AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 284: H1048-H1056, 2003. First published December 5, 2002; doi:10.1152/ajpheart.00847.2002
0363-6135/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
284/4/H1048    most recent
00847.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (35)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dzeja, P. P.
Right arrow Articles by Terzic, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dzeja, P. P.
Right arrow Articles by Terzic, A.
Vol. 284, Issue 4, H1048-H1056, April 2003

Targeting nucleotide-requiring enzymes: implications for diazoxide-induced cardioprotection

Petras P. Dzeja, Peter Bast, Cevher Ozcan, Arturo Valverde, Ekshon L. Holmuhamedov, David G. L. Van Wylen, and Andre Terzic

Division of Cardiovascular Diseases, Department of Medicine, and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Mayo Foundation, Rochester, Minnesota 55905


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Modulation of mitochondrial respiratory chain, dehydrogenase, and nucleotide-metabolizing enzyme activities is fundamental to cellular protection. Here, we demonstrate that the potassium channel opener diazoxide, within its cardioprotective concentration range, modulated the activity of flavin adenine dinucleotide-dependent succinate dehydrogenase with an IC50 of 32 µM and reduced the rate of succinate-supported generation of reactive oxygen species (ROS) in heart mitochondria. 5-Hydroxydecanoic fatty acid circumvented diazoxide-inhibited succinate dehydrogenase-driven electron flow, indicating a metabolism-dependent supply of redox equivalents to the respiratory chain. In perfused rat hearts, diazoxide diminished the generation of malondialdehyde, a marker of oxidative stress, which, however, increased on diazoxide washout. This effect of diazoxide mimicked ischemic preconditioning and was associated with reduced oxidative damage on ischemia-reperfusion. Diazoxide reduced cellular and mitochondrial ATPase activities, along with nucleotide degradation, contributing to preservation of myocardial ATP levels during ischemia. Thus, by targeting nucleotide-requiring enzymes, particularly mitochondrial succinate dehydrogenase and cellular ATPases, diazoxide reduces ROS generation and nucleotide degradation, resulting in preservation of myocardial energetics under stress.

potassium channel openers; mitochondria; ATP-sensitive potassium channel; dehydrogenase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ADJUSTMENTS IN CELLULAR ENERGETIC and ionic status are essential in the maintenance of cell tolerance to metabolic stress (11, 51, 53, 60). In cardiac muscle, during an ischemia-reperfusion challenge, adenine nucleotide homeostasis depends critically on the activity of nucleotide-metabolizing enzymes and the extent of tissue oxidative stress closely relates to the rate of dehydrogenase-catalyzed reactions (10, 30, 40, 46, 54). Although the nucleotide binding properties and regulatory mechanisms of dehydrogenases and ATPases/kinases are rather similar (14, 21, 62), their integral role in cellular protection is poorly understood.

Protection of mitochondrial energetics, attenuation of oxidative damage, and reduction of infarct size can be induced by ATP-sensitive potassium (KATP) channel openers, such as diazoxide (19, 20, 43, 46, 63). Diazoxide targets the mitochondrial respiratory chain and potassium conductances of the inner membrane (18, 23, 24, 28, 43, 45, 57, 58). Opening of sarcolemmal KATP channels and shortening of the cardiac action potential appear not to be a prerequisite for diazoxide-mediated cardioprotection (16, 20, 35), and the effect of diazoxide on the mitochondrial redox state is preserved in KATP channel-deficient cardiomyocytes (61). Thus the search for the mechanism responsible for diazoxide-induced cardioprotection has focused on mitochondrial function (9, 24, 29, 46). Indeed, a number of strategies that modulate mitochondrial respiration, ATP production, ion transport, and/or free radical generation have been proven effective in tissue preservation (39, 52, 53, 60).

Early studies indicated that diazoxide could inhibit mitochondrial succinate and alpha -glycerophosphate dehydrogenases, reduce succinate-supported Ca2+ uptake, and induce flavoprotein oxidation, effects mimicked by succinate dehydrogenase inhibitors (36, 50, 57, 58). Recently, the ability of diazoxide to modulate mitochondrial succinate oxidation was confirmed (18, 23, 45) and proposed as a potential mechanism for cardioprotection (63). Regulation of the flavoprotein redox state is of importance because components of the mitochondrial respiratory chain, in particular succinate dehydrogenase and the coenzyme Q cycle, are sites of free radical production contributing to oxidative injury (8, 30, 67). Short-term tissue exposure to inhibitors of succinate dehydrogenase produces an antiischemic, preconditioning-like effect (44, 69). In fact, a number of cardioprotective drugs, including certain volatile anesthetics, target both mitochondrial dehydrogenases and ATPases (1, 26, 68), producing an effect additive to diazoxide on flavoprotein oxidation (72). Moreover, protection by diazoxide has been associated with attenuation of mitochondrial oxidant stress at reoxygenation, an effect mimicked by free radical scavenging systems or by malonate, an inhibitor of succinate dehydrogenase (46). Thus, although diazoxide can regulate ion fluxes across the inner mitochondrial membrane and mitochondrial volume (20, 24, 60), these studies suggest an alternative K+-independent mechanism of mitochondrial protection. It is, however, unknown whether modulation of nucleotide-requiring enzymes, such as cellular ATPases and dehydrogenases, by diazoxide (3, 50, 58) contributes to cellular protection.

The purpose of the present study was to determine the effects of diazoxide on succinate dehydrogenase activity and succinate-supported generation of reactive oxygen species (ROS) as well as on cellular and mitochondrial ATPases and nucleotide degradation. We demonstrate that modulation of dehydrogenase activity-dependent free radical generation and nucleotide-consuming ATPase activities contribute to the cardioprotective and energy-sparing effects of diazoxide.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The investigation conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (1996) and was approved by the Institutional Animal Care and Use Committee.

Cardiac mitochondria. Mitochondria were isolated from hearts of pentobarbital sodium-anesthetized rats (Harlan Sprague Dawley) as described previously (24, 46). Ventricles were removed into ice-cold isolation buffer (in mM: 50 sucrose, 200 mannitol, 5 KH2PO4, 1 EGTA, and 5 MOPS, pH 7.3, with 0.2% BSA) and homogenized, and the mitochondrial fraction was obtained by differential centrifugation. Mitochondria were washed and suspended in isolation buffer (without EGTA and BSA) and kept on ice or frozen at -20°C.

