Channel Opener Cromakalim Research Articles

Supplemental treatment to atropine improves the efficacy to reverse nerve agent induced bronchoconstriction

Exposure to highly toxic organophosphorus compounds causes inhibition of the enzyme acetylcholinesterase resulting in a cholinergic toxidrome and innervation of receptors in the neuromuscular junction may cause life-threatening respiratory effects. The involvement of several receptor systems was therefore examined for their impact on bronchoconstriction using an ex vivo rat precision-cut lung slice (PCLS) model. The ability to recover airways with therapeutics following nerve agent exposure was determined by quantitative analyses of muscle contraction.PCLS exposed to nicotine resulted in a dose-dependent bronchoconstriction. The neuromuscular nicotinic antagonist tubocurarine counteracted the nicotine-induced bronchoconstriction but not the ganglion blocker mecamylamine or the common muscarinic antagonist atropine. Correspondingly, atropine demonstrated a significant airway relaxation following ACh-exposure while tubocurarine did not. Atropine, the M3 muscarinic receptor antagonist 4-DAMP, tubocurarine, the β2-adrenergic receptor agonist formoterol, the Na+-channel blocker tetrodotoxin and the K+ATP-channel opener cromakalim all significantly decreased airway contractions induced by electric field stimulation. Following VX-exposure, treatment with atropine and the Ca2+-channel blocker magnesium sulfate resulted in significant airway relaxation. Formoterol, cromakalim and magnesium sulfate administered in combinations with atropine demonstrated an additive effect.In conclusion, the present study demonstrated improved airway function following nerve agent exposure by adjunct treatment to the standard therapy of atropine.

KATP Channel Openers Inhibit Lymphatic Contractions and Lymph Flow as a Possible Mechanism of Peripheral Edema.

Pharmacological openers of ATP-sensitive potassium (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema, limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01–3 µmol/l) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01–100 µmol/l) had similar effects at clinically relevant plasma concentrations. High-speed in vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01–10 µmol/l) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time polymerase chain reaction and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory sulfonylurea receptor 2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, which is a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema.SIGNIFICANCE STATEMENTATP-sensitive potassium (KATP) channel openers (KCOs) are potent antihypertensive medications, but off-target effects, including severe peripheral edema, limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the KATP channels in lymphatic muscle cells may be unique from their counterparts in arterial muscle implies that designing arterial-selective KCOs may avoid activation of lymphatic KATP channels and peripheral edema.

Modulation of mitochondrial respiration rate and calcium-induced swelling by new cromakalim analogues

A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.

Study on the Characteristics of ATP-Sensitive Potassium Channels in Neonatal Rat Hypoxic-Ischemic Encephalopathy

KATP channels are present on the surface of cardiac internal membranes, skeletal muscle, brain, and mitochondrial membranes. KATP channels can be activated with selective drugs including diazoxide and cromakalim that may be able to limit ischemia-induced damage or brain cell death. KATP channels have no relationship with a homogenous group, while other channels do not have the same combinations. This study was to characterize the effects of the neuro-protective KATP channel openers diazoxide and cromakalim in neonatal rats with hypoxic-ischemic encephalopathy (HIE) induced by intrauterine asphyxia. Seventy-two hours-old Sprague Dawley rat pups were subjected to intrauterine asphyxia and hypoxia for 30 min, and then randomly assigned to seven groups: Vehicle, Hypoxic-ischemia (HI), Diazoxide (Dia), Cromakalim (Cro), Diazoxide+Glibenclamide (Dia + Gli), Cromakalim+Glibenclamide (Cro + Gli), and Solvent (NaOH). Brain injury was maintained by neurologic deficit or mass injury 72 hours after HI. Moreover, gene expression of KATP channels was measured by RT-PCR 72 hours after HI. Results showed that the hippocampus and cortex had the most serious injury after HI, next highest injury was in the cerebellum, and the least amount of injury was in the thalamus and brainstem. Diazoxide and cromakalim showed a neuroprotective effect to the five brain regions tested after intrauterine asphyxia. The neuroprotective effect between diazoxide and cromakalim was not statistically significant and could be inhibited by glibenclamide. Diazoxide and cromakalim had the strongest neuroprotective effect in the hippocampus and cortex, which showed the most serious injury after intrauterine asphyxia.

