In the present study, it was aimed to further identify the intracellular action mechanism of cromakalim and levcromakaliin in the porcine coronary artery. In intact porcine coronary arterial strips loaded with fura-2/AM, acetylcholine caused an increase in intracellular free Ca2+ ((Ca2+)-i) in association with a contraction in a concentration-dependent manner. Cromakalim (1 micrometer) caused a reduction in acetylcholine-induced increased (Ca2+)-i not only in the normal physiological salt solution (PSS) but also in Ca2+ -free PSS (containing 1mM EGTA). In the skinned strips prepared by exposure of tissue to 20 micrometer beta-escin, inositol 1,4,5-trisphosphate (IP-3) evoked an increase in (Ca2+)-i but it was without effect on the intact strips. The IP-3-induced increase in (Ca2+)-i was inhibited by cromakalim by 78% and levcromakalim by 59% (1 micrometer, each). Pretreatment with glibenclamide (a blocker of ATP-sensitive K+ channels, 10 micrometer and apamin (a blocker of small conductance Ca2+/-activated K+ channels, 1 micrometer strongly blocked the effect of cromakalim and levcromakalim. However, charybdotoxin (a blocker of large conductance Ca2+ -activated K+ channels, 1-micrometer) was without effect. In addition, cromakalim inhibited the GTP-gamma-S (100 micrometer, nonhydrolysable analogue of GTP)-induced increase in (Ca2+)-i. Based on these results, it is suggested that cromakalim and levcromakalim exert a potent vasorelaxation, in part, by acting on the K+ channels of the intracellular sites (e.g., sarcoplasmic reticulum membrane), thereby, resulting in decrease in release of Ca2+ from the intracellular storage site.
Acetylcholine ; Apamin ; Charybdotoxin ; Coronary Vessels* ; Cromakalim* ; Escin ; Glyburide ; Inositol 1,4,5-Trisphosphate ; Sarcoplasmic Reticulum ; Skin ; Vasodilation

In the present study, we characterized the non-vascular smooth muscle relaxant effects of a novel benzoyran derivative, SKP-450 (2-(2"(1",3"-dioxolone)-2-methyl-4-(2'-oxo-1'-pyrrolidinyl)-6-nitro- 2H-1-benzopyran) and its metabolite, SKP-310, in comparison with levcromakalim (LCRK). In the rat stomach fundus, the spontaneous motility stimulated by 10-6.5 M bethanechol was completely eliminated not only by 10(-7) M SKP-450 but also by 10(-6) M LCRK, which were blocked by 10(-6) M glibenclamide. The inhibitory effect of SKP-450 (pD2, 3.94 +/- 0.66) was much less than LCRK (pD2, 5.73 +/- 0.38, P < 0.05). In the bethanechol (10(-6.5) M)-stimulated urinary bladder, the tonus was decreased in association with elimination of spontaneous motility by 10(-7) M SKP-450 and 10-6 M LCRK (pD2, 6.77 +/- 0.06) (P < 0.05), which were inhibitable by 10-6 M glibenclamide. The inhibitory effect of SKP-450 (pD2, 7.66 +/- 0.05) was significantly more potent than that of LCRK (pD2, 6.77 +/- 0.06, P < 0.05). In the rat uterus stimulated by PGF2alpha (10(-7) M), both increased tonus and spontaneous motility were eliminated by 10(-6) M LCRK with slight depression of the tonus, but not by SKP-450 (10(-5) M). The stimulated trachea of guinea-pig by 10-6.5 M bethanechol was moderately suppressed by SKP-450 (10(-6)~10(-5) M) but little by SKP-310. In association with the relaxant effects, SKP-450 (10(-6) M) and LCRK (10(-5) M) caused a significant stimulation of the 86Rb efflux from rat urinary bladder and stomach fundus, which were antagonized by 10(-5) M glibenclamide, whereas the K+ channel openers could not exert a stimulation of the 86Rb efflux from rat uterus. In conclusion, it is suggested that SKP-450 exerts potent relaxant effects on the urinary bladder detrusor muscle and duodenum, whereas it shows much less effect on stomach fundus and uterus as contrasted to LCRK.
Animals ; Bethanechol ; Cromakalim* ; Depression ; Dinoprost ; Duodenum ; Glyburide ; Muscle, Smooth* ; Rats ; Stomach ; Trachea ; Urinary Bladder ; Uterus

