Stimulation of Na+/H+ exchange induced by angiotensin II in cultured rat vascular smooth muscle cells: role of Ca2+ and C-kinase.
Changes in cytosolic calcium concentration ([Ca2+]i) have been implicated in the regulation of intracellular pH (pHi) in several cell types. In the present study we investigated the regulatory mechanism of Na+/H+ exchange induced by angiotensin II (AII) in cultured rat vascular smooth muscle cells (VSMCs). Serially passaged VSMCs from Sprague-Dawley rat thoracic aorta were grown on coverslips and loaded with the pH-sensitive fluorescent indicator 2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein (BCECF). In HCO(3-)-free Ringer solution, pH 7.40, the resting pHi was 7.21 +/- 0.02 (n = 21). A biphasic response was seen after exposure of these cells to AII: an initial transient a acidification, followed by sustained alkalization. The magnitude of alkalization was dose-dependent. AII-mediated acidification was completely inhibited by [Sar1-IIe5-Gly8]AII, but amiloride had no effect. In contrast, the alkalization induced by AII was abolished by both amiloride and Na(+)-free medium. In Ca(2+)-free medium, the AII-induced alkalization was partially blocked and verapamil also caused partial inhibition. Since AII activates phospholipase C in VSMCs, we examined whether AII would increase Na+/H+ exchange by activation of protein kinase C. An inhibitor of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), partially inhibited the alkalization induced by AII. These results indicate that AII stimulates cytoplasmic alkalization via an amiloride-sensitive Na+/H+ exchange system in cultured rat VSMCs, and that this AII-stimulated Na+/H+ exchange is mediated by Ca(2+)-dependent and protein kinase C-dependent mechanisms.
Ohara
T
T
Department of Nephrology, Jichi Medical School, Tochigi, Japan.
Takeda
K
K
Asano
Y
Y
D000584:Amiloride / Q000494:pharmacology; D000704:Analysis of Variance; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D005452:Fluoresceins; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000378:metabolism*
D005452:Fluoresceins; D000804:Angiotensin II; D000584:Amiloride; D006859:Hydrogen; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D011493:Protein Kinase C; D002118:Calcium