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PubMed1999

Cell-type specific expression of ATP-sensitive potassium channels in the rat hippocampus.

1. The distribution of ATP-sensitive K+ channels (KATP channels) was investigated in four cell types in hippocampal slices prepared from 10- to 13-day-old rats: CA1 pyramidal cells, interneurones of stratum radiatum in CA1, complex glial cells of the same area and granule cells of the dentate gyrus. The neuronal cell types were identified visually and characterized by the shapes and patterns of their action potentials and by neurobiotin labelling. 2. The patch-clamp technique was used to study the sensitivity of whole-cell currents to diazoxide (0.3 mM), a KATP channel opener, and to tolbutamide (0.5 mM) or glibenclamide (20 microM), two KATP channel inhibitors. The fraction of cells in which whole-cell currents were activated by diazoxide and inhibited by tolbutamide was 26% of pyramidal cells, 89 % of interneurones, 100% of glial cells and 89% of granule cells. The reversal potential of the diazoxide-induced current was at the K+ equilibrium potential and a similar current activated spontaneously when cells were dialysed with an ATP-free pipette solution. 3. Using the single-cell RT-PCR method, the presence of mRNA encoding KATP channel subunits (Kir6.1, Kir6.2, SUR1 and SUR2) was examined in CA1 pyramidal cells and interneurones. Subunit mRNA combinations that can result in functional KATP channels (Kir6.1 together with SUR1, Kir6.2 together with SUR1 or SUR2) were detected in only 17% of the pyramidal cells. On the other hand, KATP channels may be formed in 75% of the interneurones, mainly by the combination of Kir6.2 with SUR1 (58% of all interneurones). 4. The results of these combined analyses indicate that functional KATP channels are present in principal neurones, interneurones and glial cells of the rat hippocampus, but at highly different densities in the four cell types studied.

ATP-Binding Cassette Transporters↗

PubMed1977

Evidence for a visceral smooth muscle abnormality in Okamoto spontaneous hypertension.

1. In order to discover whether the changes in reactivity are related to the primary cause of hypertension in spontaneously hypertensive rats (SHR) or are just an adaptation induced by the high arterial blood pressure we tested the contractile response of a visceral smooth muscle from such rats. 2. Longitudinal strips of the fundus from 20 week old male and female SHR and Wistar normotensive (NW) rats were used. Dose-response curves to Ba2+ in SHR strips were displaced to the right as compared to NW rats. Maximal responses were identical. Male SHR fundus strips contracted much more with Sr2+ (SHR: 42+/-3% of maximum response to Ba2+, n=10; NW: 19+/-4%, n=10, P less than 0.01) than NW strips. There was no difference in the response to both BaCl2 and SrCl2 between female SHR and NW fundus strips, and MnCl2 and LaCl3 were relaxant in all cases. 3. Dose-response curves to Ca2+ of depolarized SHR and NW fundus strips were obtained and the effect of diazoxide on Ca2+ contractions was observed. The contractile action of Ca2+ in depolarized preparations was enhanced in both male and female SHR strips. The effect of diazoxide was more marked in SHR strips than in NW fundus strips. 4. SHR fundus smooth muscle shows the same modification of reactivity to Ba2+, Sr2+, Ca2+ and diazoxide that was previously described in arterial smooth muscle. This indicates that the cellular modification responsible for the increase of vascular tonus in SHR is not an adaptive reaction to high blood pressure. The differences between female SHR and male SHR responses are not unexpected, considering the natural evolution of hypertension in Okamoto rats which is milder in the female.

Animals↗

PubMed1977

Coronary reactions to cardiac hyperactivity and to hypoxia in isolated perfused heart of rat.