The purpose of this study was to investigate the characteristics or the potassium channels existing in the rat urinary bladders. Smooth muscle strips of rat detrusor urinae were examined by isometric myography. Relaxation responses of detrusor muscle strips to the three potassium channel openers pinacidil, a cyanoguanidine derivative, BRL 38227, a benzopyran derivative and RP 52891, a tertrahydrothiopyran derivative were examined. The potassium channel openers reduced the basal tone, and the rank order of potency was RP 52891>pincidil>BRL 38227. Procaine, an inhibitor of the voltage-sensitive potassium channel tended to increase the basal tone, but it did not affect the relaxant effects of the calcium-activated potassium channel opener did not antagonize the relaxant effects, but it reduced the Emax of RP 52891 and BRL 38227. Glibenclamide, an inhibitor of the ATP-sensitive potassium channel, antagonized the relaxant effects of pinacidil, RP 52891 and BRL 38227 reducing the Emax of RP 52891 and BRl 38227. Galanin which inhibits secretion of insulin through opening the ATP-sensitive potassium channels in pancreatic β-cells rather increased the basal tone of the isolated detrusor strips. These results suggest that the urinary bladder of the rat has mainly the ATP-sensitive, glibenclamide sensitive potassium channel, which is a different type from that in the pancreatic β-islet cells.
Animals ; Cromakalim ; Galanin ; Glyburide ; Insulin ; KATP Channels ; Muscle, Smooth ; Myography ; Pinacidil ; Potassium Channels* ; Potassium Channels, Calcium-Activated ; Potassium* ; Procaine ; Rats* ; Relaxation ; Urinary Bladder

Glutamate Is the predominant excitatory neurotransmitter in the mammalian CNS. To elucidate the influence of glutamate on the noradrenergic neurotransmission in rat cortex, we examined the effects of agents that act in several steps of neurotransmission on [3H]norepinephrine ([3H])NE) release evoked by glutamate. Glutamate (1 mM) evoked significant release of [3H]NE from rat cortex slices in the absence of Mg2+in the incubation media. This effect was attenuated by cromakalime (10 nM) and lemakalime (10 nM), and the inhibitory effect of cromakalime was abolished by glipizide. Inhibitory effect of muscimol (30 uM) and baclofen (3 uM, 30 uM) was antagonized by biccuculine (3 uM), respectively. Nipecotic acid(10 uM), DABA(300 uM), and beta-alanine(100 uM) attenuated the glutamate-induced release of [3H]NE. Dihydrokinate (300 uM) PDC (100 nM) increased the glutamate-induced release of [3H]NE. Ifenprodile (10 nM) and arcaine (1 uN), blockers of polyamine site, attenuated the release of ("H)NE. The stimulatory effect of spermine was abolished by arcaine. CPA(100 nM) and CPCA(100 nM), EHNA(30 uN) and NBTI(1 uN) attenuated the release of ("H)NE. Verapamil(S uN), nitredipine(10 uN), u- conotoxin (100 nM) and flunarizine (5 uM) attenuated the release of (3H)NE. Dantrolene(30 uM), KT-362(3 uM), and ryanodine(10 nM), attenuated the glutamate-induced release of [3H]NE. Glycine (10 uM) increased the release of [3H]NE. DCQX (30 uN) attenuated the release of [3H]NE. These results suggest that glutamate-evoked release of norepinephrine can be modulated by GABAergic, adenosinergic neurotransmitters, and by various drugs which modulate ion channel activities in rat cortex.
Animals ; Baclofen ; Cerebral Cortex ; Conotoxins ; Cromakalim ; Flunarizine ; Glipizide ; Glutamic Acid ; Glycine ; Ion Channels ; Muscimol ; Neurotransmitter Agents ; Norepinephrine* ; Rats* ; Spermine ; Synaptic Transmission

