(Hypertension. 1997;30:1440-1447.)
© 1997 American Heart Association, Inc.
Articles |
Growth Factors Mediate Intracellular Signaling in Vascular Smooth Muscle Cells Through Protein Kinase C–Linked Pathways
From the Experimental Hypertension Laboratory, MRC Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal and Université de Montréal, Montreal, Quebec, Canada.
Correspondence to Rhian M. Touyz, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal (Quebec) Canada H2W 1R7. E-mail touyz{at}ircm.umontreal.ca
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)-56-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.
Key Words: growth factors • calcium • pH, intracellular • calphostin C • chelerythrine chloride • cultured cells
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