Protein kinase C and calcium activate p42/p44 MAPK in lacrimal gland using non‐receptor tyrosine kinases.

Cholinergic agonists are potent stimuli of protein secretion from rat lacrimal gland and do so through the activation of protein kinase C (PKC) and an increase in intracellular Ca 2+ . In addition, these agonists also activate p42/p44 MAPK (MAPK) that negatively modulates protein secretion. We previously showed that the cholinergic agonist carbachol (Cch) activated pyk2 and src to stimulate MAPK. However the effects of PKC or intracellular Ca 2+ ([Ca 2+ ] i ) on MAPK in this gland have not been investigated. To investigate the role of PKC and Ca 2+ on MAPK activation, lacrimal gland acini were isolated by collagenase digestion and stimulated for 5 minutes with the phorbol ester PMA to activate PKC, and ionomycin to increase [Ca 2+ ] i . In some experiments, acini were preincubated for 15 minutes with the PKC inhibitors Ro‐31‐8220 or calphostin C prior to stimulation with Cch (10 −4 M). After stimulation, acini were sonicated and proteins separated by SDS‐PAGE. The amount of phosphorylated and total MAPK, pyk2 and src were determined by western blotting analysis. Both PMA and ionomycin activated MAPK in a concentration dependent manner with a maximum activation 2.3 ± 0.5 fold and 4.0 ± 1.0 fold above basal both at 10 −6 M, respectively. In addition, PMA activated pyk2 and src 2.0 ± 0.3 and 1.3 ± 0.6 fold above basal, respectively. Ionomycin activated pyk2 1.6 ± 0.7 fold above basal, but did not activate src. Inhibitors of PKC at 10 −6 M completely inhibited Cch‐induced MAPK activation at 10 −6 M. We conclude that in the lacrimal gland PKC and Ca 2+ play an integral role in cholinergic agonist‐activation of MAPK by differentially using pyk2 and src.