BACKGROUND: To evaluate the role of free fatty acids on the ischemic myocardium, influences of various free fatty acids upon transmembrane action potential and ATP-sensitive K+(KATP) channel activity were examined in the ventricular myocardium and single cardiac myocytes. METHODS: KATP channel activities were measured in the enzymatically (collagenase) isolated single rat ventricular cardiac myocytes by the method of the excised inside-out and the cell-attached patch clamp, and transmembrane action potentials were recorded using the conventional 3M-KCl microelectode techniques in the rat ventricular myocardium. RESULTS: Free fatty acids [FFAs; arachidonic acid (AA), linoleic acid (LA) and lysophosphatidylcholine (LPC)] reduced the KATP channel activity in a dose-dependent manner in the inside-out patch, and 50%-inhibition concentrations (IC50) were 88 +/- 11.2, 49 +/- 12.5, and 188 +/- 17.4 M respectively. Both frequency of channel opening and the mean open-burst duration were markedly decreased, but the amplitude of single channel currents were not changed by the FFAs. AA (50 micrometer) and LPC (50 micrometer) did not affect the dinitrophenol (DNP, 50 micrometer)-induced KATP channel activity, whereas LA (50 micrometer) had a tendency to reduce the activity. The channel inhibition effects by 10 micrometer AA in the inside-out patch were significantly augmented by diclofenac (10 micrometer), but was not changed by nordihydroguaiaretic acid. FFAs never stimulated KATP channel activity, even in the inside-out patch where KATP channel activity reduced in the presence of internal ATP (100 micrometer). Time for 90% repolarization (APD90) significantly increased during superfusion of the FFAs, to 22 (50 micrometer AA), 24 (50 micrometer LA), and 18 (50 micrometer LPC) % from those of the contol at the time of 10 min superfusion, but the other action potential characteristics were not changed by the FFAs. AA (10 micrometer) attenuated cromakalim (10 micrometer)-induced APD90 shortening effects. CONCLUSION: It was inferred that FFAs inhibit the KATP channel activity directly by themselves and/or indirectly by their metabolites in the rat ventricular cardiomyocytes, and therefore, duration of action potential lengthens to be a burden over the ischemic myocardium accounting for the injury of myocardium at the late stage of ischemia.
Action Potentials* ; Adenosine Triphosphate ; Animals ; Arachidonic Acid ; Cromakalim ; Diclofenac ; Fatty Acids, Nonesterified* ; Ischemia ; Linoleic Acid ; Lysophosphatidylcholines ; Masoprocol ; Myocardium* ; Myocytes, Cardiac ; Potassium Channels* ; Potassium* ; Rats*

