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Jennifer S Lawton - One of the best experts on this subject based on the ideXlab platform.
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inhibition of succinate dehydrogenase by Diazoxide is independent of the atp sensitive potassium channel subunit sulfonylurea type 1 receptor
Journal of The American College of Surgeons, 2013Co-Authors: Melissa M. Anastacio, Richard B. Schuessler, Evelyn M. Kanter, Angela D. Keith, Colin G. Nichols, Jennifer S LawtonAbstract:Background Diazoxide maintains myocyte volume and contractility during stress via an unknown mechanism. The mechanism of action may involve an undefined (genotype unknown) mitochondrial ATP-sensitive potassium channel and is dependent on the ATP-sensitive potassium channel subunit sulfonylurea type 1 receptor (SUR1). The ATP-sensitive potassium channel openers have been shown to inhibit succinate dehydrogenase (SDH) and a gene for a portion of SDH has been found in the SUR intron. Diazoxide may be cardioprotective via inhibition of SDH, which can form part of an ATP-sensitive potassium channel or share its genetic material. This study investigated the role of inhibition of SDH by Diazoxide and its relationship to the SUR1 subunit. Study Design Mitochondria were isolated from wild-type and SUR1 knockout mice. Succinate dehydrogenase activity was measured by spectrophotometric analysis of 2,6-dichloroindophenol reduction for 20 minutes as the relative change in absorbance over time. Mitochondria were treated with succinate (20 mM), succinate + 1% dimethylsulfoxide, succinate + malonate (8 mM) (competitive inhibitor of SDH), or succinate + Diazoxide (100 μM). Results Both malonate and Diazoxide inhibit SDH activity in mitochondria of wild-type mice and in mice lacking the SUR1 subunit (p Conclusions The ability of DZX to inhibit SDH persists even after deletion of the SUR1 gene. Therefore, the enzyme complex SDH is not dependent on the SUR1 gene. The inhibition of SDH by DZX can play a role in the cardioprotection afforded by DZX; however, this role is independent of the ATP-sensitive potassium channel subunit SUR1.
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Cardioprotective Mechanism of Diazoxide Involves the Inhibition of Succinate Dehydrogenase
The Annals of thoracic surgery, 2013Co-Authors: Melissa M. Anastacio, Richard B. Schuessler, Evelyn M. Kanter, Carol M. Makepeace, Angela D. Keith, Haixia Zhang, Colin G. Nichols, Jennifer S LawtonAbstract:Background The adenosine triphosphate-sensitive potassium (K ATP ) channel opener, Diazoxide, preserves myocyte volume homeostasis and contractility during stress via an unknown mechanism. Pharmacologic overlap has been suggested between succinate dehydrogenase (SDH) activity and K ATP channel modulators. Diazoxide may be cardioprotective due to the inhibition of SDH which may form a portion of the mitochondrial K ATP channel. To determine the role of inhibition of SDH in Diazoxide's cardioprotection, this study utilized glutathione to prevent the inhibition of SDH. Methods SDH activity was measured in isolated mitochondria exposed to succinate (control), malonate (inhibitor of succinate dehydrogenase), Diazoxide, and varying concentrations of glutathione alone or in combination with Diazoxide. Enzyme activity was measured by spectrophotometric analysis. To evaluate myocyte volume and contractility, cardiac myocytes were superfused with Tyrode's physiologic solution (Tyrode's) (20 minutes), followed by test solution (20 minutes), including Tyrode's, hyperkalemic cardioplegia (stress), cardioplegia + Diazoxide, cardioplegia + Diazoxide + glutathione, or glutathione alone; followed by Tyrode's (20 minutes). Myocyte volume and contractility were recorded using image grabbing software. Results Both malonate and Diazoxide inhibited succinate dehydrogenase. Glutathione prevented the inhibition of succinate dehydrogenase by Diazoxide in a dose-dependent manner. The addition of Diazoxide prevented the detrimental myocyte swelling due to cardioplegia alone and this benefit was lost with the addition of glutathione. However, glutathione elicited an independent cardioprotective effect on myocyte contractility. Conclusions The ability of Diazoxide to provide beneficial myocyte homeostasis during stress involves the inhibition of succinate dehydrogenase, which may also involve the opening of a purported mitochondrial adenosine triphosphate sensitive potassium channel.