Muscarinic suppression of ATP-sensitive K+ channels mediated by the M3/Gq/11/phospholipase C pathway contributes to mouse ileal smooth muscle contractions.

ATP-sensitive K+ (KATP) channels are expressed in gastrointestinal smooth muscles, and their activity is regulated by muscarinic receptor stimulation. However, the physiological significance and mechanisms of muscarinic regulation of KATP channels are not fully understood. We examined the effects of the KATP channel opener cromakalim and the KATP channel blocker glibenclamide on electrical activity of single mouse ileal myocytes and on mechanical activity in ileal segment preparations. To explore muscarinic regulation of KATP channel activity and its underlying mechanisms, the effect of carbachol (CCh) on cromakalim-induced KATP channel currents ( IKATP) was studied in myocytes of M2 or M3 muscarinic receptor-knockout (KO) and wild-type (WT) mice. Cromakalim (10 µM) induced membrane hyperpolarization in single myocytes and relaxation in segment preparations from WT mice, whereas glibenclamide (10 µM) caused membrane depolarization and contraction. CCh (100 µM) induced sustained suppression of IKATP in cells from both WT and M2KO mice. However, CCh had a minimal effect on IKATP in M3KO and M2/M3 double-KO cells. The Gq/11 inhibitor YM-254890 (10 μM) and PLC inhibitor U73122 (1 μM), but not the PKC inhibitor calphostin C (1 μM), markedly decreased CCh-induced suppression of IKATP in WT cells. These results indicated that KATP channels are constitutively active and contribute to the setting of resting membrane potential in mouse ileal smooth muscles. M3 receptors inhibit the activity of these channels via a Gq/11/PLC-dependent but PKC-independent pathways, thereby contributing to membrane depolarization and contraction of smooth muscles. NEW & NOTEWORTHY We systematically investigated the regulation of ATP-sensitive K+ channels by muscarinic receptors expressed on mouse ileal smooth muscles. We found that M3 receptors inhibit the activity of ATP-sensitive K+ channels via a Gq/11/PLC-dependent, but PKC-independent, pathway. This muscarinic suppression of ATP-sensitive K+ channels contributes to membrane depolarization and contraction of smooth muscles.

Editorial Comment

Editorial Comment

Analogs of the ATP-Sensitive Potassium (KATP) Channel Opener Cromakalim with in Vivo Ocular Hypotensive Activity.

ATP-sensitive potassium (KATP) channel openers have emerged as potential therapeutics for the treatment of glaucoma, lowering intraocular pressure (IOP) in animal models and cultured human anterior segments. We have prepared water-soluble phosphate and dipeptide derivatives of the KATP channel opener cromakalim and evaluated their IOP lowering capabilities in vivo. In general, the phosphate derivatives proved to be more chemically robust and efficacious at lowering IOP with once daily dosing in a normotensive mouse model. Two of these phosphate derivatives were further evaluated in a normotensive rabbit model, with a significant difference in activity observed. No toxic effects on cell structure or alterations in morphology of the aqueous humor outflow pathway were observed after treatment with the most efficacious compound, (3S,4R)-2, suggesting that it is a strong candidate for development as an ocular hypotensive agent.

