Regulation of chloride in quiescent sheep‐heart Purkinje fibres studied using intracellular chloride and pH‐sensitive micro‐electrodes.

Abstract

The intracellular Cl activity, .**GRAPHIC**. was measured inside quiescent sheep cardiac Purkinje fibers bathed in normal Tyrode at pH 7.40 and buffered with .apprxeq. 22 mM bicarbonate/.apprxeq. 5% CO2 + 95% O2. The measurements were made using liquid ion-exchanger Cl--sensitive micro-electrodes. After internal Cl levels were depleted by prolonged exposure to Cl-free media (glucuronate-substituted) when external Cl was restored there was a rapid re-accumulation of Cl inside the fibers to levels that were much higher than those expected for a passive Cl distribution. Such a process can be conveniently defined as an active inward Cl pump. The inward-pumping was noticeably temperature-sensitive (Q10 .apprxeq. 2.6); its rate was reduced about 18-fold in the nominal absence of external bicarbonate/CO2 and it was substantially inhibited by the drug SITS (4-acetamido-4''-isothiocyanatostilbene-2,2''-disulfonic acid). The fall of .**GRAPHIC**. in Cl-free solution was slow and was also equally temperature-sensitive and substantially inhibited by SITS but was only slightly impaired in the nominal absence of external bicarbonate/CO2. pHi [intracellular] was measured using recessed-tip pH-sensitive micro-electrodes and in some experiments pHi and .**GRAPHIC**. were monitored simultaneously. When .**GRAPHIC**. slowly declined in Cl-free solution, pHi slowly became alkaline. Upon restoring external Cl there was the usual rapid recovery of a high .**GRAPHIC**. this was accompanied by a rapid reacidification of pHi. The recovery of .**GRAPHIC**. and pHi were exponential with virtually the same time constant. The slow alkalinization of pHi in Cl-free solution and the rapid reacidification upon restoring external Cl were substantially inhibited by the drug SITS. When [K]o was raised to 45 mM or more (by removing equivalent amounts of [Na]o), there was a large depolarization of Em [resting membrane potential] and a slow rise of .**GRAPHIC**. which was not accompanied by a large change of pHi. The rise of .**GRAPHIC**. appeared to be unaffected by SITS. A Cl/HCO3- exchange mechanism can operate reversibly across the membrane of quiescent Purkinje fibers: it can partly account for the high levels of .**GRAPHIC**. measured in the resting state. Cl can cross the membrane in other ways, especially in high K solution, possibly by moving passively conductance channels that are open under these conditions.