Potassium channel openers exhibit cross-tolerance with morphine in two experimental models of pain / Los abridores de canales de potasio exhiben tolerancia cruzada con la morfina en dos modelos experimentales de dolor

OBJECTIVE: The study was performed to assess the effect of potassium channel openers on morphine tolerance and vice-versa. METHODS: Swiss albino mice of either gender weighing between 25-30 g were used for the study. The study assesses the effect of potassium channel openers (cromakalim, diazoxide and minoxidil) on morphine tolerance and vice-versa, using formalin and tail-flick tests. RESULTS: The antinociceptive effect of cromakalim and minoxidil was significantly reduced when administered to morphine-tolerant mice, in both the behavioural tests. However, reduced analgesic effect of diazoxide was observed on morphine-tolerance in the formalin test but not in the tail-flick test. Tolerance was observed when morphine was administered to animals chronically treated with any of the potassium channel openers. The same effect was observed when morphine was injected into a group treated with a combination of morphine and any of the potassium channel openers. CONCLUSIONS: This study, therefore, suggests that both morphine and potassium channel openers are cross-tolerant. However, such interaction occurs at the level of potassium channels rather than at the level of receptors.

OBJETIVO: El estudio fue realizado para evaluar el efecto de los abridores de canales de potasio en la tolerancia a la morfina, y viceversa. MÉTODOS: Para el estudio, se usaron ratones albinos suizos de ambos sexos que pesaban entre 25-30 g. El estudio evalúa el efecto de los abridores de canales de potasio (cromacalina, diazóxido y minoxidil) en la tolerancia a la morfina, y viceversa, usando la prueba de la sacudida de la cola y la prueba de la formalina. RESULTADOS: El efecto antinociceptivo de la cromacalina y el minoxidil fue significativamente reducido cuando se le administró a los ratones tolerantes a la morfina, en ambas pruebas conductuales. Sin embargo, se observó un efecto analgésico reducido de diazóxido sobre la tolerancia a la morfina en la prueba de la formalina, pero no en la prueba de la sacudida de la cola. Se observó tolerancia al administrar morfina a animales crónicamente tratados con cualquiera de los abridores de canales de potasio. El mismo efecto fue observado cuando se inyectó la morfina al grupo tratado con una combinación de morfina y cualquiera de los abridores de canales de potasio. CONCLUSIONES: Por consiguiente, este estudio sugiere que tanto la morfina como los abridores de canales de potasio son tolerantes cruzados. Sin embargo, tal interacción ocurre a nivel de los canales de potasio más bien que a nivel de los receptores.

[Role of ATP-sensitive potassium channels in the clonic seizure threshold induced by pentylenetetrazole in mice with diabetes type I]

Although there is evidence that diabetes affects seizure susceptibility, the underlying mechanism has not been completely understood. On the other hand, several studies have suggested a pivotal role for K[ATP] channels in the seizure modulation. So, the present study was designed to evaluate the seizure threshold induced by pentylenetetrazole [PTZ] in diabetic mice at different times and to examine the possible role of ATP-sensitive potassium [K[ATP]] channels. In this experimental study, NMRI were diabetic with streptozocine. Then clonic seizure thresholds were determined at different times after induction of diabetes compared with corresponding non-diabetic groups. Each experimental group consisted of ten mice. There was a time-dependent alteration in the threshold in diabetic mice, reaching a peak on week 2 after STZ injection and declining significantly afterwards. The seizure threshold in 8-week diabetic mice was even lower than control levels, though the difference was not significant. The non-effective dose of K[ATP] channel blocker glibenclamide [1 mg/kg, i.p.], but not the voltage-dependent K+ channel blocker 4-aminopyridine [1 mg/kg, i.p.], decreased the seizure threshold in 2-week diabetic mice to the control levels which was blocked by pre-treatment with the K[ATP] channel opener cromakalim [10 micro g/kg, i.p.]. Moreover, the non-effective dose of cromakalim [10 micro g/kg, i.p.] increased significantly the seizure threshold in 8-week diabetic mice which was inhibited by pre-treatment with glibenclamide [1 mg/kg, i.p.] but not with 4-aminopyridine [1 mg/kg, i.p.]. This study indicated that the PTZ-induced seizure threshold is altered in diabetic mice in a time-dependent manner which could be due to the probable alteration in the K[ATP] channel functioning during diabetes

Effects of potassium channel activators on transient inward current in guinea pig ventricular myocytes / 中华儿科杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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).</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSION</b>CRK 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).</p>

Effect of KATP channel openers on myogenic and neurogenic responses in goat urinary bladder.

