Abstract
This study investigated fluctuations of cytosolic pH (pHi) of cultured rat vascular smooth muscle cells (VSMCs) in reaction to metabolic alterations induced by angiotensin II (AII). Serially passed VSMCs from Wistar rat aortae were grown on coverslips and loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. A biphasic reaction was seen after exposure of these cells to AII (1 nM to 1 microM); an initial and relatively brief phase of acidification was followed by sustained alkalinization. The rate of acidification and magnitude of alkalinization were dose-dependent. This biphasic effect of AII was also demonstrated in Ca2+-free medium and was mimicked by subjecting VSMCs to the calcium ionophore A23187 (5 microM) in Ca2+-containing medium but not in Ca2+-free medium. Verapamil (10 microM) almost entirely eliminated the AII-induced acidification, whereas amiloride analogues 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride (100 microM) as well as Na+-deficient medium abolished the subsequent (alkalinization) phase produced by the hormone. Activation of the Na+/H+ antiport by subjecting VSMCs to phorbol 12-myristate 13-acetate (100 nM) prevented a subsequent effect of AII on the pHi profile. This resistance to a further action of the hormone was not mediated via cytoplasmic alkalinization. AII produced a dramatic redistribution in the cellular compartments of 45Ca2+ associated with accelerated 45Ca2+ washout. These findings suggest that the AII-induced acidification phase may relate to activation of the Ca2+ pump (Ca2+/H+ exchange) and that this process can take place in the presence and absence of extracellular Ca2+. The alkalinization phase is the consequence of stimulation of the Na+/H+ antiport, which in cultured VSMCs can be activated by a rise in cytosolic free Ca2+ as well as other mechanisms.
Highlights
This study investigated fluctuationsof cytosolic pH A coherent accountof the mechanismby which angiotensin I1 (AII)’ exerts of cultured r a t vascular smooth muscle cells its action on the vascular smooth muscle cell (VSMC) has (VSMCs) in reaction to metabolic alterations induced started to emerge only within the last several years
AI1 pro- whereas activation of the Na+/H+ antiport was studied by duced a dramatic redistribution in the cellular com- changes in theamiloride-sensitive Na’ transport (11).In the partments of 4SCa2+ associated with accelerat4eSdCa2+ present paper we describe the use of continuous monitoring washout. These findings suggest that the AII-induced of cytosolic pH(pH,), which gives a different perspective acidification phase may relate to activatoiof nthe Ca2+ concerning the actionof AI1 on the VSMC
We suggest that pump (Ca2+/H+ exchangaen)d that thpirsocess cantake the prompt and brief risein cytosolic free Ca2+associated place in the presence and absoefnecxetracellular Ca2+. with the effect of the hormone on the VSMC activates the
Summary
This study investigated fluctuationsof cytosolic pH A coherent accountof the mechanismby which AII’ exerts (pHi) of cultured r a t vascular smooth muscle cells its action on the vascular smooth muscle cell (VSMC) has (VSMCs) in reaction to metabolic alterations induced started to emerge only within the last several years. To examine whether AII-induced acidification relates to a change in the cytosolic buffer capacity, cells were subjected to (1.6 or 5 mM)NH: or (5 or 10 mM) propionate- under basal condition and at 30, 60, and 90 s after the introduction of 100 nM AI1 into the medium.
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