CROSS-TALK BETWEEN SUBSTANCE-P AND MELITTIN-ACTIVATED CELLULAR SIGNALING PATHWAYS IN RAT LACTOTROPH-ENRICHED CELL-CULTURES

We have investigated the possible interaction (cross talk) between the phospholipase A(2) (PLA(2)) and inositol 1,4,5-trisphosphate/protein kinase C (PKC) signaling pathways in rat lactotroph-enriched cell cult ures. Melittin, a bee venom peptide, stimulated release of [H-3]-arach idonic acid ([H-3]AA) from [H-3]AA-labeled enriched lactotrophs in a d ose-dependent manner. Moreover, melittin and exogenous AA induced a re distribution of PKC catalytic activity and PKC alpha and beta immunore activity from the soluble to the particulate fraction in resting and s ubstance P (SP)-stimulated cells. Melittin had no effect on phospholip ase C (PLC) activity. Pretreatment of cell cultures with the PLA(2) in hibitors quinacrine and aristolochic acid resulted in a dose-dependent inhibition of melittin-stimulated PKC isozyme translocation as did th e inhibitor of lipoxygenase, nordihydroguaiaretic acid, whereas the cy clooxygenase inhibitor indomethacin had no effect. SP and the phorbol ester 1 2-O-tetradecanoylphorbol 13-acetate (TPA) dose-dependently inc reased levels of [H-3]AA released from cells. Pretreatment of cell cul tures with quinacrine reduced the effect of SP on [H-3]AA formation. A fter long-term treatment (24 h) of cells with TPA, the effect of TPA o n [H-3]AA production was not different from control, whereas SP still displayed [H-3]AA-releasing abilities although not at full scale. Pret reatment of cells with thapsigargin, U 73122, methoxyverapamil, and RH C 80267, an inhibitor of diacylglycerol lipase, all resulted in reduce d SP-stimulated [H-3]AA liberation. Treatment of cell cultures with pe rtussis toxin (PTX) reduced the release of [H-3]AA induced by SP, wher eas PTX had no effect on SP-stimulated generation of H-3-inositol phos phates. On the basis of these results, it is concluded that (1) the PL A(2) pathways interfere with the phosphoinositide-PLC signaling system at the level of PKC isozymes alpha and beta, the product responsible for this interaction being either AA or a metabolite produced by the a ction of lipoxygenase; (2) SP and TPA are able to activate the PLA(2) pathway at a level at or beyond PLA(2), and this effect is mediated, i n part, through PKC alpha and beta species and (for SP) intracellular Ca2+ recruited from internal stores as well as from external sources; and (3) SP also activates PLA(2) through a PTX-sensitive pathway disti nct from the one coupled to phosphoinositide-PLC, which is PTX insensi tive.