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COPI-based quality control of native ATP-sensitive potassium channelsArakel, Eric January 2011 (has links)
Quality control of heteromultimeric membrane proteins includes mechanisms to ensure that only properly assembled complexes reach the cell surface. ER retrieval signals that are recognised by the COPI vesicle coat play a central role in such trafficking checkpoints. In many cases R-based ER retrieval signals are presented on one or all subunits of the heteromultimeric complexes. ATP-sensitive potassium (KATP) channels provide an example of a heteromultimeric cargo protein presenting eight R-based signals (one in each Kir6.X and SUR subunit of the 4+4 heterooctamer). KATP channels are metabolic sensors that couple cellular energy metabolism to electrical excitability by controlling the membrane potential at the plasma membrane. By this mechanism they are involved in glucose-stimulated insulin secretion, regulation of skeletal muscle excitability, cardioprotective ischemic preconditioning, neurotransmitter release and smooth muscle relaxation. In this thesis, we have established Blue Native Poly-Acrylamide Gel Electrophoresis (BN PAGE) as a method to characterize KATP channel complexes from different rodent tissues. Knock-out mice lacking individual subunits were employed as controls to probe the composition of the observed macromolecular complexes. This approach was complemented by density gradient centrifugation, subcellular fractionation and glycosylation analysis. We demonstrate the existence of distinct macromolecular complexes containing the pore-forming subunit Kir6.2 in pancreatic islets, the brain and the heart. Furthermore, we confirm assembly-dependent forward transport of channel subunits for endogenously expressed KATP channels. We report that the steady-state levels of Kir6.2 are drastically reduced in the brains of SUR1 knock-out mice, whereas we identify both fully assembled KATP channels and putative biogenetic intermediates in the hearts of SUR1 knock-out mice. Our results also suggest that SUR1, in native neuronal and cardiac KATP channels, is differentially glycosylated. Finally we have analyzed COPI-based recognition of Arg-based signals and KATP channel assembly in a mouse model (nur17) with a point mutation in the gene encoding the delta (delta)-subunit of COPI, in close vicinity to the reported binding site of Arg-based signals. We demonstrate that both the recognition of Arg-based signals and the assembly of several multimeric cargo proteins, including the KATP channel, are unaffected in the nur17 mouse. When analyzing the subunit composition of the nur17 COPI coat by two-dimensional BN SDS PAGE we discovered the presence of distinct COPI complexes in the mutant. We have also provided evidence that the association of COPI with a specific membrane compartment is compromised in the nur17 mouse. In conclusion, we have extended the study of a model COPI cargo, the KATP channel complex, to the physiologically relevant context. Furthermore, we have also provided initial insights into the molecular mechanism underlying the physiological consequences of the nur17 delta-COP mutation.
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Design, synthesis and biological evaluation of original cromakalim analogues as ATP-sensitive potassium channel openers/Conception, synthèse et évaluation pharmacologique danalogues originaux du cromakalim en tant quactivateurs des canaux potassiques ATP-dépendantsFlorence, Xavier 16 December 2009 (has links)
Conception, synthèse et évaluation pharmacologique danalogues originaux du cromakalim en tant quactivateurs des canaux potassiques ATP-dépendants
Les canaux potassiques sensibles à lATP (canaux KATP) sont des structures transmembranaires impliquées dans le passage des ions potassium de lintérieur vers lextérieur de la cellule. Ils jouent un rôle essentiel dans le contrôle du potentiel membranaire des cellules excitables et sont donc impliqués dans la régulation de nombreux processus physiologiques tels que la sécrétion dinsuline au niveau des cellules B pancréatiques ou encore le contrôle du tonus des muscles lisses.
Certaines molécules, notées PCOs (« Potassium Channel Openers »), sont capables dinduire louverture des canaux KATP. Les potentialités thérapeutiques de tels composés sont nombreuses pour autant que lon dispose de produits puissants et sélectifs envers un tissu donné de manière à éviter les effets indésirables. Les PCOs présentant une réelle sélectivité pour le tissu pancréatique sont susceptibles dêtre développés en tant que nouveaux agents thérapeutiques dans le cadre, notamment, du traitement de lhyperinsulinémie, du diabète et de lobésité. Nos recherches visent à développer des molécules analogues au (±)-cromakalim capables dactiver sélectivement les canaux KATP des cellules B pancréatiques.
