Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle

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Abstract
Ion-sensitive electrodes (made with a Cl--sensitive ion-exchange resin) were used to measure the internal Cl- activity (.alpha.iCl) of frog satorius muscle fibers at 25.degree. C. The internal pH (pHi) of other sartorius muscle fibers was measured with a recessed tip pH-sensitive electrode (made with pH-sensitive glass). In normal HCO3--free solution (containing 2.5 mM K), the average Cl- equilibrium potential, ECl (calculated from .alpha.iCl) was 87.7 .+-. 1.7 mV (mean .+-. SE; n = 16) in fibers where the average membrane potential, Em, was 88.3 .+-. 1.5 mV (mean .+-. SE; n = 16). In experiments where .alpha.iCl was varied between about 1-10 mM (which corresponds to values of Em between about -105 and -50 mV) ECl was within 1-3 mV of Em at equilibrium. These measurements of .alpha.iCl were obtained from the potential difference between the Cl--sensitive electrode and an intracellular indifferent micro-electrode filled with KCl. If a potassium sulfate-filled indifferent micro-electrode was used, then values of .alpha.iCl below .apprx. 5 mM were erroneously high, probably due to interference from other sarcoplasmic ions at the indifferent electrode. In solutions containing 15 mM HCO3- and gassed with 5% CO2, pHi was 6.9, corresponding to an internal HCO3- concentration of 7.6 mM. ECl measured in this solution was some 4 mV positive to Em. Most of the difference between ECl and Em could be ascribed to interference by sarcoplasmic bicarbonate on the basis of selectivity measurements of Cl- against HCO3- made on the ion-exchange resin in the relevant range of .alpha.Cl. If bicarbonate/CO2 in the external solution was replaced by HEPES[2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesfulonic acid]/pure O2 at constant pH, then pHi rose from 6.88 .+-. 0.02 (mean .+-. SE) to 7.05 .+-. 0.02. A change in external pH of 1 unit caused pHi to change by .apprx. 0.02 unit and the intracellular buffering power was .apprx. 35. In solution made hypertonic by the addition of sucrose, Em changed little or depolarized and ECl and Em remained close. In solution made hypertonic by the addition of solid NaCl (high-Cl- solution) ECl became negative to Em. In low Cl- solution ECl became positive to Em. When Cl- permeability (PCl) was reduced by the use of acid solution, ECl moved positive to Em indicating an accumulation of internal Cl-. When PCl was measured again by returning to more alkaline solution, Em depolarized to ECl. Results are consistent with the existence of a small, active movement of Cl-, the effects of which are normally obscured by large passive movements of Cl- when PCl is large.