STIMULATION OF Na⁺/H⁺ EXCHANGE INDUCED BY ANGIOTENSIN II IN CULTURED RAT VASCULAR SMOOTH MUSCLE CELLS : ROLE OF Ca²⁺ AND C-KINASE

Abstract

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 HCO₃-free Ringer solution, pH 7.40, the resting pHi was 7.21±0.01 (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 Ca²⁺-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 Ca²⁺-dependent and protein kinase C-dependent mechanisms.