Les précédents travaux entrepris au sein du laboratoire visant à identifier de nouveaux PCOs sélectifs des cellules B-pancréatiques ont permis la synthèse danalogues du (±)-cromakalim Certains se sont montrés très puissants et sélectifs des cellules B pancréatiques. Cette thèse sinscrit dans la continuité de ces recherches et poursuit les pharmacomodulations autour du noyau cromane afin de développer des analogues originaux du (±)-cromakalim plus puissants et plus sélectifs de la cellule B pancréatique tout en améliorant leurs propriétés physico-chimiques et en particulier leur hydrosolubilité.
Nous avons tout dabord exploré trois groupements potentiellement plus polaires en replacement de latome dhalogène de la position 6 du noyau cromane. Lors de cette première exploration, nous avons également évalué pharmacologiquement un intermédiaire de synthèse.
Ensuite, vu les résultats prometteurs obtenus avec les molécules de première génération portant un groupement tert-butyloxycarbonylamino (-NHCOOC(CH3)3) nous ont conduit à explorer linfluence de la présence dune fonction carbamate et de son encombrement stérique. Les résultats pharmacologiques obtenus avec les composés de seconde génération furent particulièrement intéressant. Un de ces composés fut choisi afin de déterminer le mécanisme daction de ces analogues.
Par la suite, les molécules synthétisées possédant un centre chiral en position 4 du noyau benzopyrane et se présentant donc toutes sous forme dun mélange racémique des deux énantiomères, nous avons réalisé la synthèse des énantiomères pures de la molécule de deuxième génération la plus intéressante afin détudier linfluence de la stéréochimie sur lactivité pancréatique et vasculaire de ces composés. Pour compléter létude de ce centre chiral, la synthèse de benzoxazines fut réalisée en vue de supprimer le carbone chiral en position 4. Ce remplacement de latome de carbone en positon 4 par un atome dazote fut lui aussi envisagé comme une amélioration possible de lhydrosolubilité de nos molécules.
Finalement, afin dévaluer linfluence de différents groupements potentiellement plus polaires non encore étudiés, nous avons réalisé la synthèse de molécules portant sur la position 6 un groupement méthanesulfonamide ou éthanesulfonamide. Nous avons également étudié linfluence de lintroduction dun atome de fluor sur le cycle benzénique de la phénylthiourée ou de la phénylurée ainsi que sur le noyau benzopyrane en position 6.
Les résultats obtenus avec ces molécules originales testées sur différents modèles pharmacologiques in vitro sont très prometteurs. Plusieurs produits élaborés au cours de ce travail se sont révélés être de puissants inhibiteurs de la sécrétion dinsuline et certains dentre eux exprimaient une nette sélectivité daction en faveur du tissu. Il apparaît donc clairement que les modifications structurales autour de la position 6 du noyau cromane du (±)-cromakalim, peuvent fortement influencer le profil dactivité pharmacologique de ces composés. De plus, les expérimentations radioisotopiques réalisées afin de déterminer le mécanisme daction de ces analogues originaux du cromakalim confirment que ceux-ci agissent principalement comme activateurs des canaux potassiques ATP-dépendants.
Les résultats pharmacologiques générés au cours de ce travail nous encouragent à poursuivre les pharmacomodulations entamées autour du (±)-cromakalim afin daccroître tant lactivité que la sélectivité tissulaire de nos molécules. Les axes de recherche chimiques pour lavenir sont nombreux et variés car le noyau benzopyrane est encore loin dêtre épuisé.
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ATP-sensitive potassium channel subcellular trafficking during ischemia, reperfusion, and preconditioningHo, Joanne Cin-Yee 22 January 2016 (has links)
Ischemic preconditioning is an endogenous cardioprotective mechanism in which short periods of ischemia and reperfusion provide protection when given before a subsequent ischemic event. Early mechanistic studies showed ATP-sensitive potassium (KATP) channels to play an important role in ischemic preconditioning. KATP channels link intracellular energy metabolism to membrane excitability and contractility. It is thought that KATP channels provide a cardioprotective role during ischemia by inducing action potential shortening, reducing an excessive Ca^2+ influx, and by preventing arrhythmias. However, the mechanisms by which KATP channels protect during ischemic preconditioning are not known. In this study, we investigated a novel potential mechanism in which alterations in subcellular KATP channel trafficking during ischemia and ischemic preconditioning may result in altered levels of surface channel density, and therefore, a greater degree of cardioprotection.
