Abstract
The regulation of intracellular Ca2+ concentration ([Ca2+]i) plays a critical role in a variety of cellular processes, including transcription, protein activation, vesicle trafficking, and ion movement across epithelial cells. In many cells, the activation of phospholipase C-coupled receptors hydrolyzes membrane phosphoinositides and produces the depletion of endoplasmic reticulum Ca2+ stores, followed by the sustained elevation of [Ca2+]i from Ca2+ entry across the plasma membrane via store-operated Ca2+ entry (SOCE). Ca2+ entry is also increased in a store-independent manner by arachidonate-regulated Ca2+ (ARC) channels. Using rat parotid salivary gland cells, we examined multiple pathways of Ca2+ entry/elevation to determine if they activated cell signaling proteins and whether this occurred in a pathway-dependent manner. We observed that SOCE activates extracellular signal-related kinases 1 and 2 (ERK1/2) to ∼3-times basal levels via a receptor-independent mechanism when SOCE was initiated by depleting Ca2+ stores using the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (TG). TG-initiated ERK1/2 phosphorylation increased as rapidly as that initiated by the muscarinic receptor agonist carbachol, which promoted an increase to ∼5-times basal levels. Notably, ERK1/2 phosphorylation was not increased by the global elevation of [Ca2+]i by Ca2+ ionophore or by Ca2+ entry via ARC channels in native cells, although ERK1/2 phosphorylation was increased by Ca2+ ionophore in Par-C10 and HSY salivary cell lines. Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB), SKF96363, and removal of extracellular Ca2+, also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation. TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC) were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol. These observations demonstrate that ERK1/2 is activated by a selective mechanism of Ca2+ entry (SOCE) in these cells, and suggest that ERK1/2 may contribute to events downstream of SOCE.
Highlights
Receptor-mediated increases in [Ca2+]i promote a variety of physiological events in many cells, including the stimulation of fluid and electrolyte secretion in salivary gland epithelial cells [1,2,3]
storeoperated Ca2+ entry (SOCE), but not arachidonate-regulated Ca2+ (ARC)- and Ionophore-mediated Ca2+ Entry, Activates extracellular signal-related kinases 1 and 2 (ERK1/2) in Rat Parotid Acinar Cells
The increases in ERK1/2 phosphorylation by SOCE were dependent on Protein Kinases C (PKC) activity, similar to what we previously demonstrated for the activation of the M3 muscarinic receptor and P2X7R/channel [30,35]
Summary
Receptor-mediated increases in [Ca2+]i promote a variety of physiological events in many cells, including the stimulation of fluid and electrolyte secretion in salivary gland epithelial cells [1,2,3]. In various non-excitable cells, the increases in [Ca2+]i involve extracellular Ca2+ entry into the cell via SOCE, which is initiated by the release of Ca2+ stores from the endoplasmic reticulum via inositol 1,4,5trisphosphate receptors (IP3R)/Ca2+ channels when the activation of G-protein-coupled receptors produces IP3 and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis by phospholipase C [4]. In addition to Ca2+sensitive ion movements that occur in response to Ca2+ entry via the SOCE pathway in salivary gland cells, the entry of extracellular Ca2+ via the P2X7 receptor/ion channel activates Ca2+-sensitive ion channels and initiates fluid secretion and saliva formation [7,8,9]. SOCE can be activated in a receptorindependent manner using agents such as TG that block the Ca2+-
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