1. Continuous recording of cardiac force of contraction, heart rate and coronary flow from isolated perfused hearts of rats was used to study coronary reactions: (a) to cardiostimulation with noradrenaline, CaCl2, or electrically induced tachycardia; (b) to short duration stoppage of coronary inflow (hypoxia). 2. The heart rate was controlled by electrical pacing. Coronary vasodilatation resulted from cardiostimulation or hypoxia. This coronary response was greater at higher heart rates. 3. In parallel experiments administration of noradrenaline to hearts paced at different frequencies resulted in a rate-dependent elevation of adenosine-3',5'-cyclic monophosphate (cyclic AMP). 4. Duration of hypoxia leading to different degrees of reactive hyperaemia did not change the cardiac cyclic AMP levels. 5. Coronary vasodilatation due to increased cardiac metabolism produced by noradrenaline, Ca2+ or tachycardia was enhanced by the phosphodiesterase inhibitors diazoxide and papaverine while it was inhibited during the administration of prostaglandin E2.6. Reactive hyperaemia was unaffected by diazoxide, papaverine or prostaglandin E2. 7. Catecholamine depletion by reserpine did not influence metabolic coronary dilatation nor the reactive hyperaemic responses. 8. We postulate that there are at least two types of coronary reactions: one in response to hypoxia, 'reactive hyperaemia', and another resulting from cardiac hyperactivity, 'metabolic coronary dilatation'. The latter, blocked by prostaglandin E2 and enhanced by diazoxide or papaverine, would be triggered by cyclic AMP while reactive hyperaemia would result from other mechanisms.

Animals↗

PubMed1996

The effect of L-type calcium channel modulators on the mobilization of intracellular calcium stores in guinea-pig intestinal smooth muscle.

1. The action of Ca2+ channel modulators has been examined on the intracellular Ca2+ signal in the longitudinal smooth muscle cells of the guinea-pig intestine after exposure to histamine and to agents known to affect intracellular Ca2+ stores. Isometric contraction has been measured simultaneously with front-surface fluorometry of fura 2-loaded preparations. 2. Histamine (10 microM) evoked a phasic and tonic increase in [Ca2+]i and contraction which were both sensitive to the Ca2+ channel blockers, nimodipine and D600. 3. Caffeine (10 mM) evoked in rapid increase in [Ca2+]i which was sustained as long as the preparation was exposed to the drug, whereas the contractile response was only phasic. In the presence of nimodipine 1 microM, the phasic contraction was absent although the fura 2-Ca2+ signal amounted to 32% of the control. 4. Ryanodine (10 microM) evoked a slow increase in [Ca2+]i and a contraction, both of which were reversed after exposure to nimodipine (1 microM) or D600 (10 microM). In the presence of diazoxide (500 microM), a hyperpolarizing agent, the ryanodine-evoked increase in [Ca2+]i and in muscle tone were inhibited. 5. Thapsigargin (1 microM) also produced an increase in [Ca2+]i and a contraction both of which were blocked by nimodipine (1 microM). 6. In Ca2+-free solution, histamine 10 microM evoked non-reproducible phasic Ca2+ signal and contraction. This response was recovered after refilling in Ca2+ containing solution. The recovery was blocked by nimodipine, D600 or diazoxide and was facilitated by the Ca2+ channel activator, Bay K 8644. When the refilling medium was supplemented with thapsigargin, the recovered response was significantly reduced, but Bay K 8644 still had some action. 7. The present results show that blockage of L-type Ca2+ channels inhibited changes in [Ca2+]i evoked by histamine, caffeine and ryanodine which are generally attributed to Ca2+ mobilization from intracellular stores. They also show that when the tissue was exposed to nimodipine, D600 and diazoxide during the procedure of refilling after depletion of intracellular stores, the action of histamine on [Ca2+]i and contraction was blocked. Bay K 8644 had an opposite effect even when the Ca2+ pumping activity of the sarcoplasmic reticulum was reduced by thapsigargin. This indicates that refilling of intracellular Ca2+ stores depleted by histamine in guinea-pig intestine mainly occurred through L-type Ca2+ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1999

A transmembrane domain of the sulfonylurea receptor mediates activation of ATP-sensitive K(+) channels by K(+) channel openers.

