Interaction of angiotensin II and atrial natriuretic peptide on pHi regulation in MDCK cells

Abstract

The effect of ANG II and atrial natriuretic peptide (ANP) on intracellular pH (pHi) and cytosolic free calcium concentration ([Ca2+]i) was investigated in Madin-Darby canine kidney cells by using the fluorescent probes 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (AM) and fura 2-AM or fluo 4-AM. pHi recovery rate was examined in the first 2 min after the acidification of pHi with a NH4Cl pulse. In the control situation, the pHi recovery rate was 0.088 ± 0.014 pH units/min ( n = 14); in the absence of external Na+, this value was decreased. ANG II (10−12or 10−9 M) caused an increase in this value, but ANG II (10−7 M) decreased it. ANP (10−6 M) or dimethyl-1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA)-AM (50 μM) alone did not affect this value but impaired both stimulatory and inhibitory effects of ANG II. ANG II (10−12, 10−9, or 10−7 M) increased [Ca2+]i progressively from 99 ± 10 ( n = 20) to 234 ± 7 mM ( n = 10). ANP or dimethyl-BAPTA-AM decreases [Ca2+]i, and the subsequent addition of ANG II caused a recovery of [Ca2+]i but without reaching ANG II values found in the absence of these agents. The results indicate a role for [Ca2+]i in regulating the process of pHi recovery mediated by the Na+/H+ exchanger, stimulated/impaired by ANG II, and not affected by ANP or ANG II plus ANP. This hormonal interaction may represent physiologically relevant regulation in conditions of volume alterations in the intact animal.