In the optimization of our experiments, we compared various antibodies for their specificity and sensitivity for channel subunit detection in immunoblotting. In addition, we examined the effects of varying salt concentrations during tissue homogenization in order to determine the optimal conditions for protein isolation. Furthermore, we examined the effect of heating the samples prior to SDS-PAGE for improved detection of channel proteins by immunoblotting.
The subcellular trafficking of some membrane proteins is altered by ischemia. For example, the glucose transporter, Glut4, translocates from endosomal compartments to the sarcolemma (Sun, Nguyen, DeGrado, Schwaiger, & Brosius, 1994). Conflicting data exists regarding the effects of ischemia on KATP channel subcellular trafficking and the regulation of KATP channel surface density (Edwards et al., 2009 and Bao, Hadjiolova, Coetzee, & Rindler, 2011). We therefore, sought to test our hypothesis that KATP channels are internalized from the surface of cardiomyocytes to endosomal compartments during ischemia, and this internalization can be reduced and/or reversed by ischemic preconditioning. We subjected isolated Langendorff-perfused mouse hearts to ischemia, reperfusion, or ischemic preconditioning events and measured the density of KATP channels in the sarcolemmal and endosomal compartments. We also determined the degree of injury by staining heart slices with triphenyltetrazolium chloride and compared infarct sizes between hearts subjected to ischemia and ischemic preconditioning.
Our data demonstrated that KATP channels are, in fact, internalized during ischemia and that reperfusion led to a slow recovery of surface KATP channel density. Interestingly, ischemic preconditioning reduced the size of infarcts induced by ischemia and also prevented the ischemia-induced decrease of KATP channel surface density, thereby, contributing to cardioprotection.
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Loss of KATP Channel Activity in Mouse FDB Leads to an Impairment in Energy Metabolism During FatigueScott, Kyle 03 May 2012 (has links)
Recently, it has been postulated that fatigue is a mechanism to protect the muscle fiber from deleterious ATP depletion and cell death. The ATP-sensitive potassium (KATP) channel is believed to play a major role in this mechanism. Under metabolic stress, the channels open, reducing membrane excitability, Ca2+ release and force production. This alleviates energy demand within the fiber, as activation of the channel reduces ATP consumption from cellular ATPases. Loss of KATP channel activity during fatigue results in excessive intracellular Ca2+ ([Ca2+]i) levels, likely entering the fiber through L-type Ca2+ channels. It has been demonstrated that when mouse muscle lacking functional KATP channels are stimulated to fatigue, ATP levels become significantly lower than wild type levels. Thus, it was hypothesized that a lack of KATP channel activity impairs energy metabolism, resulting in insufficient ATP production. The focus of work for this M.Sc. project was to test this hypothesis. Fatigue was elicited in Kir6.2-/- FDB muscles for three min followed by 15 min recovery. After 60 sec, a 2.6-fold greater glycogen breakdown was observed in Kir6.2-/- FDB compared to wild type FDB. However, this effect disappeared thereafter, as there were no longer any differences between wild type and Kir6.2-/- FDB in glycogen breakdown by 180 sec. Glucose oxidation after 60 sec was also greater in Kir6.2-/- FDB compared to wild type FDB. However, levels of oxidation failed to increase in Kir6.2-/- FDB from 60 to 180 sec. Calculated ATP production during the fatigue period was 2.7-times greater in Kir6.2-/- FDB, yet measured ATP levels during fatigue are much lower in Kir6.2-/- FDB compared to wild type FDB. Taken together, it appears that muscle energy metabolism is impaired in the absence KATP channel activity.