ATP-sensitive K(+) (K(ATP)) channels are a complex of an ATP-binding cassette transporter, the sulfonylurea receptor (SUR), and an inward rectifier K(+) channel subunit, Kir6.2. The diverse pharmacological responsiveness of K(ATP) channels from various tissues are thought to arise from distinct SUR isoforms. Thus, when assembled with Kir6. 2, the pancreatic beta cell isoform SUR1 is activated by the hyperglycemic drug diazoxide but not by hypotensive drugs like cromakalim, whereas the cardiac muscle isoform SUR2A is activated by cromakalim and not by diazoxide. We exploited these differences between SUR1 and SUR2A to pursue a chimeric approach designed to identify the structural determinants of SUR involved in the pharmacological activation of K(ATP) channels. Wild-type and chimeric SUR were coexpressed with Kir6.2 in Xenopus oocytes, and we studied the resulting channels with the patch-clamp technique in the excised inside-out configuration. The third transmembrane domain of SUR is found to be an important determinant of the response to cromakalim, which possibly harbors at least part of its binding site. Contrary to expectations, diazoxide sensitivity could not be linked specifically to the carboxyl-terminal end (nucleotide-binding domain 2) of SUR but appeared to involve complex allosteric interactions between transmembrane and nucleotide-binding domains. In addition to providing direct evidence for the structure-function relationship governing K(ATP) channel activation by potassium channel-opening drugs, a family of drugs of the highest therapeutic interest, these findings delineate the determinants of ligand specificity within the modular ATP-binding cassette-transporter architecture of SUR.

ATP-Binding Cassette Transporters↗

PubMed1992

Impact of uncoupling glucose stimulus from secretion on B-cell release and biosynthesis.

We studied the impact of a defined degree of long-term hyperglycemia with or without blockade of attendant insulin release on subsequent B-cell secretory responsiveness and biosynthesis. Nondiabetic rats were infused for 48 h with glucose to produce marked hyperglycemia (21.3 +/- 0.5 mmol/l). Comparable levels of hyperglycemia were upheld when additions were made to this protocol. Hyperglycemia increased plasma insulin 12-fold but depressed glucose (27 mmol/l)-induced insulin secretion in vitro (isolated islets) by 67% compared with saline-infused rats. Addition of diazoxide infusion during hyperglycemia completely inhibited the hyperglycemia-induced rise in plasma insulin but enhanced glucose-induced insulin release in vitro eightfold compared with islets from rats infused with glucose alone. Addition of insulin (2 U/day) to the diazoxide plus hyperglycemia protocol inhibited the secretory response to glucose in vitro by 46% (P less than 0.05). Proinsulin biosynthesis was enhanced by 67% in islets from rats infused with glucose alone; this effect was paralleled by a similar increase in preproinsulin mRNA. Diazoxide in vivo did not affect these stimulatory effects of hyperglycemia on insulin biosynthesis; however, insulin infusion in vivo abolished the hyperglycemia-induced increase in proinsulin biosynthesis. We conclude that impairment by hyperglycemia of glucose-induced insulin secretion occurs concomitant with stimulation of biosynthesis. Uncoupling of glucose stimulus from secretion crucially affects subsequent secretory responsiveness but not biosynthesis. Insulin biosynthesis is depressed by direct or indirect effects of circulating insulin.

Animals↗

PubMed2001

Amino acids and insulin are both required to regulate assembly of the eIF4E. eIF4G complex in rat skeletal muscle.

The respective roles of insulin and amino acids in regulation of skeletal muscle protein synthesis and degradation after feeding were examined in rats fasted for 17 h and refed over 1 h with either a 25 or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained (control groups: C(25) and C(0)) or blocked with diazoxide injections (diazoxide groups: DZ(25) and DZ(0)). Muscle protein metabolism was examined in vitro in epitrochlearis muscles. Only feeding the 25% amino acid/protein meal in the presence of increased plasma insulin concentration (C(25) group) stimulated protein synthesis and inhibited proteolysis in skeletal muscle compared with the postabsorptive state. The stimulation of protein synthesis was associated with increased phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1), reduced binding of eIF4E to 4E-BP1, and increased assembly of the active eIF4E. eIF4G complex. The p70 S6 kinase (p70(S6k)) was also hyperphosphorylated in response to the 25% amino acid/protein meal. Acute postprandial insulin deficiency induced by diazoxide injections totally abolished these effects. Feeding the 0% amino acid/protein meal with or without postprandial insulin deficiency did not stimulate muscle protein synthesis, reduce proteolysis, or regulate initiation factors and p70(S6k) compared with fasted rats. Taken together, our results suggest that both insulin and amino acids are required to stimulate protein synthesis, inhibit protein degradation, and regulate the interactions between eIF4E and 4E-BP1 or eIF4G in response to feeding.

Amino Acids↗

PubMed2002

Mechanisms by which opening the mitochondrial ATP- sensitive K(+) channel protects the ischemic heart.

Diazoxide opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the heart against ischemia-reperfusion injury by unknown mechanisms. We investigated the mechanisms by which mitoK(ATP) channel opening may act as an end effector of cardioprotection in the perfused rat heart model, in permeabilized fibers, and in rat heart mitochondria. We show that diazoxide pretreatment preserves the normal low outer membrane permeability to nucleotides and cytochrome c and that these beneficial effects are abolished by the mitoK(ATP) channel inhibitor 5-hydroxydecanoate. We hypothesize that an open mitoK(ATP) channel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP. This hypothesis is supported by findings in mitochondria showing that small decreases in intermembrane space volume, induced by either osmotic swelling or diazoxide, increased the half-saturation constant for ADP stimulation of respiration and sharply reduced ATP hydrolysis. These effects are proposed to lead to preservation of adenine nucleotides during ischemia and efficient energy transfer upon reperfusion.

Adenosine Diphosphate↗

PubMed2006

First molecular evidence that inositol trisphosphate signaling contributes to infarct size reduction with preconditioning.

Considerable attention has focused on the role of protein kinase C (PKC) in triggering the profound infarct-sparing effect of ischemic preconditioning (PC). In contrast, the involvement of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], the second messenger generated in parallel with the diacylglycerol-PKC pathway, remains poorly understood. We hypothesized that, if Ins(1,4,5)P(3) signaling [i.e., release of Ins(1,4,5)P(3) and subsequent binding to Ins(1,4,5)P(3) receptors] contributes to PC-induced cardioprotection, then the reduction of infarct size achieved with PC would be attenuated in mice that are deficient in Ins(1,4,5)P(3) receptor protein. To test this concept, hearts were harvested from 1) B6C3Fe-a/a-Itpr-1(opt+/-)/J mutants displaying reduced expression of Ins(1,4,5)P(3) receptor-1 protein, 2) Itpr-1(opt+/+) wild types from the colony, and 3) C57BL/6J mice. All hearts were buffer-perfused and randomized to receive two 5-min episodes of PC ischemia, pretreatment with d-myo-Ins(1,4,5)P(3) [sodium salt of native Ins(1,4,5)P(3)], the mitochondrial ATP-sensitive K(+) channel opener diazoxide, or no intervention (controls). After the treatment phase, all hearts underwent 30-min global ischemia followed by 2 h of reperfusion, and infarct size was delineated by tetrazolium staining. In both wild-type and C57BL/6J cohorts, area of necrosis in hearts that received PC, d-myo-Ins(1,4,5)P(3), and diazoxide averaged 28-35% of the total left ventricle (LV), significantly smaller than the values of 52-53% seen in controls (P < 0.05). In contrast, in Itpr-1(opt+/-) mutants, protection was only seen with diazoxide: neither PC nor d-myo-Ins(1,4,5)P(3) limited infarct size (52-58% vs. 56% of the LV in mutant controls). These data provide novel evidence that Ins(1,4,5)P(3) signaling contributes to infarct size reduction with PC.

Animals↗

PubMed2002

Preconditioning blocks cardiocyte apoptosis: role of K(ATP) channels and PKC-epsilon.

