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PubMed2002

Diazoxide opens the mitochondrial permeability transition pore and alters Ca2+ transients in rat ventricular myocytes.

BACKGROUND: The mitochondrial K(ATP) channel (mitoK(ATP)) has been implicated as an end effector or trigger of ischemic preconditioning (IP). Although a mitoK(ATP) opener, diazoxide, mimics IP, mechanisms for the cardioprotective action remain unclear. METHODS AND RESULTS: We measured Ca2+ transients (CaTs) and mitochondrial inner membrane potential (Deltapsi(m)) with confocal microscopy and the fluorescent probes fluo-4 and tetramethylrhodamine ethyl ester perchlorate in rat ventricular myocytes. Diazoxide increased the amplitudes and diastolic levels of CaTs dose dependently. The effects of diazoxide on CaTs were inhibited by the mitoK(ATP) antagonist sodium 5-hydroxydecanoic acid (100 micromol/L), whereas application of diazoxide caused little change in Deltapsi(m). After sarcoplasmic reticulum function was disabled with ryanodine and thapsigargin, the effects of diazoxide on CaTs were still observed. The opening of the mitochondrial permeability transition pore was monitored with fluorescent calcein. Diazoxide accelerated the leakage of calcein from mitochondrial matrix (16% of control; P<0.05), and this effect was inhibited by cyclosporin A (2 micromol/L). Cyclosporin A also abolished the effects of diazoxide on CaTs. Diazoxide oxidized flavoprotein fluorescence reversibly, and this effect was partially blunted by cyclosporin A (by 24%; P<0.05). CONCLUSIONS: We conclude that in rat ventricular myocytes, diazoxide modulates the opening of the mitochondrial permeability transition pore, resulting in an increase in CaTs independent of the changes in Deltapsi(m). The action of diazoxide on the mitochondrial permeability transition pore also affects the mitochondrial redox state.

Animals↗

PubMed2001

Diazoxide-induced cardioprotection requires signaling through a redox-sensitive mechanism.

Diazoxide, a selective opener of the mitochondrial ATP-sensitive potassium channel, has been shown to elicit tolerance to ischemia in cardiac myocytes and in perfused heart. However, the mechanism of this cardioprotection is poorly understood. Because reactive oxygen species (ROS) are recognized as important intracellular signaling molecules and have been implicated in ischemic preconditioning, we examined diazoxide-induced ROS production in adult cardiomyocytes. Cells treated with 50 micromol/L diazoxide showed a 173% increase in ROS production relative to baseline. 5-Hydroxydecanoate was found to attenuate the diazoxide-induced increase in ROS generation. The diazoxide-induced increase in ROS also was abrogated by the addition of either the antioxidant N-acetylcysteine (NAC) or N-mercaptopropionylglycine. We also examined the ability of NAC to block the protective effects of diazoxide in the perfused rat heart. After 20 minutes of global ischemia and 20 minutes of reflow, hearts perfused with 100 micromol/L diazoxide before ischemia showed significantly improved postischemic contractile function relative to untreated hearts (84% versus 29% of initial left ventricular developed pressure, respectively). Hearts treated with diazoxide in the presence of 4 mmol/L NAC recovered 53% of initial left ventricular developed pressure, whereas hearts treated with NAC alone recovered 46% of preischemic function. Using (31)P NMR spectroscopy, we found that, similar to preconditioning, diazoxide significantly attenuated ischemia-induced intracellular acidification and enhanced post- ischemic recovery of phosphocreatine levels, both of which were blocked by cotreatment with NAC. These data suggest that the cardioprotective actions of diazoxide are mediated by generation of a pro-oxidant environment.

Acetylcysteine↗

PubMed1991

Caffeine potentiates the renin response to diazoxide in man. Evidence for a regulatory role of endogenous adenosine.