Studies of Ischemic Preconditioning Mechanisms in Langendorff Rat Heart Model the Impact of Phosphocreatin Kinase and ATP Sensitive Potasium Channels Pharmachological Openers and Blockers on Cardioprotection

Abstract Ischemic preconditioning (IPC) induced by administration of brief episodes of ischemia-reperfusion represents a protective mechanism of the heart against prolonged episodes of ischemia. Although the mechanism of ischemic preconditioning has been extensively studied, however, until now it is insufficient elucidated. Using a Langendorff rat heart model with 45 minutes ischemia followed by 120 minute reperfusion we aimed to evaluate the role of Phosphocreatin Kinase-C (PCK) in ischemic rat myocardium and demonstration of its involvment in the path of pharmachological preconditioning (PP) by using PKC activators 1,2-dioctanoyl-sn-glycerol (DOG) and inhibitors chelerythrine (CHE) and evaluation of the role of K ATP channels in pharmachological preconditioning (PP) mechanism by administration of a K ATP channel opener (Cromakalim) (CRK) or by blocking the opening of K ATP channels with glibenclamide(GLB). The activators of PCK(DOG) and of K ATP channel (CRK) limited the infarct size when perfused before lethal ischemia, mimicking the ischemic preconditioning in rat heart When activator of PCK DOG + GLB, a K ATP channel inhibitor were coperfused before the lethal ischemia, there was an increase in myocardial infarct size, expressed as a percentage of the area at risk, versus control, the cardioprotective effect of DOG being abolished by GLB. The same results were obtained when CHE the inhibitor of PCK was coperfused with CRK the activator of K ATP channel. The effect of CHE and GLB perfused at the beginning of IPC resulted in loss of protection accompanied by a significant increase in infarct size area. The finding that treatment with a DOG PKC activator and CRK activator of K ATP channel gives a similar degree of protection against infarction as that seen after ischemic preconditioning and that this protection can be blocked by CHE a PCK pharmachological inhibitor provides support for the hypothesis that PKC plays a pivotal role in ischemic preconditioning. Our data may have a significance in pharmachological preconditioning (PP) with decrease in infarct size as an end point.

Correction: Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems

Elevated intraocular pressure (IOP) is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001) when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89). In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002). Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/-) mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm’s canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development.

Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems.

Elevated intraocular pressure (IOP) is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001) when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89). In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002). Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/-) mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm’s canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development.

MP43-19 EFFECT OF PHARMACOLOGICAL POST CONDITIONING ON ISCHEMIA-REPERFUSION INJURY IN THE RAT TESTIS

INTRODUCTION AND OBJECTIVES: The effects of administered KATP channel openers or blockers during ischemia are still controversial in many organs/tissues. To examine whether the administration of KATP channel openers or blockers before or during ischemia ameliorates IR injury in the testis. METHODS: Eight-week-old male SD rats were subjected to 2 h right testicular ischemia followed by 24 h reperfusion. The selective mitochondrial (mito) KATP channel blocker, 5-hydroxydecanoate (5-HD) (40 mg/kg), non-selective KATP channel blocker glibenclamide (Glc) (5 mg/kg) or selective mito KATP channel opener diazoxide (DZ) (10 mg/kg), non-selective KATP channel opener cromakalim (Crom) (300 mg/kg) were administered intraperitoneally 15 min prior to the ischemia or 45 min before the induction of reperfusion. Tissue damage was evaluated by malondialdehyde concentration, myeloperoxidase activity, histological evaluation and TUNEL assay in the testis. RESULTS: There was a significant reduction in oxidative stress, neutrophil infiltration, histological score and apoptosis in the IR model in the testis when DZ and Crom were administrated before ischemia. However, this reduction was not seen with the administration of DZ or Crom during ischemia. In addition, there was a significant reduction in the testicular IR injury with the administration of Glc, but not 5-HD during ischemia. CONCLUSIONS: The pre-conditioning effect on IR injury in the testis was confirmed by treatment with DZ. The administration of non-selective KATP channel blocker Glc during ischemia ameliorated against the testicular IR injury. On the other hand, the selective mito KATP channel blocker, 5-HD and KATP channel openers during ischemia did not reduce the testicular IR injury. These data suggest that opening of KATP channel before ischemia, and blocking of the sarcolemmal KATP channel during the testicular ischemia may have a protective effect. Glc could be an effective drug to manage the post-ischemic injury after testicular torsiondetorsion injury. Source of Funding: A grant in aid from the Ministry of Education, Science, and Culture of Japan (#23118)