BACKGROUND: Although hypoxic pulmonary vasoconstriction (HPC) and hypoxic coronary vasodilatation (HCD) have been recognized by many researchers, the precise mechanism remains unknown. As isolated arteries will constrict or relax in vitro in response to hypoxia, the oxygen sensor/transduction mechanism must reside in the arterial smooth muscle, the endothelium, or both. Unfortunately, much of the current evidence is conflicting, especially concerning to the dependency of HPC and HCD on the endothelium and the role of the K+ channel. Therefore, this experiment was attempted to clarify the dependency of HPC and HCD on the endothelium and the role of the K+ channel on HPC and HCD. METHODS: HPC was investigated in isolated main pulmonary arteries precontracted with norepinephrine (NE). HCD was investigated in isolated left circumflex coronary artery precontracted with prostaglandin F2 alpha. Vascular rings were suspended for isometric tension recording in an organ chamber filled with Krebs-Henseleit solution. Hypoxia was induced by gassing the chamber with 95% N2 +5% CO2, which was maintained for 15 - 25 min. RESULTS: 1)Hypoxia elicited a vasoconstriction in NE-precontracted pulmonary arteries with endothelium, but a vasodilatation in PGF 2 alpha-precontracted coronary arteries with and without endothelium. There was no difference between the amplitude of the HPC and HCD induced by two consecutive hypoxic challenges and the effect of normoxic and hyperoxic control Krebs-Henseleit solution on subsequent response to hypoxia. 2)Inhibition of NO synthesis by the treatment with Nw-nitro-L-arginine reduced HPC in pulmonary arteries, but inhibition of the cyclooxygenase pathway by treatment with indomethacin had no effect on HPC and HCD, respectively. 3)Blockades of the TEA-sensitive K+ channel abolished HPC and HCD. 4)Apamin, a small conductance Ca2+/-activated K+ (KCa) channel blocker, and iberiotoxin, a large conductance KCa channel blocker, had no effect on the HCD. 5)Glibenclamide, an ATP-sensitive K+ (KATP) channel blocker, reduced HCD. 6)Cromakalim, an K(ATP) channel opener, relaxed the coronary artery precontracted with prostaglandin F2 alpha. The degree of relaxation by cromakalim was similar to that by hypoxia and glibenclamide reduced both hypoxia- and cromakalim-induced vasodilations. 7)Verapamil, a Ca2+ entry blocker, caffeine, a Ca2+ emptying drug; and ryanodine, an inhibitor of Ca2+ release from SR, reduced HPC, respectively. CONCLUSION: HPC is dependent on the endothelium and is considered to be induced by inhibition of the mechanisms of NO-dependent vasodilation while HCD is independent of the endothelium and is considered to be induced by activation of the K(ATP) channel.
Anoxia ; Arteries ; Caffeine ; Coronary Vessels ; Cromakalim ; Dinoprost ; Endothelium ; Glyburide ; Indomethacin ; Muscle, Smooth ; Norepinephrine ; Oxygen ; Prostaglandin-Endoperoxide Synthases ; Prostaglandins F ; Pulmonary Artery ; Relaxation ; Ryanodine ; Vasoconstriction* ; Vasodilation*

OBJECTIVETo investigate the mechanism of ATP-sensitive potassium channel (K(ATP)) activator cromakalim (CRK) on action potentials and transient inward current (I(ti)) in isolated guinea pig papillary and ventricular myocytes and to explore the mechanisms of effects of I(ti) and K(ATP) treatment in idiopathic ventricular tachycardia.

METHODSThe whole-cell patch clamp recording technique was used to detect the action potentials and I(ti) and K(ATP) current alterations during the stimulated and triggered activity. Myocytes were isolated from guinea pig ventricle by enzyme digestion. The experiment was divided into four groups: (1) Control; (2) Control + Ouabain; (3) Control + CRK; (4) Control + Ouabain + CRK. (5) Control + Ouabain + CRK + glibenclamide (GLB). The action potential of guinea pig papillary muscles was measured by using standard microelectrode. The parameters in the experiment included the amplitude (APA), resting potentials (RP), action potentials duration (APD), as well as maximum rise of the action potential (Vmax).

RESULTS(1) When the guinea pig ventricular papillary myocytes were pretreated with Ouabain 0.5 micromol/L, APD prolonged significantly, especially APD(20), APD(50), APD(90). Delayed after depolorazion (DAD) and triggered activity were elicited. I(ti) currents and DAD as well as triggered activity increased. I(ti) current was (126.9 +/- 10.8) pA, lagT (1173.0 +/- 70.9) ms (n = 10, P < 0.01). (2) When guinea pig ventricular myocytes were pretreated with CRK (10 micromol/L), APD was shortened and the amplitude of DAD was lowered. The coupling time in CRK group was significantly prolonged compared with Ouabain group (n = 10, P < 0.01). (3) CRK 50 micromol/L pretreatment of the ventricular myocytes led to an increase of K(ATP) up to (342 +/- 89) pA, which was statistically significant as compared with the control group (P < 0.01). ATP-sensitive potassium channel blocker glibenclamide (10 micromol/L) could antagonize the effects of CRK on APD and I(ti) currents.

