Properties of the intracellular pH-regulating systems of frog skeletal muscle

Abstract

1. The properties of the systems that regulate intracellular pH (pHi) in frog muscle (Rana pipiens) were studied in semitendinosus fibres using pH-sensitive micro-electrodes. All experiments were done at 22 degrees C and at external pH (pHo) 7.35. 2. Normally polarized fibres acidified to pHi approximately 6.8 by an NH4Cl pre-pulse (nominal absence of CO2) recovered at a rate of 0.26 +/- 0.04 delta pHi h-1 (n = 10). This corresponds to a net equivalent H ion efflux, JH, of 5.0 pmol cm-2 s-1. This rate was not affected by depolarizing the fibres to -20 mV in 50 mM-K, constant Cl (0.29 +/- 0.03 delta pHi h-1, JH = 4.9 pmol cm-2 s-1, n = 13). Amiloride (1 mM) reduced recovery by almost 90%, while 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS, 0.1 mM) reduced recovery by only 18%. Removal of external Na (substitution by N-methyl-D-glucammonium) abolished recovery. Thus, Na-H exchange is responsible for most of the recovery from acidification induced by an NH4Cl pre-pulse. 3. The rate of recovery after an NH4Cl pulse increased linearly as pHi was reduced from 7.25 to 6.55. The dependence of this recovery upon external Na (at pHi 6.90) can be described by Michaelis-Menten kinetics; the apparent Michaelis constant (Km) is 12 +/- 3 mM. 4. Recovery of normally polarized fibres from acidification induced by 5% CO2 is very slow (about 0.03 delta pHi h-1). This recovery could be converted into an acidification of 0.06-0.07 delta pHi h-1 either by removal of Na (as previously described) or by amiloride. We ascribe this acidification of the polarized fibres to HCO3- efflux. 5. In fibres depolarized in 50 mM-K, at constant external Cl concentration, recovery from CO2 acidification was brisk (0.28 +/- 0.01 delta pHi h-1, JH = 9.4 pmol cm-2 s-1, n = 66). It was reduced by about 50% with either SITS or amiloride, and abolished by removal of Na. In the absence of Cl (substituted by gluconate), recovery was also reduced by about 50% and was unaffected by SITS, but nearly abolished by amiloride. Thus, in depolarized fibres, in addition to Na-H exchange, there is an active, SITS-sensitive component of recovery that requires Na, Cl and HCO3.(ABSTRACT TRUNCATED AT 400 WORDS)