H+-Dependent ATPase and K+ channel activities in the rat thyroid cell strain FRTL-5

Using the fluorescent indicators 2',7'-bis(2-carboxyethyl)-5'-(6')-carboxyfluorescein and Oxonol V to monitor intracellular pH (pH(i)) and cell membrane potential respectively, we have investigated the involvement of H+-dependent ATPase and H+-dependent K+ channels in the recovery of the rat thyroid cell strain FRTL-5 from experimentally induced cytosolic acidification and membrane hyperpolarization events. Following exposure of cells to the weak acid sodium butyrate (24 mmol/l) under bicarbonate-free incubation conditions, cytoplasmic acidification was maximal after 3 min, attaining a pH(i) of 6.42. The subsequent recovery of pH(i) was unimpaired by the absence of extracellular K+, but was reduced in the presence of the Na+ antagonist amiloride (1 mmol/l), recovering by 0.11 ± 0.003 units, compared with 0.27 ± 0.02 units under amiloride-free conditions. In the presence of the H+-dependent ATPase antagonist N,N'-dicyclohexylcarbodiimide (DCC), the pH(i) recovery observed in amiloride-containing, K+-free buffer was abolished. The recovery of pH(i) in Na+- and K+-containing buffer was accompanied by hyperpolarization of the cell membrane, the latter stage of which was reduced after blockade of K+ channels with BaCl2, implying a major contribution of transmembrane K+ movement to such events. In contrast to its attenuating effect on pH(i) recovery, DCC was ineffective in reducing butyrate-dependent membrane hyperpolarization, suggesting that H+-dependent ATPase may not be a major contributory factor to this event. However, when K+ channels were blocked by addition of BaCl2, addition of DCC abolished the butyrate-induced membrane depolarization. These findings are consistent with the presence of two independent hyperpolarizing transport processes in the FRTL-5 cell membrane which appear to involve (i) a H+-dependent ATPase, activated in response to cytosolic acidification, and allowing partial recovery of pH(i) in the absence of extracellular Na+ and HCO3-, and (ii) H+-dependent K+ channels which, while contributing to membrane hyperpolarization, may not play a major role in the normal maintenance of pH(i).