Blocking of the ATP sensitive potassium channel ameliorates the ischaemia‐reperfusion injury in the rat testis

There is increasing evidence that the effects of administered ATP sensitive potassium (KATP ) channel openers or blockers during ischaemia are still controversial in many organs/tissues. Testicular torsion detorsion which causes ischaemia-reperfusion (IR) injury, cannot be predicted, thus an effective drug should be administered during or after the ischaemia. The aim of this study was to examine whether the administration of KATP channel openers or blockers during ischaemia ameliorates IR injury in the testis. Eight-week-old male Sprague-Dawley rats were subjected to 2 h right testicular ischaemia followed by 24 h reperfusion. The selective mitochondrial (mito) KATP channel blocker, 5-hydroxydecanoate (5-HD) (40 mg/kg), the non-selective KATP channel blocker glibenclamide (5 mg/kg), the selective mito KATP channel opener diazoxide (10 mg/kg) and the non-selective KATP channel opener cromakalim (300 μg/kg) were administered intraperitoneally 15 min prior to the ischaemia or 75 min after the induction of ischaemia. Tissue damage was evaluated by malondialdehyde concentration, myeloperoxidase activity, histological evaluation and TdT-mediated dUTP nick end labelling assay in the testis. There was a significant increase in oxidative stress, neutrophil infiltration, histological damage and apoptosis in the testicular IR model. A significant reduction in the testicular IR injury was observed with the administration of glibenclamide, but not 5-HD, diazoxide or cromakalim during ischaemia. The administration of non-selective KATP channel blocker glibenclamide ameliorated the testicular IR injury. On the other hand, the selective mito KATP channel blocker, 5-HD and KATP channel openers did not reduce the testicular IR injury. These data suggest that blocking of the membrane KATP channel may have a protective effect during the testicular ischaemia. Glibenclamide could be an effective drug to manage the post-ischaemic injury caused by the testicular torsion-detorsion.

Does the combination of hyperkalemia and KATP activation determine excitation rate gradient and electrical failure in the globally ischemic fibrillating heart?

Ventricular fibrillation (VF) in the globally ischemic heart is characterized by a progressive electrical depression manifested as a decline in the VF excitation rate (VFR) and loss of excitability, which occur first in the subepicardium (Epi) and spread to the subendocardium (Endo). Early electrical failure is detrimental to successful defibrillation and resuscitation during cardiac arrest. Hyperkalemia and/or the activation of ATP-sensitive K(+) (KATP) channels have been implicated in electrical failure, but the role of these factors in ischemic VF is poorly understood. We determined the VFR-extracellular K(+) concentration ([K(+)]o) relationship in the Endo and Epi of the left ventricle during VF in globally ischemic hearts (Isch group) and normoxic hearts subjected to hyperkalemia (HighK group) or a combination of hyperkalemia and the KATP channel opener cromakalim (HighK-Crom group). In the Isch group, Endo and Epi values of [K(+)]o and VFR were compared in the early (0-6 min), middle (7-13 min), and late (14-20 min) phases of ischemic VF. A significant transmural gradient in VFR (Endo > Epi) was observed in all three phases, whereas a significant transmural gradient in [K(+)]o (Epi > Endo) occurred only in the late phase of ischemic VF. In the Isch group, the VFR decrease and inexcitability started to occur at much lower [K(+)]o than in the HighK group, especially in the Epi. Combining KATP activation with hyperkalemia only shifted the VFR-[K(+)]o curve upward (an effect opposite to real ischemia) without changing the [K(+)]o threshold for asystole. We conclude that hyperkalemia and/or KATP activation cannot adequately explain the heterogeneous electrical depression and electrical failure during ischemic VF.