The aims of this study were to determine whether preconditioning blocks cardiocyte apoptosis and to determine the role of mitochondrial ATP-sensitive K(+) (K(ATP)) channels and the protein kinase C epsilon-isoform (PKC-epsilon) in this effect. Ventricular myocytes from 10-day-old chick embryos were used. In the control series, 10 h of simulated ischemia followed by 12 h of reoxygenation resulted in 42 +/- 3% apoptosis (n = 8). These results were consistent with DNA laddering and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Preconditioning, elicited with three cycles of 1 min of ischemia separated by 5 min of reoxygenation before subjection to prolonged simulated ischemia, markedly attenuated the apoptotic process (28 +/- 4%, n = 8). The selective mitochondrial K(ATP) channel opener diazoxide (400 micromol/l), given before ischemia, mimicked preconditioning effects to prevent apoptosis (22 +/- 4%, n = 6). Pretreatment with 5-hydroxydecanoate (100 micromol/l), a selective mitochondrial K(ATP) channel blocker, abolished preconditioning (42 +/- 2%, n = 6). In addition, the effects of preconditioning and diazoxide were blocked with the specific PKC inhibitors Gö-6976 (0.1 micromol/l) or chelerythrine (4 micromol/l), given at simulated ischemia and reoxygenation. Furthermore, preconditioning and diazoxide selectively activated PKC-epsilon in the particulate fraction before simulated ischemia without effect on the total fraction, cytosolic fraction, and PKC delta-isoform. The specific PKC activator phorbol 12-myristate 13-acetate (0.2 micromol/l), added during simulated ischemia and reoxygenation, mimicked preconditioning to block apoptosis. Opening mitochondrial K(ATP) channels blocks cardiocyte apoptosis via activating PKC-epsilon in cultured ventricular myocytes. Through this signal transduction, preconditioning blocks apoptosis and preserves cardiac function in ischemia-reperfusion.

Alkaloids↗

PubMed2002

Potassium channel openers protect cardiac mitochondria by attenuating oxidant stress at reoxygenation.

K(+) channel openers have been recently recognized for their ability to protect mitochondria from anoxic injury. Yet the mechanism responsible for mitochondrial preservation under oxidative stress is not fully understood. Here, mitochondria were isolated from rat hearts and subjected to 20-min anoxia, followed by reoxygenation. At reoxygenation, increased generation of reactive oxygen species (ROS) was associated with reduced ADP-stimulated oxygen consumption, blunted ATP production, and disrupted mitochondrial structural integrity coupled with cytochrome c release. The prototype K(+) channel opener diazoxide markedly reduced mitochondrial ROS production at reoxygenation with a half-maximal effect of 29 microM. Diazoxide also preserved oxidative phosphorylation and mitochondrial membrane integrity, as indicated by electron microscopy and reduced cytochrome c release. The protective effect of diazoxide was reproduced by the structurally distinct K(+) channel opener nicorandil and antagonized by 5-hydroxydecanoic acid, a short-chain fatty acid derivative and presumed blocker of mitochondrial ATP-sensitive K(+) channels. Opener-mediated mitochondrial protection was simulated by the free radical scavenger system composed of superoxide dismutase and catalase. However, the effect of openers on ROS production was maintained in nominally K(+)-free medium in the presence or absence of the K(+) ionophore valinomycin and was mimicked by malonate, a modulator of the mitochondrial redox state. This suggests the existence of a K(+) conductance-independent pathway for mitochondrial protection targeted by K(+) channel openers. Thus the cardioprotecive mechanism of K(+) channel openers includes direct attenuation of mitochondrial oxidant stress at reoxygenation.

Adenosine Diphosphate↗

PubMed2002

Mitochondrial Ca2+ uptake is important over low [Ca2+]i range in arterial smooth muscle.

Mitochondrial Ca(2+) uptake is usually thought to occur only when intracellular Ca(2+) concentration ([Ca(2+)](i)) is high. We investigated whether mitochondrial Ca(2+) removal participates in shaping [Ca(2+)](i) signals in arterial smooth muscle over a low [Ca(2+)](i) range. [Ca(2+)](i) was measured using fura 2-loaded, voltage-clamped cells from rat femoral arteries. Both diazoxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized the mitochondria. Diazoxide application increased resting [Ca(2+)](i), suggesting that Ca(2+) is sequestered in mitochondria. Over a low [Ca(2+)](i) range, diazoxide and CCCP slowed Ca(2+) removal rate, determined after a brief depolarization. When [Ca(2+)](i) was measured during sustained depolarization to -30 mV, CCCP application increased [Ca(2+)](i). When Ca(2+) transients were repeatedly evoked by caffeine applications, CCCP application elevated resting [Ca(2+)](i). Caffeine-induced Ca(2+) transients were compared before and after CCCP application using the half decay time, or time required to reduce increase in [Ca(2+)](i) by 50% (t((1/2))). CCCP treatment significantly increased t((1/2)). These results suggest that Ca(2+) removal to mitochondria in arterial smooth muscle cells may be important at a low [Ca(2+)](i).