In the rat and dog, exogenous adenosine inhibits renin release and adenosine receptor blockade augments stimulated renin release, suggesting that endogenous adenosine contributes to the regulation of renin release. The present study examines the role of endogenous adenosine in the regulation of renin in humans. The ability of the adenosine receptor blocker, caffeine, to augment renin release in response to the vasodilator, diazoxide, has been investigated in eight normal subjects in a double-blind, placebo-controlled, cross-over study. During each arm of the study, subjects on a 150 mEq of sodium, xanthine-free diet received caffeine (250 mg 3 times daily) or placebo for 3 days before and on the study day, when they were given an i.v. loading dose of diazoxide (4 mg/kg) followed by a 3-hr continuous infusion (0.67 mg/kg/hour). PRA, caffeine and diazoxide levels were measured before, during and after the diazoxide infusion. PRA measurements were repeated with subjects standing, 6 hr after starting diazoxide. Administration of diazoxide resulted in a modest tachycardia and a small, but significant, decrease in BP. Supine PRA was elevated during and after the diazoxide infusion and rose further with standing. Although there was no difference in plasma diazoxide levels, maximal pulse or BP response to diazoxide between the two arms of the study, the renin response was significantly greater in the presence of caffeine. These data confirm that caffeine augments the PRA response to diazoxide and suggest that endogenous adenosine inhibits stimulated renin response in humans.

Adenosine↗

PubMed1985

Effects of diazoxide-induced reversible diabetes on chemically induced autochthonous mammary carcinomas in Sprague-Dawley rats.

The effect of oral administration of diazoxide on rats bearing mammary carcinomas induced by dimethylbenzanthracene (7,12-DMBA) or methylnitrosourea (MNU) was investigated. Administration of 300 mg/kg diazoxide caused mild reversible diabetes with maximum glucose levels of 305 +/- 74 (control: 119 +/- 12) mg/dl and related insulin levels of 15 +/- 5 (control: 24 +/- 11) microU/ml after 4 hr in tumor-bearing animals. Following the same dose of diazoxide a more than 90% inhibition of tumor growth was observed in 7,12-DMBA- and MNU-induced autochthonous rat mammary carcinomas as well as remission of the median total tumor volume per group in 7,12-DMBA-induced lesions. Frequently, onset of remissions and median remission duration proved to be dose-dependent in 7,12-DMBA-induced mammary carcinoma and, with the exception of the median remission duration, in MNU-induced tumors too. After cessation of diazoxide application, 30% rebound responses were observed in 7,12-DMBA-induced tumors of animals that had had a first remission due to diazoxide. Application of insulin (2 IU per rat) together with diazoxide (300 mg/kg) reversed the tumor-inhibiting effect of diazoxide in MNU-induced tumors. The diazoxide effect might in part be due to a decrease in the percentage of proliferating cells caused by insulin depletion as indicated by a lower amount of cells in S-phase, as measured by DNA-flow cytometry. Marked toxicity was observed after effective doses of diazoxide; the experiments indicate that induction of reversible diabetes might be a useful tool in the treatment of hormone-dependent mammary carcinoma.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1998

Long-term treatment of persistent hyperinsulinaemic hypoglycaemia of infancy with diazoxide: a retrospective review of 77 cases and analysis of efficacy-predicting criteria.

UNLABELLED: Primary persistent hyperinsulinaemic hypoglycaemia of infancy is rare. Diazoxide treatment remains the mainstay of medical therapy in long-term management. We reviewed 77 cases of primary persistent hyperinsulinism in neonates and infants who were treated with diazoxide and studied criteria predictive of therapeutic efficacy. The only criterion identified was age at manifestation. All but 1 of the 31 neonatal cases were unresponsive to diazoxide. Responsiveness increased with age: 12 of 39 early-infantile cases, and all seven late-infantile cases were diazoxide-responsive. In responders, a diazoxide dose of 10-15 mg/kg per day was always effective, suggesting an "all or none" response. Diazoxide-resistant hyperinsulinism is characterized by its severity with higher plasma insulin levels. The analysis of 46 surgically treated patients showed that the efficacy of diazoxide is not related to the aetiology of the pancreatic lesions. In six cases, after many years of management, diazoxide treatment was stopped without recurrence of hypoglycaemia. CONCLUSION: Diazoxide is an efficient treatment in the long-term management of most persistent hyperinsulinaemic hypoglycaemia of infancy revealed in infants and children but is usually ineffective in neonatal forms. Drug efficacy does not correlate with anatomical lesions. Medical treatment can sometimes be stopped after many years of management without recurrence of disease manifestations.

Diazoxide↗

PubMed2000

Treatment with diazoxide causes prolonged improvement of beta-cell function in rat islets transplanted to a diabetic environment.

