GROWTH-FACTORS MEDIATE INTRACELLULAR SIGNALING IN VASCULAR SMOOTH-MUSCLE CELLS THROUGH PROTEIN-KINASE C-LINKED PATHWAYS

Citation
Rm. Touyz et El. Schiffrin, GROWTH-FACTORS MEDIATE INTRACELLULAR SIGNALING IN VASCULAR SMOOTH-MUSCLE CELLS THROUGH PROTEIN-KINASE C-LINKED PATHWAYS, Hypertension, 30(6), 1997, pp. 1440-1447
Citations number
37
Language
INGLESE
art.tipo
Article
Journal title
ISSN journal
0194-911X
Volume
30
Issue
6
Year of publication
1997
Pages
1440 - 1447
Database
ISI
SICI code
0194-911X(1997)30:6<1440:GMISIV>2.0.ZU;2-J
Abstract
Intracellular Ca2+ and pH are potent modulators of growth factor-induc ed mitogenesis and contraction. This study examined platelet-derived g rowth factor-(PDGF-BB) and insulin-like growth factor (IGF-1)-mediated signal transduction in primary cultured unpassaged vascular smooth mu scle cells (VSMC) from mesenteric arteries of Sprague-Dawley rats. Int racellular free Ca2+ concentration ([Ca2+](i)) and intracellular pH (p H(i)) were measured by fluorescence digital imaging using fura-2 AM an d 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescsin, respectively. Char acteristics of [Ca2+](i) transients were determined by pre-exposing ce lls to Ca2+-free buffer, and involvement of the Na+/Ca2+ exchanger was assessed by withdrawal of extracellular Na+ and by exposure to dimeth ylbenzamil (Na+/Ca2+ exchange blocker). To determine whether pH(i) res ponses 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 k inases in growth factor signaling was assessed by pre-exposing cells t o calphostin C and chelerythrine chloride (selective PKC inhibitors; 1 0(-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 nmo l/L to 328+/-28 and 251+/-18 nmol/L, respectively. Ca2+ withdrawal abo lished the second phase of [Ca2+](i) elevation. Agonist-induced [Ca2+] (i) responses were similarly altered by Na+ withdrawal, by Na+/Ca2+ ex change blockade, and by PKC inhibition; latency, the period from stimu lus application to the first [Ca2+](i) peak, was increased, the initia l [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. EI PA and calphostin C completely inhibited agonist-elicited alkalinizati on. Tyrphostin A-23 abolished second-messenger responses to PDGF-BB an d 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 partia lly dependent on Na+/Ca2+ transporters and the Na+/H+ exchanger, both of which are linked to PKC activation.