Animals↗

PubMed2000

Preconditioning reduces myocardial complement gene expression in vivo.

This investigation examined the effect of preconditioning in an in vivo model of ischemia-reperfusion injury. Anesthetized New Zealand White rabbits underwent 30 min of regional myocardial ischemia followed by 2 h of reperfusion. Hearts preconditioned with two cycles of 5 min ischemia-10 min reperfusion (IPC) or with the ATP-sensitive K (K(ATP)) channel opener, diazoxide (10 mg/kg), exhibited significantly (P < 0.05) smaller infarcts compared with control. These treatments also significantly (P < 0.001 to P < 0.05) reduced C1q, C1r, C3, C8, and C9 mRNA in the areas at risk (AAR). The K(ATP) channel blocker 5-hydroxydecanoate (5-HD; 10 mg/kg) attenuated infarct size reduction elicited by IPC and diazoxide treatment. 5-HD partially reversed the decrease in complement expression caused by IPC but not diazoxide. There were no significant differences in complement gene expression in the nonrisk regions and livers of all groups. Western blot analysis revealed that IPC also reduced membrane attack complex expression in the AAR. The data demonstrate that preconditioning significantly decreases reperfusion-induced myocardial complement expression in vivo.

Animals↗

PubMed2001

Diabetes and hyperglycemia impair activation of mitochondrial K(ATP) channels.

Hyperglycemia is an important predictor of cardiovascular mortality in patients with diabetes. We investigated the hypothesis that diabetes or acute hyperglycemia attenuates the reduction of myocardial infarct size produced by activation of mitochondrial ATP-regulated potassium (K(ATP)) channels. Acutely instrumented barbiturate-anesthetized dogs were subjected to a 60-min period of coronary artery occlusion and 3 h of reperfusion. Myocardial infarct size (triphenyltetrazolium chloride staining) was 25 +/- 1, 28 +/- 3, and 25 +/- 1% of the area at risk (AAR) for infarction in control, diabetic (3 wk after streptozotocin-alloxan), and hyperglycemic (15% intravenous dextrose) dogs, respectively. Diazoxide (2.5 mg/kg iv) significantly decreased infarct size (10 +/- 1% of AAR, P < 0.05) but did not produce protection in the presence of diabetes (28 +/- 5%) or moderate hyperglycemia (blood glucose 310 +/- 10 mg/dl; 23 +/- 2%). The dose of diazoxide and the degree of hyperglycemia were interactive. Profound (blood glucose 574 +/- 23 mg/dl) but not moderate hyperglycemia blocked the effects of high-dose (5.0 mg/kg) diazoxide [26 +/- 3, 15 +/- 3 (P < 0.05), and 11 +/- 2% (P < 0.05), respectively]. There were no differences in systemic hemodynamics, AAR, or coronary collateral blood flow (by radioactive microspheres) between groups. The results indicate that diabetes or hyperglycemia impairs activation of mitochondrial K(ATP) channels.

Analysis of Variance↗

PubMed2003

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.

Adenosine Triphosphate↗

PubMed2000

Adenosine primes the opening of mitochondrial ATP-sensitive potassium channels: a key step in ischemic preconditioning?

BACKGROUND: Adenosine can initiate ischemic preconditioning, and mitochondrial ATP-sensitive potassium (K(ATP)) channels have emerged as the likely effectors. We sought to determine the mechanistic interactions between these 2 observations. METHODS AND RESULTS: The mitochondrial flavoprotein oxidation induced by diazoxide (100 micromol/L) was used to quantify mitochondrial K(ATP) channel activity in intact rabbit ventricular myocytes. Adenosine (100 micromol/L) increased mitochondrial K(ATP) channel activity and abbreviated the latency to mitochondrial K(ATP) channel opening. These potentiating effects were entirely prevented by the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline (100 micromol/L) or by the protein kinase C inhibitor polymyxin B (50 micromol/L). The effects of adenosine and diazoxide reflected mitochondrial K(ATP) channel activation, because they could be blocked by the mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (500 micromol/L). In a cellular model of simulated ischemia, adenosine mitigated cell injury; this cardioprotective effect was blocked by 5-hydroxydecanoate but not by the surface-selective K(ATP) channel blocker HMR1098. Moreover, adenosine augmented the cardioprotective effect of diazoxide. A quantitative model of mitochondrial K(ATP) channel gating reproduced the major experimental findings. CONCLUSIONS: Our results support the hypothesis that adenosine receptor activation primes the opening of mitochondrial K(ATP) channels in a protein kinase C-dependent manner. The findings provide tangible links among various key elements in the preconditioning cascade.

