Proliferation of vascular smooth muscle (VSM) involves gene expression dependent upon the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and the transcription factor cAMP response element-binding protein (CREB). Growth factors stimulate these pathways and can also increase intracellular Ca²⁺ ([Ca²⁺]_i) which may play an important regulatory role in this process. This study examines the Ca²⁺ dependency of platelet derived growth factor-BB (PDGF) and endothelin-1 (ET-1)-induced CREB and ERK1/2 activation in proliferating (Tasker et al. 1999), compared to fully differentiated VSM.

Portal veins (PV) from 2- to 4-day-old neonatal and 6-week-old Sprague-Dawley rats humanely killed by cervical dislocation were stimulated with PDGF (50 ng ml^-1) or ET-1 (50 nM). Following stimulation, homogenised tissues were subjected to SDS-PAGE and activation of ERK1/2 or CREB detected by immunoblotting with phospho-specific antibodies. PDGF- and ET-1-induced increases in [Ca²⁺]_i from dispersed PV cells loaded with Fura-2 were determined by fluorescence Ca²⁺ imaging.

Following a 15 min incubation, PDGF stimulated CREB phosphorylation (2 ± 0.3-fold increase compared to control; n = 5, P < 0.05, mean ± S.E.M., Student’s unpaired t test) and ERK1/2 phosphorylation (2.3 ± 0.5-fold; n = 4, P < 0.05) in the neonatal PV. PDGF did not produce a significant increase in CREB activation in developed PV but significantly increased ERK1/2 activation (2.4 ± 0.8-fold; n = 4, P < 0.05). BAPTA (which buffers [Ca²⁺]_i) and 2-APB (inhibits InsP₃-induced Ca²⁺ release) were used to examine the dependence of ERK_1/2 and PDGF activation on [Ca²⁺]_i. BAPTA (30 µM) and 2-APB (10 µM) completely inhibited ERK1/2 activation induced by PDGF in developed PV (n = 4; P < 0.05). However, in neonatal PV (n = 3), BAPTA and 2-APB did not reduce ERK1/2 or CREB activation. PDGF produced an increase in [Ca²⁺]_i in myocytes isolated from adult PV (n = 32, P < 0.01) but not from neonates. These experiments were repeated following stimulation with ET-1. In both neonatal and developed PV, ET-1 produced an activation of CREB and ERK1/2 that was significantly inhibited by BAPTA or 2-APB. Stimulation with ET-1 increased the [Ca²⁺]_i in isolated myocytes from both neonatal (n = 53) and developed rats (n = 19).

In conclusion, PDGF stimulation of these proliferative pathways is Ca²⁺ dependent in developed, but not in proliferating, PV. This is, at least partly, agonist specific. Although the intracellular mechanism of this altered Ca²⁺ dependency in proliferating VSM is unknown, it may be directly correlated with a concurrent agonist-induced increase in [Ca²⁺]_i.

This study was supported by the British Heart Foundation.