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Mechanisms Underlying the Pathogenesis of Atrial Arrhythmias in RGS4-deficient MiceMighiu, Alexandra Sorana 19 March 2014 (has links)
Atrial arrhythmias are very common clinically relevant conditions that are strongly associated with aging and parasympathetic tone. Additionally, ATP-sensitive K+ (KATP) channel activation has been reported to facilitate the development of re-entrant atrial arrhythmias. Since KATP channels are direct effectors of Gαi/o and RGS4 is an inhibitor of Gαi/o-signaling, we here investigate whether KATP channel activity is increased under decreased RGS4 activity in a manner that enhances susceptibility to AF. We show that loss of RGS4 facilitates the induction of atrial arrhythmias under parasympathetic challenge both in whole animals and isolated atrial tissues. Furthermore, using both genetic disruption (Kir6.2 ablation) and pharmacologic blockade (tolbutamide), we show that loss of functional KATP channels decreases the incidence of pacing-induced re-entry and prolongs repolarization in RGS4-deficient atria. Our findings are consistent with the conclusion that enhanced KATP channel activity may contribute to pacing-induced re-entrant rotors in the RGS4-deficient mouse model.
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Mechanisms Underlying the Pathogenesis of Atrial Arrhythmias in RGS4-deficient MiceMighiu, Alexandra Sorana 19 March 2014 (has links)
Atrial arrhythmias are very common clinically relevant conditions that are strongly associated with aging and parasympathetic tone. Additionally, ATP-sensitive K+ (KATP) channel activation has been reported to facilitate the development of re-entrant atrial arrhythmias. Since KATP channels are direct effectors of Gαi/o and RGS4 is an inhibitor of Gαi/o-signaling, we here investigate whether KATP channel activity is increased under decreased RGS4 activity in a manner that enhances susceptibility to AF. We show that loss of RGS4 facilitates the induction of atrial arrhythmias under parasympathetic challenge both in whole animals and isolated atrial tissues. Furthermore, using both genetic disruption (Kir6.2 ablation) and pharmacologic blockade (tolbutamide), we show that loss of functional KATP channels decreases the incidence of pacing-induced re-entry and prolongs repolarization in RGS4-deficient atria. Our findings are consistent with the conclusion that enhanced KATP channel activity may contribute to pacing-induced re-entrant rotors in the RGS4-deficient mouse model.
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Loss of KATP Channel Activity in Mouse FDB Leads to an Impairment in Energy Metabolism During FatigueScott, Kyle 03 May 2012 (has links)
Recently, it has been postulated that fatigue is a mechanism to protect the muscle fiber from deleterious ATP depletion and cell death. The ATP-sensitive potassium (KATP) channel is believed to play a major role in this mechanism. Under metabolic stress, the channels open, reducing membrane excitability, Ca2+ release and force production. This alleviates energy demand within the fiber, as activation of the channel reduces ATP consumption from cellular ATPases. Loss of KATP channel activity during fatigue results in excessive intracellular Ca2+ ([Ca2+]i) levels, likely entering the fiber through L-type Ca2+ channels. It has been demonstrated that when mouse muscle lacking functional KATP channels are stimulated to fatigue, ATP levels become significantly lower than wild type levels. Thus, it was hypothesized that a lack of KATP channel activity impairs energy metabolism, resulting in insufficient ATP production. The focus of work for this M.Sc. project was to test this hypothesis. Fatigue was elicited in Kir6.2-/- FDB muscles for three min followed by 15 min recovery. After 60 sec, a 2.6-fold greater glycogen breakdown was observed in Kir6.2-/- FDB compared to wild type FDB. However, this effect disappeared thereafter, as there were no longer any differences between wild type and Kir6.2-/- FDB in glycogen breakdown by 180 sec. Glucose oxidation after 60 sec was also greater in Kir6.2-/- FDB compared to wild type FDB. However, levels of oxidation failed to increase in Kir6.2-/- FDB from 60 to 180 sec. Calculated ATP production during the fatigue period was 2.7-times greater in Kir6.2-/- FDB, yet measured ATP levels during fatigue are much lower in Kir6.2-/- FDB compared to wild type FDB. Taken together, it appears that muscle energy metabolism is impaired in the absence KATP channel activity.