Prolonged hyperglycemia desensitizes beta cells. A role for hyperglycemia-induced excessive stimulation can be tested by diazoxide, which inhibits glucose-induced insulin secretion. Using diazoxide, we have investigated in a rat transplantation model whether excessive stimulation can induce lasting effects on beta cells. One batch with 150 islets and another with 20 islets isolated from Wistar-Furth rats were transplanted under the left-kidney capsule of syngeneic streptozotocin-diabetic recipients. In a first series, recipients were treated for 8 weeks with or without 0.2% diazoxide in the food. Graft-bearing kidneys were then perfused and excised. Diazoxide treatment increased by 5.5-fold the insulin response to 10 mmol/L arginine, by 4.1-fold the graft insulin content, and by 2.3-fold the preproinsulin mRNA versus nontreated diabetic controls. The persistence of these effects was assessed in a second series in which 8 weeks of diazoxide treatment was followed by 1 week of no treatment. Again, perfusion experiments showed a higher insulin response to arginine in diazoxide-treated rats (136.0 +/- 25.7 v 62.3 +/- 11.8 fmol/min, P < .05). Also, the response to 27.8 mmol/L glucose was increased (54.0 +/- 17.1 v 13.6 +/- 7.8 fmol/min, P < .05). The insulin content was increased (2.2 +/- 0.6 v 1.0 +/- 0.4 pmol/islet, P < .05), as well as the preproinsulin mRNA (0.60 +/- 0.08 v0.22 +/- 0.02 pg/islet, P < .05). In a third series, we tested the impact of diazoxide treatment when given only during the first 2 weeks following transplantation. When tested 6 weeks later, insulin secretion was unaffected, whereas there was a strong tendency for a higher preproinsulin mRNA and insulin content in grafts of diazoxide-treated rats. In conclusion, this study demonstrates that beta-cell function in transplanted islets is improved by diazoxide long after the end of treatment, an effect that is likely due to removal of hyperglycemia-induced excessive stimulation.

Animals↗

PubMed2003

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.

Animals↗

PubMed1992

Diazoxide-sensitivity of the adenosine 5'-triphosphate-dependent K+ channel in mouse pancreatic beta-cells.

1. In mouse pancreatic beta-cells the regulation of the diazoxide-sensitivity of the adenosine 5'-triphosphate-dependent K+ channel (K-ATP-channel) was examined by use of the patch-clamp technique. 2. In intact beta-cells incubated at 37 degrees C in the presence of 3 mM D-glucose, diazoxide did not affect the single channel conductance but stimulated channel-opening activity. Diazoxide produced half-maximal effects at 82 microM and 13 fold activation at maximally effective concentrations (300-400 microM). The response to diazoxide (300 microM) was not completely suppressed by saturating tolbutamide concentrations (1 or 5 mM). 3. Inside-out patch-clamp experiments were carried out using an experimental protocol favouring phosphorylation of membrane proteins. Under these conditions diazoxide was ineffective in the absence of any nucleotides, weakly effective in the presence of MgATP (26 or 87 microM) and strongly effective in the presence of the Mg complexes of adenosine 5'-diphosphate, 2'-deoxyadenosine 5'-diphosphate or guanosine 5'-diphosphate (MgADP, MgdADP or MgGDP). 4. In inside-out patches exposed to nucleotide-free solutions, saturating concentrations of tolbutamide did not cause complete block of K-ATP-channels. When the channels were activated by MgdADP (48 microM), tolbutamide was even less effective. Sensitization of MgdADP-induced channel activation by diazoxide further weakened the effects of tolbutamide. 5. Diazoxide (50 or 300 microM) prevented the complete channel block induced by saturating tolbutamide concentrations in the presence of Mg2+ and ADP (1 mM). 6. In the presence of Mg2", the K-ATP-channel-blocking potency of cytosolic ATP decreased in the order inside-out> outside-out> whole-cell configuration of the patch-clamp technique.7. It is concluded that the K-ATP-channel is controlled via four separate binding sites for inhibitory nucleotides (e.g. free ATP and ADP), stimulatory nucleotides (MgADP, MgdADP, MgGDP), sulphonylureas and diazoxide. Strong inhibition of the channel openings by sulphonylureas results from occupation of both sites for nucleotides. Diazoxide is only effective when the site for stimulatory nucleotides is occupied.

Adenine Nucleotides↗

PubMed1993

B-cell insensitivity in vitro: reversal by diazoxide entails more than one event in stimulus-secretion coupling.