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Diazoxide maintenance of myocyte volume and contractility during stress evidence for a non sarcolemmal katp channel location
The Journal of Thoracic and Cardiovascular Surgery, 2010Co-Authors: Angela D Sellitto, Richard B. Schuessler, Haixia Zhang, Colin G. Nichols, Sarah K Maffit, Ashraf S Aldadah, Jennifer S LawtonAbstract:Objective Animal and human myocytes demonstrate significant swelling and reduced contractility during exposure to stress (metabolic inhibition, hyposmotic stress, or hyperkalemic cardioplegia), and these detrimental consequences may be inhibited by the addition of Diazoxide (adenosine triphosphate-sensitive potassium channel opener) via an unknown mechanism. Both SUR1 and SUR2A subunits have been localized to the heart, and mouse sarcolemmal adenosine triphosphate-sensitive potassium channels are composed of SUR2A/Kir6.2 subunits in the ventricle and SUR1/Kir6.2 subunits in the atria. This study was performed to localize the mechanism of Diazoxide by direct probing of sarcolemmal adenosine triphosphate-sensitive potassium channel current and by genetic deletion of channel subunits. Methods Sarcolemmal adenosine triphosphate-sensitive potassium channel current was recorded in isolated wild-type ventricular mouse myocytes during exposure to Tyrode's solution, Tyrode's + 100 μmol/L Diazoxide, hyperkalemic cardioplegia, cardioplegia + Diazoxide, cardioplegia + 100 μmol/L pinacidil, or metabolic inhibition using whole-cell voltage clamp (N = 7–12 cells per group). Ventricular myocyte volume was measured from SUR1(-/-) and wild-type mice during exposure to control solution, hyperkalemic cardioplegia, or cardioplegia + 100 μmol/L Diazoxide (N = 7–10 cells per group). Results Diazoxide did not increase sarcolemmal adenosine triphosphate-sensitive potassium current in wild-type myocytes, although they demonstrated significant swelling during exposure to cardioplegia that was prevented by Diazoxide. SUR1(-/-) myocytes also demonstrated significant swelling during exposure to cardioplegia, but this was not altered by Diazoxide. Conclusions Diazoxide does not open the ventricular sarcolemmal adenosine triphosphate-sensitive potassium channel but provides volume homeostasis via an SUR1-dependent pathway in mouse ventricular myocytes, supporting a mechanism of action distinct from sarcolemmal adenosine triphosphate-sensitive potassium channel activation.
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maintenance of myocyte volume homeostasis during stress by Diazoxide is cardioprotective
The Annals of Thoracic Surgery, 2007Co-Authors: Ashraf S Aldadah, Richard B. Schuessler, Ralph J Damiano, Rochus K Voeller, Jennifer S LawtonAbstract:Background. We previously demonstrated that myocyte swelling and reduced contractility secondary to hyperkalemic cardioplegia and hyposmotic stress are attenuated by the addition of Diazoxide, an adenosine triphosphate–sensitive potassium channel (KATP) opener. The goal of this study was to investigate the effect of Diazoxide on myocyte swelling and reduced contractility after metabolic inhibition and to attempt to summarize the potential mechanisms involved. Methods. Isolated rabbit myocytes were perfused with Tyrode’s control solution for 20 minutes, followed by test solution for 20 minutes. Test solutions included (1) Tyrode’s control, (2) a metabolic inhibition solution containing sodium cyanide and 2-deoxyglucose, (3) metabolic inhibition plus Diazoxide, (4) metabolic inhibition plus Diazoxide plus HMR1098 (a sarcolemmal KATPchannel blocker), or (5) metabolic inhibition plus Diazoxide plus 5-hydroxydeconoate (a mitochondrial KATPchannel blocker). Myocytes were then reexposed to Tyrode’s solution for 20 minutes. Volume measurements were taken every 5 minutes. Contractility was recorded using edge-detection software at baseline and at 10 and 20 minutes of reexposure to Tyrode’s solution. Results. Simulated ischemia (metabolic inhibition) caused significant myocyte swelling and associated reduced contractility. The addition of Diazoxide abolished myocyte swelling and attenuated the associated reduced contractility. Observations with Diazoxide were unchanged by the addition of HMR 1098 or 5hydroxydeconoate. Conclusions. Diazoxide, with or without either KATPchannel blocker, attenuated the significant myocyte swelling and reduced contractility secondary to metabolic inhibition. These data suggest a role for Diazoxide, independent of the KATP channel, in myocyte volume homeostasis. In addition, the prevention of myocyte swelling resulted in improved contractility, consistent with previous data and the hypothesis that myocyte swelling may participate in the phenomenon of myocardial stunning. (Ann Thorac Surg 2007;84:857– 63) © 2007 by The Society of Thoracic Surgeons
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hyperkalemic cardioplegia induced myocyte swelling and contractile dysfunction prevention by Diazoxide
The Annals of Thoracic Surgery, 2006Co-Authors: Shinichi Mizutani, Sandip M Prasad, Richard B. Schuessler, Ralph J Damiano, Ashraf S Aldadah, Jeffrey B Bloch, Michael D Diodato, Jennifer S LawtonAbstract:Background Hyperkalemic cardioplegia (9°C) results in significant myocyte swelling and reduced contractility, representing a possible mechanism of myocardial stunning. Adenosine triphosphate–sensitive potassium channel (K ATP ) openers have been shown to ameliorate stunning. This study evaluated the hypothesis that a K ATP opener would prevent hyperkalemic cardioplegia-induced myocyte swelling and reduced contractility. Methods Isolated rabbit myocytes were perfused with 37°C Tyrode's solution for 20 minutes, followed by test solution (9°C or 37°C) including control Tyrode's, Tyrode's + 100 μmol/L Diazoxide (K ATP opener), St. Thomas's solution; or 9°C St. Thomas's + 100 μmol/L Diazoxide or St. Thomas's + 100 μmol/L Diazoxide + 20 μmol/L HMR1098 or 50 μmol/L 5-hydroxydeconoate (K ATP blockers) for 20 minutes (n = 8 per group). Myocytes were then reexposed to 37°C Tyrode's solution for 20 minutes. Volume and contractility were measured by videomicroscopy and video-based edge detection, respectively. Results St. Thomas's solution (9°C) caused significant myocyte swelling and associated reduced contractility ( p p p Conclusions Diazoxide prevented myocyte swelling and reduced contractility secondary to hyperkalemic cardioplegia, and this was unchanged by the addition of either K ATP channel blocker. Prevention of myocyte swelling was associated with improved contractility, consistent with the hypothesis that myocyte swelling may be a mechanism of myocardial stunning. Diazoxide may play a role in myocyte volume homeostasis by means of a mechanism separate from opening the K ATP channel.
