title: Growth factors mediate intracellular signaling in vascular smooth muscle cells through protein kinase C-linked pathways.
abstract: 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 (pHi) were measured by fluorescence digital imaging using fura-2 AM and 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, 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 pHi 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 pHi 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 pHi 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 pHi 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.
authors.affiliation: Experimental Hypertension Laboratory, MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal and Université de Montréal, Quebec, Canada. touyz@ircm.umontreal.ca
authors.forename: R M
authors.initials: RM
authors.lastname: Touyz
authors.forename: E L
authors.initials: EL
authors.lastname: Schiffrin
mesh_terms: D000470:Alkaloids; D000818:Animals; D000077214:Becaplermin; D053119:Benzophenanthridines; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D006863:Hydrogen-Ion Concentration; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008638:Mesenteric Arteries / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D009281:Naphthalenes / Q000494:pharmacology; D010617:Phenanthridines / Q000494:pharmacology; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D012964:Sodium / Q000494:pharmacology
chemical_list: D000470:Alkaloids; D053119:Benzophenanthridines; D009281:Naphthalenes; D010617:Phenanthridines; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D000077214:Becaplermin; D007334:Insulin-Like Growth Factor I; D012964:Sodium; C016299:chelerythrine; D011493:Protein Kinase C; C058819:calphostin C; D002118:Calcium