Properties of the intracellular pH‐regulating systems of frog skeletal muscle.

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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.degree. C and at external pH (pHo) 7.35. 2. Normally polarized fibres acidified to pHi .apprx. 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 ony 18%. Removal of external Na (subtitution 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 NHCl 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 CO3 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. 6. Fibres depolarized to .apprx. -20 mV, either by 50 mM-K, constant [K] .times. [Cl], or by 0.5 mM-Ba in the presence of 2.5 mM-K, 5.9 mM-Cl, showed a slower recovery from CO2 acidification (0.20 .+-. 0.01 .DELTA.pHi h-1, JH = 6.7 pmol cm-2 s-1, n = 59 and 0.14 .+-. 0.02 .DELTA.pHi 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 CO2 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 HCO3- efflux, and in part to the appearance of a SITS-sensitive component of acid extrusion most likely due to the elevated intracellular Cl.