Inhibition of Na+/H+ exchange preserves viability, restores mechanical function, and prevents the pH paradox in reperfusion injury to rat neonatal myocytes.
Rat neonatal myocytes exposed to 2.5 mM CaCN and 20 mM 2-deoxyglucose at pH 6.2 (chemical hypoxia) quickly lose viability when pH is increased to 7.4, with or without washout of inhibitors--a 'pH paradox'. In this study, we evaluated the effect of two Na+/H+ exchange inhibitors (dimethylamiloride and HOE694) and a Na+/Ca2+ exchange inhibitor (dichlorobenzamil) on pH-dependent reperfusion injury. Intracellular free Ca2+ and electrical potential were monitored by laser scanning confocal microscopy of rat neonatal cardiac myocytes grown on coverslips and co-loaded with Fluo-3 and tetramethylrhodamine methylester. After 30-60 min of chemical hypoxia at pH 6.2, mitochondria depolarized and Ca2+ began to increase uniformly throughout the cell. Free Ca2+ reached levels estimated to exceed 2 microM by 4 h. Washout of inhibitors at pH 7.4 (reperfusion), with or without dichlorobenzamil, killed most cells within 60 min, despite a marked reduction of Ca2+ in dichlorobenzamil-treated cells. Reperfusion at pH 7.4 in the presence of 75 microM dimethylamiloride or 20 microM HOE694, or at pH 6.2, prevented cell death. HOE694-treated cells placed into culture medium recovered mitochondrial membrane potential. In most cells, this occurred before normal Ca2+ was restored. Contracted myocytes re-extended over a 24-h-period. By 48 hours, most cells contracted spontaneously and showed normal Ca2+ transients. Our results indicate that Na+/H+ exchange inhibition protects against pH-dependent reperfusion injury and facilitates full recovery of cell function.
Harper
I S
IS
Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina at Chapel Hill.
Bond
J M
JM
Chacon
E
E
Reece
J M
JM
Herman
B
B
Lemasters
J J
JJ
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000814:Aniline Compounds; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002470:Cell Survival; D004247:DNA / Q000494:pharmacology; D004594:Electrophysiology; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D000860:Hypoxia / Q000378:metabolism; D017202:Myocardial Ischemia / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D019831:Sodium-Calcium Exchanger; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D014966:Xanthenes
D000814:Aniline Compounds; D002352:Carrier Proteins; D006146:Guanidines; D019831:Sodium-Calcium Exchanger; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; D014966:Xanthenes; C041470:3',4'-dichlorobenzamil; C074107:3-methylsulfonyl-4-piperidinobenzoyl guanidine; C059715:Fluo-3; C039465:5-dimethylamiloride; D000584:Amiloride; D004247:DNA; D002118:Calcium