Role of L-type calcium channel blocking in epidermal growth factor receptor-independent activation of extracellular signal regulated kinase 1/2

OBJECTIVE: Vascular smooth muscle cell (VSMC) differentiation, growth and survival, key events in the development of cardiovascular diseases, are under the control of signaling enzymes including extracellular signal regulated kinase 1/2 (ERK 1/2), Akt and epidermal growth factor receptor (EGFR) activation. EGFR trans-activation is known to mediate thrombin- or angiotensin II (AII)-stimulated ERK 1/2 activation. However, our laboratory has demonstrated, in thrombin-stimulated VSMC, that the prevention of intracellular Ca2+ elevation ([Ca2+]i) by BAPTA-AM pretreatment unveiled EGFR-independent ERK 1/2 activation. Since calcium channel blockers (CCBs) also impair agonist-induced [Ca2+]i elevation, we investigated whether EGFR-independent ERK 1/2 activation could occur in VSMCs treated by CCBs such as amlodipine, isradipine and verapamil, and examined the possible role of Akt. METHODS: Cultured VSMCs were pretreated or not with CCBs and with various inhibitors of the signaling pathways under study, prior to stimulation by thrombin or AII, and the phosphorylation/activation status of EGFR, Akt and ERK 1/2 was determined by Western blotting using phospho-specific antibodies. RESULTS AND CONCLUSION: Unlike BAPTA, CCBs did not impair stimulus-induced EGFR trans-activation, hence ERK1/2 phosphorylation. However, when EGFR kinase was inhibited, CCBs and BAPTA dose-dependently protected stimulus-induced ERK1/2 phosphorylation. The effect of amlodipine could not be mimicked by its R+ enantiomer, which is devoid of CCB activity, suggesting that the effects of CCBs were accounted for by their L-type Ca2+ channel-blocking property. Altogether, our results indicated that in G-protein-coupled receptor (GPCR)-stimulated VSMCs, the prevention of [Ca2+]i elevation by CCBs unmasked an EGFR kinase-independent phosphorylation of ERK 1/2. Since EGFR kinase inhibitors are supposed to be efficient in the treatment of some cancers, such a mechanism might be clinically relevant in hypertensive patients with cancer.