Isolated goat detrusor muscle exhibited spontaneous contractility with an irregular amplitude and frequency. The spontaneity of detrusor muscle exhibited a mean amplitude as 11.99 +/- 0.83 mm and frequency as 1.37 +/- 0.16/min. KATP-channel openers namely, cromakalim or pinacidil (10(-7) - 10(-4) M) added cumulatively, elicited a concentration-related inhibition of both amplitude and rate of spontaneous contractions. The mean IC50 values for both amplitude and frequency for cromakalim were 3.3 x 10(-6) M and 2.9 x 10(-6) M, respectively; and for pinacidil were 2.0 x 10(-5) M and 1.5 x 10(-5) M, respectively. Glibenclamide, a KATP-channel blocker inhibited the cromakalim-induced concentration-related relaxation of spontaneous contractions with a significant increase in its mean IC50. ACh-induced concentration-related contractile response was inhibited in the presence of either cromakalim (10(-4) M) or pinacidil (10(-4) M). The mean EC50 value of ACh, in the presence of cromakalim (2.5 x 10(-3) M) was significantly increased as compared to the control (1.2 x 10(-6) M). In the presence of glibenclamide (10(-5) M) the inhibitory effect of cromakalim was significantly reduced with consequent decrease in the EC50 value (1.9 x 10(-5) M). Application of EFS (30 V and 5 ms) on goat urinary bladder strips at 1, 2, 5, 10, 20 and 30 Hz elicited frequency-related contractile responses. Both cromakalim and pinacidil caused a rightward shift in the frequency-related contractile response curve with significant increase in the mean EF25 and EF50 values, respectively. In the presence of glibenclamide (10(-4) M), the frequency-related inhibitory response curve was shifted to left with significant (P < 0.001) increase in the mean EF25, EF50 and EF75. The present results suggest that in the goat detrusor muscle, agonist and EFS-induced contractile responses were more potently inhibited by cromakalim than pinacidil with activation of glibenclamide sensitive KATP channels.

High throughput screening method of potassium channel regulators / 药学学报

<p><b>AIM</b>To discover new regulators of potassium channel, an in vitro assay based on DiBAC4 (3) to determine the fluorescence was established for high throughput screening.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>Extracellular 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.</p><p><b>CONCLUSION</b>The DiBAC4 (3)-based assay is easily operated, economical and repeatable. So, it can be performed by high throughput screening for potassium channel regulators.</p>

Vasomotor Reactivity of the Spasm Model by the Polystyrene Latex Bead

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.

Vasomotor Reactivity of the Spasm Model by the Polystyrene Latex Bead

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.

Vascular Smooth Muscle Adenosine Triphosphate (ATP)-Sensitive Potassium Channel and Anesthetics / 대한마취과학회지

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.

Salt-induced hypertension in rats alters the response of isolated aortic rings to cromakalim

The effect of cromakalim, an opener of ATP-sensitive potassium (KATP) channel, on precontracted aortic rings from control and salt-loaded rats was studied in Sprague-Dawley rats. Salt-loading experiments involved the induction of hypertension by 6-week feeding of 80 g sodium chloride (NaCl) per kilogram (kg) diet while the control diet had 3 g NaCl per kg diet. Blood pressure and heart rate were determined by cannulation of a femoral artery under urethane/alpha-chloralose anaesthesia. Isolated aortic rings were mounted in tissue baths for isometric tension measurement. The sodium-potassium adenosine triphosphatase (Na-K ATPase) pump activity was measured by potassium (K+)-induced relaxation (with or without ouabain) following precontraction with 10(-7) M noradrenaline. The KATP channel was studied by measuring the relaxation response to cromakalim, precontracted with either 10(-7) M noradrenaline or 60 mM potassium chloride (KCl). The Na-K ATPase pump appeared to be inhibited during salt loading. ATPase inactivation was found to be ouabain sensitive but did not seem to affect subsequent K(+)-induced contraction. Cromakalim produced relaxation of noradrenaline-precontracted rings from the control rats; rings from salt-loaded rats showed significantly less relaxation than control (p < 0.05) under similar conditions. During K(+)-induced precontraction, cromakalim produced a weak biphasic response in the control rings--an initial relaxation and then a reversal. Cromakalim produced further contraction of K(+)-induced precontraction in the salt-loaded group. The results suggest that ATP-sensitive potassium channels and Na-K ATPase pumps on the vascular smooth muscle membrane may be deactivated in the development of hypertension during salt loading.

Role of KATP channels in reduced antinociceptive effect of morphine in streptozotocin-induced diabetic mice.

The nociceptive effect was measured using withdrawal latency in tail flick test in mice rendered diabetic by administering streptozotocin (200 mg/kg, i.p.). The antinociceptive effect of morphine (4 and 8 mg/kg, s.c.) and cromakalim, a KATP channel opener, (0.3, 1 and 2 micrograms, i.c.v.) was significantly reduced in diabetic mice. Moreover, co-administration of cromakalim(0.3 microgram) did not alter the reduced antinociceptive effect of morphine(4 mg/kg) in diabetic mice. Spleenectomy in diabetic mice restored the decrease in antinociceptive effect of morphine and cromakalim. Multiple dose treatment with insulin to maintain euglycaemia for 3 days in diabetic mice prevented the decrease in antinociceptive effect of morphine and cromakalim. However, hyperglycaemic tyrode's buffer did not alter the pD2 value of morphine in isolated guinea pig ileum suggesting that hyperglycaemia does not interfere with mu receptor mediated responses in vitro. The results suggest that hyperglycaemia induced decrease in antinociceptive effect of morphine and cromakalim may be due to alteration in KATP channels. Some unknown factor from spleen in diabetic mice may be responsible for this alteration in KATP channels in diabetic mice.