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Loss of KATP Channel Activity in Mouse FDB Leads to an Impairment in Energy Metabolism During FatigueScott, Kyle January 2012 (has links)
Recently, it has been postulated that fatigue is a mechanism to protect the muscle fiber from deleterious ATP depletion and cell death. The ATP-sensitive potassium (KATP) channel is believed to play a major role in this mechanism. Under metabolic stress, the channels open, reducing membrane excitability, Ca2+ release and force production. This alleviates energy demand within the fiber, as activation of the channel reduces ATP consumption from cellular ATPases. Loss of KATP channel activity during fatigue results in excessive intracellular Ca2+ ([Ca2+]i) levels, likely entering the fiber through L-type Ca2+ channels. It has been demonstrated that when mouse muscle lacking functional KATP channels are stimulated to fatigue, ATP levels become significantly lower than wild type levels. Thus, it was hypothesized that a lack of KATP channel activity impairs energy metabolism, resulting in insufficient ATP production. The focus of work for this M.Sc. project was to test this hypothesis. Fatigue was elicited in Kir6.2-/- FDB muscles for three min followed by 15 min recovery. After 60 sec, a 2.6-fold greater glycogen breakdown was observed in Kir6.2-/- FDB compared to wild type FDB. However, this effect disappeared thereafter, as there were no longer any differences between wild type and Kir6.2-/- FDB in glycogen breakdown by 180 sec. Glucose oxidation after 60 sec was also greater in Kir6.2-/- FDB compared to wild type FDB. However, levels of oxidation failed to increase in Kir6.2-/- FDB from 60 to 180 sec. Calculated ATP production during the fatigue period was 2.7-times greater in Kir6.2-/- FDB, yet measured ATP levels during fatigue are much lower in Kir6.2-/- FDB compared to wild type FDB. Taken together, it appears that muscle energy metabolism is impaired in the absence KATP channel activity.
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Modulation of ATP-sensitive potassium channels by hydrogen sulfide and hydroxylamineTang, Guanghua 04 January 2005
ATP-sensitive potassium (K+) channels (KATP) in vascular smooth muscle cells (VSMC) play a major role in the regulation of vascular tone by coupling cell contractility and K+ fluxes to cellular metabolism. They are composed of the regulatory sulphonylurea receptors (SUR) and the pore-forming inwardly rectifying K+ (Kir) channels. SUR subunits interact closely with Kir subunits by conferring their sensitivity to nucleotide or sulphonylurea. However, the modulatory mechanisms of KATP channels in VSMC are largely unknown. In particular, the effects of hydrogen sulfide (H2S) and hydroxylamine (HA) on KATP channels and underlying mechanisms have not been addressed in VSMC of resistance arteries. The combined approaches including molecular biology, biochemical assays, and patch-clamp techniques were applied. The electrophysiological and pharmacological features of native KATP channels in VSMC and cloned KATP channels in HEK-293 cells, and the modulation of KATP channels by H2S and HA in single freshly isolated VSMC from rat mesenteric arteries were characterized. In the present study, only small conductance KATP channels of 13 pS were found in rat mesenteric artery VSMC. The recorded macroscopic and unitary KATP currents were activated by nucleoside diphosphate in the presence of magnesium and K+ channel openers, inhibited by a specific KATP channel blocker glibenclamide, but were insensitive to ATP inhibition. The reversal potential shifted rightward in response to the elevation of extracellular K+ and matched the calculated K+ equilibrium potential, indicating the basal currents in both VSMC and HEK-293 cells are carried by K+ ions. Heterologous expression of Kir6.1 with SUR2B in HEK-293 cells formed functional channels and elicited whole-cell K+ currents, which shared some similar biophysical characteristics of native KATP channels in VSMC. Basal KATP currents and resting membrane potential in VSMC were reduced by glibenclamide, demonstrating that KATP channels contribute to background K+ conductance and in the setting of resting membrane potential in this resistance artery. Exogenous H2S enhanced macroscopic and unitary KATP currents with an EC50 of 116 ± 8.3 µM and hyperpolarized membrane potential. H2S activated KATP channels by increasing the open probability of single channels, but not single channel conductance. The reduced endogenous H2S production by D, L-propargylglycine resulted in the attenuation of KATP currents. H2S-induced activation of KATP channels and resultant hyperpolarization were not mediated by cGMP signaling pathway. HA enhanced reversibly KATP currents in a dose-dependent fashion with an EC50 of 54±3.4 µM and also hyperpolarized the cell membrane. HA-stimulated KATP currents were blocked by free radical scavengers (superoxide dismutase and N-acetyl-L-cysteine), and KATP channels were stimulated by a free radical generating system (hypoxanthine/xanthine oxidase), indicating the involvement of superoxide (O2-) in HA effects. Sodium nitroprusside and 8-Br-cGMP did not affect basal KATP currents and HA-stimulated KATP currents, disproving the involvement of NO-sGC-cGMP-mediated signaling pathway in the HA effects. Therefore, HA-induced KATP channel activation and hyperpolarization are likely due to the generation of O2-. In conclusion, KATP channels in resistance artery VSMC serve as the regulatory targets of H2S and HA. These two endogenous molecules modulate KATP channels via different mechanisms. H2S may directly act on KATP channel proteins while HA oxidized them via the formation of O2-, leading to the activation of KATP channels.
