H+/Ca2+ exchange in rabbit renal cortical endosomes

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Abstract
Summary We have examined the effect of second messengers on ATP-driven H+ transport in an H+ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H+ transport. Acridine orange fluorescence in the presence of 0.5mm Ca2+ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H+ transport by Ca2+ was concentration dependent; 0.25 and 0.5mm Ca2+ (+1mm EGTA) inhibited acridine orange fluorescence by ∼50 and ∼80%, respectively. Ca2+ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca2+ did not affect ATP hydrolysis. ATP-dependent Br− uptake was virtually unchanged in the presence of 0.5mm Ca2+ (+1mm EGTA). These vesicles were also shown to transport Ca2+ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca2+ uptake. These results are consistent with the co-existence of an H+ ATPase and an H+/Ca2+ exchanger on these endosomes, the latter transport system using the H+ gradient to energize Ca2+ uptake. Attempts to demonstrate an H+/Ca2+ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca2+ uptake could be demonstrated. A Ca2+-dependent increase in H+ permeability and an ATP-dependent Ca2+ uptake might have important implications for the regulation of vacuolar H+ ATPase activity as well as the homeostasis of cytosolic Ca2+ concentration.