Influences of Free Fatty Acids on Transmembrane Action Potential and ATP-sensitive Potassium Channel Activity in Rat Myocardium

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.

Effect of the K+ Channel Modulations on Glutamate and K+ Concentrations in Rabbit Hippocampus during Transient Global Ischemia / 대한마취과학회지

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.

Cromakalim blocks membrane phosphoinositide activated signals in the guinea pig lung mast cells stimulated with antigen-antibody reactions

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.

ATP-sensitive K+ currents in gastric myocytes isolated from guinea-pig

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.

Study on the Mechanism of Hypoxic Induced Vasodilatation and Vasoconstriction

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.

Pharmacological evidence that cromakalim inhibits Ca2+ release from intracellular stores in porcine coronary artery

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.

In vitro pharmacological characteristics of SKP-450, a novel K+ channel opener, in non-vascular smooth muscles in comparison with levcromakalim

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.

The Mechanism on the Glutamate-evoked Release of 3H) norepinephrine in Rat Cortex Slices

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.

The effects of cromakalim on the mediator releases from guinea pig lung mast cell activated by specific antigen-antibody reactions

The inhibitory effect of cromakalim on the mediator release from mast cells caused by antigenantibody reactions was in controversy with the specific antigen used. However, it has recently been observed that cromakalim inhibits the release of mediators from superfused tracheal and parenchymal strips or lung mast cells after passive sensitization with the IgG1 antibody. An attempt, therefore, was made to determine the inhibitory mechanisms of cromakalim on the release of mediators such as histamine and leukotriene released by the activation of enzymes during mast cell activation. Guinea pig lung mast cells were purified through enzyme digestion, rough percoll and continuous percoll density gradients. The purified mast cells were prelabeled with [3H]palmitic acid. PLD activity was assessed more directly by the production of labeled phosphatidylethanol by PLD-mediated transphosphatidylation in the presence of ethanol. In the cells labelled with [3H]myristic acid, [3H] DAG production was measured. The methyltransferase activity was assessed by measuring the incorporation of [3H]methyl moiety into phospholipids in sensitized mast cells labelled with L-[3H] methylmethionine. cAMP level was measured by radioimmunoassay. Cromakalim resulted in a decrease in the amount of histamine and leukotrienes releases by 30% in the ovalumin-induced mast cell. Cromakalim had little effect on phospholipase D activity enhanced by the activated mast cell. Cromakalim inhibited the initial increase of diacylglycerol production during mast cell activations. Cromakalim inhibited the phospholipid methylation increased in the activated mast cell. These results show that cromakalim decreases histamine release by inhibiting the initial increase of 1,2-diacylglycerol during the mast cell activation, which is mediated via the phosphatidylinositide-phospholipase C system rather than the phosphatidylcholine-phospholipase D system. Furthermore, cromakalim reduces phosphatidylcholine production by inhibiting the methyltransferase, which decreases the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrienes.

The effects of cromakalim on the mediator releases from guinea pig lung mast cell activated by specific antigen-antibody reactions

The inhibitory effect of cromakalim on the mediator release from mast cells caused by antigenantibody reactions was in controversy with the specific antigen used. However, it has recently been observed that cromakalim inhibits the release of mediators from superfused tracheal and parenchymal strips or lung mast cells after passive sensitization with the IgG1 antibody. An attempt, therefore, was made to determine the inhibitory mechanisms of cromakalim on the release of mediators such as histamine and leukotriene released by the activation of enzymes during mast cell activation. Guinea pig lung mast cells were purified through enzyme digestion, rough percoll and continuous percoll density gradients. The purified mast cells were prelabeled with [3H]palmitic acid. PLD activity was assessed more directly by the production of labeled phosphatidylethanol by PLD-mediated transphosphatidylation in the presence of ethanol. In the cells labelled with [3H]myristic acid, [3H] DAG production was measured. The methyltransferase activity was assessed by measuring the incorporation of [3H]methyl moiety into phospholipids in sensitized mast cells labelled with L-[3H] methylmethionine. cAMP level was measured by radioimmunoassay. Cromakalim resulted in a decrease in the amount of histamine and leukotrienes releases by 30% in the ovalumin-induced mast cell. Cromakalim had little effect on phospholipase D activity enhanced by the activated mast cell. Cromakalim inhibited the initial increase of diacylglycerol production during mast cell activations. Cromakalim inhibited the phospholipid methylation increased in the activated mast cell. These results show that cromakalim decreases histamine release by inhibiting the initial increase of 1,2-diacylglycerol during the mast cell activation, which is mediated via the phosphatidylinositide-phospholipase C system rather than the phosphatidylcholine-phospholipase D system. Furthermore, cromakalim reduces phosphatidylcholine production by inhibiting the methyltransferase, which decreases the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrienes.