Effect of vacuolar proton ATPase on pHi, Ca2+, and apoptosis in neonatal cardiomyocytes during metabolic inhibition/recovery.
Recently, we found that vacuolar proton ATPase (VPATPase) operates in cardiomyocytes as a complementary proton-extruding mechanism. Its activity was increased by preconditioning with resultant attenuation of intracellular acidification during ischemia. In this study, we examined whether VPATPase-mediated proton efflux during metabolic inhibition/recovery may spare Na+ overload via Na+-H+ exchange, attenuate Na+-Ca2+ exchange, and decrease apoptosis. Neonatal rat cardiomyocytes were subjected to 2- to 3-hour metabolic inhibition with cyanide and 2-deoxyglucose and 24-hour recovery. The effect of VPATPase inhibition by 50 nmol/L bafilomycin A1 on apoptosis, pHi, and [Ca2+]i was studied by flow cytometry with propidium iodide, seminaphthorhodafluor (SNARF)-1-AM, and indo-1-AM staining, respectively. VPATPase inhibition increased the amount of apoptosis measured after 24 hours of recovery and abrogated the protective effect of inhibition of Na+-H+ exchange by (5-N-ethyl-N-isopropyl)amiloride (EIPA). Dual blockade of VPATPase and Na+-H+ exchange was additive in effect with EIPA on pHi during metabolic inhibition/recovery and recovery from the acid challenge with sodium propionate. VPATPase blockade increased the rate of accumulation of intracellular Ca2+ at the beginning of metabolic inhibition and abrogated the delaying effect of EIPA on intracellular Ca2+ accumulation. These results indicate that VPATPase plays an important accessory role in cardiomyocyte protection by reducing acidosis and Na+-H+ exchange-induced Ca2+ overload.
Karwatowska-Prokopczuk
E
E
Research Service, Veterans Affairs San Diego Healthcare System, University of California, San Diego, School of Medicine, 92161, USA.
Nordberg
J A
JA
Li
H L
HL
Engler
R L
RL
Gottlieb
R A
RA
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D018942:Macrolides; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology
D000889:Anti-Arrhythmia Agents; D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D018942:Macrolides; D019831:Sodium-Calcium Exchanger; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases; D002118:Calcium; C039614:ethylisopropylamiloride