Neuropeptide Y binding and inhibition of cAMP accumulation in human neuroepithelioma cells.
The specific binding of 125I-labeled neuropeptide Y (NPY) and the biological response to NPY receptor activation were measured in cultured human neuroepithelioma (SK-N-MC) cells. A single class of high-affinity binding sites [dissociation constant (KD) = 0.2 nM] was characterized both by equilibrium binding of 125I-NPY concentrations less than 1 nM and kinetically by the initial rates of 125I-NPY association and dissociation. Specific 125I-NPY binding was decreased in a concentration-dependent manner by inclusion of guanine nucleotides in the incubation medium. The existence of multiple affinity states or NPY receptor subtypes was suggested by 1) a Hill coefficient of less than 1.0 obtained when analyzing equilibrium binding with 125I-NPY concentrations greater than 1 nM, 2) biphasic dissociation of 125I-NPY, 3) an increase in the component of rapid dissociation and decrease in the component of slow dissociation when guanine nucleotides were present during dissociation of 125I-NPY, and 4) displacement of 125I-NPY by unlabeled peptide with a slope factor of 0.6. Exposure of intact cells to NPY caused a concentration-dependent pertussis toxin-sensitive inhibition of forskolin-stimulated cellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation [50% effective concentration (EC50) = 0.4 nM]. In contrast, NPY had no effect on cellular inositol phosphate content or protein kinase C activation. These results demonstrate that NPY binds specifically to a G protein-linked receptor that inhibits adenylate cyclase in SK-N-MC cells.
Lobaugh
L A
LA
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Blackshear
P J
PJ
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001667:Binding, Competitive; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D018241:Neuroectodermal Tumors, Primitive, Peripheral; D009478:Neuropeptide Y / Q000378:metabolism* / Q000494:pharmacology; D037342:Pertussis Toxin; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D007295:Inositol Phosphates; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D002217:Carbachol; D000242:Cyclic AMP; D037342:Pertussis Toxin; D007545:Isoproterenol