Adenosine↗

PubMed2005

Mitochondria-derived reactive oxygen species dilate cerebral arteries by activating Ca2+ sparks.

Mitochondria regulate intracellular calcium (Ca2+) signals in smooth muscle cells, but mechanisms mediating these effects, and the functional relevance, are poorly understood. Similarly, antihypertensive ATP-sensitive potassium (KATP) channel openers (KCOs) activate plasma membrane KATP channels and depolarize mitochondria in several cell types, but the contribution of each of these mechanisms to vasodilation is unclear. Here, we show that cerebral artery smooth muscle cell mitochondria are most effectively depolarized by diazoxide (-15%, tetramethylrhodamine [TMRM]), less so by levcromakalim, and not depolarized by pinacidil. KCO-induced mitochondrial depolarization increased the generation of mitochondria-derived reactive oxygen species (ROS) that stimulated Ca2+ sparks and large-conductance Ca2+-activated potassium (KCa) channels, leading to transient KCa current activation. KCO-induced mitochondrial depolarization and transient KCa current activation were attenuated by 5-HD and glibenclamide, KATP channel blockers. MnTMPyP, an antioxidant, and Ca2+ spark and KCa channel blockers reduced diazoxide-induced vasodilations by >60%, but did not alter dilations induced by pinacidil, which did not elevate ROS. Data suggest diazoxide drives ROS generation by inducing a small mitochondrial depolarization, because nanomolar CCCP, a protonophore, similarly depolarized mitochondria, elevated ROS, and activated transient KCa currents. In contrast, micromolar CCCP, or rotenone, an electron transport chain blocker, induced a large mitochondrial depolarization (-84%, TMRM), reduced ROS, and inhibited transient KCa currents. In summary, data demonstrate that mitochondria-derived ROS dilate cerebral arteries by activating Ca2+ sparks, that some antihypertensive KCOs dilate by stimulating this pathway, and that small and large mitochondrial depolarizations lead to differential regulation of ROS and Ca2+ sparks.

Adenosine Triphosphate↗

PubMed1998

Modulation of mitochondrial ATP-dependent K+ channels by protein kinase C.

Pharmacological openers of mitochondrial ATP-dependent K+ (mitoKATP) channels mimic ischemic preconditioning, and such cardioprotection can be prevented by mitoKATP channel blockers. It is also known that protein kinase C (PKC) plays a key role in the induction and maintenance of preconditioning. To look for possible mechanistic links between these 2 sets of observations, we measured mitochondrial matrix redox potential as an index of mitoKATP channel activity in rabbit ventricular myocytes. The mitoKATP channel opener diazoxide (100 micromol/L) partially oxidized the matrix redox potential. Exposure to phorbol 12-myristate 13-acetate (PMA, 100 nmol/L) potentiated and accelerated the effect of diazoxide. These effects of PMA were blocked by the mitoKATP channel blocker 5-hydroxydecanoate, which we verified to be a selective blocker of the mitoKATP channel in simultaneous recordings of membrane current and flavoprotein fluorescence. The inactive control compound 4alpha-phorbol (100 nmol/L) did not alter the effects of diazoxide. We conclude that the activity of mitoKATP channels can be regulated by PKC in intact heart cells. Potentiation of mitoKATP channel opening by PKC provides a direct mechanistic link between the signal transduction of ischemic preconditioning and pharmacological cardioprotection targeted at ATP-dependent K+ channels.

Adenosine Triphosphate↗