Effect of metabolic inhibitors and second messengers upon Na(+)-H+ exchange in the sheep cardiac Purkinje fibre.

1. Acid extrusion through Na(+)-H+ exchange was studied in the sheep cardiac Purkinje fibre (bathed in Hepes-buffered solution, nominally free of CO2-HCO3-) by examining (i) intracellular pH (pHi) recovery from an intracellular acid load (induced by 20 mM NH4Cl prepulse) and (ii) the rate of rise of intracellular Na+ activity (aiNa) following the ammonium prepulse (used as an estimate of apparent Na+ influx on Na(+)-H+ exchange). The pHi and aiNa were recorded using ion-selective microelectrodes. 2. The pHi recovery and rise of aiNa were both greatly slowed in the presence of 2-deoxyglucose (DOG; glucose-free solution), an inhibitor of glycolysis, indicating inhibition of Na(+)-H+ exchange. 3. Cyanide moderately slowed pHi recovery rate but did not significantly affect the rise of aiNa. Estimates of beta 1 (intracellular buffering power) indicated an increase of approximately 50% in the presence of cyanide; such an increase accounts for most of the observed slowing of pHi recovery. It is concluded that oxidative inhibition with cyanide does not inhibit Na(+)-H+ exchange. 4. Intracellular ATP, measured from luciferin-luciferase luminescence, was reduced by a similar amount (approximately 70%) by either DOG or cyanide. This suggests that, if intracellular ATP (ATPi) reduction is the cause of exchanger inhibition by metabolic inhibitors, then ATPi generated glycolytically is more important for activation of the exchange. 5. 3-Isobutyl-1-methylxanthine (IBMX; a non-specific phosphodiesterase inhibitor which can elevate intracellular [cAMP]) slowed acid extrusion and reduced apparent Na+ influx by a similar amount, whereas addition of sodium nitroprusside (to elevate intracellular [cGMP]) had no effect, suggesting that raising intracellular [cAMP] downregulates Na(+)-H+ exchange, whereas raising intracellular [cGMP] does not. 6. Application of trifluorperazine (TFP; a non-specific calcium-calmodulin inhibitor) completely reversed the inhibitory effects of IBMX upon pHi recovery and aiNa. Under control conditions (no IBMX), TFP had no effect on pHi recovery or upon resting pHi. 7. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) had no significant effect on pHi recovery or apparent Na+ efflux. 8. We conclude that inhibition of glycolysis or elevation of cAMP produces downregulation of Na(+)-H+ exchange in the cardiac Purkinje fibre. Possible reasons for the lack of inhibitory effect of oxidative inhibitors are discussed.