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

1. The properties of the systems that regulate intracellular pH (pH _i ) 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 (pH ₀ ) 7·35.

2. Normally polarized fibres acidified to pH _i ~ 6·8 by an NH ₄ Cl pre-pulse (nominal absence of C0 ₂ ) recovered at a rate of 0·26±0·04 ΔpH _i h ^-1 ( n = 10). This corresponds to a net equivalent H ion efflux, J _H , 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 ΔpH _i h ^-1 , J _H = 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 NH ₄ Cl pre-pulse.

3. The rate of recovery after an NH ₄ Cl pulse increased linearly as pH _i was reduced from 7·25 to 6·55. The dependence of this recovery upon external Na (at pH _i 6·90) can be described by Michaelis-Menten kinetics; the apparent Michaelis constant (K _m ) is 12±3 mM.

4. Recovery of normally polarized fibres from acidification induced by 5% C0 ₂ is very slow (about 0·03 ΔpH _i h ^-1 ). This recovery could be converted into an acidification of 0·06—0·07 ΔpH _i h ^-1 either by removal of Na (as previously described) or by amiloride. We ascribe this acidification of the polarized fibres to HC0 ₃ - efflux.

5. In fibres depolarized in 50 mM-K, at constant external Cl concentration, recovery from C0 ₂ acidification was brisk (0·28±0·01 ΔpH _i h ^-1 , J _H = 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 HC0 ₃ .

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 C0 ₂ acidification (0·20±0·01 ΔpH _i h ^-1 , J _H = 6·7 pmol cm ^-2 s ^-1 , n = 59 and 0·14±0·02 ΔpH _i h ^-1 , J _H = 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 pH _i recovery from C0 ₂ 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 HC0 ₃ - efflux, and in part to the appearance of a SITS-sensitive component of acid extrusion most likely due to the elevated intracellular Cl.