Heart perfusion. Excised hearts from heparinized (500 units ip) and anesthetized (75 mg/kg pentobarbital ip) rats were perfused on a Langendorff apparatus with a 95% O2-5% CO2 saturated Krebs-Henseleit solution (in mM: 118 NaCl, 5.3 KCl, 2.0 CaCl2, 19 NaHCO3, 1.2 MgSO4, 11.0 glucose, and 0.5 EDTA; 37°C) at a perfusion pressure of 70 mmHg (51). Left ventricular developed pressure, left ventricular end-diastolic pressure, rate-pressure product (RPP), and heart rate were determined with a fluid-filled balloon-tipped pressure transducer (Harvard Apparatus). After 20 min of stabilization, hearts were perfused for 6 min with medium containing diazoxide (30 µM) or vehicle containing 0.03% of the solvent DMSO followed by a 5-min washout. Hearts were then subjected to 30 min of zero-flow normothermic ischemia followed by a 30-min reperfusion. Preconditioned hearts were perfused for 20 min and then subjected to two "conditioning" cycles, 5 min of ischemia plus 5 min of reperfusion each, followed by 30-min ischemia and 30-min reperfusion.

Succinate dehydrogenase. The activity of succinate dehydrogenase was determined in isolated mitochondria with a spectrophotometric method (12, 21). The assay mixture contained (in mM) 50 Tris · HCl buffer (pH 8.0), 3 KCN, 0.42 phenazine methosulfonate, and 0.042 2,6-dichloroindophenol (DCIP) with 6 µM rotenone and 0.1 mg/ml protein of intact or freeze-thawed mitochondria. The reaction was initiated by 10-20 mM succinate, and the rate of DCIP reduction was followed at 600 nm.

Reactive oxygen species. The rate of superoxide anion (O<UP><SUB>2</SUB><SUP>−</SUP></UP>·) generation was measured as SOD-inhibitable reduction of acetylated ferricytochrome c (4). The reaction mixture contained 0.1 M potassium phosphate buffer (pH 7.4), 7.2 µM acetylated cytochrome c, 2 µM antimycin A, 6 µM rotenone, 10 mM succinate, and 20-30 µg/ml mitochondrial protein; 100 units of SOD/ml were added to the reference cuvette. The reduction of acetylated cytochrome c was monitored at 550-540 nm. The production of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· was calculated with the extinction coefficient of 19.0 mM-1 · cm-1. To measure the production of H2O2 and other ROS, mitochondria were preloaded with 10 µM of the ROS-sensitive dye 2',7'-dichlorofluorescein (DCF) diacetate for 30 min in aerated cell culture flasks (46, 65). Mitochondrial aliquots (100 µl) were diluted with 1 ml of H2O, and DCF fluorescence in mitochondrial suspensions was measured with a spectrofluorometer at excitation and emission wavelengths of 500 and 530 nm, respectively. DCF fluorescence was normalized to maximal values obtained by exposing mitochondrial suspension to 1 mM H2O2. The rate of ROS generation in perfused hearts was determined by measuring release of malondialdehyde (MDA), a lipid peroxidation product, into the coronary perfusate. The amount of MDA was determined by a thiobarbituric acid-based method (47).

ATPase activity. Myocardial oligomycin-insensitive Ca2+/Mg2+-ATPase activity was measured in heart homogenates (55) with an EnzChek inorganic phosphate assay kit (Molecular Probes). Tissue homogenates were prepared from hearts pulverized under liquid nitrogen and diluted with 10 volumes of buffer containing 0.25 M sucrose, 20 mM MOPS, pH 7.5, and the protease inhibitor cocktail Complete (Roche). The resulting suspension was homogenized with a Polytron PT 10/35 for 2 × 5 s. The ATPase assay mixture contained (in mM) 100 KCl, 20 HEPES (pH 7.35), 1 EGTA, 5 MgCl2, 1 CaCl2, and 5 NaN3 with 1 µg/ml oligomycin, and the reaction was initiated by 1 mM ATP. Mitochondrial ATPase activity was measured by liberation of inorganic phosphate (54) with the EnzChek kit (Molecular Probes). Incubation medium contained 50 mM Tris-Cl (pH 8.0), 1 mM ATP, and 30 µg/ml of mitochondrial protein, and the reaction was started with 1 mM MgCl2.

ATP levels and nucleotide degradation. ATP levels were determined in perchloric acid extracts of freeze-clamped control or ischemic tissue with HPLC (51). Nucleotide degradation products, namely, adenosine, inosine, hypoxanthine, xanthine, and uric acid, were measured with reverse-phase HPLC (40). Ischemia was simulated by incubating myocardial tissue (0.04-0.06 g) for 10 min at 37°C in deoxygenated heart perfusion medium (in mM: 137 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 1 NaH2PO4, 0.05 EDTA, and 24 NaHCO3; pH 7.45) gassed with 95% N2-5% CO2. Control tissue was incubated for the same time in oxygenated medium gassed with 95% O2-5% CO2.

Chemicals. Diazoxide was purchased from RBI and dissolved as a concentrated stock solution in DMSO. The maximal concentration of DMSO within the incubation medium was kept under 0.1%, and control experiments were performed with corresponding DMSO concentrations. 5-Hydroxydecanoic fatty acid (5-HD) was purchased from ICN Biomedicals and dissolved in incubation medium. All other chemicals were obtained from Sigma.

Statistical analysis. Data are presented as means ± SE. Group comparisons were performed with Student's t-test. A P value of <0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Diazoxide inhibits succinate dehydrogenase activity and succinate-supported generation of ROS in heart mitochondria. The effects of diazoxide on cardiac mitochondria were previously related to regulation of ion fluxes, substrate oxidation, and matrix volume (9, 20, 24, 29, 60). Here, we demonstrate that diazoxide is also a potent inhibitor of succinate dehydrogenase in isolated heart mitochondria (Fig. 1A). At baseline, mitochondrial succinate dehydrogenase activity was 68.8 ± 1.5 nmol · min-1 · mg protein-1 (n = 6) and was inhibited by diazoxide in a concentration-dependent manner with an IC50 of 32 ± 7 µM (n = 6; Fig. 1A). Mitochondrial succinate dehydrogenase, coupled with the coenzyme Q cycle, is a site of generation of ROS, and its activation has been implicated in tissue oxidative damage (8, 10, 21, 30). Treatment of mitochondria with diazoxide reduced succinate-supported ROS generation, assessed by the decrease in fluorescence of DCF (Fig. 1B), which detects H2O2 along with other reactive species (46, 65). On average, in heart mitochondria treated with 20 and 50 µM diazoxide, ROS generation was reduced by 34% and 43% (P < 0.05, n = 6), respectively, compared with nontreated controls (Fig. 1B). Diazoxide was effective in inhibiting succinate-dependent O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production (Fig. 1C), measured with acetylated cytochrome c, a sensitive detection probe (4). Superoxide-induced reduction of acetylated cytochrome c was blocked by the O<UP><SUB>2</SUB><SUP>−</SUP></UP>· scavenger SOD, indicating the specificity of the probe (Fig. 1C). On average, diazoxide (50 µM) significantly reduced the rate of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generation from 11.1 ± 1.6 to 6.4 ± 1.0 nmol · min-1 · mg protein-1 (P < 0.05, n = 5) in control and diazoxide-treated mitochondria, respectively (Fig. 1D). This effect was mimicked by malonate (5 mM), an inhibitor of succinate dehydrogenase, which reduced the rate of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production to 2.5 ± 0.4 nmol · min-1 · mg protein-1 (P < 0.01, n = 5; Fig. 1D). Thus modulation of succinate dehydrogenase activity by diazoxide has a profound effect on the ability of mitochondria to generate an excess of ROS.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 1.   Diazoxide inhibits succinate dehydrogenase (SDH) activity and succinate-supported reactive oxygen species (ROS) generation. A: concentration dependence of diazoxide inhibition of SDH activity in heart mitochondria. B: effect of diazoxide on ROS production in heart mitochondria measured by 2',7'-dichlorofluorescein (DCF) fluorescence, with 10 mM succinate as substrate. C: effect of diazoxide (50 µM) on kinetics of superoxide generation by heart mitochondria measured by reduction of acetylated cytochrome c. Substrate, 10 mM succinate; SOD, 500 U/ml. D: effect of diazoxide (50 µM) and malonate (5 mM), an inhibitor of SDH, on succinate-supported O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production in heart mitochondria. * Significantly different from control.

