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
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.