CHANGES IN INTRACELLULAR PH CAUSED BY HIGH-K IN NORMAL AND ACIDIFIED FROG-MUSCLE - RELATION TO METABOLIC CHANGES

We examined the effect of depolarization on intracellular pH (pHi) of normal (pHi ~7.37) and acidified (pHi 5.90-6.70) frog semitendinosus muscle using microelectrodes. A small bundle was superfused with a Na+-free buffered solution (10 mM HEPES, 100% O2, pH 7.35) containing either 2.5 or 25 mM K+. An NH4CI prepLdse was used to lower pHi. At normal phi, depolarization usually produced a slight (0.04) alkalinization, followed by a fall in pHi. of ~0.2. In contrast, in all 25 acidified bundles pHi rose by 0.1-0.7. The rise was greater the lower the initial pH i. It could be imitated by caffeine and blocked by tetracaine and thus was, most likely, initiated by release of calcium. We ascribed the alkalinization to hydrolysis of phosphocreatine (PCr); 2,4-dinitrofluorobenzene abolished it. Biochemical analysis on fibers at the peak of alkalinization showed PCr to be reduced by one-half, while PCr in normal fibers that had been depolarized for the same period (4-6 rain) showed no change. We postulated that low pHi slows glycolysis with its associated ATP formation by reducing glycogenolysis and particularly by reducing conversion of fructose-6-phosphate to fructose-l,6-diphosphate through inhibition of phosphofructokinase (PFK), an enzyme which is known to be highly pH sensitive. Thus PCr hydrolysis would be required to replace much of the hydrolyzed ATP. This postulated effect on PFK is in agreement with the finding that glucose-6-phosphate (in near-equilibrium with fructose-6-phosphate) was increased nearly fivefold in the depolarized acid fibers, but not in the depolarized normal fibers. However, fructose-l,6-diphosphate also increased significantly; 3-phosphoglycerate was not affected. This suggests an additional acid-induced botdeneck between the latter two substrates. We measured the intrinsic buffering power, Β, of frog semitendinosus muscle with small pulses of NH4C1. It was found to vary with pHi according to Β = 144.6 - 17.2 (pHi). © 1990, Rockefeller University Press., All rights reserved.