Richard B. Schuessler - One of the best experts on this subject based on the ideXlab platform.
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inhibition of succinate dehydrogenase by Diazoxide is independent of the atp sensitive potassium channel subunit sulfonylurea type 1 receptor
Journal of The American College of Surgeons, 2013Co-Authors: Melissa M. Anastacio, Richard B. Schuessler, Evelyn M. Kanter, Angela D. Keith, Colin G. Nichols, Jennifer S LawtonAbstract:Background Diazoxide maintains myocyte volume and contractility during stress via an unknown mechanism. The mechanism of action may involve an undefined (genotype unknown) mitochondrial ATP-sensitive potassium channel and is dependent on the ATP-sensitive potassium channel subunit sulfonylurea type 1 receptor (SUR1). The ATP-sensitive potassium channel openers have been shown to inhibit succinate dehydrogenase (SDH) and a gene for a portion of SDH has been found in the SUR intron. Diazoxide may be cardioprotective via inhibition of SDH, which can form part of an ATP-sensitive potassium channel or share its genetic material. This study investigated the role of inhibition of SDH by Diazoxide and its relationship to the SUR1 subunit. Study Design Mitochondria were isolated from wild-type and SUR1 knockout mice. Succinate dehydrogenase activity was measured by spectrophotometric analysis of 2,6-dichloroindophenol reduction for 20 minutes as the relative change in absorbance over time. Mitochondria were treated with succinate (20 mM), succinate + 1% dimethylsulfoxide, succinate + malonate (8 mM) (competitive inhibitor of SDH), or succinate + Diazoxide (100 μM). Results Both malonate and Diazoxide inhibit SDH activity in mitochondria of wild-type mice and in mice lacking the SUR1 subunit (p Conclusions The ability of DZX to inhibit SDH persists even after deletion of the SUR1 gene. Therefore, the enzyme complex SDH is not dependent on the SUR1 gene. The inhibition of SDH by DZX can play a role in the cardioprotection afforded by DZX; however, this role is independent of the ATP-sensitive potassium channel subunit SUR1.
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Cardioprotective Mechanism of Diazoxide Involves the Inhibition of Succinate Dehydrogenase
The Annals of thoracic surgery, 2013Co-Authors: Melissa M. Anastacio, Richard B. Schuessler, Evelyn M. Kanter, Carol M. Makepeace, Angela D. Keith, Haixia Zhang, Colin G. Nichols, Jennifer S LawtonAbstract:Background The adenosine triphosphate-sensitive potassium (K ATP ) channel opener, Diazoxide, preserves myocyte volume homeostasis and contractility during stress via an unknown mechanism. Pharmacologic overlap has been suggested between succinate dehydrogenase (SDH) activity and K ATP channel modulators. Diazoxide may be cardioprotective due to the inhibition of SDH which may form a portion of the mitochondrial K ATP channel. To determine the role of inhibition of SDH in Diazoxide's cardioprotection, this study utilized glutathione to prevent the inhibition of SDH. Methods SDH activity was measured in isolated mitochondria exposed to succinate (control), malonate (inhibitor of succinate dehydrogenase), Diazoxide, and varying concentrations of glutathione alone or in combination with Diazoxide. Enzyme activity was measured by spectrophotometric analysis. To evaluate myocyte volume and contractility, cardiac myocytes were superfused with Tyrode's physiologic solution (Tyrode's) (20 minutes), followed by test solution (20 minutes), including Tyrode's, hyperkalemic cardioplegia (stress), cardioplegia + Diazoxide, cardioplegia + Diazoxide + glutathione, or glutathione alone; followed by Tyrode's (20 minutes). Myocyte volume and contractility were recorded using image grabbing software. Results Both malonate and Diazoxide inhibited succinate dehydrogenase. Glutathione prevented the inhibition of succinate dehydrogenase by Diazoxide in a dose-dependent manner. The addition of Diazoxide prevented the detrimental myocyte swelling due to cardioplegia alone and this benefit was lost with the addition of glutathione. However, glutathione elicited an independent cardioprotective effect on myocyte contractility. Conclusions The ability of Diazoxide to provide beneficial myocyte homeostasis during stress involves the inhibition of succinate dehydrogenase, which may also involve the opening of a purported mitochondrial adenosine triphosphate sensitive potassium channel.
