakalim, a KATP channel opener, reduced IS to 11.8+/-1.8% and 13.4+/-2.6% (P<0. 05 versus control). Digoxin abolished the reduction in IS induced by IPC (33.5+/-3.3%), whereas it did not change that induced by cromakalim (18.8+/-3.0%). In patch-clamp experiments, digoxin was found to inhibit the opening of KATP channels in single ventricular myocytes in which ATP depletion had been induced by metabolic stress. In contrast, digoxin had little effect on the channel opening induced by cromakalim. Moreover, the inhibitory action of digoxin on channel activities was dependent on subsarcolemmal ATP concentration. CONCLUSIONS: The IS-limiting effect of IPC is modulated by an interaction between KATP channels and Na+,K+-ATPase through subsarcolemmal ATP.

Antagonism by class I antiarrhythmic drugs of levcromakalim-induced relaxation in isolated rat aorta.

We have analyzed the effects of several class I antiarrhythmic drugs (propafenone, quinidine, its enantiomer quinine, disopyramide, flecainide and mexiletine), tetraethylammonium (TEA) and glibenclamide on the vasodilator effects of the adenosine 5'-triphosphate-dependent K+ channels channel opener levcromakalim in isolated rat aorta precontracted by 30 mM KCl. TEA (>1 mM) and disopyramide (>/=10 microM), induced a sustained contraction in resting aortic rings. Propafenone (>/=3 microM), quinidine (>/=30 microM), disopyramide (>/=100 microM) and flecainide (>/=100 microM) but not the other drugs decreased the contraction induced by 30 mM KCl in a concentration-dependent manner. Propafenone (>/=1 microM), quinidine (>/=10 microM), quinine (>/=1 microM), disopyramide (>/=3 microM), flecainide (>/=100 microM), mexiletine (>/=3 microM), TEA (>/=0.3 mM) and glibenclamide (>/=0.1 microM) caused a concentration-dependent inhibition of the vasodilation induced by levcromakalim in rat aortic rings. The order of potency of the drugs, expressed as pD2 values, to inhibit the vasodilation induced by 0.3 microM levcromakalim was the following: glibenclamide (6.84) > quinine (6.14) > propafenone (5.27) > disopyramide (5.03) > quinidine (4.80) > mexiletine (4.68) > flecainide (3.37) > TEA (3. 20). With the exception of flecainide and mexiletine, the slopes of the Schild plots were similar to unity. Based on the mode of antagonism these drugs could be classified in four groups: 1) glibenclamide which only shifted the curves to the right, 2) quinidine and disopyramide that, at low concentrations, shifted the curve to the right but, at higher concentrations, it also reduced the maximal relaxant effect, 3) propafenone, quinine and TEA that shifted the curve rightwards and reduced the maximal relaxation at all concentrations and 4) flecainide and mexiletine whose Schild slopes were clearly different from unity. In conclusion, class I antiarrhythmic drugs inhibited levcromakalim-induced relaxation in isolated rat aorta. The concentrations at which these effects were observed were within the therapeutic range (except for flecainide) and similar to those reported to inhibit adenosine 5'-triphosphate-dependent K+ channel currents. Analysis of the concentration-response curves revealed that these drugs produced a noncompetitive antagonism of levcromakalim-induced relaxations.

A K+ single channel and whole-cell clamp study on the effects of levocromakalim in guinea pig portal vein cells.

