Lack of manifestations of diazoxide/5-hydroxydecanoate-sensitive KATP channel in rat brain nonsynaptosomal mitochondria.
Pharmacological modulation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) sensitive to diazoxide and 5-hydroxydecanoate (5-HD) represents an attractive strategy to protect cells against ischaemia/reperfusion- and stroke-related injury. To re-evaluate a functional role for the mitoKATP in brain, we used Percoll-gradient-purified brain nonsynaptosomal mitochondria in a light absorbance assay, in radioisotope measurements of matrix volume, and in measurements of respiration, membrane potential (DeltaPsi) and depolarization-induced K+ efflux. The changes in mitochondrial morphology were evaluated by transmission electron microscopy (TEM). Polyclonal antibodies raised against certain fragments of known sulphonylurea receptor subunits, SUR1 and SUR2, and against different epitopes of K+ inward rectifier subunits Kir 6.1 and Kir 6.2 of the ATP-sensitive K+ channel of the plasma membrane (cellKATP), were employed to detect similar subunits in brain mitochondria. A variety of plausible blockers (ATP, 5-hydroxydecanoate, glibenclamide, tetraphenylphosphonium cation) and openers (diazoxide, pinacidil, chromakalim, minoxidil, testosterone) of the putative mitoKATP were applied to show the role of the channel in regulating matrix volume, respiration, and DeltaPsi and K+ fluxes across the inner mitochondrial membrane. None of the pharmacological agents applied to brain mitochondria in the various assays pinpointed processes that could be unequivocally associated with mitoKATP activity. In addition, immunoblotting analysis did not provide explicit evidence for the presence of the mitoKATP, similar to the cellKATP, in brain mitochondria. On the other hand, the depolarization-evoked release of K+ suppressed by ATP could be re-activated by carboxyatractyloside, an inhibitor of the adenine nucleotide translocase (ANT). Moreover, bongkrekic acid, another inhibitor of the ANT, inhibited K+ efflux similarly to ATP. These observations implicate the ANT in ATP-sensitive K+ transport in brain mitochondria.
Brustovetsky
Tatiana
T
Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Dr., Medical Science Building 549, Indianapolis, IN 46202, USA.
Shalbuyeva
Natalia
N
Brustovetsky
Nickolay
N
D018528:ATP-Binding Cassette Transporters / Q000032:analysis; D000255:Adenosine Triphosphate; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D003470:Culture Media; D003652:Decanoic Acids / Q000494:pharmacology*; D003981:Diazoxide / Q000494:pharmacology*; D006880:Hydroxy Acids / Q000494:pharmacology*; D066298:In Vitro Techniques; D054086:KATP Channels; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors; D010101:Oxygen Consumption; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000032:analysis; D011189:Potassium Chloride; D051381:Rats; D017207:Rats, Sprague-Dawley; D011955:Receptors, Drug / Q000032:analysis; D064233:Sulfonylurea Receptors; D000099161:Kcnj11 Channel
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D001278:Atractyloside; D003470:Culture Media; D003652:Decanoic Acids; D003981:Diazoxide; D006880:Hydroxy Acids; D054086:KATP Channels; D000226:Mitochondrial ADP, ATP Translocases; D011188:Potassium; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011189:Potassium Chloride; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D000099161:Kcnj11 Channel; C577509:Abcc8 protein, rat; C577512:Abcc9 protein, rat; C117780:mitochondrial K(ATP) channel; C092520:uK-ATP-1 potassium channel; C052853:5-hydroxydecanoic acid; C003853:carboxyatractyloside