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Diazoxide maintenance of myocyte volume and contractility during stress evidence for a non sarcolemmal katp channel location
The Journal of Thoracic and Cardiovascular Surgery, 2010Co-Authors: Angela D Sellitto, Richard B. Schuessler, Haixia Zhang, Colin G. Nichols, Sarah K Maffit, Ashraf S Aldadah, Jennifer S LawtonAbstract:Objective Animal and human myocytes demonstrate significant swelling and reduced contractility during exposure to stress (metabolic inhibition, hyposmotic stress, or hyperkalemic cardioplegia), and these detrimental consequences may be inhibited by the addition of Diazoxide (adenosine triphosphate-sensitive potassium channel opener) via an unknown mechanism. Both SUR1 and SUR2A subunits have been localized to the heart, and mouse sarcolemmal adenosine triphosphate-sensitive potassium channels are composed of SUR2A/Kir6.2 subunits in the ventricle and SUR1/Kir6.2 subunits in the atria. This study was performed to localize the mechanism of Diazoxide by direct probing of sarcolemmal adenosine triphosphate-sensitive potassium channel current and by genetic deletion of channel subunits. Methods Sarcolemmal adenosine triphosphate-sensitive potassium channel current was recorded in isolated wild-type ventricular mouse myocytes during exposure to Tyrode's solution, Tyrode's + 100 μmol/L Diazoxide, hyperkalemic cardioplegia, cardioplegia + Diazoxide, cardioplegia + 100 μmol/L pinacidil, or metabolic inhibition using whole-cell voltage clamp (N = 7–12 cells per group). Ventricular myocyte volume was measured from SUR1(-/-) and wild-type mice during exposure to control solution, hyperkalemic cardioplegia, or cardioplegia + 100 μmol/L Diazoxide (N = 7–10 cells per group). Results Diazoxide did not increase sarcolemmal adenosine triphosphate-sensitive potassium current in wild-type myocytes, although they demonstrated significant swelling during exposure to cardioplegia that was prevented by Diazoxide. SUR1(-/-) myocytes also demonstrated significant swelling during exposure to cardioplegia, but this was not altered by Diazoxide. Conclusions Diazoxide does not open the ventricular sarcolemmal adenosine triphosphate-sensitive potassium channel but provides volume homeostasis via an SUR1-dependent pathway in mouse ventricular myocytes, supporting a mechanism of action distinct from sarcolemmal adenosine triphosphate-sensitive potassium channel activation.
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maintenance of myocyte volume homeostasis during stress by Diazoxide is cardioprotective
The Annals of Thoracic Surgery, 2007Co-Authors: Ashraf S Aldadah, Richard B. Schuessler, Ralph J Damiano, Rochus K Voeller, Jennifer S LawtonAbstract:Background. We previously demonstrated that myocyte swelling and reduced contractility secondary to hyperkalemic cardioplegia and hyposmotic stress are attenuated by the addition of Diazoxide, an adenosine triphosphate–sensitive potassium channel (KATP) opener. The goal of this study was to investigate the effect of Diazoxide on myocyte swelling and reduced contractility after metabolic inhibition and to attempt to summarize the potential mechanisms involved. Methods. Isolated rabbit myocytes were perfused with Tyrode’s control solution for 20 minutes, followed by test solution for 20 minutes. Test solutions included (1) Tyrode’s control, (2) a metabolic inhibition solution containing sodium cyanide and 2-deoxyglucose, (3) metabolic inhibition plus Diazoxide, (4) metabolic inhibition plus Diazoxide plus HMR1098 (a sarcolemmal KATPchannel blocker), or (5) metabolic inhibition plus Diazoxide plus 5-hydroxydeconoate (a mitochondrial KATPchannel blocker). Myocytes were then reexposed to Tyrode’s solution for 20 minutes. Volume measurements were taken every 5 minutes. Contractility was recorded using edge-detection software at baseline and at 10 and 20 minutes of reexposure to Tyrode’s solution. Results. Simulated ischemia (metabolic inhibition) caused significant myocyte swelling and associated reduced contractility. The addition of Diazoxide abolished myocyte swelling and attenuated the associated reduced contractility. Observations with Diazoxide were unchanged by the addition of HMR 1098 or 5hydroxydeconoate. Conclusions. Diazoxide, with or without either KATPchannel blocker, attenuated the significant myocyte swelling and reduced contractility secondary to metabolic inhibition. These data suggest a role for Diazoxide, independent of the KATP channel, in myocyte volume homeostasis. In addition, the prevention of myocyte swelling resulted in improved contractility, consistent with previous data and the hypothesis that myocyte swelling may participate in the phenomenon of myocardial stunning. (Ann Thorac Surg 2007;84:857– 63) © 2007 by The Society of Thoracic Surgeons
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hyperkalemic cardioplegia induced myocyte swelling and contractile dysfunction prevention by Diazoxide
The Annals of Thoracic Surgery, 2006Co-Authors: Shinichi Mizutani, Sandip M Prasad, Richard B. Schuessler, Ralph J Damiano, Ashraf S Aldadah, Jeffrey B Bloch, Michael D Diodato, Jennifer S LawtonAbstract:Background Hyperkalemic cardioplegia (9°C) results in significant myocyte swelling and reduced contractility, representing a possible mechanism of myocardial stunning. Adenosine triphosphate–sensitive potassium channel (K ATP ) openers have been shown to ameliorate stunning. This study evaluated the hypothesis that a K ATP opener would prevent hyperkalemic cardioplegia-induced myocyte swelling and reduced contractility. Methods Isolated rabbit myocytes were perfused with 37°C Tyrode's solution for 20 minutes, followed by test solution (9°C or 37°C) including control Tyrode's, Tyrode's + 100 μmol/L Diazoxide (K ATP opener), St. Thomas's solution; or 9°C St. Thomas's + 100 μmol/L Diazoxide or St. Thomas's + 100 μmol/L Diazoxide + 20 μmol/L HMR1098 or 50 μmol/L 5-hydroxydeconoate (K ATP blockers) for 20 minutes (n = 8 per group). Myocytes were then reexposed to 37°C Tyrode's solution for 20 minutes. Volume and contractility were measured by videomicroscopy and video-based edge detection, respectively. Results St. Thomas's solution (9°C) caused significant myocyte swelling and associated reduced contractility ( p p p Conclusions Diazoxide prevented myocyte swelling and reduced contractility secondary to hyperkalemic cardioplegia, and this was unchanged by the addition of either K ATP channel blocker. Prevention of myocyte swelling was associated with improved contractility, consistent with the hypothesis that myocyte swelling may be a mechanism of myocardial stunning. Diazoxide may play a role in myocyte volume homeostasis by means of a mechanism separate from opening the K ATP channel.
