Synergistic interaction of Y1-neuropeptide Y and alpha 1b-adrenergic receptors in the regulation of phospholipase C, protein kinase C, and arachidonic acid production.
Neuropeptide Y (NPY) and norepinephrine, found colocalized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have established 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 microM phenylephrine (PE), respectively, resulted in a rapid increase in the cytoplasmic free calcium 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 pertussis toxin. Both responses were largely maintained in the absence of extracellular Ca2+, but abolished by prior depletion of intracellular Ca2+ pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled with myo-[3H]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as analyzed by anion-exchange high pressure liquid chromatography, whereas the response to PYY (first significant at > 15 s post-stimulation) was 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 additive, whereas they promoted a clearly synergistic rise in Ins(1,4,5)P3 at both 15 and 60 s. Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [3H]arachidonic acid release. In either instance PYY alone was without effect. The potentiation 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)P3-independent fashion minimizes the functional importance of the capacity to potentiate PE-stimulated Ins(1,4,5)P3 generation. Instead the major consequences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.
Selbie
L A
LA
Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales, Australia.
Darby
K
K
Schmitz-Peiffer
C
C
Browne
C L
CL
Herzog
H
H
Shine
J
J
Biden
T J
TJ
D058646:Adrenergic alpha-1 Receptor Agonists; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D006873:Hydroquinones / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D019894:Peptide YY; D010455:Peptides / Q000494:pharmacology*; D037342:Pertussis Toxin; D010656:Phenylephrine / Q000494:pharmacology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D018340:Receptors, Adrenergic, alpha-1 / Q000502:physiology*; D017476:Receptors, Neuropeptide Y / Q000819:agonists / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D058646:Adrenergic alpha-1 Receptor Agonists; D006873:Hydroquinones; D010455:Peptides; D018340:Receptors, Adrenergic, alpha-1; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; D013729:Terpenes; D010566:Virulence Factors, Bordetella; D019894:Peptide YY; D010656:Phenylephrine; C019359:2,5-di-tert-butylhydroquinone; D016718:Arachidonic Acid; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D011493:Protein Kinase C; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium