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- (GTPγS sensitive) and low-affinity binding sites with calculated IC50 values of 0.27 nM and 0.14 μM, 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 was 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 G(i1/i2)α1 recognized two species of which a 41-kDa protein comigrated with the PTX substrate. These results demonstrate that NPY inhibits adenylate cyclase activity in cultured bovine adrenal chromaffin cells through a receptor- linked PTX-sensitive G-protein, presumably G(i)2.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - Dec 1 1992|
ASJC Scopus subject areas
- Molecular Medicine