ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H+-ATPase subcellular vesicle trafficking

The effect of angiotensin II (ANG II) or arginine vasopressin (AVP) alone or plus atrial natriuretic peptide (ANP) on H-ATPase subcellular vesicle trafficking was investigated in MDCK cells following intracellular pH (pHi) acidification by exposure to 20 mM NH4Cl for 2 min in a Na-free solution containing Schering 28080, conditions under which H-ATPase is the only cell mechanism for pHi recovery. Using the acridine orange fluorescent probe (5 M) and confocal microscopy, the vesicle movement was quantified by determining, for each experimental group, the mean slope of the line indicating the changes in apical/basolateral fluorescence density ratio over time during the first 5.30 min of the pHi recovery period. Under the control conditions, the mean slope was 0.079  0.0033 min (14) and it increased significantly with ANG II [10 and 10 M, respectively to 0.322  0.038 min (13) and 0.578  0.061 min (12)] or AVP [10 and 10 M, respectively to 0.301  0.018 min (12) and 0.687  0.049 min (11)]. However, in presence of ANP (10 M, decreases cytosolic free calcium), dimethyl-BAPTA/AM (5 × 10 M, chelates intracellular calcium) or colchicine (10 M, 2-h preincubation; inhibits microtubule-dependent vesicular trafficking) alone or plus ANG II or AVP the mean slopes were similar to the control values, indicating that such agents blocked the stimulatory effect of ANG II or AVP on vesicle trafficking. The results suggest that the pathway responsible for the increase in cytosolic free calcium and the microtu-bule-dependent vesicular trafficking are involved in this hormonal stimulating effect. Whether cytosolic free calcium reduction represents an important direct mechanism for ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H-ATPase subcellular vesicle trafficking, or is a side effect of other signaling pathways which will require additional studies.