Sustained hyperglycemia desensitizes pancreatic B-cells to glucose. Infusion of diazoxide protects against this effect, i.e. an insulin response to glucose is preserved after exposure to the drug in vivo. The mechanisms behind this aftereffect were presently investigated in vitro. In one type of experiment, rat pancreatic islets were exposed to diazoxide (75 micrograms/ml) for 20-22 h in tissue culture. Previous exposure to diazoxide increased 2.4-fold the response to 16.7 mM glucose in final batch-type incubations. This effect was not altered by adding insulin (10 mU/ml) to the tissue culture medium. In other experiments, islets were perifused for 180 min with 27 mM glucose and, usually, 0.2 mM 3-isobutyl-1-methylxanthine. A second stimulation, 30 min long, was used to assess desensitization, which ranged between 52-80%. Diazoxide treatment during the first stimulation profoundly inhibited secretion and, as an aftereffect, completely prevented restimulation-assessed desensitization. Neither desensitization nor aftereffect were seen after low (6 mM) glucose treatment. Previous diazoxide treatment enhanced the response not only to glucose but also to 2.5 mM barium or 10 mM arginine. Desensitization induced by 30 mM potassium was not affected by diazoxide. Cooling (22 C) was employed to block glucose-induced exocytosis of insulin, but not proximal events in stimulus secretion coupling. Diazoxide treatment during cooling exerted an aftereffect that was attenuated, but not abolished (1.7-fold of the control, as assessed by restimulation at 37 C). When calcium was omitted from the perifusion medium during cooling, no aftereffect of diazoxide was seen. The results indicate that modulation of ATP-sensitive potassium channel activity is not closely linked to induction and expression of diazoxide's aftereffect, and furthermore, that protection from desensitization includes both proximal and distal events in stimulus-secretion coupling.

1-Methyl-3-isobutylxanthine↗

PubMed2004

[The protective effect of diazoxide on long-term heart preservation].

Prolongation of the duration of heart preservation in vitro is very important in clinical heart transplantation. Previous studies have shown that mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) plays an important role in cardioprotective effect. The purpose of this study was to assess whether the mitoK(ATP) opener diazoxide as an additive to cardioplegia solution could enhance myocardial protection during long-term hypothermic preservation of the rat heart. Langendorff model of isolated rat heart was used. After 30 min stabilization of perfusion, the hearts were stored in Celsior cardioplegia solution at 4 degrees C with (15, 30 and 45 micromol/L) or without diazoxide, a mitoK(ATP) channel opener, for 10 h followed by 60 min reperfusion. The recovery of cardiac contractile function, myocardial enzyme leakage in the coronary effluent, and myocardial water content were determined. The myocardial ultrastructure was also observed. We found that: (1) Diazoxide treatment improved the recovery of left ventricular developed pressure and +/-dp/dt(max) dose-dependently. Left ventricular end-diastolic pressure was significantly lower in diazoxide-treated hearts than that of hearts in Celsior solution after hypothermic preservation for 10 h. (2) Diazoxide at 30 and 45 micromol/L significantly decreased the water content of myocardium and increased coronary flow of the hearts compared to those in control. (3) The leakage of myocardial enzymes (lactate dehydrogenase, creatine kinase and glutamate-oxaloacetate transaminase) in the coronary effluent was significantly reduced in diazoxide-treated hearts. (4) Impairment of myocardial ultrastructure after 10 h hypothermic preservation was alleviated in hearts treated with 30 micromol/L diazoxide. (5) The cardiac effects of 30 micromol/L diazoxide were attenuated by a mitoK(ATP) blocker 5-hydroxydecanoate (5-HD, 100 micromol/L). These results indicate that diazoxide as a supplementation in cardioplegia solution could enhance myocardial protection during long-term hypothermic heart preservation via opening of mitochondrial K(ATP) channel.

Animals↗

PubMed1987

Biochemical determination of islet lysosomal enzyme activities following crinophagy-stimulating treatment with diazoxide in mice.

