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.

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.

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.

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.

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.

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.

Cibenzoline inhibits diazoxide- and 2,4-dinitrophenol-activated ATP-sensitive K+ channels in guinea-pig ventricular cells.

1. We have investigated the effects of diazoxide (a sulphonamide derivative) and cibenzoline (a class I antiarrhythmic drug) on ATP-sensitive K+ currents in guinea-pig ventricular cells, using whole-cell clamp techniques. 2. Diazoxide (50 microM) produced a marked shortening of action potential duration which was antagonized by 1 microM glibenclamide, an ATP-sensitive K+ channel blocker. 3. Diazoxide (50 microM) increased the quasi-steady state outward current elicited by a ramp voltage protocol (-20 mV s-1) at potentials positive to about -70 mV. This effect was completely prevented in the presence of glibenclamide (1 microM), thereby suggesting that diazoxide opens ATP-sensitive K+ channels. 4. Cibenzoline (5 microM) depressed the diazoxide-induced increases in the outward current and the pretreatment with this agent prevented the development of the diazoxide-induced outward current. 5. Cibenzoline (10 microM) reversed the 2,4-dinitrophenol (50 microM)-induced shortening of the action potential duration partially but significantly. 6. These results suggest that diazoxide activates ATP-sensitive K+ channels of guinea-pig ventricular cells and that cibenzoline, at therapeutic concentrations, inhibits this channel.

Effects of the K+ channel activators, RP 52891, cromakalim and diazoxide, on the plasma insulin level, plasma renin activity and blood pressure in rats.

In normo- or hyperglycemic (i.v. infusion of 50 mg/kg/min glucose over 30 min) pithed rats, diazoxide (1 mg/kg/min i.v. over 20 min) significantly reduced plasma insulin content. By contrast, cromakalim, nicorandil or RP 52891 even at doses 40-fold higher than those producing the same hypotensive effect as diazoxide in intact anesthetized normotensive rats, failed to change insulin plasma levels. Glibenclamide (0.01-0.3 mg/kg i.v.) pretreatment antagonized dose-dependently the hypoinsulinemic activity of diazoxide with an i.v. ED50 value of 49 +/- 1 microgram/kg. In pithed rats, diazoxide increased markedly plasma renin activity. This effect was almost inhibited completely by 20 mg/kg i.v., but not at all by a 1-mg/kg i.v. dose of glibenclamide. In pentobarbital-anesthetized rats, diazoxide (0.5-2 mg/kg/min i.v. over 20 min) produced decreases in mean carotid artery blood pressure which were antagonized dose-dependently by glibenclamide (5-20 mg/kg i.v.). This sulfonylurea (20 mg/kg i.v.) also prevented the hypotensive effects of several i.v. administered K+ channel activators (cromakalim, RP 52891 and nicorandil) but not those of numerous hypotensive agents such as acetylcholine, adenosine, bradykinin, clonidine, histamine, salbutamol, dihydralazine, papaverine, platelet aggregating factor, nitroglycerin, nitroprusside, nitrendipine and diltiazem. Although glibenclamide lowered plasma glucose levels, its blocking activity vis-à-vis the hypotension evoked by cromakalim was not affected when its hypoglycemic effects were reversed with an i.v. injection of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Decrease of some metabolic and physiologic effects of diazoxide by propranolol in rat.

Diazoxide significantly decreased the blood pressure and relaxed the uterine muscle in anaesthetized normotensive rats. A marked elevation of blood glucose followed the intravenous injection of diazoxide. The hyperglycemic and the uterine relaxing response could be significantly decreased by injection of propranolol prior to diazoxide. The hypotensive effect was not diminished by propranolol, however. In liver and uterus the content of cAMP was increased following diazoxide treatment in vivo. The rise in cAMP could be completely inhibited by propranolol, indicating a beta-receptor stimulation being the cause of the cAMP elevation.

Mechanical and metabolic effects of diazoxide in rat uterus.

Diazoxide relaxed both polarized and depolarized rat uterus. The drug also conteracted the contractions elicited by Ca2+ in a competitive manner. The relaxing effect was associated with an increase in the tissue level of cyclic AMP. This metabolic effect of diazoxide was inhibited by propranolol-treatment and in preparations from reserpinized animals, while the mechanical effects were only partially reduced. Diazoxide was also found to increase the release of tritium from preparations preloaded with [3H]-noradrenaline. It is suggested that diazoxide may induce some of its mechanical and metabolic effects by releasing the adrenergic transmittor substance noradrenaline. An effect of diazoxide on the Ca2+-metabolism is also probable.