CONCLUSIONCRK might reduce the toxic effect of Ouabain on cardiomyocytes, shorten APD, terminate DAD and trigger excitation, and have protective effect on cardiomyocytes. The effects of CRK, may be associated with the inhibiting I(ti) current and increasing K(ATP).

Action Potentials ; drug effects ; Animals ; Cromakalim ; pharmacology ; Guinea Pigs ; Heart Ventricles ; drug effects ; Myocytes, Cardiac ; drug effects ; physiology ; Patch-Clamp Techniques ; Potassium Channels, Inwardly Rectifying ; agonists

ATP-sensitive K+ channels (KATP) were not identified in gastric smooth muscle cells. However, in tension recording of intact gastric circular muscle, lemakalim of KATP channels opener in other tissues suppressed mechanical contractions and this effect was blocked by glibenclamide, a specific inhibitor of KATP channels. The aims of this study were to investigate whether KATP channels exist in gastric smooth muscle of guinea-pig and to know its physiological role. Whole cell K+ currents activated by lemakalim were recorded from freshly isolated cells with a 0.1 mM ATP, 140 mM KCl pipette solutions. Lemakalim (10 muM) increased inward currents of -224 +/- 34 pA (n = 13) at -80 mV of holding potential in bath solution contained 90 mM K+. Bath-applied glibenclamide (10 muM) inhibited the lemakalim-activated inward currents by 91 +/- 6% (n = 5). These lemakalim-activated inward currents were reduced by increased intracellular ATP from 0.1 to 3 mM (-41 +/- 12 pA) (n = 5). The reversal potential of the glibenclamide-sensitive inward currents was -5.2 +/- 2.4 mV (n = 3) in external 90 mM K+ and shifted to -14.8 +/- 3.6 mV (n = 3) in external 60 mM K+, which close to equilibrium potential of K+ (EK). External barium and cesium inhibited the lemakalim-activated inward currents dose-dependently. The half-inhibitory dose (IC50) of barium and cesium were 2.3 muM (n = 5) and 0.38 mM (n = 4), respectively. 10 mM tetraethylammonium (TEA) also inhibited the lemakalim-activated inward currents by 66 +/- 15% (n = 5). Both substance P (SP) (5 muM) and acetylcholine (ACh) (5 muM) inhibited lemakalim-activated inward currents. These results suggest that KATP channels exist in the gastric smooth muscle and its modulation by neurotransmitters may play an important role in regulating gastric motility.
Acetylcholine ; Adenosine Triphosphate ; Barium ; Baths ; Cesium ; Cromakalim ; Glyburide ; KATP Channels ; Muscle Cells* ; Muscle, Smooth ; Myocytes, Smooth Muscle ; Neurotransmitter Agents ; Substance P ; Tetraethylammonium

