Depolarization‐induced ERK phosphorylation depends on the cytosolic Ca²⁺ level rather than on the Ca²⁺ channel subtype of chromaffin cells

Abstract

The contribution of Ca 2+ entry through different voltage-activated Ca 2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K + depolarization (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 AMPdependent 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 depended on Ca 2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca 2+ in the cytosol rather than on activation of a given Ca 2+ channel subtype. The expression of L, N, P/Q, R and T subtypes of voltageactivated Ca 2+ channels (VACC) in neurons (Olivera et al. 1994;García et al. 2000) poses the interesting question of their specialization to control different cell functions. For instance, N and P/Q-type channels, which are predominantly 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;García et al. 2000). In contrast, L-type channels located on proximal dendrites and neuronal cell bodies Ahlijanian et al. 1990;Westenbroek et al. 1992;Waterman 1997;Westenbroek et al. 1998; Timmerman et al. 2002) have been associated with the regulation of gene expression 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 specialization to accomplish such specific functions in various neuronal cell types. However, specialization of the different

Calcium Signaling and Exocytosis in Adrenal Chromaffin Cells

“…O'Farrell and Marley (293) observed that the individual blockade of a given calcium channel subtype was not enough to prevent the activation of tyrosine hydroxylase by K ϩ depolarization; however, blocking various calcium channel subtypes abolished the enzyme activation. This was also true for the Ca 2ϩ -dependent K ϩ -evoked increase in the expression of the exocytotic protein SNAP-25 (170) as well as for ERK phosphorylation, which seems to depend on a critical [Ca 2ϩ ] c rather than on activation of a given calcium channel subtype (260). We have also found that secretory control by mitochondrial Ca 2ϩ fluxes critically depends on the ability of K ϩ depolarization to create high [Ca 2ϩ ] c microdomains, rather than on Ca 2ϩ entry through a given calcium channel subtype (268).…”

Calcium channel subtypes differentially regulate fusion pore stability and expansion

“…1998; Polo‐Parada et al. 2006), protein kinase activation (Mendoza et al. 2003), and gene expression (García‐Palomero et al.…”

The F-Actin Binding Protein Cortactin Regulates the Dynamics of the Exocytotic Fusion Pore through its SH3 Domain

“…Therefore, ACCs were incubated with 10 μM of U0126, or its inactive analog U0124, 15 min prior to experimentation and throughout the test. In these conditions, U0126 efficiently inhibits ERK1/2 phosphorylation (Evans et al, 2002; Mendoza et al, 2003). …”

“…Both types of phosphorylation determine cortactin activity as well as its association with proteins such as the N-WASP (Martinez-Quiles et al, 2004; Tehrani et al, 2007; Kelley et al, 2011), an actin NPF that accumulates at exocytosis sites together with the Arp2/3 nucleation complex and F-actin (Gasman et al, 2004). In ACCs, both ERK1/2 and Src kinases are activated by secretagogues and regulate exocytosis (Allen et al, 1996; Cox and Parsons, 1997; Mendoza et al, 2003). As we recently reported, Src kinases also control Ca 2+ -dependent actin polymerization and fusion pore lifetime in ACCs (Olivares et al, 2014).…”