Properties of the Intracellular pH-Regulating Systems of Frog Skeletal Muscle

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 °C and at external pH (pH0) 7·35. 2. Normally polarized fibres acidified to pHi ~ 6·8 by an NH4Cl pre-pulse (nominal absence of C02) recovered at a rate of 0·26±0·04 ΔpHi 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 ΔpHi h-1, JH = 4·9 pmol cm-2 s-1, n = 13). Amiloride (1 mM) reduced recovery by almost 90%, while 4-acetamido-4\u27 -isothiocyanostilbene-2,2\u27 -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% C02 is very slow (about 0·03 ΔpHi h-1). This recovery could be converted into an acidification of 0·06—0·07 ΔpHi h-1 either by removal of Na (as previously described) or by amiloride. We ascribe this acidification of the polarized fibres to HC03- efflux. 5. In fibres depolarized in 50 mM-K, at constant external Cl concentration, recovery from C02 acidification was brisk (0·28±0·01 ΔpHi 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 HC03. 6. Fibres depolarized to ~ —20 mV, either by 50 mM-K, constant [K] x [Cl], or by 0·5 mM-Ba in the presence of 2·5 mM-K, 5·9 mM-Cl, showed a slower recovery from C02 acidification (0·20±0·01 ΔpHi h-1, JH = 6·7 pmol cm-2 s-1 , n = 59 and 0·14±0·02 ΔpHi h-1, JH = 4·7 pmol cm-2 s-1, n = 22, respectively). Under both conditions, internal Cl should be low, while in 50 mM-K, constant Cl, internal Cl has previously been shown to be markedly raised. 7. The marked increase in pHi recovery from C02 acidification of fibres depolarized in 50 mM-K, constant Cl, as compared to normally polarized fibres, can be ascribed in part to the elimination of the driving force for the acidifying HC03- efflux, and in part to the appearance of a SITS-sensitive component of acid extrusion most likely due to the elevated intracellular Cl