David W Busija - One of the best experts on this subject based on the ideXlab platform.
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effects of atp sensitive potassium channel activators Diazoxide and bms 191095 on membrane potential and reactive oxygen species production in isolated piglet mitochondria
Brain Research Bulletin, 2005Co-Authors: David W Busija, Gary J Grover, Bela Kis, Nishadi Rajapakse, Prasad V G Katakam, Ferenc Domoki, Ferenc BariAbstract:Abstract Mitochondrial ATP-sensitive potassium (mitoK ATP ) channel openers protect the piglet brain against ischemic stress. Effects of mitoK ATP channel agonists on isolated mitochondria, however, have not been directly examined. We investigated the effects of K ATP channel openers and blockers on membrane potential and on the production of reactive oxygen species (ROS) in isolated piglet mitochondria. Diazoxide and BMS-191095, putative selective openers of mitoK ATP , decreased the mitochondrial membrane potential (Δ Ψ m ). On a molar basis, Diazoxide was less effective than BMS-191095. In contrast, Diazoxide but not BMS-191095 increased ROS production by mitochondria. Since Diazoxide also inhibits succinate dehydrogenase (SDH), we examined the effects of 3-nitropropionic acid (3-NPA), an inhibitor of SDH. 3-NPA failed to change the Δ Ψ m but increased ROS production. Inhibitors of K ATP channels did not affect resting Δ Ψ m or ROS production, but glibenclamide and 5-hydroxydecanoate (5-HD) blocked effects of Diazoxide and BMS-191095 on Δ Ψ m and Diazoxide effects on ROS production. We conclude that BMS-191095 has selective effects on mitoK ATP channels while Diazoxide also increases ROS production probably via inhibition of SDH.
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Diazoxide preconditioning attenuates global cerebral ischemia induced blood brain barrier permeability
Brain Research, 2005Co-Authors: Gabor Lenzser, Bela Kis, Ferenc Bari, David W BusijaAbstract:Brain edema formation due to blood-brain barrier (BBB) disruption is a major consequence of cerebral ischemia. Previously, we demonstrated that targeting mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) protects neuronal tissues in vivo and in vitro, however, the effects of mitoK(ATP) openers on cerebral endothelial cells and on BBB functions have never been examined. We investigated the effects of mitoK(ATP) channel opener Diazoxide on BBB functions during ischemia/reperfusion injury (I/R). Rats were treated with 6, 20 or 40 mg/kg Diazoxide ip for 3 days then exposed to global cerebral ischemia for 30 min. BBB permeability was assessed by administering Evan's-blue (EB) and Na-fluorescein (NaF) at the beginning of the 30 min reperfusion. I/R increased BBB permeability for the large molecular weight EB (ng/mg) in the cortex (control: 146 +/- 12, n = 7; I/R: 1049 +/- 152, n = 11) which was significantly attenuated in Diazoxide-treated rats (575 +/- 99, n = 9; 582 +/- 104, n = 8; 20 and 40 mg/kg doses). Diazoxide pretreatment also significantly inhibited the extravasation of the low molecular weight NaF. Edema formation in the cortex was also decreased after Diazoxide pretreatment. In cultured cerebral endothelial cells, Diazoxide depolarized the mitochondrial membrane, suggesting a direct Diazoxide effect on the endothelial mitochondria. Our results demonstrate that preconditioning of cerebral endothelium with Diazoxide protects the BBB against ischemic stress.
