Neuropeptide Y inhibits forskolin-stimulated adenylate cyclase in bovine adrenal chromaffin cells via a pertussis toxin-sensitive process.
The effect of neuropeptide Y (NPY) on adenylate cyclase activity and the role of G-proteins mediating NPY's effect were investigated in cultured bovine adrenal chromaffin cells. The equilibrium binding of [125I]NPY to sucrose gradient purified bovine adrenal medulla plasma membranes revealed high- (GTP gamma S sensitive) and low-affinity binding sites with calculated IC50 values of 0.27 nM and 0.14 microM, respectively. Inhibition of forskolin-stimulated cyclic AMP accumulation was dependent upon the NPY concentration (IC50 = 0.9 nM) and independent of cyclic AMP (cAMP) phosphodiesterase activity. NPY-related peptides, except peptide YY, and NPY fragments exhibited minimal inhibitory activity. The inhibitory effect of NPY on forskolin-stimulated adenylate cyclase activity was completely abolished by pretreatment of the cells with pertussis toxin (PTX). Incubation of membranes with PTX and [32P]nicotinamide adenine dinucleotide revealed a protein band with an apparent molecular mass of 41 kDa. The time course and dose dependence of PTX pretreatment for in vitro ADP-ribosylation were similar to those for PTX to attenuate the NPY effect on forskolin-stimulated adenylate cyclase activity. The direct relation between the NPY receptor and the PTX-sensitive G-protein was further shown by the ability of NPY to inhibit PTX-catalyzed in vitro ADP-ribosylation. ADP-ribosylation of the 41-kDa protein was partially inhibited by 5'-guanylylimidodiphosphate and further inhibited by high concentrations of NPY. An antibody against Gi1/i2 alpha 1 recognized two species of which a 41-kDa protein comigrated with the PTX substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhu
J
J
Department of Pharmacology, University of Nebraska Medical Center, Omaha.
Li
W
W
Toews
M L
ML
Hexum
T D
TD
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D000313:Adrenal Medulla / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000201:enzymology*; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000096:biosynthesis; D019204:GTP-Binding Proteins / Q000502:physiology; D009478:Neuropeptide Y / Q000378:metabolism / Q000494:pharmacology*; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D000067956:Adenylyl Cyclase Inhibitors; D009478:Neuropeptide Y; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D000246:Adenosine Diphosphate Ribose; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins