[Ontogeny of sulphonylurea-binding regulatory subunits of K(ATP) channels in the pregnant rat myometrium]. / A K(ATP) csatorna szulfonilurea alegységeinek ontogenezise a terhes patkány miometriumban.

K(ATP) channels are composed of sulphonylurea receptors (SURs) and potassium inward rectifiers (Kir(6.x)) that assemble to form a large octameric channel. This study was designed to examine the expression and role of sulphonylurea-binding regulatory subunits 1 [SUR1 (ABCC8)] and 2 [SUR2 (ABCC9)] of the K(ATP) channels in the pregnant rat myometrium with particular regard to the contractility. RT-PCR and Western blot analysis were performed to detect the presence of SUR1 and SUR2. The SUR1 levels were markedly increased in the early stages of pregnancy. The highest level was detected on day 6 of pregnancy, while in the late stages the levels of SUR1 were significantly decreased. The SUR2 level remained unchanged throughout pregnancy. The SUR-non-selective diazoxide and the SUR2-selective pinacidil inhibited oxytocin-induced contractions. Glibenclamide, a K(ATP) channel blocker, antagonized both pinacidil and diazoxide-induced relaxations. It was established that SURs are responsible for pharmacological reactivity of K(ATP) channel openers. We conclude that, both SURs are involved in the K(ATP) channel in the pregnant rat myometrium. It may further be concluded that "pinacidil-like" K(ATP) channel openers may be of therapeutic relevance as tocolytic agents in the future.

Ontogeny of sulfonylurea-binding regulatory subunits of K(ATP) channels in the pregnant rat myometrium.

ATP-sensitive potassium channels (K(ATP) channels) are composed of sulfonylurea receptors (SURs) and potassium inward rectifiers (Kir(6.x)) that assemble to form a large octameric channel. This study was designed to examine the expression and role of sulfonylurea-binding regulatory subunits 1 (SUR1 (ABCC8)) and 2 (SUR2 (ABCC9)) of the K(ATP) channels in the pregnant rat myometrium with particular regard to the contractility. RT-PCR and western blot analyses were performed to detect the presence of SUR1 and SUR2. The SUR1 levels were markedly increased in the early stages of pregnancy. The highest level was detected on day 6 of pregnancy, whereas in the late stages, the levels of SUR1 were significantly decreased. The SUR2 level remained unchanged throughout pregnancy. The SUR non-selective diazoxide and the SUR2-selective pinacidil inhibited oxytocin-induced contractions. Glibenclamide, a K(ATP) channel blocker, antagonized both pinacidil- and diazoxide-induced relaxations. It was established that SURs are responsible for pharmacological reactivity of K(ATP) channel openers. We conclude that both SURs are involved in the K(ATP) channel in the pregnant rat myometrium. It may further be concluded that 'pinacidil-like' K(ATP) channel openers may be of therapeutic relevance as tocolytic agents in the future.

Neuroprotective effects of diazoxide and its antagonism by glibenclamide in pyramidal neurons of rat hippocampus subjected to ischemia-reperfusion-induced injury.

Mitochondrial ATP-sensitive potassium channel opener, diazoxide, is shown to have protective effect on the heart and brain following ischemia-reperfusion-induced injury (IR/II). However, the detailed effect of diazoxide and its antagonist on neuronal death, mitochondrial changes, and apoptosis in cerebral IR/II has not fully studied. IR/II was induced in rats by the 4-vessel occlusion model. Neuronal cell death and mitochondrial changes in CA1-CA4 pyramidal cells of the hippocampus were studied by light and electron microscopy, respectively. Apoptosis was assessed by measuring the amount of protein expressed by Bax and Bcl-2 genes. In light microscopy studies, the number of total and normal cells were increased only following 18 mg/kg of diazoxide. Lower doses (2 and 6 mg/kg) failed to change the cell numbers. All three doses of glibenclamide (1, 5, and 25 mg/kg) decreased the number of total and normal cell populations. In electron microscopy studies, different doses of diazoxide and glibenclamide prevented and aggravated the IR-induced morphological changes, respectively. Western blot analysis showed that diazoxide and glibenclamide inhibited and enhanced Bax protein expression respectively. Regarding Bcl-2 expression, only diazoxide showed a significant enhancement of gene expression. In conclusion, the results show that diazoxide can exhibit neuroprotective effects against IR/II in hippocampal regions, possibly through the opening of mitochondrial ATP-sensitive K(+) channels.

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

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

Myocardial preconditioning against ischemia-reperfusion injury is abolished in Zucker obese rats with insulin resistance.

Insulin resistance (IR) precedes the onset of Type 2 diabetes, but its impact on preconditioning against myocardial ischemia-reperfusion injury is unexplored. We examined the effects of diazoxide and ischemic preconditioning (IPC; 5-min ischemia and 5-min reperfusion) on ischemia (30 min)-reperfusion (240 min) injury in young IR Zucker obese (ZO) and lean (ZL) rats. ZO hearts developed larger infarcts than ZL hearts (infarct size: 57.3 +/- 3% in ZO vs. 39.2 +/- 3.2% in ZL; P < 0.05) and also failed to respond to cardioprotection by IPC or diazoxide (47.2 +/- 4.3% and 52.5 +/- 5.8%, respectively; P = not significant). In contrast, IPC and diazoxide treatment reduced the infarct size in ZL hearts (12.7 +/- 2% and 16.3 +/- 6.7%, respectively; P < 0.05). The mitochondrial ATP-activated potassium channel (K(ATP)) antagonist 5-hydroxydecanoic acid inhibited IPC and diazoxide-induced preconditioning in ZL hearts, whereas it had no effect on ZO hearts. Diazoxide elicited reduced depolarization of isolated mitochondria from ZO hearts compared with ZL (73 +/- 9% in ZL vs. 39 +/- 9% in ZO; P < 0.05). Diazoxide also failed to enhance superoxide generation in isolated mitochondria from ZO compared with ZL hearts. Electron micrographs of ZO hearts revealed a decreased number of mitochondria accompanied by swelling, disorganized cristae, and vacuolation. Immunoblots of mitochondrial protein showed a modest increase in manganese superoxide dismutase in ZO hearts. Thus obesity accompanied by IR is associated with the inability to precondition against ischemic cardiac injury, which is mediated by enhanced mitochondrial oxidative stress and impaired activation of mitochondrial K(ATP).

Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.

We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.

It is AMPA receptor, not kainate receptor, that contributes to the NBQX-induced antinociception in the spinal cord of rats.

Studies demonstrated that intrathecal 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo[f]quinoxaline-7-sulfonamide disodium (NBQX), an antagonist of AMPA/kainate receptors, induced antinociception in the spinal cord of rats. The present study demonstrated that the NBQX-induced increases in hindpaw withdrawal latencies (HWLs) were dose-dependently attenuated by intrathecal pretreatment of the AMPA receptor desensitization inhibitor, diazoxide. The effect was unrelated to the opening of K+ channels by diazoxide. On the other hand, intrathecal pretreatment of concanavalin A, which selectively inhibits the desensitization of kainate receptor, produced no significant influence on the NBQX-induced antinociception. The results suggest that the NBQX-induced antinociception was mediated by AMPA receptors, not by kainate receptors, in the spinal cord of rats.

ATP-sensitive potassium channels mediate the anti-ischemic properties of ischemic and pharmacologic preconditioning in rat random-pattern skin flap.

Ischemic preconditioning (IPC) and pharmacologic preconditioning by morphine and adenosine may significantly decrease the amount of necrosis in rat random pattern skin flaps. We examined the role of ATP-sensitive potassium channels (K(ATP) channels) in mediating these protective phenomenon by using glibenclamide a nonspecific blocker of these channels. We also investigated whether administration of diazoxide an opener of the K(ATP) channels could mimic the same protective effect. Ninety male Sprague-Dawley rats were randomly divided into either control or treatment groups (n = 6 each). Bipedicled dorsal skin flaps (2 x 8 cm) were elevated at the midline. In pharmacologic preconditioning groups, 1 mL of morphine (5 mg/flap), adenosine (0.5 mg/flap), or different doses of diazoxide (0.5, 1, 5, and 15 mg/flap) were administered locally in the cranial half of the flap, respectively. One milliliter of saline was locally injected in the control group. In the IPC group, 1 hour after local saline injection the cranial pedicle was clamped for 20 minutes, and then 40 minutes' reperfusion was performed. In another experiment, 0.3 mg/kg of glibenclamide was injected intraperitoneally 30 minutes before local administration of saline or drug in ischemic or pharmacologic preconditioning groups. Regardless of the group, all flaps were cut at the cranial side 2 hours after elevation and were sutured back. Flap survival area was evaluated on the seventh postoperative day. IPC and pharmacologic preconditioning with morphine, adenosine, and diazoxide (in higher doses; 1, 5, and 15 mg/flap) improved survival area compared with the control group. Glibenclamide abolished their protective effect. K(ATP) channels may have a key role in anti-ischemic properties of IPC and pharmacologic preconditioning.

K+-independent actions of diazoxide question the role of inner membrane KATP channels in mitochondrial cytoprotective signaling.

Activation by diazoxide and inhibition by 5-hydroxydecanoate are the hallmarks of mitochondrial ATP-sensitive K+ (K(ATP)) channels. Opening of these channels is thought to trigger cytoprotection (preconditioning) through the generation of reactive oxygen species. However, we found that diazoxide-induced oxidation of the widely used reactive oxygen species indicator 2',7'-dichlorodihydrofluorescein in isolated liver and heart mitochondria was observed in the absence of ATP or K+ and therefore independent of K(ATP) channels. The response was blocked by stigmatellin, implying a role for the cytochrome bc1 complex (complex III). Diazoxide, though, did not increase hydrogen peroxide (H2O2) production (quantitatively measured with Amplex Red) in intact mitochondria, submitochondrial particles, or purified cytochrome bc1 complex. We confirmed that diazoxide inhibited succinate oxidation, but it also weakly stimulated state 4 respiration even in K+-free buffer, excluding a role for K(ATP) channels. Furthermore, we have shown previously that 5-hydroxydecanoate is partially metabolized, and we hypothesized that fatty acid metabolism may explain the ability of this putative mitochondrial K(ATP) channel blocker to inhibit diazoxide-induced flavoprotein fluorescence, commonly used as an assay of K(ATP) channel activity. Indeed, consistent with our hypothesis, we found that decanoate inhibited diazoxide-induced flavoprotein oxidation. Taken together, our data question the "mitochondrial K(ATP) channel" hypothesis of preconditioning. Diazoxide did not evoke superoxide (which dismutates to H2O2) from the respiratory chain by a direct mechanism, and the stimulatory effects of this compound on mitochondrial respiration and 2',7'-dichlorodihydrofluorescein oxidation were not due to the opening of K(ATP) channels.

Initiation of remote microvascular preconditioning requires K(ATP) channel activity.

The purpose of this study was to investigate vascular preconditioning of individual microvascular networks. Prior work shows that exposure of downstream arterioles to specific agonists preconditions upstream arterioles so that they exhibit an altered local vasoactive response [remote microvascular preconditioning (RMP)]. We hypothesized that mitochondrial ATP-sensitive K+ (K(ATP)) channels were involved in stimulation of RMP. Arteriolar diameter (approximately 15 microm) was observed approximately 1,000 microm upstream of the remote exposure site in the cheek pouch of pentobarbital sodium-anesthetized (70 mg/kg) male hamsters (n = 104); all agonists were applied via micropipette. RMP was initiated by application of pinacidil (Pin), diazoxide (DZ), sodium nitroprusside (SNP), or bradykinin (BK) to the downstream vessel. After 15 min, RMP was apparent at the upstream observation site from testing of local vasoactive responses to L-arginine. Pin, DZ, SNP, and BK each stimulated RMP. To evaluate a specific role for mitochondrial K(ATP) channels in this response, 5-hydroxydecanoate was applied (via a 2nd pipette) during downstream stimulation with agonist. 5-Hydroxydecanoate blocked RMP initiated by Pin, DZ, or SNP, suggesting that mitochondrial K(ATP) channels are involved before SNP signal transduction. To verify this, we applied N(omega)-nitro-L-arginine during DZ or SNP stimulation. RMP was blocked during SNP, but not during DZ, stimulation. Thus stimulation of the RMP response requires mitochondrial K(ATP) channel activity after stimulation by nitric oxide donors.

Short-term intermittent exposure to diazoxide improves functional performance of beta-cells in a high-glucose environment.

Prolonged periods of "beta-cell rest" exert beneficial effects on insulin secretion from pancreatic islets subjected to a high-glucose environment. Here, we tested for effects of short-term intermittent rest achieved by diazoxide. Rat islets were cultured for 48 h with 27 mmol/l glucose alone, with diazoxide present for 2 h every 12 h or with continuous 48-h presence of diazoxide. Both protocols with diazoxide enhanced the postculture insulin response to 27 mmol/l glucose, to 200 mumol/l tolbutamide, and to 20 mmol/l KCl. Intermittent diazoxide did not affect islet insulin content and enhanced only K(ATP)-dependent secretion, whereas continuous diazoxide increased islet insulin contents and enhanced both K(ATP)-dependent and -independent secretory effects of glucose. Intermittent and continuous diazoxide alike increased postculture ATP-to-ADP ratios, failed to affect [(14)C]glucose oxidation, but decreased oxidation of [(14)C]oleate. Neither of the two protocols affected gene expression of the ion channel-associated proteins Kir6.2, sulfonylurea receptor 1, voltage-dependent calcium channel-alpha1, or Kv2.1. Continuous, but not intermittent, diazoxide decreased significantly mRNA for uncoupling protein-2. A 2-h exposure to 20 mmol/l KCl or 10 mumol/l cycloheximide abrogated the postculture effects of intermittent, but not of continuous, diazoxide. Intermittent diazoxide decreased islet levels of the SNARE protein SNAP-25, and KCl antagonized this effect. Thus short-term intermittent diazoxide treatment has beneficial functional effects that encompass some but not all characteristics of continuous diazoxide treatment. The results support the soundness of intermittent beta-cell rest as a treatment strategy in type 2 diabetes.

Diazoxide induces delayed pre-conditioning in cultured rat cortical neurons.

We investigated the effect of diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygen-glucose deprivation (OGD). Diazoxide pre-treatment induced delayed pre-conditioning and almost entirely attenuated the OGD-induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate abolished the protective effect of diazoxide while the non-selective KATP channel blocker glibenclamide did not. The non-selective KATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of diazoxide. Diazoxide did not increase reduced glutathione and manganese-superoxide dismutase levels but we found significantly higher reduced glutathione levels in diazoxide-pre-conditioned neurons after OGD. In pre-conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3-nitropropionic acid also induced pre-conditioning and free radical production in neurons. Here, we provide the first evidence that diazoxide induces delayed pre-conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase-inhibiting effect of diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP-sensitive potassium channels.

MCC-134, a single pharmacophore, opens surface ATP-sensitive potassium channels, blocks mitochondrial ATP-sensitive potassium channels, and suppresses preconditioning.

BACKGROUND: MCC-134 (1-[4-(H-imidazol-1-yl)benzoyl]-N-methylcyclobutane-carbothioamide), a newly developed analog of aprikalim, opens surface smooth muscle-type ATP-sensitive potassium (K(ATP)) channels but inhibits pancreatic K(ATP) channels. However, the effects of MCC-134 on cardiac surface K(ATP) channels and mitochondrial K(ATP) (mitoK(ATP)) channels are unknown. A mixed agonist/blocker with differential effects on the two channel types would help to clarify the role of K(ATP) channels in cardioprotection. METHODS AND RESULTS: To index mitoK(ATP) channels, we measured mitochondrial flavoprotein fluorescence in rabbit ventricular myocytes. MCC-134 alone had little effect on basal flavoprotein fluorescence. However, MCC-134 inhibited diazoxide-induced flavoprotein oxidation in a dose-dependent manner (EC(50)=27 micro mol/L). When ATP was included in the pipette solution, MCC-134 slowly activated surface K(ATP) currents with some delay (>10 minutes). These results indicate that MCC-134 is a mitoK(ATP) channel inhibitor and a surface K(ATP) channel opener in native cardiac cells. In cell-pelleting ischemia assays, coapplication of MCC-134 with diazoxide abolished the cardioprotective effect of diazoxide, whereas MCC-134 alone did not alter cell death. These results were reproducible in both rabbit and mouse myocytes. MCC-134 also attenuated the effect of ischemic preconditioning against myocardial infarction in mice, consistent with the results of cell-pelleting ischemia assays. CONCLUSIONS: A single drug, MCC-134, opens surface K(ATP) channels but blocks mitoK(ATP) channels; the fact that this drug inhibits preconditioning reaffirms the primacy of mitoK(ATP) rather than surface K(ATP), channels in the mechanism of cardioprotection.

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

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

Differential role of K(ATP) channels in late preconditioning against myocardial stunning and infarction in rabbits.

The role of ATP-sensitive potassium (K(ATP)) channels in the late phase of ischemic preconditioning (PC) remains unclear. Furthermore, it is unknown whether K(ATP) channels serve as end effectors both for late PC against infarction and against stunning. Thus, in phase I of this study, conscious rabbits underwent a 30-min coronary occlusion (O) followed by 72 h of reperfusion (R) with or without ischemic PC (6 4-min O/4-min R cycles) 24 h earlier. Late PC reduced infarct size approximately 46% versus controls. The K(ATP) channel blocker 5-hydroxydecanoic acid (5-HD), given 5 min before the 30-min O, abrogated the infarct-sparing effect of late PC but did not alter infarct size in non-PC rabbits. In phase II, rabbits underwent six 4-min O/4-min R cycles for 3 consecutive days (days 1, 2, and 3). In controls, the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 46% on day 2 and 54% on day 3 compared with day 1, indicating a late PC effect against myocardial stunning. Neither 5-HD nor glibenclamide, given on day 2, abrogated late PC. The K(ATP) channel opener diazoxide, given on day 1, attenuated stunning, and this effect was completely blocked by 5-HD. Thus the same dose of 5-HD that blocked the antistunning effect of diazoxide failed to block the antistunning effects of late PC. Furthermore, when diazoxide was administered in PC rabbits on day 2, myocardial stunning was further attenuated, indicating that diazoxide and late PC have additive anti-stunning effects. We conclude that K(ATP) channels play an essential role in late PC against infarction but not in late PC against stunning, revealing an important pathogenetic difference between these two forms of cardioprotection.

Mitochondrial potassium channel opener diazoxide preserves neuronal-vascular function after cerebral ischemia in newborn pigs.

BACKGROUND AND PURPOSE: N-Methyl-D-aspartate (NMDA) elicits neuronally mediated cerebral arteriolar vasodilation that is reduced by ischemia/reperfusion (I/R). This sequence has been preserved by pretreatment with the ATP-sensitive potassium (K(ATP)) channel opener aprikalim, although the mechanism was unclear. In the heart, mitochondrial K(ATP) channels (mitoK(ATP)) are involved in the ischemic preconditioning-like effect of K(+) channel openers. We determined whether the selective mitoK(ATP) channel opener diazoxide preserves the vascular dilation to NMDA after I/R. METHODS: Pial arteriolar diameters were determined with the use of closed cranial window/intravital microscopy in anesthetized piglets. Vascular responses to NMDA were assessed before and 1 hour after 10 minutes of global cerebral ischemia induced by raising intracranial pressure. Subgroups received 1 of the following pretreatments before I/R: vehicle; 1 to 10 micromol/L diazoxide; and coapplication of 100 micromol/L 5-hydroxydecanoic acid (5-HD), a K(ATP) antagonist with diazoxide. RESULTS: NMDA-induced dose-dependent pial arteriolar dilation was not affected by diazoxide treatment only but was severely attenuated by I/R. In contrast, diazoxide dose-dependently preserved the NMDA vascular response after I/R; at 10 micromol/L, diazoxide arteriolar responses were unaltered by I/R. The effect of diazoxide was antagonized by coapplication of 5-HD with diazoxide. Percent preservation of 100 micromol/L NMDA-induced vasodilation after I/R was 53+/-19% (mean+/-SEM, n=8) in vehicle-treated controls versus 55+/-10%, 85+/-5%, and 99+/-15% in animals pretreated with 1, 5, and 10 micromol/L diazoxide (n=8, n=8, and n=12, respectively) and 60+/-15% in the group treated with 5-HD+diazoxide (n=5). CONCLUSIONS: The mitoK(ATP) channel opener diazoxide in vivo preserves neuronal function after I/R, shown by pial arteriolar responses to NMDA, in a dose-dependent manner. Thus, activation of mitoK(ATP) channels may play a role in mediating the protective effect of other K(+) channel openers.

ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria.