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Diazoxide preconditioning protects against neuronal cell death by attenuation of oxidative stress upon glutamate stimulation
Journal of Neuroscience Research, 2004Co-Authors: Bela Kis, Nishadi Rajapakse, Krisztina Nagy, Ferenc Bari, David W BusijaAbstract:We examined the effects of Diazoxide, the putative mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channel opener, against glutamate excitotoxicity in primary cultures of rat cortical neurons. Cells were treated with Diazoxide for 24 hr and then exposed to 200 μM glutamate. Cell viability was measured 24 hr after glutamate exposure. We found that treatment 24 hr before glutamate exposure with 250 and 500 μM Diazoxide but not with another mitoKATP channel opener, nicorandil, increased neuronal viability from 54 ± 2% to 84 ± 2% and 92 ± 3%, respectively (n = 25–40). These effects were not inhibited by the putative mitoKATP channel blocker 5-hydroxydecanoic acid. Diazoxide application increased production of reactive oxygen species (ROS) and coapplication of M40401, a superoxide dismutase mimetic, prevented delayed preconditioning. The 24 hr preconditioned neurons showed significantly reduced ROS production upon glutamate stimulation compared to that in untreated cells. These results suggest that Diazoxide induces delayed preconditioning in cultured cortical neurons via increased ROS production and attenuation of oxidative stress upon glutamate stimulation. © 2004 Wiley-Liss, Inc.
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Diazoxide induces delayed pre conditioning in cultured rat cortical neurons
Journal of Neurochemistry, 2004Co-Authors: Bela Kis, Nishadi Rajapakse, James A Snipes, Krisztina Nagy, Takashi Horiguchi, David W BusijaAbstract:We investigated the effect of Diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygen-glucose deprivation (OGD). Diazoxide pre-treatment induced delayed pre-conditioning and almost entirely attenuated the OGD-induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate abolished the protective effect of Diazoxide while the non-selective KATP channel blocker glibenclamide did not. The non-selective KATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of Diazoxide. Diazoxide did not increase reduced glutathione and manganese-superoxide dismutase levels but we found significantly higher reduced glutathione levels in Diazoxide-pre-conditioned neurons after OGD. In pre-conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3-nitropropionic acid also induced pre-conditioning and free radical production in neurons. Here, we provide the first evidence that Diazoxide induces delayed pre-conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase-inhibiting effect of Diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP-sensitive potassium channels.
Colin G. Nichols - One of the best experts on this subject based on the ideXlab platform.
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focal congenital hyperinsulinism managed by medical treatment a diagnostic algorithm based on molecular genetic screening
Clinical Endocrinology, 2014Co-Authors: Arianna Maiorana, Colin G. Nichols, Fabrizio Barbetti, Arianna Boiani, Vittoria Rufini, Milena Pizzoferro, Paola Francalanci, Flavio Faletra, Chiara GrimaldiAbstract:Objective: Congenital hyperinsulinism (CHI) requires rapid diagnosis and treatment to avoid irreversible neurological sequelae due to hypoglycaemia. Aetiological diagnosis is instrumental in directing the appropriate therapy. Current diagnostic algorithms provide a complete set of diagnostic tools including (i) biochemical assays, (ii) genetic facility and (iii) state-of-the-art imaging. They consider the response to a therapeutic Diazoxide trial an early, crucial step before proceeding (or not) to specific genetic testing and eventually imaging, aimed at distinguishing diffuse vs focal CHI. However, interpretation of the Diazoxide test is not trivial and can vary between research groups, which may lead to inappropriate decisions. Objective of this report is proposing a new algorithm in which early genetic screening, rather than Diazoxide trial, dictates subsequent clinical decisions. Patients, Methods and Results: Two CHI patients weaned from parenteral glucose infusion and glucagon after starting Diazoxide. No hypoglycaemia was registered during a 72-h continuous glucose monitoring (CGMS), or hypoglycaemic episodes were present for no longer than 3% of 72-h. Normoglycaemia was obtained by low–medium dose Diazoxide combined with frequent carbohydrate feeds for several years. We identified monoallelic, paternally inherited mutations in KATP channel genes, and 18F-DOPA PET-CT revealed a focal lesion that was surgically resected, resulting in complete remission of hypoglycaemia. Conclusions: Although rare, some patients with focal lesions may be responsive to Diazoxide. As a consequence, we propose an algorithm that is not based on a ‘formal’ Diazoxide response but on genetic testing, in which patients carrying paternally inherited ABCC8 or KCNJ11 mutations should always be subjected to 18F-DOPA PET-CT.
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inhibition of succinate dehydrogenase by Diazoxide is independent of the atp sensitive potassium channel subunit sulfonylurea type 1 receptor
Journal of The American College of Surgeons, 2013Co-Authors: Melissa M. Anastacio, Richard B. Schuessler, Evelyn M. Kanter, Angela D. Keith, Colin G. Nichols, Jennifer S LawtonAbstract:Background Diazoxide maintains myocyte volume and contractility during stress via an unknown mechanism. The mechanism of action may involve an undefined (genotype unknown) mitochondrial ATP-sensitive potassium channel and is dependent on the ATP-sensitive potassium channel subunit sulfonylurea type 1 receptor (SUR1). The ATP-sensitive potassium channel openers have been shown to inhibit succinate dehydrogenase (SDH) and a gene for a portion of SDH has been found in the SUR intron. Diazoxide may be cardioprotective via inhibition of SDH, which can form part of an ATP-sensitive potassium channel or share its genetic material. This study investigated the role of inhibition of SDH by Diazoxide and its relationship to the SUR1 subunit. Study Design Mitochondria were isolated from wild-type and SUR1 knockout mice. Succinate dehydrogenase activity was measured by spectrophotometric analysis of 2,6-dichloroindophenol reduction for 20 minutes as the relative change in absorbance over time. Mitochondria were treated with succinate (20 mM), succinate + 1% dimethylsulfoxide, succinate + malonate (8 mM) (competitive inhibitor of SDH), or succinate + Diazoxide (100 μM). Results Both malonate and Diazoxide inhibit SDH activity in mitochondria of wild-type mice and in mice lacking the SUR1 subunit (p Conclusions The ability of DZX to inhibit SDH persists even after deletion of the SUR1 gene. Therefore, the enzyme complex SDH is not dependent on the SUR1 gene. The inhibition of SDH by DZX can play a role in the cardioprotection afforded by DZX; however, this role is independent of the ATP-sensitive potassium channel subunit SUR1.