The pattern of islet lysosomal enzyme activities, islet insulin concentration and the plasma levels of insulin and glucose were studied in freely fed mice after the in vivo administration of diazoxide in doses known to induce crinophagy in islet beta-cells. After diazoxide treatment at time 0 and at 18 hr, the plasma glucose levels at 20 hr were markedly enhanced from 6.6 +/- 0.2 mmol/l (controls) to 27.2 +/- 2.7 mmol/l (diazoxide). Inhibition of insulin secretion by diazoxide was reflected in the insulinogenic index, which was reduced by approximately 40% (p less than 0.01) in the diazoxide-treated animals, who also displayed an increased concentration of islet insulin (+50%; p less than 0.01). Moreover, we found that the activities of certain lysosomal enzymes in islet tissue were markedly increased following diazoxide treatment. Thus the activities of the acid phosphatase, (+57%; p less than 0.02) the hexosaminidase N-acetyl-beta-D-glucosaminidase, (+52%; p less than 0.001), and the carboxyl proteinase cathepsin D (+41%; p less than 0.001), were all enhanced after diazoxide, whereas the activity of another lysosomal enzyme, the glycogen hydrolysing acid amyloglucosidase, was not altered by diazoxide treatment. The present data thus indicate that the morphological observation of diazoxide-induced crinophagy in pancreatic beta-cells has a biochemical correlate in enhanced levels of certain islet lysosomal enzyme activities known to participate in degradative processes. The results also suggest that islet lysosomal enzyme activities and/or lysosome populations can be modulated by a relative independence from each other.

Acetylglucosaminidase↗

PubMed1980

[Comparison of oral diazoxide and minoxidil in refractory hypertension].

The efficacy and side effects of two potent vasodilatators, namely diazoxide and minoxidil given orally, were compared in 11 patients who had hypertension refractory to conventional drug treatment. The latter included diuretics, betablockers and/or sympatholytics, and either dihydralazine or prazosin. In a crossover approach, dihydralazine and prazosin were withdrawn and replaced by diazoxide or minoxidil. In nine patients the diazoxide phase preceded the minoxidil treatment, while in two it followed minoxidil treatment. Before introduction of the more potent vasodilators blood pressure averaged 181/107 mm Hg in the supine and 161/103 mm Hg in the upright position. Both, oral diazoxide (median dose 400 mg/d( and minoxidil (medium dose 17.5 mg/d) produced similar decreases (p < 0.02) in mean arterial pressure (-15%) in the supine and (-11 vs. -12%) in the upright position. They caused a comparable tendency for sodium retention and weight gain which could be satisfactorily controlled with increased diuretic therapy, except in one patient. Hypertrichosis occurred with both drugs, but tended to be somewhat milder with diazoxide. Electrocardiograms remained generally unchanged. Plasma glucose levels were increased during diazoxide treatment in six patients necessitating interruption of this therapy in four patients. It is concluded that the antihypertensive potency and most side effect of orally administered diazoxide are comparable to those of minoxidil, except for diazoxide-related hyperglycemia which may limit the use of this substance.

Blood Glucose↗

PubMed2005

Diazoxide preconditioning attenuates global cerebral ischemia-induced blood-brain barrier permeability.

Brain edema formation due to blood-brain barrier (BBB) disruption is a major consequence of cerebral ischemia. Previously, we demonstrated that targeting mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) protects neuronal tissues in vivo and in vitro, however, the effects of mitoK(ATP) openers on cerebral endothelial cells and on BBB functions have never been examined. We investigated the effects of mitoK(ATP) channel opener diazoxide on BBB functions during ischemia/reperfusion injury (I/R). Rats were treated with 6, 20 or 40 mg/kg diazoxide ip for 3 days then exposed to global cerebral ischemia for 30 min. BBB permeability was assessed by administering Evan's-blue (EB) and Na-fluorescein (NaF) at the beginning of the 30 min reperfusion. I/R increased BBB permeability for the large molecular weight EB (ng/mg) in the cortex (control: 146 +/- 12, n = 7; I/R: 1049 +/- 152, n = 11) which was significantly attenuated in diazoxide-treated rats (575 +/- 99, n = 9; 582 +/- 104, n = 8; 20 and 40 mg/kg doses). Diazoxide pretreatment also significantly inhibited the extravasation of the low molecular weight NaF. Edema formation in the cortex was also decreased after diazoxide pretreatment. In cultured cerebral endothelial cells, diazoxide depolarized the mitochondrial membrane, suggesting a direct diazoxide effect on the endothelial mitochondria. Our results demonstrate that preconditioning of cerebral endothelium with diazoxide protects the BBB against ischemic stress.

Animals↗

PubMed2006

The effect of pre- and posttreatment with diazoxide on the early phase of chronic cerebral hypoperfusion in the rat.