5-HD circumvents diazoxide-inhibited succinate dehydrogenase-driven electron flow. 5-HD antagonizes the effect of diazoxide on mitochondrial flavoprotein oxidation and cardioprotection (20, 35). Here, the reduced rate of electron transfer through the respiratory chain complex II, after succinate dehydrogenase inhibition by diazoxide, was accelerated in the presence of 5-HD (Table 1), indicating that this medium-chain fatty acid could counteract the effect of diazoxide on succinate dehydrogenase activity and/or be metabolized in heart mitochondria, generating electron flow. 5-HD per se did not reverse diazoxide-mediated inhibition of the activity of soluble succinate dehydrogenase (42.8 ± 2.2 and 43.6 ± 3.1 nmol · min-1 · mg protein-1 at 50 µM diazoxide in the absence and presence of 500 µM 5-HD, respectively; n = 3 each), suggesting a metabolism-related effect (23, 60). Thus 5-HD apparently supplies redox equivalents to the respiratory chain, thereby bypassing diazoxide inhibition of succinate dehydrogenase.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Modulation of electron flow at mitochondrial respiratory chain complex II by diazoxide and 5-HD

Diazoxide mimics ischemic preconditioning in regulating ROS generation in perfused heart. Diazoxide reduces ROS generation in isolated heart mitochondria and myocardium on reoxygenation (43, 46) and, depending on experimental conditions, could also increase ROS release (5, 15, 22). Here, in perfused hearts, a brief (6 min) exposure to diazoxide (30 µM) reduced, by 42 ± 4.3% (n = 5, P < 0.05), release of MDA (Fig. 2A), a product of ROS interaction with cellular constituents and an indicator of oxidative stress (47). On diazoxide washout, MDA release in the coronary effluent was augmented by 43 ± 3.8% (n = 5, P < 0.05; Fig. 3A). After ischemia-reperfusion, in diazoxide-pretreated hearts, MDA release was diminished, by 48 ± 4.6% (n = 5), compared with nontreated controls (P < 0.01; Fig. 2A). Reduction of oxidative stress by diazoxide on reperfusion was associated with improved postischemic contractile recovery, with the RPP at 6,658 ± 1,517 mmHg/min in nontreated (a 22 ± 5.0% recovery from preischemic value) vs. 18,570 ± 5,062 mmHg/min in diazoxide-pretreated (a 57 ± 6.7% recovery from preischemic value; P < 0.05, n = 5 each) hearts. These cyclical effects of diazoxide on MDA generation were similar to those produced by ischemic preconditioning induced by two conditioning cycles of short ischemia-reperfusion (Fig. 2B). Compared with nonpreconditioned hearts, MDA release was increased by 46 ± 3.6% and 32 ± 2.8% (n = 3, P < 0.05) after the first and second 5-min preconditioning cycle, respectively, but significantly decreased, by 59 ± 3.1% (n = 3, P < 0.01), on reperfusion after prolonged (45 min) ischemia (Fig. 2B). Thus exposure of heart muscle to diazoxide reduces production of free radical species and on washout, through an ischemic preconditioning-like effect on ROS generation, induces protection of the myocardium at reperfusion.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2.   Diazoxide reduces tissue oxidative stress and mimics ischemic preconditioning (IPC)-induced cycles of ROS generation in perfused heart. A: effect of short-term (6 min) application of diazoxide (30 µM) on the generation of malondialdehyde (MDA), a marker of oxidative stress, on diazoxide washout (5 min) and after ischemia (30 min) and reperfusion (30 min). B: effect of ischemic preconditioning (2 cycles of 5-min ischemia and 5-min reperfusion) on MDA generation before and after ischemia-reperfusion. * Significantly different from respective controls.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   Diazoxide attenuates cellular and mitochondrial ATPase activities and nucleotide degradation, preserving ATP levels during ischemia. A: effect of diazoxide (50 µM) on cellular oligomycin-insensitive Ca2+/Mg2+-ATPase in heart homogenates. B: effect of diazoxide (50 µM) on mitochondrial ATPase activity. C: effect of diazoxide (50 µM) on preservation of ATP levels during myocardial ischemia (10 min). D: effect of diazoxide (50 µM) on adenine nucleotide degradation during myocardial ischemia (10 min), measured by production of total purine nucleosides. * Significantly different from control.

Diazoxide reduces cellular and mitochondrial ATPase activities and preserves ATP levels in ischemia. Potassium channel openers reduce the drop in ATP levels in myocardial ischemia, thereby preserving cellular high-energy phosphate pools (16, 20, 38). However, the mechanism underlying this energy-sparing effect remains obscure, because it is uncertain how it can be related to modulation of a mitochondrial potassium conductance and/or regulation of the mitochondrial redox state and ROS production (19, 31, 35, 63). Here, in myocardial homogenates, diazoxide (50 µM) reduced the activity of cellular, oligomycin-insensitive Ca2+/Mg2+-ATPases by 28%, from 564 ± 24 to 408 ± 27 nmol · min-1 · mg protein-1 (P < 0.05, n = 5; Fig. 3A). Oligomycin was included to separate cytosolic from oligomycin-sensitive mitochondrial ATPases. Diazoxide also inhibited ATPase activity in isolated cardiac mitochondria (Fig. 3B). Specifically, in hypotonically permeabilized mitochondria (54), diazoxide (50 µM) reduced the activity of oligomycin-sensitive FoF1- ATPase by 27%, from 1,947 ± 50 to 1,429 ± 88 nmol · min-1 · mg protein-1 (P < 0.05, n = 3). Thus, besides sulfonylurea receptors, which belong to the family of ATP-binding cassette proteins (3, 41), diazoxide also targets other adenine nucleotide binding proteins, such as cellular ATPases. In this regard, treatment with diazoxide (50 µM) significantly ameliorated myocardial ATP during ischemia (Fig. 3C). After 10 min of ischemic conditions, myocardial ATP levels were 5.4 ± 0.4 and 17.1 ± 1.2 nmol/mg protein in control and diazoxide-treated tissue, respectively (P < 0.05, n = 5 each). Diazoxide also markedly reduced adenine nucleotide degradation during simulated ischemia, as indicated by lower myocardial levels of total purine nucleosides in diazoxide-treated compared with nontreated hearts (Fig. 3D). Purine nucleoside levels, at 10 min of ischemia, were 12.4 ± 1.2 and 7.4 ± 1.0 nmol/mg protein in nontreated and diazoxide-treated myocardium, (P < 0.05, n = 5 each), respectively (Fig. 3D). In addition, in the ischemic myocardium, diazoxide reduced to a greater extent the xanthine compared with the hypoxanthine content, i.e., 59 ± 4.8% vs. 12 ± 2.1% (P < 0.05, n = 3). This suggests that conversion of hypoxanthine to xanthine, catalyzed by the flavoprotein-dependent xanthine oxidase/dehydrogenase (48), is reduced after diazoxide treatment. Thus, by attenuating cytosolic and mitochondrial ATPase activities and targeting downstream nucleotide degradation pathways, diazoxide contributes to the preservation of the cellular adenine nucleotide pool.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Myocardial resistance to metabolic challenge depends on maintained cellular energetic and ionic homeostasis (11, 20, 40, 51, 60). In this regard, potassium channel openers, such as diazoxide, that target mitochondrial functions have emerged as potent cytoprotective agents; yet the mechanisms responsible for their cardioprotective effect are not fully understood (19, 25, 46, 63). Potassium conductance of the mitochondrial inner membrane plays a significant role in mitochondrial physiology (2, 60), but the significance of modulating mitochondrial membrane permeability in cardiac protection remains elusive (23, 46, 60, 63). In fact, the substrate dependence of diazoxide effects on mitochondrial respiration (18, 23, 28), calcium transport (36), and flavoprotein oxidation (23, 34, 57, 61) argues against the involvement of changes in inner membrane potassium conductance as a sole mechanism of protection.

Here, diazoxide, in its cardioprotective range of concentrations, inhibited flavin adenine dinucleotide-dependent succinate dehydrogenase activity and reduced succinate-supported generation of ROS in heart mitochondria. Diazoxide attenuated cellular and mitochondrial ATPase activities and nucleotide degradation pathways, limiting the decline in myocardial ATP during ischemia. Diazoxide also mimicked ischemic preconditioning-induced cycles of increase and decrease in ROS generation in perfused heart, resulting in improved contractile recovery. Thus, although changes in potassium conductance of the inner mitochondrial membrane could affect mitochondrial membrane potential, calcium transport, and nucleotide hydrolysis (6-8, 31, 32), by targeting nucleotide-requiring enzymes, diazoxide reduces tissue oxidative damage, preserves cellular ATP, and triggers preconditioning events.

Succinate dehydrogenase is a four-subunit membrane molecular complex integrated with the mitochondrial respiratory chain that links the tricarbocylic acid and coenzyme Q cycles promoting electron flow and energy-dependent mitochondrial functions, such as ATP production and Ca2+ transport (21, 33). Vigorous succinate oxidation, associated with high activity of succinate dehydrogenase, hyperpolarizes mitochondrial inner membrane to sustain higher rates of ATP production compared with oxidation of NAD-dependent substrates (12, 21, 64). However, excessive succinate oxidation, which occurs on reoxygenation after ischemia as succinate accumulates (70), could be a major source of ROS and tissue damage (7, 30, 67). This mandates tight control of succinate dehydrogenase activity, which is achieved by endogenous inhibitors such as oxaloacetate (12, 21). Thus the inhibition of heart mitochondrial succinate dehydrogenase and succinate-supported ROS generation observed here by diazoxide underscores the role of dehydrogenases in mitochondrial physiology and cell protection (12, 21, 30, 69).

Original reports demonstrated that low micromolar concentrations of diazoxide modulate succinate oxidation and dehydrogenase activity, with an estimated IC50 in the 50 µM range (58), close to the 32 ± 7 µM obtained here. The extent of modulation of flavoprotein dehydrogenase activity in the intact heart by diazoxide could differ from that observed in soluble or membrane preparations; yet, cardioprotective concentrations (<100 µM) of diazoxide do affect succinate oxidation in heart mitochondria as confirmed in recent reports (23). Higher concentrations of diazoxide may be required for full inhibition of succinate oxidation (31), possibly depending on the mitochondrial preparation, the presence of albumin, or other substrates. Because complete inhibition of dehydrogenases is cytotoxic, only partial modulation of succinate dehydrogenase activity is apparently sufficient for induction of cytoprotection (23, 44, 53, 69).

Excessive ROS production is a major contributor to myocardial damage (67); yet, short-term free radical bursts can modulate enzyme activities, including succinate dehydrogenase (10), and initiate signal transduction events promoting preconditioning (66). In fact, although diazoxide suppresses ROS generation in perfused hearts, removal of diazoxide before ischemic insult was associated with a burst in ROS generation. The effect of diazoxide mimicked the pattern of ischemic preconditioning-induced regulation of ROS generation before prolonged ischemia and was associated with attenuated oxidant stress and improved performance on reperfusion, as observed with other potassium channel openers (65). Reduction, by diazoxide, of mitochondrial ROS generation after anoxia-reoxygenation can proceed in a potassium-independent manner and can be mimicked by inhibition of succinate dehydrogenase (46). Calculations indicate that opening of mitochondrial KATP channels would reduce membrane potential by <4 mV because of moderate potassium influx (31). However, this is not accompanied by reduction in total proton-motive force and deenergization of mitochondria, as reduction in membrane potential is compensated for by an increase in the pH gradient (Delta pH) (6, 7). In fact, the increase in Delta pH could be beneficial for mitochondrial substrate transport and oxidation. Because mitochondrial ROS production depends primarily on the total proton-motive force rather than on any of its components (7, 8, 59), the effect of diazoxide on succinate dehydrogenase inhibition and ROS generation cannot be readily explained by changes in mitochondrial potassium conductance and reduction in membrane potential. Conversely, ROS generation is very sensitive to modulation of dehydrogenase or respiratory chain activity and subsequent changes in proton-motive force or redox state (8, 30, 59, 67).

