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

Abstract

The coupling between H+ transport (JH) and anaerobic glycolysis was examined in vitro in an anaerobic preparation of turtle urinary bladder. JH 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 (PO2 less than 0.5 mm Hg) and contained 10 mM glucose. Under these conditions, JH was not inhibited by 3 mM serosal (S) cyanide or by 0.1 mM mucosal (M) dinitrophenol. Control anaerobic lactate production (Jlac) of 47 bladders was plotted as a function of simultaneously measured JH. The slope of Jlac on JH was 0.58 0.12 with an intercept for Jlac at JH = 0 of 0.55 micromol/hr. Values for delta Jlac/delta JH were determined in groups of individual bladders when JH 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 delto Jlac/ delta JH indicates a high degree of coupling between JH and Jlac. Since the anaerobic metabolism of glucose produces one ATP for each lactate formed, the delta Jlac/ delta JH values can be used to estimate the stoichiometry of H+ translocation. The movement of slightly less than 2H+ ions is coupled to the hydrolysis of one ATP. During anaerobiosis (absence of mitochondrial ATPase function) the acidification pump was not inhibited by M addition of oligomycin but was inhibited by M addition of DCCD and Dio-9, inhibitors of H+ flow in the proteolipid portion of H+-translocating ATPases. DCCD inhibited anaerobic JH without change in delta Jlac/delta JH or basal Jlac and, therefore, acted primarily on the H+ pump. S addition of vanadate also inhibited JH, but the inhibition was associated with an increase in Jlac. 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.