Diazoxide has been identified as a mitochondrial, ATP-dependent K(+) channel opener, and a potentially neuroprotective compound under ischemic conditions. We set out to characterize the consequences of various treatment strategies with diazoxide in a rat model of chronic cerebral hypoperfusion. Cerebral hypoperfusion was induced by permanent, bilateral occlusion of the common carotid arteries (2VO, n = 36), sham-operated rats serving as controls (SHAM, n = 29). Diazoxide or its vehicle was administered i.p. daily (5 x 0.5 mg/kg/0.25 ml) or as a bolus injection (5 mg/kg/0.25 ml) before surgery or daily after surgery (5 x 0.5 mg/kg/0.25 ml). Spatial learning performance was assessed 1 week after 2VO in the Morris maze. Hippocampal pyramidal cell loss was assessed on cresyl violet-stained sections, while glial reactivity was labeled immunocytochemically. Daily or bolus pretreatment with diazoxide significantly improved 2VO-related learning impairment, whereas posttreatment was ineffective. The number of CA1 pyramidal neurons was reduced by 2VO, which was prevented by repeated or bolus pretreatment with diazoxide. Astrocyte proliferation and microglial activation were enhanced by posttreatment with diazoxide in the hippocampus CA1 area of 2VO animals as compared with SHAM. These data demonstrate that the neuroprotective effect exerted by diazoxide depends on the time of administration with respect to the onset of ischemia; pretreatment but not posttreatment with the compound has proved to be neuroprotective in chronic cerebral hypoperfusion. Thus, pretreatment with diazoxide offers therapeutical prospects for the treatment of cerebral ischemia.

Animals↗

PubMed2004

Diazoxide causes early activation of cardiac sarcolemmal KATP channels during metabolic inhibition by an indirect mechanism.

OBJECTIVE: We have used isolated myocytes to investigate the effects of diazoxide on sarcolemmal KATP channel (sarcoKATP) activity and action potential failure during metabolic inhibition, and the role of these channels in protection of functional recovery on reperfusion. MATERIALS AND METHODS: Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion. Functional recovery was assessed from the ability of cells to contract on electrical stimulation and to recover calcium homeostasis, measured with fura-2. Action potentials and KATP currents were measured using patch clamp. RESULTS: Pretreatment with diazoxide (100 microM, 5 min) increased the proportion of cells that recovered contractile function after MI and reperfusion from 16.8 +/- 2.4% to 65.0 +/- 2.2% (p<0.001) and the proportion of cells in which [Ca2+]i recovered to <250 nM. Pretreatment also accelerated action potential and contractile failure during MI. In cell-attached patches, MI activated sarcoKATP channels after 224 +/- 11 s, and diazoxide pretreatment decreased this to 145 +/- 24 s (p<0.01). However, diazoxide present in the patch pipette did not accelerate sarcoKATP channel activation. Intracellular Mg2+ rose earlier in diazoxide-pretreated cells. The sarcoKATP blocker HMR 1883 delayed action potential failure and reduced diazoxide protection. CONCLUSIONS: Diazoxide pretreatment increases recovery of function and [Ca2+]i following reperfusion. Protection is coupled with early action potential failure, due to early activation of sarcoKATP channels during metabolic inhibition (MI), which is likely to involve an indirect effect of diazoxide.

Action Potentials↗

PubMed2004

Additive antinociceptive effect of the combination of diazoxide, an activator of ATP-sensitive K+ channels, and sodium nitroprusside and dibutyryl-cGMP.