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Cardioprotective Mechanism of Diazoxide Involves the Inhibition of Succinate Dehydrogenase
The Annals of thoracic surgery, 2013Co-Authors: Melissa M. Anastacio, Richard B. Schuessler, Evelyn M. Kanter, Carol M. Makepeace, Angela D. Keith, Haixia Zhang, Colin G. Nichols, Jennifer S LawtonAbstract:Background The adenosine triphosphate-sensitive potassium (K ATP ) channel opener, Diazoxide, preserves myocyte volume homeostasis and contractility during stress via an unknown mechanism. Pharmacologic overlap has been suggested between succinate dehydrogenase (SDH) activity and K ATP channel modulators. Diazoxide may be cardioprotective due to the inhibition of SDH which may form a portion of the mitochondrial K ATP channel. To determine the role of inhibition of SDH in Diazoxide's cardioprotection, this study utilized glutathione to prevent the inhibition of SDH. Methods SDH activity was measured in isolated mitochondria exposed to succinate (control), malonate (inhibitor of succinate dehydrogenase), Diazoxide, and varying concentrations of glutathione alone or in combination with Diazoxide. Enzyme activity was measured by spectrophotometric analysis. To evaluate myocyte volume and contractility, cardiac myocytes were superfused with Tyrode's physiologic solution (Tyrode's) (20 minutes), followed by test solution (20 minutes), including Tyrode's, hyperkalemic cardioplegia (stress), cardioplegia + Diazoxide, cardioplegia + Diazoxide + glutathione, or glutathione alone; followed by Tyrode's (20 minutes). Myocyte volume and contractility were recorded using image grabbing software. Results Both malonate and Diazoxide inhibited succinate dehydrogenase. Glutathione prevented the inhibition of succinate dehydrogenase by Diazoxide in a dose-dependent manner. The addition of Diazoxide prevented the detrimental myocyte swelling due to cardioplegia alone and this benefit was lost with the addition of glutathione. However, glutathione elicited an independent cardioprotective effect on myocyte contractility. Conclusions The ability of Diazoxide to provide beneficial myocyte homeostasis during stress involves the inhibition of succinate dehydrogenase, which may also involve the opening of a purported mitochondrial adenosine triphosphate sensitive potassium channel.
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Diazoxide maintenance of myocyte volume and contractility during stress evidence for a non sarcolemmal katp channel location
The Journal of Thoracic and Cardiovascular Surgery, 2010Co-Authors: Angela D Sellitto, Richard B. Schuessler, Haixia Zhang, Colin G. Nichols, Sarah K Maffit, Ashraf S Aldadah, Jennifer S LawtonAbstract:Objective Animal and human myocytes demonstrate significant swelling and reduced contractility during exposure to stress (metabolic inhibition, hyposmotic stress, or hyperkalemic cardioplegia), and these detrimental consequences may be inhibited by the addition of Diazoxide (adenosine triphosphate-sensitive potassium channel opener) via an unknown mechanism. Both SUR1 and SUR2A subunits have been localized to the heart, and mouse sarcolemmal adenosine triphosphate-sensitive potassium channels are composed of SUR2A/Kir6.2 subunits in the ventricle and SUR1/Kir6.2 subunits in the atria. This study was performed to localize the mechanism of Diazoxide by direct probing of sarcolemmal adenosine triphosphate-sensitive potassium channel current and by genetic deletion of channel subunits. Methods Sarcolemmal adenosine triphosphate-sensitive potassium channel current was recorded in isolated wild-type ventricular mouse myocytes during exposure to Tyrode's solution, Tyrode's + 100 μmol/L Diazoxide, hyperkalemic cardioplegia, cardioplegia + Diazoxide, cardioplegia + 100 μmol/L pinacidil, or metabolic inhibition using whole-cell voltage clamp (N = 7–12 cells per group). Ventricular myocyte volume was measured from SUR1(-/-) and wild-type mice during exposure to control solution, hyperkalemic cardioplegia, or cardioplegia + 100 μmol/L Diazoxide (N = 7–10 cells per group). Results Diazoxide did not increase sarcolemmal adenosine triphosphate-sensitive potassium current in wild-type myocytes, although they demonstrated significant swelling during exposure to cardioplegia that was prevented by Diazoxide. SUR1(-/-) myocytes also demonstrated significant swelling during exposure to cardioplegia, but this was not altered by Diazoxide. Conclusions Diazoxide does not open the ventricular sarcolemmal adenosine triphosphate-sensitive potassium channel but provides volume homeostasis via an SUR1-dependent pathway in mouse ventricular myocytes, supporting a mechanism of action distinct from sarcolemmal adenosine triphosphate-sensitive potassium channel activation.
