THE TEMPERATURE-DEPENDENCE OF INTRACELLULAR PH IN ISOLATED FROG SKELETAL-MUSCLE - LESSONS CONCERNING THE NA-H+ EXCHANGER()

We used P-31 NMR to investigate the temperature-dependence of intracel lular pH (pH(i)) in isolated frog skeletal muscles. We found that ln[H -i(+)] is a linear function of 1/T-abs paralleling those of neutral wa ter (i.e., H+ = OH-) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was un affected by inhibition of glycolysis and was not dependent upon the pH or [Na+] in the bathing solution. Insulin stimulation of Na+-H+ excha nge shifted the intercept in the alkaline direction but had no effect on the slope. Acid loading followed by washout resulted in an amilorid e-sensitive return to the (temperature dependent) basal pH(i). These r esults show that the temperature dependence of activation of Na+-H+ ex change is similar to that of the intracellular buffers, and suggest th at constancy of [H+]/ [OH-] with changing temperature is achieved in t he short term by intracellular buffering and in the long term by the s et-point of the Na+-H+ exchanger. Proton activation of the exchanger h as an apparent standard enthalpy change (Delta H degrees) under both c ontrol and insulin-stimulated conditions that is similar to the Delta H degrees of the intracellular buffers and approximately half of the D elta H degrees for the dissociation of water. Thus, the temperature-de pendent component of the standard free-energy change (Delta F degrees) is unaffected by insulin stimulation, suggesting that changes in Arrh enius activation energy (E-a) may not be a part of the mechanism of ho rmone stimulation.