PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) ACTIONS ONALPHA-T3-1 GONADOTROPHS SHOW DESENSITIZATION

PACAP is a hypothalamic hypophysiotropic factor that acts upon a numbe r of pituitary cells, including gonadotrophs. In the gonadotroph-deriv ed alpha T3-1 cell line, PACAP acts via PVR1 receptors to stimulate ad enylyl cyclase and phosphoinositidase C. PACAP-stimulated cAMP accumul ation is inhibited by protein kinase C-activating phorbol esters in th ese cells and the current work was undertaken primarily to establish w hether it is also subject to homologous regulation, In acute experimen ts, PACAP27-stimulated cAMP accumulation (intracellular plus extracell ular) was measured (in the presence of phosphodiesterase inhibitor) bo th in intact cells and in cell membranes. The peptide increased cAMP a ccumulation, but initial rates of PACAP27-stimulated cAMP accumulation were reduced to between 10 and 50% within 10 min of stimulation in bo th cells and membranes. The initial rate of forskolin-stimulated cAMP accumulation was maintained in membranes but not in intact cells (alth ough the deviation from linearity was less pronounced than with PACAP2 7), Thus, rapid homologous desensitization to PACAP27 occurs in intact alpha T3-1 cells, but is not entirely receptor specific. Rapid homolo gous desensitization of PACAP27-stimulated cAMP accumulation also occu rred in the presence of a protein kinase C activating phorbol ester, w hich inhibited cAMP accumulation without altering the kinetics of the PACAP27 effect. Brief pre-treatment (3 min) with PACAP27 also reduced the ability of PACAP27, but not gonadotrophin-releasing hormone, to ca use a spike-type elevation of cytosolic Ca2+ concentration (a conseque nce of phosphoinositidase C activation). In chronic desensitization st udies, pre-treatment for 6 h with PACAP27 caused a dose-dependent (IC5 0 approximately 10 nM) reduction of PACAP-stimulated cAMP accumulation and down regulated cell surface PVR1 receptors (to approximately 50%) . Thus, it appears that PACAP27-stimulated (PVR-1 receptor mediated) a denylyl cyclase undergoes rapid homologous desensitization in alpha T3 -1 cells, which is paralleled by homologous desensitization of PACAP27 stimulated phosphoinositidase C activity and involves mechanisms dist inct from those underlying heterologous desensitization by phorbol est ers, Chronic desensitization of PACAP-stimulated cAMP accumulation and down-regulation of cell surface PVR-1 receptors also occurs in these cells although the receptor loss may not entirely explain the observed desensitization.