Single channel cell-attached patch and whole-cell clamp experiments on the mode of action of the K+ channel opener (KCO), levcromakalim, were performed in guinea pig isolated portal vein cells. At +20 mV (135/23 mM K+ in bath/pipette), 10 microM levcromakalim activated K+ channels with a chord conductance of 23.2 pS (K(KCO)), which were sensitive to the blocker of ATP-dependent K+ channels (K(ATP)), glibenclamide. Voltage steps from -80 mV to +20 mV activated 4-aminopyridine-sensitive K+ channels of 6.5 pS with properties of delayed rectifier K+ channels (Kv). In patches which upon a previous voltage step had revealed the existence of Kv, levcromakalim reduced the open-probability of Kv, but it did not concomitantly activate K(KCO). During the course of the experiments, but unrelated to the presence of levcromakalim, large conductance K+ channels (BK(Ca)) appeared which could be inhibited by iberiotoxin, a selective blocker of BK(Ca), and by the membrane-permeant calcium buffer, BAPTA/AM, but not by glibenclamide. Whole-cell current-voltage (i-V) relations were established in response to voltage ramps from +50 mV to -100 mV; on subtraction of control i-V curves from i-V curves obtained in the presence of 10 microM levcromakalim, the KCO-induced K+ current remained which was proportional to voltage. This is not compatible with the upward-bent curvature predicted by the GHK current equation for purely resistive channels at high [K+]i versus low [K+]o. In conclusion, in the guinea pig portal vein cells, no evidence could be established for the hypotheses that KCOs may act via conversion of Kv to K(ATP) (Beech and Bolton 1989; Edwards et al. 1993) or by activation of BK(Ca) (Balwierczak et al. 1995). In these cells, mild inward rectification of the levcromakalim-induced current was observed which underlines their relationship to K(ATP) in other tissues.

Thiopental and propofol impair relaxation produced by ATP-sensitive potassium channel openers in the rat aorta.

ATP-sensitive potassium channel openers are used as vasodilators in the treatment of cardiovascular disorders. The effects of i.v. anaesthetics on arterial relaxation induced by ATP-sensitive potassium channel openers have not been studied. Therefore, in this study, we have examined if thiopental (thiopentone) and propofol affect the vascular response to the ATP-sensitive potassium channel openers, cromakalim and pinacidil, in the isolated rat aorta. Rings of rat thoracic aortas without endothelium were suspended for isometric force recording. Concentration-response curves were obtained in a cumulative manner. During submaximal contractions with phenylephrine 0.3 mumol litre-1, relaxation after cromakalim 0.1-30 mumol litre-1, pinacidil 0.1-30 mumol litre-1 and papaverine 0.1-300 mumol litre-1 was demonstrated. Thiopental 30-300 mumol litre-1, propofol 10-100 mumol litre-1, 10% Intralipid 45 microliters or glibenclamide 5 mumol litre-1 were applied 15 min before addition of phenylephrine. During contractions with phenylephrine, cromakalim and pinacidil induced concentration-dependent relaxation. A selective ATP-sensitive potassium channel antagonist, glibenclamide 5 mumol litre-1, abolished this relaxation, whereas it did not affect relaxation produced by papaverine. Thiopental concentrations > 30 mumol litre-1 significantly impaired relaxation produced by cromakalim or pinacidil. Propofol concentrations > 10 mumol litre-1 also significantly reduced relaxation produced by cromakalim or pinacidil, whereas Intralipid was ineffective. Thiopental 300 mumol litre-1 and propofol 100 mumol litre-1 did not alter relaxation produced by papaverine. These results suggest that the i.v. anaesthetics, thiopental and propofol, impaired vasodilatation mediated by ATP-sensitive potassium channels in vascular smooth muscle cells.

The potassium channel opener (-)-cromakalim prevents glutamate-induced cell death in hippocampal neurons.

(-)-Cromakalim, a typical K+-channel opener, prevents neuronal death induced by either glucose and oxygen privation or by high (100 microM) extracellular glutamate in primary cultures of hippocampus. (-)-Cromakalim has no effect on the earliest events associated with exposure to glutamate. It does not prevent the rapid rise of intracellular Ca2+, the initial swelling of neurons, or the induction of c-fos mRNA transcription. (-)-Cromakalim inhibits all delayed effects associated with the excitotoxic effect of glutamate: (a) (-)-cromakalim inhibits the late and major phase of intracellular Ca2+ increase occurring up to hours after glutamate application; and (b) although (-)-cromakalim cannot prevent the initial cellular swelling induced by glutamate, cells that have been pretreated with (-)-cromakalim return to their original size in a few hours, whereas non-(-)-cromakalim-treated cells remain swollen for more prolonged periods. Many neurons surviving the initial necrotic phase of glutamate-induced cell death undergo progressive DNA cleavage leading to apoptosis. This apoptotic process is prevented completely by (-)-cromakalim. Glibenclamide, a potent blocker of the ATP-sensitive K+ channel, abolishes all the beneficial effects of (-)-cromakalim. These findings strongly suggest that (-)-cromakalim has postsynaptic effects that are closely related to the regulation of Ca2+ homeostasis and cell volume.