Reduced angiotensin II levels cause generalized vascular dysfunction via oxidant stress in hamster cheek pouch arterioles

ObjectivesWe investigated the effect of suppressing plasma angiotensin II (ANG II) levels on arteriolar relaxation in the hamster cheek pouch. MethodsArteriolar diameters were measured via television microscopy during short-term (3–6days) high salt (HS; 4% NaCl) diet and angiotensin converting enzyme (ACE) inhibition with captopril (100mg/kg/day). ResultsACE inhibition and/or HS diet eliminated endothelium-dependent arteriolar dilation to acetylcholine, endothelium-independent dilation to the NO donor sodium nitroprusside, the prostacyclin analogs carbacyclin and iloprost, and the KATP channel opener cromakalim; and eliminated arteriolar constriction during KATP channel blockade with glibenclamide. Scavenging of superoxide radicals and low dose ANG II infusion (25ng/kg/min, subcutaneous) reduced oxidant stress and restored arteriolar dilation in arterioles of HS-fed hamsters. Vasoconstriction to topically-applied ANG II was unaffected by HS diet while arteriolar responses to elevation of superfusion solution PO2 were unaffected (5% O2, 10% O2) or reduced (21% O2) by HS diet. ConclusionsThese findings indicate that sustained exposure to low levels of circulating ANG II leads to widespread dysfunction in endothelium-dependent and independent vascular relaxation mechanisms in cheek pouch arterioles by increasing vascular oxidant stress, but does not potentiate O2- or ANG II-induced constriction of arterioles in the distal microcirculation of normotensive hamsters.

Significance of KATP channels, L-type Ca2+ channels and CYP450-4A enzymes in oxygen sensing in mouse cremaster muscle arterioles In vivo

BackgroundATP-sensitive K+ channels (KATP channels), NO, prostaglandins, 20-HETE and L-type Ca2+ channels have all been suggested to be involved in oxygen sensing in skeletal muscle arterioles, but the role of the individual mechanisms remain controversial. We aimed to establish the importance of these mechanisms for oxygen sensing in arterioles in an in vivo model of metabolically active skeletal muscle. For this purpose we utilized the exteriorized cremaster muscle of anesthetized mice, in which the cremaster muscle was exposed to controlled perturbation of tissue PO2.ResultsChange from “high” oxygen tension (PO2 = 153.4 ± 3.4 mmHg) to “low” oxygen tension (PO2 = 13.8 ± 1.3 mmHg) dilated cremaster muscle arterioles from 11.0 ± 0.4 μm to 32.9 ± 0.9 μm (n = 28, P < 0.05). Glibenclamide (KATP channel blocker) caused maximal vasoconstriction, and abolished the dilation to low oxygen, whereas the KATP channel opener cromakalim caused maximal dilation and prevented the constriction to high oxygen. When adding cromakalim on top of glibenclamide or vice versa, the reactivity to oxygen was gradually restored. Inhibition of L-type Ca2+ channels using 3 μM nifedipine did not fully block basal tone in the arterioles, but rendered them unresponsive to changes in PO2. Inhibition of the CYP450-4A enzyme using DDMS blocked vasoconstriction to an increase in PO2, but had no effect on dilation to low PO2.ConclusionsWe conclude that: 1) L-type Ca2+ channels are central to oxygen sensing, 2) KATP channels are permissive for the arteriolar response to oxygen, but are not directly involved in the oxygen sensing mechanism and 3) CYP450-4A mediated 20-HETE production is involved in vasoconstriction to high PO2.