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regulation of katp channel activity by Diazoxide and mgadp distinct functions of the two nucleotide binding folds of the sulfonylurea receptor
The Journal of General Physiology, 1997Co-Authors: Show Ling Shyng, T Ferrigni, Colin G. NicholsAbstract:KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and Diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945–951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and Diazoxide, with the exception of G1479R, which showed a small stimulatory response to Diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by Diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4−. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and Diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and Diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation.
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effects of atp sensitive potassium channel activators Diazoxide and bms 191095 on membrane potential and reactive oxygen species production in isolated piglet mitochondria
Brain Research Bulletin, 2005Co-Authors: David W Busija, Gary J Grover, Bela Kis, Nishadi Rajapakse, Prasad V G Katakam, Ferenc Domoki, Ferenc BariAbstract:Abstract Mitochondrial ATP-sensitive potassium (mitoK ATP ) channel openers protect the piglet brain against ischemic stress. Effects of mitoK ATP channel agonists on isolated mitochondria, however, have not been directly examined. We investigated the effects of K ATP channel openers and blockers on membrane potential and on the production of reactive oxygen species (ROS) in isolated piglet mitochondria. Diazoxide and BMS-191095, putative selective openers of mitoK ATP , decreased the mitochondrial membrane potential (Δ Ψ m ). On a molar basis, Diazoxide was less effective than BMS-191095. In contrast, Diazoxide but not BMS-191095 increased ROS production by mitochondria. Since Diazoxide also inhibits succinate dehydrogenase (SDH), we examined the effects of 3-nitropropionic acid (3-NPA), an inhibitor of SDH. 3-NPA failed to change the Δ Ψ m but increased ROS production. Inhibitors of K ATP channels did not affect resting Δ Ψ m or ROS production, but glibenclamide and 5-hydroxydecanoate (5-HD) blocked effects of Diazoxide and BMS-191095 on Δ Ψ m and Diazoxide effects on ROS production. We conclude that BMS-191095 has selective effects on mitoK ATP channels while Diazoxide also increases ROS production probably via inhibition of SDH.
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Diazoxide preconditioning attenuates global cerebral ischemia induced blood brain barrier permeability
Brain Research, 2005Co-Authors: Gabor Lenzser, Bela Kis, Ferenc Bari, David W BusijaAbstract:Brain edema formation due to blood-brain barrier (BBB) disruption is a major consequence of cerebral ischemia. Previously, we demonstrated that targeting mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) protects neuronal tissues in vivo and in vitro, however, the effects of mitoK(ATP) openers on cerebral endothelial cells and on BBB functions have never been examined. We investigated the effects of mitoK(ATP) channel opener Diazoxide on BBB functions during ischemia/reperfusion injury (I/R). Rats were treated with 6, 20 or 40 mg/kg Diazoxide ip for 3 days then exposed to global cerebral ischemia for 30 min. BBB permeability was assessed by administering Evan's-blue (EB) and Na-fluorescein (NaF) at the beginning of the 30 min reperfusion. I/R increased BBB permeability for the large molecular weight EB (ng/mg) in the cortex (control: 146 +/- 12, n = 7; I/R: 1049 +/- 152, n = 11) which was significantly attenuated in Diazoxide-treated rats (575 +/- 99, n = 9; 582 +/- 104, n = 8; 20 and 40 mg/kg doses). Diazoxide pretreatment also significantly inhibited the extravasation of the low molecular weight NaF. Edema formation in the cortex was also decreased after Diazoxide pretreatment. In cultured cerebral endothelial cells, Diazoxide depolarized the mitochondrial membrane, suggesting a direct Diazoxide effect on the endothelial mitochondria. Our results demonstrate that preconditioning of cerebral endothelium with Diazoxide protects the BBB against ischemic stress.
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Diazoxide preconditioning protects against neuronal cell death by attenuation of oxidative stress upon glutamate stimulation
Journal of Neuroscience Research, 2004Co-Authors: Bela Kis, Nishadi Rajapakse, Krisztina Nagy, Ferenc Bari, David W BusijaAbstract:We examined the effects of Diazoxide, the putative mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channel opener, against glutamate excitotoxicity in primary cultures of rat cortical neurons. Cells were treated with Diazoxide for 24 hr and then exposed to 200 μM glutamate. Cell viability was measured 24 hr after glutamate exposure. We found that treatment 24 hr before glutamate exposure with 250 and 500 μM Diazoxide but not with another mitoKATP channel opener, nicorandil, increased neuronal viability from 54 ± 2% to 84 ± 2% and 92 ± 3%, respectively (n = 25–40). These effects were not inhibited by the putative mitoKATP channel blocker 5-hydroxydecanoic acid. Diazoxide application increased production of reactive oxygen species (ROS) and coapplication of M40401, a superoxide dismutase mimetic, prevented delayed preconditioning. The 24 hr preconditioned neurons showed significantly reduced ROS production upon glutamate stimulation compared to that in untreated cells. These results suggest that Diazoxide induces delayed preconditioning in cultured cortical neurons via increased ROS production and attenuation of oxidative stress upon glutamate stimulation. © 2004 Wiley-Liss, Inc.