CELL SIGNALING PATHWAY INVOLVED IN PACAP-INDUCED AR4-2J CELL-PROLIFERATION

The novel 38-amino acid neuropeptide PACAP (pituitary adenylate activating peptide) has recently been shown to induce the pancreatic acinar tumour AR4-2J cell growth. This growth promoting effect of PACAP was, however, independent of adenylate cyclase activation but suppressed by pertussis toxin and the somatostatin analog SMS 201-995. This study was undertaken to search for potential cell signalling pathways involved in the growth promoting effect of PACAP on AR4-2J cells. The AR4-2J cells were grown in Dulbecco's Modified Eagle's Medium containing 10% foetal calf serum. For studies on cell signalling pathways, all experiments were carried out on cells which have reached 50 to 75% confluency. At that point, they were transferred to serum free medium overnight with or without 1 mu Ci/ml myristic acid. The next morning, cells were harvested, washed and used for tyrosine kinase and phospholipase D (PLD) activities. For studies on growth, cells were grown for 2 days in the presence of 1 nM PACAP +/- the different inhibitors of tyrosine kinase and PLD. PACAP-38 and -27 caused a dose-dependent and parallel activation of tyrosine kinase and PLD, an effect prevented by the antagonist PACAP 7-38. PACAP-38-stimulated tyrosine kinase and PLD activation are both dose-dependently inhibited by SMS 201-995. Finally, PACAP-stimulated tyrosine kinase and PLD activities are both inhibited by cell's preincubation with genistein and pertussis toxin. After 2 days, the PACAP-induced increase in AR4-2J cell growth was significantly inhibited by increasing concentrations of genistein and wortmannin, inhibitors of tyrosine kinase, PLD and phosphatidylinositol 3-kinase, respectively. PACAP can induce concomitant activation of tyrosine kinase and PLD; this finding and the observation that inhibition of these two enzymes inhibited PACAP-induced AR4-2J cell growth strongly suggests that they are intimately involved in the overall process of PACAP-induced AR4-2J cell proliferation.