1. Mitochondrial dysfunction, secondary to excessive accumulation of Ca2+, has been implicated in cardiac injury. We here examined the action of potassium channel openers on mitochondrial Ca2+ homeostasis, as these cardioprotective ion channel modulators have recently been shown to target a mitochondrial ATP-sensitive K+ channel. 2. In isolated cardiac mitochondria, diazoxide and pinacidil decreased the rate and magnitude of Ca2+ uptake into the mitochondrial matrix with an IC50 of 65 and 128 microM, respectively. At all stages of Ca2+ uptake, the potassium channel openers depolarized the mitochondrial membrane thereby reducing Ca2+ influx through the potential-dependent mitochondrial uniporter. 3. Diazoxide and pinacidil, in a concentration-dependent manner, also activated release of Ca2+ from mitochondria. This was prevented by cyclosporin A, an inhibitor of Ca2+ release through the mitochondrial permeability transition pore. 4. Replacement of extramitochondrial K+ with mannitol abolished the effects of diazoxide and pinacidil on mitochondrial Ca2+, while the K+ ionophore valinomycin mimicked the effects of the potassium channel openers. 5. ATP and ADP, which block K+ flux through mitochondrial ATP-sensitive K+ channels, inhibited the effects of potassium channel openers, without preventing the action of valinomycin. 6. In intact cardiomyocytes, diazoxide also induced mitochondrial depolarization and decreased mitochondrial Ca2+ content. These effects were inhibited by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. 7. Thus, potassium channel openers prevent mitochondrial Ca2+ overload by reducing the driving force for Ca2+ uptake and by activating cyclosporin-sensitive Ca2+ release. In this regard, modulators of an ATP-sensitive mitochondrial K+ conductance may contribute to the maintenance of mitochondrial Ca2+ homeostasis.

Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury.

ATP-dependent potassium (KATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate KATP channels. In addition, the effects of the KATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N2-5% CO2 instead of 95% O2-5% CO2 for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%),, probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (30 microM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 MicroM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 microM). The ATP/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO2. However, only the ATP/ADP ratio was decreased further by diazoxide (300 microM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of KATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival viaprotein and DNA synthesis.

Pre-synaptic effect of the ATP-sensitive potassium channel opener diazoxide on rat substantia nigra pars reticulata neurons.

Spontaneous synaptic currents were recorded from visually identified substantia nigra pars reticulata (SNR) neurons in the rat brain slice preparation by whole-cell patch clamp technique. GABA neurons were distinguished from dopamine neurons by their electrophysiological characteristics. In the presence of 20 microM AP5 and CNQX, the spontaneous synaptic currents recorded from GABA neurons were sensitive to bicuculline and reversed polarity at a potential close to the equilibrium potential of Cl-, indicating that they were mediated by GABA(A) receptors. TTX at 1 microM eliminated action potential-dependent release of GABA from nerve terminals, revealing the miniature inhibitory post-synaptic currents (mIPSCs). The ATP-sensitive potassium channel (K(ATP) channel) opener diazoxide (30-300 microM) significantly reduced the frequency of the mIPSCs in a dose-dependent manner. However, diazoxide did not affect the average value and the distribution of the mIPSC amplitudes. Thus, this effect of diazoxide was pre-synaptic in nature. The K(ATP) channel blocker glibenclamide (300 microM) was able to restore the frequency of the mIPSCs. These data suggest that the striatonigral projection, which represents the major inhibitory input controlling SNR GABA neuron activities, possesses presynaptic K(ATP) channels on the nerve terminals.

Role of K+ channels in neuropeptide Y-induced vasoconstriction in rabbit cerebral basilar artery.

The role of K+ channels in the direct vasoconstrictive response induced by neuropeptide Y was investigated in isolated basilar arteries of rabbits and in vivo in rats. K+ channel openers, either BRL38227 or diazoxide, caused dose-dependent and complete relaxation of isolated arteries precontracted by neuropeptide Y. Exposure to both BRL38227 and diazoxide shifted the concentration-response curves for neuropeptide Y to the right without changing the maximal response. However, BRL38227 antagonized the angiotensin II-induced vasoconstriction noncompetitively. In vivo, the pressor responses produced by neuropeptide Y were significantly inhibited by pretreatment with BRL38227 in anesthetized rats. These results show that K+ channel openers antagonize neuropeptide Y-induced vasoconstriction in a competitive manner and suggest that blockade of K+ channels contributes, at least in part, to the direct vasoconstrictive effect of neuropeptide Y.