Vasodilator effects of cromakalim and HA 1077 in diabetic rat aorta

Impairment of the vasorelaxant responses have been reported in diabetes mellitus. In this study, the roles of the K(ATP) channel and rho kinase pathway were evaluated by using the K(ATP) channel opener cromakalim and Rho-kinase inhibitor HA 1077 in diabetic rat aorta. Adult male Wistar rats weighing (250-300 g) were divided into diabetic and control groups. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ, 55 mg/kg/i.p). Vasodilator responses induced by cromakalim (10(-7) to 10(-3M)) and HA 1077 (10(-6) to 10(-4M)) were significantly less in diabetic rings compared with control rings (p <0.01). The decrease in the relaxant effect of cromakalim was more in endothelium-denuded rings compared to the endothelium-intact rings (p <0.05). There were no significant differences between endothelium intact and non-intact rings in the presence of HA 1077. When two drugs were administered together, relaxation was significantly less than with seperate administration of each drug in the diabetic group (p <0.01). Pre-treatment with N omega-nitro-L-arginine methylester (L-NAME) (10(-6) to 10(-4 M)), an NO synthase inhibitor, significantly decreased the relaxant response to cromakalime and HA 1077 in both the control and diabetic groups (p <0.05). These results suggest that the impaired relaxant effects were further decreased depending on K(ATP) channel activity but the effects of Rho-kinase enzyme inhibitors on relaxation responses were not significantly changed in diabetes mellitus.

Inhibitory effects of the ATP-sensitive potassium channel openers cromakalim, pinacidil and minoxidil on the carbachol–response curve in porcine detrusor muscle

AimsATP-sensitive potassium channels represent promising drug targets for treating specific bladder diseases. The inhibitory effects of ATP-selective potassium channel openers (PCOs) on the carbachol–response curve in porcine detrusor muscle were examined. Materials and methodsEach of the three substances used in the study represent one prototype of a different class of PCO: cromakalim belongs to the benzopyran series, pinacidil is a cyanoguanidine derivative, and minoxidil represents a pyrimidine derivative. The porcine detrusor muscle represents one of the best models for human detrusor. Experiments were conducted on muscle strips of porcine detrusor muscle suspended in a tissue bath. Concentration–response curves of carbachol were constructed after pretreatment with cromakalim at 10−7, 10−6 and 10−5M, and with pinacidil and minoxidil at 10−6, 10−5.5 and 10−5M, respectively. Each muscle strip was only used to examine one concentration of one substance. ResultsCromakalim had the greatest inhibitory effect, significantly suppressing the carbachol–response curve at 10−6 and 10−5M. Pinacidil showed a significant inhibitory effect at 10−5.5 and 10−5M, which was smaller than that of cromakalim. Minoxidil did not significantly inhibit the contractions at all examined concentrations. ConclusionsThe examined ATP-sensitive PCOs belonging to the benzopyrans and cyanoguanidines significantly suppressed detrusor contractions. The development of derivatives of these prototypes could open new possibilities for the pharmacological treatment of selected bladder diseases.

Role of potassium channels in the antidepressant-like effect of folic acid in the forced swimming test in mice

Potassium (K+) channels have been implicated in depressive disorders and in the mechanism of action of antidepressants. Considering that several studies have indicated that folic acid plays an important role in the pathophysiology of depression, the present study investigated the involvement of potassium channels in the antidepressant-like effect of this vitamin. For this aim, the effect of the combined administration of different types of K+ channel blockers and folic acid in the forced swimming test (FST) was investigated. Treatment of mice by intracerebroventricular (i.c.v.) route with subactive doses of glibenclamide (an ATP-sensitive K+ channels blocker, 0.5pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K+ channel blocker, 25pg/site) or apamin (a small-conductance calcium-activated K+ channel blocker, 10pg/site), augmented the effect of folic acid (10mg/kg, p.o., subeffective dose) in the FST. Additionally, the administration of folic acid and the K+ channel blockers, alone or in combination, did not affect locomotion in the open-field test. Moreover, the reduction in the immobility time in the FST elicited by folic acid administered at a higher dose (50mg/kg, p.o.) was prevented by the pretreatment of mice with the K+ channel opener cromakalim (10μg/site, i.c.v.), without affecting locomotor activity. The results of this study indicate that the antidepressant-like effect of folic acid in the FST may be at least partly due to its modulatory effects on neuronal excitability, via inhibition of K+ channels.