Potassium channel openers, such as nicorandil and pinacidil, have anti-free radical effects and attenuate oxidant stress, a major damaging factor at reperfusion (49, 65). Moreover, potassium channel openers, including diazoxide, were shown to reduce oxidative damage at the cellular and mitochondrial levels (17, 37, 43, 46). Yet other reports indicate that potassium channel openers can also increase ROS generation, mostly in the cellular environment (5, 15, 22, 32). The apparent discrepancy may be due to the experimental conditions and models used. Indeed, in the present study, ROS generation was decreased on diazoxide exposure but increased upon diazoxide washout, apparently reflecting initial inhibition of dehydrogenase and follow-up reactivation. This could explain the apparent controversy in previous studies, as increase in ROS generation by potassium channel openers was observed when media changes and washing procedures, which could trigger ROS production, were used. Other factors, such as time of drug exposure and changes in the cellular redox state, can also contribute to the observed effects (7, 30, 67). In addition, because inhibition of succinate dehydrogenase by diazoxide reduces reverse electron transfer (50), this might accelerate oxidation of NADH-dependent substrates coupled with ROS generation (21, 39).

The antagonist of mitochondrial potassium channel openers 5-HD circumvented the effect of diazoxide on succinate dehydrogenase by increasing electron flow to the respiratory chain. In fact, 5-HD did not relieve diazoxide-induced inhibition of soluble succinate dehydrogenase. This is in line with the ability of heart mitochondria to metabolize 5-HD (23, 60), resulting in increased supply of electrons at the flavoprotein level of acyl CoA-dehydrogenase and alterations in myocardial substrate oxidation (23). Indeed, attenuation of ROS production in diazoxide-treated mitochondria was reversed by 5-HD (46), indicating that bypass of succinate dehydrogenase blunts the ability of diazoxide to reduce mitochondrial damage.

Other potassium channel openers structurally distinct from diazoxide, such as pinacidil, nicorandil, cromakalim, and rilmakalim, had marginal effects on succinate dehydrogenase activity (data not shown). Rather, it was reported that pinacidil inhibits NADH (23) and alpha -ketoglutarate oxidation (18), whereas nicorandil through the release of nitric oxide could modulate the activity of the mitochondrial respiratory chain (23, 52, 56). Pinacidil, similarly to diazoxide, also activates pyruvate plus malate-coupled respiration, producing an uncoupler-like effect, which was potassium independent and could be blocked by carboxyatractyloside, an inhibitor of the adenine nucleotide translocator (28). Moreover, nicorandil and pinacidil also reduce cellular oxidative stress on reoxygenation (49, 65). Therefore, potassium channel openers may target distinct components of the substrate oxidation machinery; yet, their common outcome is attenuation of oxidant stress, leading to mitochondrial protection.

The present results further demonstrate that diazoxide also attenuates the activities of cellular Ca2+/Mg2+-ATPase as well as mitochondrial FoF1-ATPase in the heart, in line with reports of diazoxide-mediated regulation of mitochondrial and plasma membrane ATPases (3, 13, 31, 50). Indeed, dehydrogenases and ATPases/kinases share similarities in nucleotide binding properties and regulatory mechanisms (14, 21, 62). Potassium channel opener-mediated activation of KATP channels requires nucleotide binding folds and transmembrane domains of the sulfonylurea receptor, a regulatory subunit of the KATP channel complex (3, 41). Indeed, potassium channel openers modulate sarcolemmal ATPase activity, presumably by targeting ATP hydrolysis intrinsic to the sulfonylurea receptor, which regulates nucleotide-dependent KATP channel gating (3, 73). In this regard, a sequence of a succinate dehydrogenase subunit was actually found within the sulfonylurea receptor gene (71). Therefore, it is conceivable that structural similarities between flavin adenine nucleotide and adenine nucleotide binding proteins, including succinate dehydrogenase, alpha -glycerophosphate dehydrogenase, NADH dehydrogenase, and ATPases, underlie the action of potassium channel openers (1, 3, 9, 14, 23, 57).

Reduction of ATPase activity and cellular ATP consumption by diazoxide contributed to preservation of ATP levels and adenine nucleotide pool during ischemia. In diazoxide-treated hearts, the larger fractional decrease in xanthine compared with hypoxanthine levels indicates that the potassium channel opener may target specific steps in nucleotide degradation (40). This may include the flavoprotein-containing enzyme xanthine oxidase, which catalyzes conversion of xanthine to hypoxanthine. The regulation of the transition from the dehydrogenase to the oxidase form of this enzyme is an important step in cellular protection against injury (48). Thus diazoxide-induced attenuation of nucleotide hydrolysis and metabolism provides a mechanistic basis for previous observations on potassium channel opener-mediated preservation of ATP levels during ischemia (20, 38). Although not universally observed (27), an ATP-sparing effect and the reduction of nucleotide degradation could contribute to improved recovery of preconditioned hearts (40, 42, 51). Among other mechanisms, changes in mitochondrial intermembrane space volume by diazoxide-induced swelling could reduce ATP/ADP import/export from mitochondria, thus sustaining the cellular nucleotide pool (9).

In summary, by targeting nucleotide-requiring enzymes, particularly mitochondrial succinate dehydrogenase and cellular ATPases, diazoxide reduces ROS generation and nucleotide degradation, resulting in preservation of tissue ATP levels during ischemia. Thus directed regulation of mitochondrial dehydrogenase-dependent substrate oxidation and adenine nucleotide metabolism is an alternative strategy in cellular protection and tolerance to stress.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-64822, the American Heart Association, Miami Heart Research Institute, the Bruce and Ruth Rappaport Program in Vascular Biology and Gene Delivery, and the Marriott Foundation. A. Terzic is an Established Investigator of the American Heart Association.


    FOOTNOTES

Present address of D. G. L. Van Wylen: Department of Biology, Saint Olaf College, Northfield, MN 55057.

Address for reprint requests and other correspondence: A. Terzic, Guggenheim 7, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: terzic.andre{at}mayo.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 5, 2002;10.1152/ajpheart.00847.2002

Received 25 September 2002; accepted in final form 1 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bernardes, CF, Meyer-Fernandes JR, Martins OB, and Vercesi AE. Inhibition of succinic dehydrogenase and F0F1-ATP synthase by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Z Naturforsch [C] 52: 799-806, 1997[Medline].

