Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

Pretreatment of the cells with the Ca2+ chelators BAPTA-AM or EGTA, as well as the Ca2+ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF2α.The present data indicate that Ca2+ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.The extracellular signal regulated kinases ERK1 (p44mapk) and ERK2 (p42mapk) are activated in response to stimulation of receptor tyrosine kinases (RTKs) as well as heptahelical G protein coupled receptors (GPCR) and transmit signals which regulate cell differentiation and growth [1-3]. The molecular steps involved in signaling from GPCRs to ERK are incompletely understood. Data obtained in various cell systems have provided evidence in support of several signaling pathways including protein kinase C (PKC) [4], Ca2+-mediated mechanisms [5-12], and transactivation of receptor tyrosine kinases [13,14]. In hepatocytes several hormones, including vasopressin, angiotensin II, norepinephrine, and PGF2α, that bind to GPCRs activate ERK [15-17]. The mechanisms mediating the ERK activation by GPCR agonists are not clarified; there is evidence that protein kinase C is involved [15,18], but a role for Ca2+ also appears likely, since all the agents above activate phospholipase C and elevate intracellular Ca2+ in hepatocytes [19,20]. Furthermore, agents that elevate intracellular Ca2+ through mechanisms bypassing receptors have been found to activate ERK [15,21]. However, agonist-stimulated phospholipase C activity is rapidly down-reg