The effect of angiotensin II on intracellular pH is mediated by AT(1) receptor translocation.
The effect of ANG II on intracellular pH (pH(i)) recovery rate and AT(1) receptor translocation was investigated in transfected MDCK cells. The pH(i) recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT(1)) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pH(i) recovery 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(-9) M) but not with ANG II (10(-6) M). Losartan (10(-7) M) and dimethyl-BAPTA-AM (10(-7) M) decreased significantly the stimulatory effect of ANG II (10(-9) M) and induced an increase in Na(+)/H(+) exchanger 1 (NHE-1) activity with ANG II (10(-6) M). Immunofluorescence studies indicated that in control situation, the hAT(1) receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10(-9) M) and internalized with ANG II (10(-6) M). Losartan (10(-7) M) induced hAT(1) translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10(-7) M) did not change the effect of ANG II (10(-9) M) on the hAT(1) receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10(-6) M). With ionomycin (10(-6) M), 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 AT(1) receptor and intracellular signaling events related to AT(1) translocation.
Thieme
Karina
K
Department of Physiology and Biophysics, Instituto de Ciências Biomédicas, University of São Paulo, São Paulo, Brazil.
Eguti
Débora Mai N
DM
Mello-Aires
Margarida
M
Oliveira-Souza
Maria
M
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000494:pharmacology / Q000502:physiology*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019808:Losartan / Q000494:pharmacology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D021381:Protein Transport; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D014162:Transfection
C460021:5,5'-dimethyl bis(o-aminophenoxy)ethane-N,N,N',N' tetraacetic acetoxymethyl ester; D047228:Angiotensin II Type 1 Receptor Blockers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D044140:Receptor, Angiotensin, Type 1; D017923:Sodium-Hydrogen Exchangers; D000804:Angiotensin II; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; D004533:Egtazic Acid; D019808:Losartan