2.   Bernardi, P. Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 79: 1127-1155, 1999[Abstract/Free Full Text].

3.   Bienengraeber, M, Alekseev AE, Abraham MR, Carrasco AJ, Moreau C, Vivaudou M, Dzeja PP, and Terzic A. ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex. FASEB J 14: 1943-1952, 2000[Abstract/Free Full Text].

4.   Boveris, A. Determination of the production of superoxide radicals and hydrogen peroxide in mitochondria. Methods Enzymol 105: 429-435, 1984[ISI][Medline].

5.   Carroll, R, Gant VA, and Yellon DM. Mitochondrial KATP channel opening protects a human atrial-derived cell line by a mechanism involving free radical generation. Cardiovasc Res 51: 691-700, 2001[Abstract/Free Full Text].

6.   Czyz, A, Szewczyk A, Nalecz MJ, and Wojtczak L. The role of mitochondrial potassium fluxes in controlling the protonmotive force in energized mitochondria. Biochem Biophys Res Commun 210: 98-104, 1995[ISI][Medline].

7.   Demin, OV, Gorianin II, Kholodenko BN, and Westerhoff HV. Kinetic modeling of energy metabolism and generation of active forms of oxygen in hepatocyte mitochondria. Mol Biol (Mosk) 35: 1095-1104, 2001[Medline].

8.   Demin, OV, Kholodenko BN, and Skulachev VP. A model of O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-generation in the complex III of the electron transport chain. Mol Cell Biochem 184: 21-33, 1998[ISI][Medline].

9.   Dos Santos, P, Kowaltowski AJ, Laclau MN, Seetharaman S, Paucek P, Boudina S, Thambo JB, Tariosse L, and Garlid KD. Mechanisms by which opening the mitochondrial ATP-sensitive K+ channel protects the ischemic heart. Am J Physiol Heart Circ Physiol 283: H284-H295, 2002[Abstract/Free Full Text].

10.   Du, G, Mouithys-Mickalad A, and Sluse FE. Generation of superoxide anion by mitochondria and impairment of their functions during anoxia and reoxygenation in vitro. Free Radic Biol Med 25: 1066-1074, 1998[ISI][Medline].

11.   Dzeja, PP, Holmuhamedov EL, Ozcan C, Pucar D, Jahangir A, and Terzic A. Mitochondria: gateway for cytoprotection. Circ Res 89: 744-746, 2001[Free Full Text].

12.   Dzeja, PP, Toleikis AI, and Prashkiavichius AK. Effect of experimental myocardial infarction on the succinate oxidation rate and succinate dehydrogenase activity in heart mitochondria. Vopr Med Khim 26: 591-594, 1980[ISI][Medline].

13.   Elmi, A, Idahl LA, and Sehlin J. Modulation of beta -cell ouabain-sensitive 86Rb+ influx Na+/K+ pump by D-glucose, glibenclamide or diazoxide. Int J Exp Diabetes Res 1: 265-274, 2001[Medline].

14.   Eventoff, W, and Rossmann MG. The evolution of dehydrogenases and kinases. CRC Crit Rev Biochem 3: 111-140, 1975[Medline].

15.   Forbes, RA, Steenbergen C, and Murphy E. Diazoxide-induced cardioprotection requires signaling through a redox-sensitive mechanism. Circ Res 88: 802-809, 2001[Abstract/Free Full Text].

16.   Fryer, RM, Eells JT, Hsu AK, Henry MM, and Gross GJ. Ischemic preconditioning in rats: role of mitochondrial KATP channel in preservation of mitochondrial function. Am J Physiol Heart Circ Physiol 278: H305-H312, 2000[Abstract/Free Full Text].

17.   Furuya, A, Kashimoto S, and Kumazawa T. Effects of nicorandil on myocardial function and metabolism in the post-ischaemic reperfused heart with or without inhalation anaesthetics. Acta Anaesthesiol Scand 46: 24-29, 2002[ISI][Medline].

18.   Grimmsmann, T, and Rustenbeck I. Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria. Br J Pharmacol 123: 781-788, 1998[ISI][Medline].

19.   Gross, GJ. The role of mitochondrial KATP channels in cardioprotection. Basic Res Cardiol 95: 280-284, 2000[ISI][Medline].

20.   Grover, GJ, and Garlid KD. ATP-sensitive potassium channels: a review of their cardioprotective pharmacology. J Mol Cell Cardiol 32: 677-695, 2000[ISI][Medline].

21.   Gutman, M. Modulation of mitochondrial succinate dehydrogenase activity, mechanism and function. Mol Cell Biochem 20: 41-60, 1978[ISI][Medline].

22.   Han, J, Kim N, Park J, Seog DH, Joo H, and Kim E. Opening of mitochondrial ATP-sensitive potassium channels evokes oxygen radical generation in rabbit heart slices. J Biochem (Tokyo) 131: 721-727, 2002[Abstract/Free Full Text].

23.   Hanley, PJ, Mickel M, Loffler M, Brandt U, and Daut J. KATP channel-independent targets of diazoxide and 5-hydroxydecanoate in the heart. J Physiol 542: 735-741, 2002[Abstract/Free Full Text].

24.   Holmuhamedov, EL, Wang L, and Terzic A. ATP-sensitive K+ channel openers prevent calcium overload in rat cardiac mitochondria. J Physiol 519: 347-360, 1999[Abstract/Free Full Text].

25.   Jilkina, O, Kuzio B, Grover GJ, and Kupriyanov VV. Effects of KATP channel openers, P-1075, pinacidil, and diazoxide, on energetics and contractile function in isolated rat hearts. J Mol Cell Cardiol 34: 427-440, 2002[ISI][Medline].

26.   Kissin, I, Aultman DF, and Smith LR. Effects of volatile anesthetics on myocardial oxidation-reduction status assessed by NADH fluorometry. Anesthesiology 59: 447-452, 1983[ISI][Medline].

27.   Kolocassides, KG, Seymour AM, Galinanes M, and Hearse DJ. Paradoxical effect of ischemic preconditioning on ischemic contracture? NMR studies of energy metabolism and intracellular pH in the rat heart. J Mol Cell Cardiol 28: 1045-1057, 1996[ISI][Medline].

28.   Kopustinskiene, D, Jovaisiene J, Liobikas J, and Toleikis A. Diazoxide and pinacidil uncouple pyruvate-malate-induced mitochondrial respiration. J Bioenerg Biomembr 34: 49-53, 2002[ISI][Medline].

29.   Korge, P, Honda HM, and Weiss JN. Protection of cardiac mitochondria by diazoxide and protein kinase C: implications for ischemic preconditioning. Proc Natl Acad Sci USA 99: 3312-3317, 2002[Abstract/Free Full Text].

30.   Korshunov, SS, Skulachev VP, and Starkov AA. High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett 416: 15-18, 1997[ISI][Medline].

