Oxidant-induced pHi/Ca2+changes in rat aortic smooth muscle cells: The role of atrial natriuretic peptide

The aim of this study was to investigate the effects of oxidative stress on PLD activity, [Ca2+]iand pHi levels and the possible relationship among them. Moreover, since atrial natriuretic peptide (ANP) protects against oxidant-induced injury, we investigated the potential protective role of the hormone in Rat Aortic Smooth Muscle (RASM) cells exposed to oxidative stress. Water-soluble 2,2′-Azobis (2hyphen;amidinopropane) dihydrochloride (AAPH) was used as free radical generating system, since it generates peroxyl radicals with defined reaction and the half time of peroxyl radicals is longer than other ROS. A significant increase of PLD activity was related to a significant decrease in pHi, while [Ca2+]ilevels showed an increase followed by a decrease after cell exposure to AAPH. [Ca2+]ichanges and pHi fall induced by AAPH were prevented by cadmium which inhibits a plasma membrane Ca2+ATPase coupled to Ca2+/H+exchanger, that operates the efflux of Ca2+coupled to H+influx. The involvement of PLD in pHi and [Ca2+]ichanges was confirmed by calphostin-c treatment, a potent inhibitor of PLD, which abolished all AAPH-induced effects. Pretreatment of RASM cells with pharmacological concentrations of ANP attenuated the AAPH effects on PLD activity as well as [Ca2+]iand pHi changes, while no effects were observed with physiological ANP concentrations, suggesting a possible role of the hormone as defensive effector against early events of the oxidative stress.