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Coupling of the three known ␣ 1 -adrenergic receptor (␣ 1 -AR) subtypes to mitogen-activated protein kinase (MAPK) pathways were studied in stably transfected PC12 cells. Subclones stably expressing ␣ 1A -, ␣ 1B -, and ␣ 1D -ARs under control of an inducible promoter, or at high and low receptor density, were isolated and characterized. Radioligand binding showed similar ranges of expression of each subtype. Norepinephrine (NE) increased inositol phosphate formation and intracellular Ca 2ϩ level in these cells in a manner dependent on receptor density. However, ␣ 1A -ARs activated these second messenger responses more effectively than ␣ 1B -ARs, whereas ␣ 1D -ARs were least effective. NE stimulated activation of extracellular signal-regulated kinases (ERKs) in cells expressing all three ␣ 1 -AR subtypes, although ␣ 1A -and ␣ 1B -ARs caused larger ERK activation than did ␣ 1D -ARs. Nerve growth factor (NGF) caused similar levels of ERK activation in all subclones. NE also activated p38 MAPK in ␣ 1A -and ␣ 1B -but not ␣ 1D -transfected cells and activated c-Jun NH 2 -terminal kinase (JNK) only in ␣ 1A -transfected cells. NE, but not NGF, strongly stimulated tyrosine phosphorylation of a 70-kDa protein only in ␣ 1A -transfected PC12 cells. NE caused neurite outgrowth only in ␣ 1A -expressing PC12 cells, but not in ␣ 1B -or ␣ 1D -transfected cells, whereas NGF caused neurite outgrowth in all cells. These studies show that ␣ 1A -ARs activate all three MAPK pathways, ␣ 1B -ARs activate ERKs and p38 but not JNKs, and ␣ 1D -ARs only activate ERKs. Only the ␣ 1A -AR-expressing cells differentiated in response to NE. The relationship of these responses to second messenger pathways activated by these subtypes is discussed. Key Words: Norepinephrine -PC12 cells-Mitogen-activated protein kinaseAdrenergic receptor.

We studied the role of Ca++ and protein kinase C (PKC) in alpha-1A adrenergic receptor (AR)-mediated activation of mitogen-activated protein kinase pathways in PC12 cells. In PC12 cells stably transfected with the human alpha-1A AR, norepinephrine (NE) strongly activated both extracellular signal regulated 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 activation of ERKs but not JNK. Emptying [Ca++]i stores by pretreatment with TG prevented the NE-stimulated increase in [Ca++]i but not ERK or JNK activation. 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 ester tumor promoting agent (TPA) caused a dose-dependent activation of ERKs that was potentiated by 10 nM TG. TPA caused only a small activation of JNK relative to that caused by NE, which was not affected by TG. The potent PKC inhibitor bisindolylmaleimide I dose dependently inhibited ERK and JNK activation by TPA, but not NE. ATP and UTP activated similar mitogen-activated protein kinase responses through endogenous P2Y2 receptors, and these responses were not blocked by BAPTA or bisindolylmaleimide I, suggesting that these results may be generalizable to other Gq/11-coupled receptors. The results suggest that Ca++ release and PKC activation are neither necessary nor sufficient for alpha-1A AR-mediated activation of mitogenic responses in PC12 cells.