31.   Kowaltowski, AJ, Seetharaman S, Paucek P, and Garlid KD. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria. Am J Physiol Heart Circ Physiol 280: H649-H657, 2001[Abstract/Free Full Text].

32.   Krenz, M, Oldenburg O, Wimpee H, Cohen MV, Garlid KD, Critz SD, Downey JM, and Benoit JN. Opening of ATP-sensitive potassium channels causes generation of free radicals in vascular smooth muscle cells. Basic Res Cardiol 97: 365-373, 2002[ISI][Medline].

33.   Lancaster, CR. Succinate:quinone oxidoreductases: an overview. Biochim Biophys Acta 1553: 1-6, 2002[Medline].

34.   Lawrence, CL, Billups B, Rodrigo GC, and Standen NB. The KATP channel opener diazoxide protects cardiac myocytes during metabolic inhibition without causing mitochondrial depolarization or flavoprotein oxidation. Br J Pharmacol 134: 535-542, 2001[ISI][Medline].

35.   Liu, Y, Sato T, O'Rourke B, and Marban E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection? Circulation 97: 2463-2469, 1998[Abstract/Free Full Text].

36.   MacDonald, MJ. The use of calcium uptake by small amounts of mitochondria from pancreatic islets to study mitochondrial respiration: the effects of diazoxide and sodium. Biochem Int 8: 771-778, 1984[ISI][Medline].

37.   Mano, T, Shinohara R, Nagasaka A, Nakagawa H, Uchimura K, Hayashi R, Nakano I, Tsugawa T, Watanabe F, Kobayashi T, Fujiwara K, Nakai A, and Itoh M. Scavenging effect of nicorandil on free radicals and lipid peroxide in streptozotocin-induced diabetic rats. Metabolism 49: 427-431, 2000[ISI][Medline].

38.   McPherson, CD, Pierce GN, and Cole WC. Ischemic cardioprotection by ATP-sensitive K+ channels involves high-energy phosphate preservation. Am J Physiol Heart Circ Physiol 265: H1809-H1818, 1993[Abstract/Free Full Text].

39.   Minners, J, van den Bos EJ, Yellon DM, Schwalb H, Opie LH, and Sack MN. Dinitrophenol, cyclosporin A, and trimetazidine modulate preconditioning in the isolated rat heart: support for a mitochondrial role in cardioprotection. Cardiovasc Res 47: 68-73, 2000[Abstract/Free Full Text].

40.   Mortimer, SL, Kodl CT, Reiling CR, and Van Wylen DG. Preconditioning-induced attenuation of purine metabolite accumulation during ischemia: memory and multiple cycles. Basic Res Cardiol 95: 119-126, 2000[ISI][Medline].

41.   Moreau, C, Jacquet H, Prost AL, D'hahan N, and Vivaudou M. The molecular basis of the specificity of action of KATP channel openers. EMBO J 19: 6644-6651, 2000[ISI][Medline].

42.   Murry, CE, Richard VJ, Reimer KA, and Jennings RB. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circ Res 66: 913-931, 1990[Abstract/Free Full Text].

43.   Narayan, P, Mentzer RM, Jr, and Lasley RD. Adenosine A1 receptor activation reduces reactive oxygen species and attenuates stunning in ventricular myocytes. J Mol Cell Cardiol 33: 121-129, 2001[ISI][Medline].

44.   Ockaili, R, Bhargava P, and Kukreja RC. Chemical preconditioning with 3-nitropropionic acid in hearts: role of mitochondrial KATP channel. Am J Physiol Heart Circ Physiol 280: H2406-H2411, 2001[Abstract/Free Full Text].

45.   Ovide-Bordeaux, S, Ventura-Clapier R, and Veksler V. Do modulators of the mitochondrial KATP channel change the function of mitochondria in situ? J Biol Chem 275: 37291-37295, 2000[Abstract/Free Full Text].

46.   Ozcan, C, Bienengraeber M, Dzeja PP, and Terzic A. Potassium channel openers protect cardiac mitochondria by attenuating oxidant stress at reoxygenation. Am J Physiol Heart Circ Physiol 282: H531-H539, 2002[Abstract/Free Full Text].

47.   Park, JW, Chun YS, Kim YH, Kim CH, and Kim MS. Ischemic preconditioning reduces ROS generation and prevents respiratory impairment in the mitochondria of post-ischemic reperfused heart of rat. Life Sci 60: 2207-2219, 1997[ISI][Medline].

48.   Peralta, C, Bulbena O, Xaus C, Prats N, Cutrin JC, Poli G, Gelpi E, and Rosello-Catafau J. Ischemic preconditioning: a defense mechanism against the reactive oxygen species generated after hepatic ischemia reperfusion. Transplantation 73: 1203-1211, 2002[ISI][Medline].

49.   Pieper, GM, and Gross GJ. Anti-free-radical and neutrophil-modulating properties of the nitrovasodilator, nicorandil. Cardiovasc Drugs Ther 6: 225-232, 1992[ISI][Medline].

50.   Portenhauser, R, Schafer G, and Trolp R. Inhibition of mitochondrial metabolism by the diabetogenic thiadiazine diazoxide. II. Interaction with energy conservation and ion transport. Biochem Pharmacol 20: 2623-2632, 1971[ISI][Medline].

51.   Pucar, D, Dzeja PP, Bast P, Juranic N, Macura S, and Terzic A. Cellular energetics in the preconditioned state: protective role for phosphotransfer reactions captured by 18O-assisted 31P NMR. J Biol Chem 276: 44812-44819, 2001[Abstract/Free Full Text].

52.   Rakhit, RD, Mojet MH, Marber MS, and Duchen MR. Mitochondria as targets for nitric oxide-induced protection during simulated ischemia and reoxygenation in isolated neonatal cardiomyocytes. Circulation 103: 2617-2623, 2001[Abstract/Free Full Text].

53.   Riepe, MW, and Ludolph AC. Chemical preconditioning: a cytoprotective strategy. Mol Cell Biochem 174: 249-254, 1997[ISI][Medline].

54.   Rouslin, W. Protonic inhibition of the mitochondrial oligomycin-sensitive adenosine 5'-triphosphatase in ischemic and autolyzing cardiac muscle. Possible mechanism for the mitigation of ATP hydrolysis under nonenergizing conditions. J Biol Chem 258: 9657-9661, 1983[Abstract/Free Full Text].

55.   Saborido, A, Delgado J, and Megias A. Measurement of sarcoplasmic reticulum Ca2+-ATPase activity and E-type Mg2+-ATPase activity in rat heart homogenates. Anal Biochem 268: 79-88, 1999[ISI][Medline].

