No role for Ca++ or protein kinase C in alpha-1A adrenergic receptor activation of mitogen-activated protein kinase pathways in transfected PC12 cells

We studied the role of Ca++ and protein kinase C (PKC) in alpha-1A adrenerg ic receptor (AR)-mediated activation of mitogen-activated protein kinase pa thways in PC12 cells. In PC12 cells stably transfected with the human alpha -1A AR, norepinephrine (NE) strongly activated both extracellular signal re gulated kinases (ERKs) and c-jun-NH2-terminal kinases (JNK). Ten nanomolar thapsigargin (TG) increased cytoplasmic Ca++ at least as much as NE but did not activate ERKs or JNK. Higher concentrations of TG caused a small activ ation of ERKs but not JNK. Emptying [Ca++](i) stores by pretreatment with T G prevented the NE-stimulated increase in [Ca++](i) but not ERK or JNK acti vation. The Ca++ chelator bis(2-aminophenoxy)ethane-N-N-N'-N'-tetraacetate (BAPTA) dose dependently abolished NE-stimulated Ca++ responses but not ERK or JNK activation. NE increased tyrosine phosphorylation of Pyk2, and this response was neither blocked by BAPTA nor mimicked by TG. The phorbol este r tumor promoting agent (TPA) caused a dose-dependent activation of ERKs th at was potentiated by 10 nM TG. TPA caused only a small activation of JNK r elative to that caused by NE, which was not affected by TG. The potent PKC inhibitor bisindolylmaleimide I dose dependently inhibited ERK and JNK acti vation by TPA, but not NE. ATP and UTP activated similar mitogen-activated protein kinase responses through endogenous P2Y2 receptors, and these respo nses were not blocked by BAPTA or bisindolylmaleimide I, suggesting that th ese results may be generalizable to other G(q/11)-coupled receptors, The re sults suggest that Ca++ release and PKC activation are neither necessary no r sufficient for alpha-1A AR-mediated activation of mitogenic responses in PC12 cells.