title: Effects of peroxynitrite on sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle.
abstract: Peroxynitrite generated in arteries from superoxide and NO may damage Ca(2+) pumps. Here, we report the effects of peroxynitrite on ATP-dependent azide-insensitive uptake of Ca(2+) into pig coronary artery vesicular membrane fractions F2 [enriched in plasma membrane (PM)] and F3 [enriched in sarcoplasmic reticulum (SR)]. Membranes were pretreated with peroxynitrite and then with DTT to quench this agent. This pretreatment inhibited Ca(2+) uptake in a peroxynitrite concentration-dependent manner, but the effect was more severe in F3 than in F2. The inhibition was thus not overcome by excess DTT used to quench peroxynitrite and was not affected if catalase, SOD, or mannitol was added along with peroxynitrite. Such damage to the pump protein would be difficult to repair if produced during ischemia-reperfusion. The acylphosphates formed with ATP in F3 corresponded mainly to the SR Ca(2+) pump (110 kDa), but in F2 both PM (140 kDa) and 110-kDa bands were observed. Peroxynitrite treatment of F2 inhibited only the 110-kDa band. Inhibition of Ca(2+) uptake and acylphosphate formation from ATP correlated well in peroxynitrite-treated F3 samples. However, inhibition of acylphosphates from orthophosphate (reverse reaction of the pump) was slightly poorer. Peroxynitrite treatment also covalently cross-linked the pump protein, yielding no dimers but only larger oligomers. In contrast, cross-linking of the SR Ca(2+) pump in skeletal and cardiac muscles gives dimers as the first oligomers. Therefore, we speculate that SERCA2 has a different quaternary structure in the coronary artery smooth muscle.
authors.affiliation: Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
authors.forename: Ashok K
authors.initials: AK
authors.lastname: Grover
authors.forename: Sue E
authors.initials: SE
authors.lastname: Samson
authors.forename: Sarah
authors.initials: S
authors.lastname: Robinson
authors.forename: Chiu Yin
authors.initials: CY
authors.lastname: Kwan
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000187:drug effects / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D000252:Calcium-Transporting ATPases / Q000187:drug effects / Q000378:metabolism*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003324:Coronary Artery Disease / Q000378:metabolism / Q000503:physiopathology; D003331:Coronary Vessels / Q000187:drug effects / Q000378:metabolism*; D004229:Dithiothreitol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D030421:Peroxynitrous Acid / Q000378:metabolism* / Q000494:pharmacology; D053499:Plasma Membrane Calcium-Transporting ATPases; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism / Q000503:physiopathology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013347:Subcellular Fractions; D034421:Sus scrofa; D000097024:Acylphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D027682:Cation Transport Proteins; D004229:Dithiothreitol; D004791:Enzyme Inhibitors; D030421:Peroxynitrous Acid; D053499:Plasma Membrane Calcium-Transporting ATPases; D017382:Reactive Oxygen Species; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000097024:Acylphosphatase; C085285:dithiothreitol tetraacetate