Reopening of ATP-sensitive potassium channels reduces neuropathic pain and regulates astroglial gap junctions in the rat spinal cord

Adenosine triphosphate–sensitive potassium (K ATP) channels are suggested to be involved in pathogenesis of neuropathic pain, but remain underinvestigated in primary afferents and in the spinal cord. We examined alterations of K ATP channels in rat spinal cord and tested whether and how they could contribute to neuropathic pain. The results showed that protein expression for K ATP channel subunits SUR1, SUR2, and Kir6.1, but not Kir6.2, were significantly downregulated and associated with thermal hyperalgesia and mechanical allodynia after sciatic nerve injury. Spinal administration of a K ATP channel opener cromakalim (CRO, 5, 10, and 20 μg, respectively) prevented or suppressed, in a dose-dependent manner, the hyperalgesia and allodynia. Nerve injury also significantly increased expression and phosphorylation of connexin 43, an astroglial gap junction protein. Such an increase of phosphorylation of connexin 43 was inhibited by CRO treatment. Furthermore, preadministration of an astroglial gap junction decoupler carbenoxolone (10 μg) completely reversed the inhibitory effects of CRO treatment on the hyperalgesia and allodynia and phosphorylation of NR1 and NR2B receptors and the subsequent activation of Ca 2+-dependent signals Ca 2+/calmodulin-dependent kinase II and cyclic adenosine monophosphate (cAMP) response element binding protein. These findings suggest that nerve injury–induced downregulation of the K ATP channels in the spinal cord may interrupt the astroglial gap junctional function and contribute to neuropathic pain, thus the K ATP channels opener can reduce neuropathic pain probably partly via regulating the astroglial gap junctions. This study may provide a new strategy for treating neuropathic pain using K ATP channel openers in the clinic.

Investigating the effects of the cromakalim pretreatment on the expression of aquaporin -4 and the blood -brain barrier permeability in acute cerebral ischemia/reperfusion injury mice

Objective To investigate the effects of ATP-sensitive potassium channel openers (Cromakalim) on the expression of aquaporin-4 and permeability of blood-brain barrer (BBB) after cerebral ischemic/repeffusion. Methods Thirty healthy male Wistar rats were randomized into three groups for different conditioning, the sham-operated group(A,n=10), the cerebral I/R group(B,n=10),and the cerebral I/R+Cromakalim group (C,n=10). Intraluminal suture methods were applied to establish the middle cerebral artery occlusion(MCAO) model with occlusion 2 h and reperfusion 24 h. The neurobehavioral function was evaluated with Bederson's test, and the pathological changes were observed with hematoxylin-eosin(HE) staining. The water content of brain was evaluated by wet-dry weight method, and the expressions of IgG and AQP-4 in brain were observed by immunohistochemistry staining. Results In comparison with group A's water content(78.2±1.3 )% and expressions of IgG and AQP-4 (0.0±0.0,13.6±1.5) ,the expressions of IgG and AQP-4(2.4±0.4,19.8±1.9) and the water content (81.3±1.2)% in group B both were significantly higher (P<0.05). However,in group C,the water content (79.5±0.6)%was similar to group A( 78.2± 1.3 )%. Interestingly, the expressions of IgG and AQP-4( 1.1 ±0.2, 15.7± 1.2 ) of group C were higher than that of group A' s (P<0.05); compared with group B, The neurobehavioral deficit scores were obviously reduced, the expressions of IgG and AQP-4 (1.1 ±0.2,15.7±1.2) and the water content (79.5 ±0.6)% were significantly lower in group C (P<0.05).Conclusion Cromakalim maybe can alleviate cerebral edema via reducing permeability of blood-brain barrier and inhibit the expression of AQP-4 in cerebral ischemia/reperfusion mice. Key words: Ischemia-reperfusion injury; Cromakalim; AQP-4; Blood brain barrier