Intracellular chloride and the mechanism for its accumulation in rat lumbrical muscle.

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Abstract
1. Double-barrelled Cl--sensitive microelectrodes have been used to measure the intracellular Cl- activity (aCli) and membrane potential (Em) in rat lumbrical muscles. The mean Cl- equilibrium potential (ECl), calculated from the measured aCli in sixty fibres, was 2.9 .+-. 2.5 mV (S.D. of an observation) less negative than Em. The value of aCli was higher than would be expected for a passive distribution, by a mean 1.4 .+-. 1.2 mM. The mean Em was -59.5 .+-. 8.2 mV. 2. Removal of external Cl- (Clo-) resulted in a rapid fall in aCli and a transient depolarization. aCli stabilized at an apparent level of 1.7 .+-. 1.0 mM (n = 24) while Em became substantially more negative than in normal Krebs solution (mean, -80.1 .+-. 12.4 mV). Readdition of Clo- caused a rapid rise in aCli and transient hyperpolarization. ECl quickly became less negative than Em and both then fell in parallel towards the levels previously recorded in normal Krebs solution. 3. If lack of selectivity of the Cl--sensitive ion exchanger and the intracellular presence of interfering anions, assumed to be responsible for the apparent aCli recorded in Cl--depleted fibres, were also responsible for the apparently non-passive Cl- distribution recorded under normal conditions, the difference between the calculated ECl and Em would increase at more negative potentials. This was not observed over a range of Em values between -46 and -84 mV. 4. Inhibition of the Cl- permeability by application of 9-anthracene carboxylic acid (9-AC) resulted in an immediate rise in aCli and hyperpolarization. An aCli up to 40 mM higher, or eleven times higher, than that predicted by a passive distribution was recorded. Application of 9-AC after depletion of intracellular Cl- in Cl--free solution had no effect on either the apparent aCli or Em. 5. It is concluded that Cl- ions are actively accumulated by the skeletal muscle fibre and that the Cl- distribution therefore normally exerts a depolarizing influence. 6. In the presence of 9-AC and nominal absence of CO2 and HCO3-, readdition of Clo- to Cl--depleted fibres resulted in a substantial rise in aCli and a small, maintained depolarization. This clear demonstration of active accumulation was used to investigate the mechanism responsible for inward transport of Cl- ions. 7. Neither application of CO2 and HCO3- nor application of DIDS (4,4''-diisothiocyanostilbene-2,2''-disulphonic acid) had any effect on the accumulation of Cl- ions. This suggests that Cl--HCO3- exchange is not involved. 8. Removal of Nao+ or Ko+ resulted in a limited rise in aCli such that ECl approximated to Em. Removal of both cations had no greater effect than the absence of only one. Readdition of the cations caused an immediate accumulation of Cl- ions and divergence of ECl from Em. This was largely inhibited by the presence of frusemide. These results suggest that a Na+, K+, Cl- co-transport mechanism is responsible for the inward transport of Cl-.