Growth factors mediate intracellular signaling in vascular smooth muscle cells through protein kinase C-linked pathways

Intracellular Ca2+ and pH are potent modulators of growth factor-induced mitogenesis and contraction. This study examined platelet-derived growth factor-(PDGF-BB) and insulin-like growth factor (IGF-1)-mediated signal transduction in primary cultured unpassaged vascular smooth muscle cells (VSMC) from mesenteric arteries of Sprague-Dawley rats. Intracellular free Ca2+ concentration ([Ca2+](i)) and intracellular pH (pH(i)) were measured by fluorescence digital imaging using fura-2 AM and 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescsin, respectively. Characteristics of [Ca2+](i) transients were determined by pre-exposing cells to Ca2+-free buffer, and involvement of the Na+/Ca2+ exchanger was assessed by withdrawal of extracellular Na+ and by exposure to dimethylbenzamil (Na+/Ca2+ exchange blocker). To determine whether pH(i) responses were mediated via the Na+/H+ exchanger, cells were preincubated with 10(-5) mol/L 5-(N-ethyl-N-isopropyl)amiloride (a selective Na+/H- exchange blocker). The role of protein kinase C (PKC) and tyrosine kinases in growth factor signaling was assessed by pre-exposing cells to calphostin C and chelerythrine chloride (selective PKC inhibitors; 10(-5) mol/L) and tyrphostin A23 (a selective tyrosine kinase inhibitor; 10(-5) mol/L). PDGF-BB and IGF-1 (1 to 10 ng/mL) increased [Ca2+](i) and pH(i) in a dose-dependent manner. At concentrations greater than 1 ng/mL both growth factors induced a biphasic [Ca2+](i) response with an initial transient peak followed by a sustained elevation. At 5 ng/mL PDGF-BB and IGF-1 significantly increased [Ca2+](i) from 95+/-3 nmol/L to 328+/-28 and 251+/-18 nmol/L, respectively. Ca2+ withdrawal abolished the second phase of [Ca2+](i) elevation. Agonist-induced [Ca2+](i) responses were similarly altered by Na+ withdrawal, by Na+/Ca2+ exchange blockade, and by PKC inhibition; latency, the period from stimulus application to the first [Ca2+](i) peak, was increased, the initial [Ca2+](i) peak was attenuated, and the sustained phase was prolonged. PDGF-BB and IGF-1 (10 ng/mL) significantly increased pH(i) from 6.89+/-0.04 nmol/L to 7.11+/-0.01 and 7.09+/-0.02 nmol/L, respectively. EIPA and calphostin C completely inhibited agonist-elicited alkalinization. Tyrphostin A-23 abolished second-messenger responses to PDGF-BB and IGF-1, whose receptors have tyrosine kinase activity. In conclusion, PDGF-BB and IGF-1 elicit significant [Ca2+](i) and pH(i) responses in VSMC. The underlying pathways that mediate these responses are partially dependent on Na+/Ca2+ transporters and the Na+/H+ exchanger, both of which are linked to PKC activation.