The effects of levcromakalim and pinacidil on the human internal mammary artery.

The present study was undertaken to examine the effects of pinacidil and levcromakalim, two potassium, channel openers, on human internal mammary artery (HIMA) obtained from patients undergoing coronary artery bypass surgery, and to clarify the contribution of different K+ channel subtypes in pinacidil and levcromakalim action in this blood vessel. Pinacidil and levcromakalim induced a concentration-dependent relaxation of the precontracted arterial segments (pEC50 = 5.77 +/- 0.05 and 6.89 +/- 0.03, respectively), 4-Aminopyridine (3 mM), a non-selective blocker of K+ channels, induced significant shifts to the right of the concentration-response curves for pinacidil and levcromakalim. Tetraethylammonium (6 mM), charybdotoxin (0.4 microM) and apamin (0.1 microM), blockers of Ca(2+)-sensitive K+ channels, had no effect on the pinacidil- and levcromakalim-evoked relaxation. Glibenclamide (0.1-10 microM), a selective blocker of adenosine triphosphate (ATP)-sensitive K+ channels, competitively antagonized the response to levcromakalim (pKB = 7.92 +/- 0.07). In contrast, glibenclamide, in significantly higher concentrations (3-30 microM), non-competitively antagonized the response to pinacidil. High concentrations of pinacidil (> 10 microM) relaxed arterial rings bathed by a medium containing 100 mM K+ with maximum response 83 +/- 6%. Under the same conditions, the maximum levcromakalim-induced relaxation on HIMA was almost abolished (15 +/- 2%). It is concluded that pinacidil and levcromakalim do not relax the HIMA through the same subtype of K+ channel. ATP-sensitive K+ channels are probably involved in levcromakalim- but not in a pinacidil-induced relaxation in the HIMA. In addition, in pinacidil-induced relaxation of the HIMA, K+ channel-independent mechanisms seem to be involved.

Potassium channel openers inhibit ATP-induced cytosolic free calcium increase in cultured rabbit aortic smooth muscle cells.

AIM: To study the effects of potassium channel openers (PCO) on cytosolic free calcium ([Ca2+]i) changes and their possible mechanisms in vascular smooth muscle cells (VSMC). METHODS: Cultured rabbit aortic VSMC were treated with Fura-2 AM 2.5 mumol.L-1 at 37 degrees C for 50 min. The PCO were pinacidil (Pin), nicorandil (Nic), lemakalim (Lem), and RP 49356 (RP). [Ca2+]i level was measured by fluorospectrometer. RESULTS: [Ca2+]i increase induced by K+ 30 mmol.L-1 was weakly inhibited by Pin, Nic, Lem, and RP (441 +/- 23, 455 +/- 48, 451 +/- 22, 370 +/- 31 vs 544 +/- 40 nmol.L-1, P < 0.01). ATP (0.1 mmol.L-1)-induced peak and sustained [Ca2+]i increase were inhibited by these agents in a concentration-dependent manner. The effects of Pin, Lem, and RP were completely canceled (peak phase: 549 +/- 39, 540 +/- 30, 564 +/- 13 vs 541 +/- 39 nmol.L-1; sustained phase: 413 +/- 25, 364 +/- 16, 377 +/- 11 vs 380 +/- 8 nmol.L-1), but that of Nic was only partially blocked (peak phase: 453 +/- 31 vs 541 nmol.L-1; sustained phase: 348 +/- 19 vs 380 +/- 8 nmol.L-1, P < 0.01) by glibenclamide (Gli, 10 mumol.L-1). Pretreated with the Pin, Nic, Lem, and RP (10 mumol.L-1), the peak [Ca2+]i elevation induced by ATP was reduced in the Ca(2+)-free solution (129 +/- 17, 142 +/- 21, 136 +/- 14, 114 +/- 9 vs 258 +/- 32 nmol.L-1, P < 0.01). CONCLUSION: Pin, Nic, Lem, and RP inhibited ATP-induced [Ca2+]i increase, associated with decreases of both Ca2+ release from intracellular store and Ca2+ influx from extracellular store.