This study examines the involvement of GTP-binding proteins (Gps) in the regulation of Na+/H+ exchange and Ca2+ influx, which are increased in vascular smooth muscle cells from spontaneously hypertensive rats. Gp activity was modulated by fluoride, GTPgammaS, GDPbetaS, and antisense oligodeoxynucleotides complementary to conserved regions of the alpha- and beta-subunits of Gps (alpha-comm and beta-comm, respectively). Beta-adrenergic-induced Gs-mediated cAMP production was used as a positive control to estimate the efficiency of these compounds. Na+/H+ exchange, measured as ethylisopropyl amiloride-sensitive 22Na influx, was activated by 5- to 6-fold by a 30-minute preincubation of cells with 10 mmol/L NaF with a K0.5 for NaF of approximately 13 mmol/L. In contrast, no activation of 45Ca influx was observed under preincubation of vascular smooth muscle cells with NaF in Ca2+-free medium, whereas at [Ca2+]o >0.5 mmol/L, simultaneous addition of 45Ca and 10 mmol/L NaF led to sharply increased isotope uptake. NaF-induced 45Ca influx did not reach saturation up to 3 mmol/L [Ca2+]o and 20 mmol/L NaF and was correlated with the formation of calcium-fluoride complexes measured by light scattering. GTPgammaS increased basal cAMP production and Na+/H+ exchange, whereas GDPbetaS decreased isoproterenol-induced cAMP production and Na+/H+ exchange. Alpha-comm reduced whereas beta-comm augmented isoproterenol-induced cAMP production by 70%. Both oligodeoxynucleotides decreased basal Na+/H+ exchange by 40% to 50%. NaF-induced Na+/H+ exchange was not sensitive to alpha-comm but was inhibited by 60% in beta-comm-loaded cells. Neither basal nor NaF-induced 45Ca uptake was affected by GTPgammaS, GDPbetaS, and the oligodeoxynucleotides. Our results show that 45Ca uptake is activated by NaF in vascular smooth muscle cells by nonspecific accumulation of calcium-fluoride complexes and is not related to modification of Gps. On the contrary, the Na+/H+ exchanger is controlled by Gps, and Gp beta-subunits are involved in [Ca2+]o-independent activation of this carrier by NaF.