Depolarization-induced ERK phosphorylation depends onthecytosolicCa 2+ levelratherthanontheCa 2+ channelsubtype of chromaffin cells Isabel E. Mendoza,* Oliver Schmachtenberg,* Ernesto Tonk,* Jorge Fuentealba,  Pamela Dı ´az-Raya,*Vero ´nicaL.Lagos,*AntonioG.Garcı ´a andAnaM.Ca ´rdenas* *Centro de Neurociencia de Valparaı ´so, Universidad de Valparaı ´so, Chile  Instituto Teo ´filo Hernando, Departamento de Farmacologı ´a, Facultad de Medicina, Universidad Auto ´noma de Madrid, Spain Abstract The contribution of Ca 2+ entry through different voltage-acti- vated Ca 2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K + depo- larization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-a (PKC-a), but not on Ca 2+ /calmodulin-dependent protein kinase nor cyclic AMP- dependent protein kinase. Blockade of L-type Ca 2+ channels by 3 lM furnidipine, or blockade of N channels by 1 lM x-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 lM x-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca 2+ elevations elicited by 40 mM K + , showed that Ca 2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca 2+ channel blockers. Fifty-eight percent of the fluorescence rise depen- ded on Ca 2+ entering through N channels. Thus, ERK phos- phorylation seems to depend on a critical level of Ca 2+ in the cytosol rather than on activation of a given Ca 2+ channel subtype. Keywords: chromaffin cells, cyclic AMP-dependent protein kinase, extracellular signal regulated kinase, intracellular calcium, protein kinase C, voltage activated calcium channel. J. Neurochem. (2003) 86, 1477–1486. TheexpressionofL,N,P/Q,RandTsubtypesofvoltage- activated Ca 2+ channels (VACC) in neurons (Olivera et al. 1994; Garcı ´a et al. 2000) poses the interesting question of their specialization to control different cell functions. For instance,NandP/Q-typechannels,whicharepredominantly found along the length of apical dendrites and in axon terminals that synapse on dendrites (Westenbroek et al. 1992), control the release of various neurotransmitters (Olivera et al. 1994; Wheeler et al. 1994; Garcı ´a et al. 2000). In contrast, L-type channels located on proximal dendritesandneuronalcellbodies(Westenbroek et al.1990; Ahlijanian et al. 1990; Westenbroek et al. 1992; Waterman 1997; Westenbroek et al. 1998; Timmerman et al. 2002) havebeenassociatedwiththeregulationofgeneexpression and enzyme activity in cortical and hippocampal neurons (Murphy et al. 1991; Bading et al. 1993; Elliot et al.1995; Westenbroek et al. 1995; Deisseroth et al.1998). The particular segregation of Ca 2+ channel subtypes to dendrites, axon terminals, or somata facilitates their special- ization to accomplish such specific functions in various neuronal cell types. However, specialization of the different ReceivedFebruary2,2003;revisedmanuscriptreceivedMay13,2003; acceptedJune3,2003. Address correspondence and reprint requests to Ana M. Ca ´rdenas, Ca ´tedra de Farmacologı ´a, Escuela de Medicina, Universidad de Val- paraı ´so,Casilla92-V,Valparaı ´so,Chile.E-mail:ana.cardenas@uv.cl Abbreviations used: BSA, bovine serum albumin; Ca 2+ /CaMK, Ca 2+ /calmodulin-dependent protein kinase; [Ca 2+ ] c , cytosolic Ca 2+ concentration; ERK, extracellular signal regulated kinase; MAPK, mitogen-actived protein kinase; MEK, MAP/ERK kinase; PBS, phos- phate-buffered saline; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SNAP- 25, synaptosomal-associated protein of 25kDa; VACC, voltage- activated Ca 2+ channel. Journal of Neurochemistry ,2003, 86, 1477–1486 doi:10.1046/j.1471-4159.2003.01965.x Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 86, 1477–1486 1477