Synergistic activation of mitogen-activated protein kinase by insulin and adenosine triphosphate in liver cells: permissive role of Ca2+.

We have previously demonstrated that insulin and G(q)-coupled receptor agonists individually activate mitogen-activated protein kinase (MAPK) in liver cells and both effects involve an influx of extracellular Ca(2+). Yet, these agonists have opposing physiological actions on hepatocyte glucose metabolism. We thus investigated the interaction between insulin and the P2Y(2) purinergic agonist adenosine triphosphate (ATP) on MAPK in HTC cells, a model hepatocyte cell line, and determined the involvement of cytosolic Ca(2+). Insulin and ATP each induced a dose-dependent phosphorylation of p44/42 MAPK that was partially inhibited by EGTA. However, pretreatment with insulin markedly increased the MAPK phosphorylation response to ATP. This potentiation was canceled by chelation of extracellular Ca(2+) with EGTA. We used patch clamp electrophysiology and fluorescence microscopy to understand the role of intracellular Ca(2+) in this effect. Insulin and ATP, respectively, induced monophasic and multiphasic changes in membrane potential and intracellular Ca(2+) as expected. Pretreatment with 10 nmol/L insulin significantly decreased the initial rapid depolarization (inward nonselective cation current [NSCC]), as well as the compounded Ca(2+) response induced by 100 micro mol/L ATP. However, in Ca(2+)-free conditions, insulin did not modify the Ca(2+) mobilized from internal pools after stimulation with ATP. Upon Ca(2+) readmission, internal store depletion by ATP or thapsigargin doubled the rate of capacitative Ca(2+) influx, whereas insulin increased this influx 1.32-fold. On the other hand, insulin pretreatment counteracted the increased rate of Ca(2+) influx induced by ATP but not by thapsigargin. In summary, insulin counteracts the membrane potential and Ca(2+) responses to ATP in HTC cells. However, insulin and ATP effects on MAPK activation are synergistic and Ca(2+) influx plays a permissive role. Therefore, the opposing metabolic actions of insulin and G(q)-coupled receptor agonists involve an interaction in signaling pathways that resides downstream of Ca(2+) influx.