Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle.
1. Ion-sensitive electrodes (made with a chloride-sensitive ion-exchange resin) were used to measure the internal chloride activity (a(i) (Cl)) of frog sartorius fibres at 25 degrees C.2. The internal pH (pH(i)) of other sartorius fibres was measured with a recessed tip pH-sensitive electrode (made with pH-sensitive glass).3. In normal bicarbonate-free solution (containing 2.5 mM potassium), the average chloride equilibrium potential, E(Cl) (calculated from a(i) (Cl) and the measured chloride activity of the external solution (a(o) (Cl)) was 87.7 +/- 1.7 mV (mean +/- S.E.; n = 16) in fibres where the average membrane potential, E(m), was 88.3 +/- 1.5 mV (mean +/- S.E.; n = 16). In experiments where a(i) (Cl) was varied between about 1 and 10 mM (which corresponds to values of E(m) between about -105 and -50 mV) E(Cl) was within 1-3 mV of E(m) at equilibrium. These measurements of a(i) (Cl) were obtained from the potential difference between the chloride-sensitive electrode and an intracellular indifferent micro-electrode filled with potassium chloride. If a potassium sulphate-filled indifferent micro-electrode was used, then values of a(i) (Cl) below about 5 mM were erroneously high, probably due to interference from other sarcoplasmic ions at the indifferent electrode.4. In solutions containing 15 mM bicarbonate and gassed with 5% CO(2), pH(i) was 6.9, corresponding to an internal bicarbonate concentration of 7.6 mM. E(Cl) measured in this solution was some 4 mV positive to E(m). Most of the difference between E(Cl) and E(m) could be ascribed to interference by sarcoplasmic bicarbonate on the basis of selectivity measurements of chloride against bicarbonate made on the ion-exchange resin in the relevant range of a(Cl).5. If bicarbonate/CO(2) in the external solution was replaced by HEPES/pure O(2) at constant pH, then pH(i) rose from 6.88 +/- 0.02 (mean +/- S.E.) to 7.05 +/- 0.02. A change in external pH of 1 unit caused pH(i) to change by about 0.02 unit and the intracellular buffering power was calculated to be about 35.6. In solution made hypertonic by the addition of sucrose, E(m) changed little or depolarized and E(Cl) and E(m) remained close. In contrast, in solution made hypertonic by the addition of solid sodium chloride (high-chloride solution) E(Cl) became negative to E(m). Conversely in low chloride solution E(Cl) became positive to E(m).7. When the chloride permeability (P(Cl)) was reduced by the use of acid solution, E(Cl) moved positive to E(m) indicating an accumulation of internal chloride. When P(Cl) was increased again by returning to more alkaline solution, E(m) depolarized to E(Cl).8. The results are consistent with the existence of a small, active movement of chloride, the effects of which are normally obscured by large passive movements of chloride when P(Cl) is large.
Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle. 1. Ion-sensitive electrodes (made with a chloride-sensitive ion-exchange resin) were used to measure the internal chloride activity (a(i) (Cl)) of frog sartorius fibres at 25 degrees C.2. The internal pH (pH(i)) of other sartorius fibres was measured with a recessed tip pH-sensitive electrode (made with pH-sensitive glass).3. In normal bicarbonate-free solution (containing 2.5 mM potassium), the average chloride equilibrium potential, E(Cl) (calculated from a(i) (Cl) and the measured chloride activity of the external solution (a(o) (Cl)) was 87.7 +/- 1.7 mV (mean +/- S.E.; n = 16) in fibres where the average membrane potential, E(m), was 88.3 +/- 1.5 mV (mean +/- S.E.; n = 16). In experiments where a(i) (Cl) was varied between about 1 and 10 mM (which corresponds to values of E(m) between about -105 and -50 mV) E(Cl) was within 1-3 mV of E(m) at equilibrium. These measurements of a(i) (Cl) were obtained from the potential difference between the chloride-sensitive electrode and an intracellular indifferent micro-electrode filled with potassium chloride. If a potassium sulphate-filled indifferent micro-electrode was used, then values of a(i) (Cl) below about 5 mM were erroneously high, probably due to interference from other sarcoplasmic ions at the indifferent electrode.4. In solutions containing 15 mM bicarbonate and gassed with 5% CO(2), pH(i) was 6.9, corresponding to an internal bicarbonate concentration of 7.6 mM. E(Cl) measured in this solution was some 4 mV positive to E(m). Most of the difference between E(Cl) and E(m) could be ascribed to interference by sarcoplasmic bicarbonate on the basis of selectivity measurements of chloride against bicarbonate made on the ion-exchange resin in the relevant range of a(Cl).5. If bicarbonate/CO(2) in the external solution was replaced by HEPES/pure O(2) at constant pH, then pH(i) rose from 6.88 +/- 0.02 (mean +/- S.E.) to 7.05 +/- 0.02. A change in external pH of 1 unit caused pH(i) to change by about 0.02 unit and the intracellular buffering power was calculated to be about 35.6. In solution made hypertonic by the addition of sucrose, E(m) changed little or depolarized and E(Cl) and E(m) remained close. In contrast, in solution made hypertonic by the addition of solid sodium chloride (high-chloride solution) E(Cl) became negative to E(m). Conversely in low chloride solution E(Cl) became positive to E(m).7. When the chloride permeability (P(Cl)) was reduced by the use of acid solution, E(Cl) moved positive to E(m) indicating an accumulation of internal chloride. When P(Cl) was increased again by returning to more alkaline solution, E(m) depolarized to E(Cl).8. The results are consistent with the existence of a small, active movement of chloride, the effects of which are normally obscured by large passive movements of chloride when P(Cl) is large.