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Modulation of ATP-sensitive potassium channels by hydrogen sulfide and hydroxylamineTang, Guanghua 04 January 2005 (has links)
ATP-sensitive potassium (K+) channels (KATP) in vascular smooth muscle cells (VSMC) play a major role in the regulation of vascular tone by coupling cell contractility and K+ fluxes to cellular metabolism. They are composed of the regulatory sulphonylurea receptors (SUR) and the pore-forming inwardly rectifying K+ (Kir) channels. SUR subunits interact closely with Kir subunits by conferring their sensitivity to nucleotide or sulphonylurea. However, the modulatory mechanisms of KATP channels in VSMC are largely unknown. In particular, the effects of hydrogen sulfide (H2S) and hydroxylamine (HA) on KATP channels and underlying mechanisms have not been addressed in VSMC of resistance arteries. The combined approaches including molecular biology, biochemical assays, and patch-clamp techniques were applied. The electrophysiological and pharmacological features of native KATP channels in VSMC and cloned KATP channels in HEK-293 cells, and the modulation of KATP channels by H2S and HA in single freshly isolated VSMC from rat mesenteric arteries were characterized. In the present study, only small conductance KATP channels of 13 pS were found in rat mesenteric artery VSMC. The recorded macroscopic and unitary KATP currents were activated by nucleoside diphosphate in the presence of magnesium and K+ channel openers, inhibited by a specific KATP channel blocker glibenclamide, but were insensitive to ATP inhibition. The reversal potential shifted rightward in response to the elevation of extracellular K+ and matched the calculated K+ equilibrium potential, indicating the basal currents in both VSMC and HEK-293 cells are carried by K+ ions. Heterologous expression of Kir6.1 with SUR2B in HEK-293 cells formed functional channels and elicited whole-cell K+ currents, which shared some similar biophysical characteristics of native KATP channels in VSMC. Basal KATP currents and resting membrane potential in VSMC were reduced by glibenclamide, demonstrating that KATP channels contribute to background K+ conductance and in the setting of resting membrane potential in this resistance artery. Exogenous H2S enhanced macroscopic and unitary KATP currents with an EC50 of 116 ± 8.3 µM and hyperpolarized membrane potential. H2S activated KATP channels by increasing the open probability of single channels, but not single channel conductance. The reduced endogenous H2S production by D, L-propargylglycine resulted in the attenuation of KATP currents. H2S-induced activation of KATP channels and resultant hyperpolarization were not mediated by cGMP signaling pathway. HA enhanced reversibly KATP currents in a dose-dependent fashion with an EC50 of 54±3.4 µM and also hyperpolarized the cell membrane. HA-stimulated KATP currents were blocked by free radical scavengers (superoxide dismutase and N-acetyl-L-cysteine), and KATP channels were stimulated by a free radical generating system (hypoxanthine/xanthine oxidase), indicating the involvement of superoxide (O2-) in HA effects. Sodium nitroprusside and 8-Br-cGMP did not affect basal KATP currents and HA-stimulated KATP currents, disproving the involvement of NO-sGC-cGMP-mediated signaling pathway in the HA effects. Therefore, HA-induced KATP channel activation and hyperpolarization are likely due to the generation of O2-. In conclusion, KATP channels in resistance artery VSMC serve as the regulatory targets of H2S and HA. These two endogenous molecules modulate KATP channels via different mechanisms. H2S may directly act on KATP channel proteins while HA oxidized them via the formation of O2-, leading to the activation of KATP channels.
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