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

AIMTo discover new regulators of potassium channel, an in vitro assay based on DiBAC4 (3) to determine the fluorescence was established for high throughput screening.

METHODSA cell-based 96-well format fluorescence assay using DiBAC4 (3) in cultured PC12 cells was described. Cells were loaded with 5 mumol.L-1 DiBAC4 (3) and incubated at 37 degrees C for 30 min before adding KCl or several known potassium channel regulators. The cellular DiBAC4 (3) fluorescence responce was then detected. The fluorescence changes can be used to evaluate membrane potential changes, which are determined mainly by potassium channels.

RESULTSExtracellular high K(+)-induced depolarization and several potassium channel blockers including 4-AP, TEA, E-4031, glibenclamide, quinidine and nifedipine all evoked increases in DiBAC4 (3) fluorescence response. The potassium channel opener, cromakalim, evoked decrease in DiBAC4 (3) fluorescence response. The fluorescence changes of 4-AP, TEA, glibenclamide, nifedipine and cromakalim were in a concentration-dependent manner. In 76 compounds screened by using the established DiBAC4 (3)-based assay, 9 compounds were found to change the fluorescence dose-dependently. Patch clamp technique is needed to further testify and screen their actions on potassium currents.

CONCLUSIONThe DiBAC4 (3)-based assay is easily operated, economical and repeatable. So, it can be performed by high throughput screening for potassium channel regulators.

4-Aminopyridine ; pharmacology ; Animals ; Barbiturates ; chemistry ; Calcium Channel Blockers ; pharmacology ; Cromakalim ; pharmacology ; Isoxazoles ; chemistry ; Membrane Potentials ; drug effects ; Nifedipine ; pharmacology ; PC12 Cells ; Patch-Clamp Techniques ; Piperidines ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; drug effects ; Pyridines ; pharmacology ; Quinidine ; pharmacology ; Rats

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

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

Adenosine triphosphate-sensitive potassium channel (KATP channel) closed by intracellular adenosine triphosphate (ATP) appears widely distributed in the vascular system. Activation of vascular smooth muscle KATP channel with hyperpolarizing agents such as lemakalim results in membrane hyperpolarization, a consequent reduction in calcium influx through voltage-dependent calcium channel, and leads to vessel relaxation. In contrast to KATP channel activation in vascular smooth muscle cell, KATP channel-induced hyperpolarization of endothelial cells results in an increase in calcium influx, which could stimulate the production of nitric oxide and prostacyclin from the endothelial cell. KATP channels response to change in the cellular metabolic status like ischemia and hypoxia, and are the target of a variety of synthetic and endogenous vasoactive substance. KATP channel openers are used as therapeutic agent for cardiovascular disease. Endogenous KATP channel-induced vasodilation is functionally important because it has been shown to modulate the pulmonary vasoconstrictor response to hypoxia and systemic hypotension in the pulmonary circulation, enhance tissue perfusion in response to hypoxia and severe hypotension in the systemic circulation. In virtro, halothane and intravenous anesthetics attenuated KATP channel agonist, lemaklim-induced vasodilation. The coronary vasodilation by volatile anesthetics such as isoflurane, enflurane and halothane was associated with activation of KATP channel in coronary artery. Further investigation is required to determine signal transduction pathway in detail stimulated by KATP channel agonist in human blood vessel and effect of anesthetics on the KATP channel-induced signal transduction, and role of KATP channel of pathophysiology of vascular disease such as hypertension, angina.
Adenosine Triphosphate* ; Adenosine* ; Anesthetics* ; Anesthetics, Intravenous ; Anoxia ; Blood Vessels ; Calcium ; Calcium Channels ; Cardiovascular Diseases ; Coronary Vessels ; Cromakalim ; Endothelial Cells ; Enflurane ; Epoprostenol ; Halothane ; Humans ; Hypertension ; Hypotension ; Ischemia ; Isoflurane ; KATP Channels ; Membranes ; Muscle, Smooth, Vascular* ; Nitric Oxide ; Perfusion ; Potassium Channels* ; Potassium* ; Pulmonary Circulation ; Relaxation ; Signal Transduction ; Vascular Diseases ; Vasodilation

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: 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

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*

Cromakalim (BRL 34915), known as an airway smooth muscle relaxant, inhibited the releases of mediators in the antigen-induced mast cell activation. It has been suggested that cromakalim, in part, inhibited mediator releases by inhibiting the initial increase of 1,2-diacylglycerol (DAG) produced by the activation of the other phospholipase system which is different from phosphatidylcholine-phospholipase D pathway. The aim of this study is to further examine the inhibitory mechanism of cromakalim on the mediator release in the mast cell activation. Guinea pig lung mast cells were purified by using enzyme digestion and percoll density gradient. In purified mast cells prelabeled with (3H)PIP2, phospholipase C (PLC) activity was assessed by the production of (3H)insitol phosphates. Protein kinase C (PKC) activity was assessed by measuring the protein phosphorylated from mast cells prelabeled with (gamma-32P)ATP, and Phospholipase A2 (PLA2) activity by measuring the lyso-phosphatidylcholine produced from mast cell prelabeled with 1-palmitoyl-2-arachidonyl phosphatidyl-(14C)choline. Histamine was assayed by fluorometric analyzer, and leukotrienes by radioimmunoassay. The PLC activity was increased by activation of the passively sensitized mast cells. This increased PLC activity was decreased by cromakalim pretreatment. The PKC activity increased by the activation of the passively sensitized mast cells was decreased by calphostin C, staurosporine and cromakalim, respectively. The PLA2 activity was increased in the activated mast cells. The pretreatment of cromakalim did not significantly decrease PLA2 activity. These data show that cromakalim inhibits histamine release by continuously inhibiting signal transduction processes which is mediated via PLC pathway during mast cell activation, but that cromakalim does not affect PLA2 activity related to leukotriene release.
Animals ; Antigen-Antibody Reactions* ; Cromakalim* ; Digestion ; Guinea Pigs* ; Guinea* ; Histamine ; Histamine Release ; Leukotrienes ; Lung* ; Mast Cells* ; Membranes* ; Muscle, Smooth ; Phosphates ; Phospholipases ; Phospholipases A2 ; Protein Kinase C ; Radioimmunoassay ; Signal Transduction ; Staurosporine ; Type C Phospholipases

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