SYNERGISTIC INTERACTION OF Y1-NEUROPEPTIDE-Y AND ALPHA(1B)-ADRENERGICRECEPTORS IN THE REGULATION OF PHOSPHOLIPASE-C, PROTEIN-KINASE-C, ANDARACHIDONIC-ACID PRODUCTION

Neuropeptide Y (NPY) and norepinephrine, found colocalized in sympathe tic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free o f complications associated with mixed receptor populations, we have es tablished a stable Chinese hamster ovary cell line expressing both Y1- NPY and alpha(1b)-adrenergic receptors. Occupation of either receptor species, with 100 nM peptide YY (PYY) or 10 mu M phenylephrine (PE), r espectively, resulted in a rapid increase in the cytoplasmic free calc ium concentration ([Ca2+](i)) as assessed with Fura-2/AM. The rise due to PYY, but not that due to PE, was abolished by pretreatment with pe rtussis toxin, Both responses were largely maintained in the absence o f extracellular Ca2+, but abolished by prior depletion of intracellula r Ca2+ pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohyd roquinone. Using cells prelabeled with myo-[H-3]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3) as analyzed by anion-exchange high pressure liquid chromatography, wherea s the response to PYY (first significant at >15 s post-stimulation) wa s too slow to play a causative role in Ca2+ mobilization. Combination of PE and PYY resulted in increases in [Ca2+](i) which were at best ad ditive, whereas they promoted a clearly synergistic rise in Ins(1,4,5) P-3 at both 15 and 60 s, Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [H-3]arachidonic acid r elease. In either instance PYY alone was without effect. The potentiat ion of arachidonic acid release was abolished by depletion of cellular PKC following chronic treatment with phorbol esters. It is suggested that the ability of PYY to mobilize Ca2+ in an Ins(1,4,5)P-3-independe nt fashion minimizes the functional importance of the capacity to pote ntiate PE-stimulated Ins(1,4,5)P-3 generation. Instead the major conse quences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.