Coupling between H+ transport and anaerobic glycolysis in turtle urinary bladder: Effects of inhibitors of H+ ATPase

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Abstract
Summary The coupling between H+ transport (J H) and anaerobic glycolysis was examinedin vitro in an anaerobic preparation of turtle urinary bladder.J H was measured as the short-circuit current after Na+ transport was abolished with ouabain and by pH stat titration. The media were gassed with N2 and 1% CO2 (PO2J H was not inhibited by 3mm serosal (S) cyanide or by 0.1mm mucosal (M) dinitrophenol. Control anerobic lactate production (J lac) of 47 bladders was plotted as a function of simultaneously measuredJ H. The slope ofJ lac onJ H was 0.58±0.12 with an intercept forJ lac atJ H=0 of 0.55 μmol/hr. Values for δJ lac/δJ H were determined in groups of individual bladders whenJ H was inhibited by an opposing pH gradient (0.55±0.16), by acetazolamide (0.58±0.19) and by dicyclohexylcarbodiimide, DCCD (0.58±0.14). The constancy of δJ lac/δJ H indicates a high degree of coupling betweenJ H andJ lac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the δJ lac/δJ H values can be used to estimate the stoichiometry of H+ translocation. The movement of slightly less than 2 H+ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited byM addition of oligomycin but was inhibited byM addition of DCCD and Dio-9, inhibitors of H+ flow in the proteolipid portion of H+-translocating ATPases. DCCD inhibited anaerobicJ H without change in δJ lac/δJ H or basalJ lac and, therefore, acted primarily on the H+ pump.S addition of vanadate also inhibitedJ H, but the inhibition was associated with an increase inJ lac. The site of this apparent uncoupling remains to be defined. The acidification pump of the luminal cell membrane of the turtle bladder has H+-ATPase characteristics that differ from mitochondrial ATPase in that H+ transport is oligomycin-resistant and vanadate-sensitive. As judged from the flows of H+ and lactate, the H+/ATP stoichiometry of the pump is about 2.