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

Touyz, R.M. and Schiffrin, E.L. (1997) Growth factors mediate intracellular signaling in vascular smooth muscle cells through protein kinase C-linked pathways. Hypertension, 30(6), pp. 1440-1447. (doi: 10.1161/01.HYP.30.6.1440)

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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.

Item Type:Articles
Glasgow Author(s) Enlighten ID:Touyz, Professor Rhian
Authors: Touyz, R.M., and Schiffrin, E.L.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
Journal Name:Hypertension
Publisher:American Heart Association
ISSN (Online):1524-4563

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