56.   Sakai, K, Akima M, Saito K, Saitoh M, and Matsubara S. Nicorandil metabolism in rat myocardial mitochondria. J Cardiovasc Pharmacol 35: 723-728, 2000[ISI][Medline].

57.   Schafer, G, Portenhauser R, and Trolp R. Inhibition of mitochondrial metabolism by the diabetogenic thiadiazine diazoxide. Action on succinate dehydrogenase and TCA-cycle oxidations. Biochem Pharmacol 20: 1271-1280, 1971[ISI][Medline].

58.   Schafer, G, Wegener C, Portenhauser R, and Bojanovski D. Diazoxide, an inhibitor of succinate oxidation. Biochem Pharmacol 18: 2678-2681, 1969[ISI][Medline].

59.   Skulachev, VP. Uncoupling: new approaches to an old problem of bioenergetics. Biochim Biophys Acta 1363: 100-124, 1998[Medline].

60.   Suleiman, MS, Halestrap AP, and Griffiths EJ. Mitochondria: a target for myocardial protection. Pharmacol Ther 89: 29-46, 2001[ISI][Medline].

61.   Suzuki, M, Sasaki N, Miki T, Sakamoto N, Ohmoto-Sekine Y, Tamagawa M, Seino S, Marban E, and Nakaya H. Role of sarcolemmal KATP channels in cardioprotection against ischemia/reperfusion injury in mice. J Clin Invest 109: 509-516, 2002[ISI][Medline].

62.   Tamura, JK, Rakov RD, and Cross RL. Affinity labeling of nucleotide-binding sites on kinases and dehydrogenases by pyridoxal 5'-diphospho-5'-adenosine. J Biol Chem 261: 4126-4133, 1986[Abstract/Free Full Text].

63.   Terzic, A, Dzeja PP, and Holmuhamedov EL. Mitochondrial KATP channels: probing molecular identity and pharmacology. J Mol Cell Cardiol 32: 1911-1915, 2000[ISI][Medline].

64.   Toleikis, A, Dzeja P, Praskevicius A, and Jasaitis A. Mitochondrial functions in ischemic myocardium. I. Proton electrochemical gradient, inner membrane permeability, calcium transport and oxidative phosphorylation in isolated mitochondria. J Mol Cell Cardiol 11: 57-76, 1979[ISI][Medline].

65.   Vanden Hoek, TL, Becker LB, Shao ZH, Li CQ, and Schumacker PT. Preconditioning in cardiomyocytes protects by attenuating oxidant stress at reperfusion. Circ Res 86: 541-548, 2000[Abstract/Free Full Text].

66.   Vanden Hoek, TL, Becker LB, Shao Z, Li C, and Schumacker PT. Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J Biol Chem 273: 18092-18098, 1998[Abstract/Free Full Text].

67.   Vanden Hoek, TL, Shao Z, Li C, Schumacker PT, and Becker LB. Mitochondrial electron transport can become a significant source of oxidative injury in cardiomyocytes. J Mol Cell Cardiol 29: 2441-2450, 1997[ISI][Medline].

68.   Vincenti, E, Branca D, Varotto ML, and Scutari G. Effects of halothane, enflurane and isoflurane on some energy-converting functions of isolated rat liver mitochondria. Agressologie 30: 517-520, 1989[Medline].

69.   Wiegand, F, Liao W, Busch C, Castell S, Knapp F, Lindauer U, Megow D, Meisel A, Redetzky A, Ruscher K, Trendelenburg G, Victorov I, Riepe M, Diener HC, and Dirnagl U. Respiratory chain inhibition induces tolerance to focal cerebral ischemia. J Cereb Blood Flow Metab 19: 1229-1237, 1999[ISI][Medline].

70.   Wiesner, RJ, Deussen A, Borst M, Schrader J, and Grieshaber MK. Glutamate degradation in the ischemic dog heart: contribution to anaerobic energy production. J Mol Cell Cardiol 21: 49-59, 1989[ISI][Medline].

71.   Wohllk, N, Thomas PM, Huang E, and Cote GJ. A human succinate-ubiquinone oxidoreductase CII-3 subunit gene ending in a polymorphic dinucleotide repeat is located within the sulfonylurea receptor (SUR) gene. Mol Genet Metab 65: 187-190, 1998[ISI][Medline].

72.   Zaugg, M, Lucchinetti E, Spahn DR, Pasch T, Garcia C, and Schaub MC. Differential effects of anesthetics on mitochondrial KATP channel activity and cardiomyocyte protection. Anesthesiology 97: 15-23, 2002[ISI][Medline].

73.   Zingman, LV, Alekseev AE, Bienengraeber M, Hodgson D, Karger AB, Dzeja PP, and Terzic A. Signaling in channel/enzyme multimers: ATPase transitions in SUR module gate ATP-sensitive K+ conductance. Neuron 31: 233-245, 2001[ISI][Medline].

Am J Physiol Heart Circ Physiol 284(4):H1048-H1056
0363-6135/03 $5.00 Copyright © 2003 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
P. Pasdois, B. Beauvoit, L. Tariosse, B. Vinassa, S. Bonoron-Adele, and P. D. Santos
Effect of diazoxide on flavoprotein oxidation and reactive oxygen species generation during ischemia-reperfusion: a study on Langendorff-perfused rat hearts using optic fibers
Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2088 - H2097.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
J. W. Starnes, B. D. Barnes, and M. E. Olsen
Exercise training decreases rat heart mitochondria free radical generation but does not prevent Ca2+-induced dysfunction
J Appl Physiol, May 1, 2007; 102(5): 1793 - 1798.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
Z. Ma, N. Portwood, D. Brodin, V. Grill, and A. Bjorklund
Effects of Diazoxide on Gene Expression in Rat Pancreatic Islets Are Largely Linked to Elevated Glucose and Potentially Serve to Enhance {beta}-Cell Sensitivity
Diabetes, April 1, 2007; 56(4): 1095 - 1106.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. I. Niagara, H. Kh. Haider, S. Jiang, and M. Ashraf
Pharmacologically Preconditioned Skeletal Myoblasts Are Resistant to Oxidative Stress and Promote Angiomyogenesis via Release of Paracrine Factors in the Infarcted Heart
Circ. Res., March 2, 2007; 100(4): 545 - 555.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. Comelli, G. Metelli, and I. Mavelli
Downmodulation of mitochondrial F0F1 ATP synthase by diazoxide in cardiac myoblasts: a dual effect of the drug
Am J Physiol Heart Circ Physiol, February 1, 2007; 292(2): H820 - H829.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Heinen, A. K. S. Camara, M. Aldakkak, S. S. Rhodes, M. L. Riess, and D. F. Stowe
Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential
Am J Physiol Cell Physiol, January 1, 2007; 292(1): C148 - C156.
[Abstract]