BACKGROUND: Cerebral ischemia causes an increase in extracellular potassium ([K+]e) through activation of the KATP channel. This increase in [K+]e could result in neuronal depolarization and a reversal of the glutamate uptake system in glia. This may further contribute to the excessive concentrations of glutamate and asparate in the extracellular space during ischemia. If the early rise in [K+]e during ischemia could be attenuated, less excitotoxic neuronal damage may be the result. However, activation of KATP channels has been shown to attenuate the anoxia induced depolarization in the hippocampus and may reduce the release of excitatory neurotransmitters during cerebral ischemia. In this study, we address the question of whether KATP channel modulation affects [K+]e and whether it is related with extracellular glutamate concentrations. METHODS: After approval by the Animal Care and Use Committee, 18 New Zealand white rabbits were anesthetized with halothane and mechanically ventilated to maintain normocarbia. Microdialysis catheters were inserted into the left dorsal hippocampus and perfused with artificial cerebrospinal fluid at 2 ml/min. K+ sensitive microelectrodes were inserted into the contralateral hippocampus. A pneumatic tourniquet was placed loosely around the neck. Animals were randomized to receive glibenclamide (n=5, KATP blocker, 3.7 mg/kg) or cromakalim (n=5, KATP opener, 0.5 mg/kg). The control group (n=6) had neither drug. Ten-minute period of global cerebral ischemia was produced by inflation of the tourniquet combined with induced hypotension. Hippocampal [K+]e was measured throughout the periischemic period and glutamate concentrations in dialysate were determined by high-performance liquid chromatography. Peak levels were compared by ANOVA. RESULTS: Glutamate concentration significantly increased during ischemia period for all groups (p<0.05). In glibenclamide treated animals, brain glutamate concentration increased markedly during early reperfusion (t=I+15) compared to other groups (p<0.05). There were no statistical differences on ischemia-induced increases in [K+]e among the three groups. CONCLUSIONS: Although it was not possible to demonstrate an effect of modulators of the ATP sensitive K+ channel on [K+]e, glibenclamide increased glutamate during reperfusion. This paradoxical increase in glutamate after administration of a K+ channel blocker suggests that the mechanism of glutamate release is not related to [K+]e change.
Adenosine Triphosphate ; Animals ; Anoxia ; Brain ; Brain Ischemia ; Catheters ; Cerebrospinal Fluid ; Chromatography, Liquid ; Cromakalim ; Extracellular Space ; Glutamic Acid* ; Glyburide ; Halothane ; Hippocampus* ; Hypotension ; Inflation, Economic ; Ischemia* ; KATP Channels ; Microdialysis ; Microelectrodes ; Neck ; Neuroglia ; Neurons ; Neurotransmitter Agents ; Potassium ; Rabbits ; Reperfusion ; Tourniquets

OBJECTIVE: It has been reported that the presence of a pharmacologically inactive foreign substance, polystyrene latex bead, in subarachnoid space activates a non-specific immunological response and elicits arterial narrowing. In vivo study was undertaken to characterize vascular reactivity of bead-induced constriction. METHODS: The spasm models similar to that by subarachnoid blood injection were created by injection of bead(5 volume% or 10 volume %) into rabbit cisterna magna. The basilar artery was visualized using transclival exposure, and its diameter was monitored using videomicroscopy on day two after cisternal injection. Consequently, many kinds of vasodilators such as papaverine, endothelin receptor antagonist, nicardipine, H7, dibutyryl-c-AMP, 8-bromo-c-GMP, nitroglycerine, forskolin, calcitonin gene-related peptide, and cromakalim were topically applied to determine what vasodilators attenuate arterial constriction induced by bead in 31 rabbits. RESULTS: Injection of bead elicited an arterial constriction, reducing arterial diameter to 78.5% of resting tone in 5 volume% and 67.7% in 10 volume%. ATP-sensitive potassium channel activator, cromakalim, inhibited 5 volume% or 10 volume% bead induced constriction. This effect achieved statistical significance (p<0.05) at a concentration of 10nM. However, other vasodilators did not make a significant vasodilatation of bead induced constriction. CONCLUSION: These results suggest that inactivation of ATP-sensitive potassium channel by inflammation is possibly responsible for the polysytrene latex bead-induced vasospasm, and support the concept that targeting vascular potassium channels can be of benefit in preventing the development of vasospasm.
Basilar Artery ; Calcitonin Gene-Related Peptide ; Cisterna Magna ; Colforsin ; Constriction ; Cromakalim ; Inflammation ; Latex* ; Microscopy, Video ; Microspheres* ; Nicardipine ; Nitroglycerin ; Papaverine ; Polystyrenes* ; Potassium Channels ; Rabbits ; Receptors, Endothelin ; Spasm* ; Subarachnoid Space ; Vasodilation ; Vasodilator Agents