The effect of angiotensin II on intracellular pH is mediated by AT1receptor translocation

Abstract

The effect of ANG II on intracellular pH (pHi) recovery rate and AT1receptor translocation was investigated in transfected MDCK cells. The pHirecovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT1) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pHirecovery rate of 0.219 ± 0.017 pH U/min ( n = 11). This value was similar to nontransfected cells [0.211 ± 0.009 pH U/min ( n = 12)]. Both values were significantly increased with ANG II (10−9M) but not with ANG II (10−6M). Losartan (10−7M) and dimethyl-BAPTA-AM (10−7M) decreased significantly the stimulatory effect of ANG II (10−9M) and induced an increase in Na+/H+exchanger 1 (NHE-1) activity with ANG II (10−6M). Immunofluorescence studies indicated that in control situation, the hAT1receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10−9M) and internalized with ANG II (10−6M). Losartan (10−7M) induced hAT1translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10−7M) did not change the effect of ANG II (10−9M) on the hAT1receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10−6M). With ionomycin (10−6M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT1receptor and intracellular signaling events related to AT1translocation.