Using the rat paw pressure test, in which increased sensitivity is induced by intraplantar injection of prostaglandin E2, we assessed the antinociceptive effect of the ATP-sensitive K+ channel opener diazoxide and the large-conductance Ca(2+)-activated K+ channel opener NS-1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one) on the peripheral hyperalgesia induced by prostaglandin E2. Diazoxide, administered locally into the right hindpaw (20, 38, 75, 150, 300 and 600 microg), elicited a dose-dependent antinociceptive effect on prostaglandin E2-induced hyperalgesia (2 microg/paw). The effect of diazoxide at the dose of 300 microg/paw was shown to be local since it did not produce any effect when administered in the contralateral paw. The action of diazoxide (300 microg/paw) as an ATP-sensitive K+ channel opener seems to be specific, since its effect was antagonized in a dose-dependent manner by glibenclamide (40, 80 and 160 microg/paw), a specific blocker of these channels, while tetraethylammonium (7.5, 15 and 30 microg/paw), dequalinium (12.5, 25 and 50 microg/paw) or charybdotoxin (0.5, 1 and 2 microg/paw), blockers of voltage-dependent K+ channels and of small- and large-conductance Ca(2+)-activated K+ channels, respectively, were not able to abolish the antinociception induced by diazoxide. The peripheral antinociceptive effect of diazoxide was not prevented by prior administration of naloxone (12.5, 25 and 50 microg/paw), an opioid receptor antagonist, or methylene blue (75, 125 and 300 microg/paw), an agent that inhibits the activation of guanylate cyclase by nitric oxide. A low dose of diazoxide (20 microg/paw) administered together with a low dose of sodium nitroprusside (125 microg/paw) or dibutyryl cGMP (db-cGMP, 50 microg/paw) induced a marked antinociceptive effect similar to that observed when each drug was administered alone. NS1619 (75, 150 and 300 microg/paw), a specific opener of large-conductance Ca(2+)-activated K+ channels, had no antinociceptive action on prostaglandin E2-induced hyperalgesia. This series of experiments provides evidence for a peripheral antinociceptive action of diazoxide and supports the suggestion that the activation of ATP-sensitive K+ channels could be the mechanism by which sodium nitroprusside and db-cGMP induce peripheral antinociception, excluding the involvement of large-contuctance Ca(2+)-activated K+ channels in the process.

Analgesics↗

PubMed2005

Post-ischemic administration of diazoxide attenuates long-term microglial activation in the rat brain after permanent carotid artery occlusion.

Diazoxide is a putative mitochondrial, ATP-sensitive potassium channel opener that has been implicated in neuroprotection in cerebral ischemia. Administered as pretreatment, diazoxide can attenuate ischemia-related neuronal injury, but little is known about the potential neuroprotective properties of the drug when it is given after the onset of an ischemic insult. In a previous study, we applied diazoxide after imposing chronic cerebral hypoperfusion by means of permanent, bilateral occlusion of the common carotid arteries (2VO) in rats. We observed that ischemia-induced learning impairment assessed in the Morris water maze, and microglial activation visualized by immunocytochemistry, were prevented by diazoxide as determined at 13 weeks after 2VO. However, dimethyl sulfoxide, the organic solvent of diazoxide also prevented memory deficits, without any effect on microglial activity. Therefore, we have repeated our experiments with the use of an inorganic solvent, aqueous NaOH solution in order to clarify the effect of diazoxide independent of dimethyl sulfoxide. The present results demonstrated that diazoxide alone did not improve learning performance, but it prevented microglial activation in the hippocampus 13 weeks after the onset of 2VO. These data provide evidence that post-treatment with diazoxide is not effective in impeding a long-term memory deficiency, but it can attenuate ischemia-induced microglial activation, independently of the solvent used.

Animals↗

PubMed2001

Distinct effect of diazoxide on insulin secretion stimulated by protein kinase A and protein kinase C in rat pancreatic islets.

Protein kinase activation is known to stimulate glucose-induced insulin secretion in the presence of diazoxide. Diazoxide opens the ATP-sensitive K(+) channel and inhibits FAD-linked glycerophosphate dehydrogenase activity in a concentration-dependent manner. In the present study, we examined the effect of lower (100 microM) and higher (250 microM) concentrations of diazoxide on insulin release by protein kinase A (PKA) and protein kinase C (PKC) activation. Forced depolarization by a high potassium concentration, augmented the intracellular Ca(2+) concentration ([Ca(2+)](i)) similarly in the presence of both concentrations of diazoxide. Under this condition, 250 microM diazoxide inhibited insulin release enhanced by PKA activation but not that by PKC. Under a basal concentration of [Ca(2+)](i), PKC activation elicited glucose-induced insulin secretion at 100 and 250 microM diazoxide, while PKA activation did so only at 100 microM. These augmentations were completely inhibited by mannoheptulose, a glucokinase inhibitor. Glyceraldehyde, in place of glucose, enhanced insulin secretion by PKC activation under both concentrations of diazoxide. On the other hand, it did not affect PKA-stimulated insulin release under either conditions, but in the case of 100 microM, glucose augmented the insulin secretion in the presence of glyceraldehyde and db-cAMP concentration-dependently. These data suggest that insulin release stimulated by PKA and PKC activation under diazoxide is dependent on glucose metabolism, and that a signal derived from proximal steps in glycolysis may be necessary for the secretion by PKA activation.

Animals↗