Neuropeptide Y is localized together with enkephalins in adrenergic granules of bovine adrenal medulla

Abstract

The subcellular localization of neuropeptide Y in the bovine adrenal medulla was studied. After differential centrifugation, a large part of total neuropeptide Y immunoreactivity (66%) was recovered in the large granule fraction. This fraction also contained 42% of the total catecholamines and 52% of the total free [Met]enkephalin. Thus neuropeptide Y co-sedimented with these chromaffin granule markers. The large granule fraction was analysed by the technique of rate zonal centrifugation in hypertonic sucrose media (molarities ranging from 1.6 to 2.2 M). Noradrenaline vesicles were preferentially enriched at high sucrose concentration. Adrenaline vesicles as well as enkephalin and neuropeptide Y immunoreactivity pelleted mainly at lower sucrose concentrations. The large granule fraction was also separated by successive centrifugation in self-generating gradients of Percoll-sucrose into two subpopulations, one containing lighter adrenergic vesicles and the other the dense noradrenergic vesicles. Like [Met]enkephalin immunoreactivity, neuropeptide Y immunoreactivity was concentrated in fractions containing the lighter adrenergic vesicles. In these fractions the molar ratio of adrenaline to free [Met]enkephalin to neuropeptide Y was 5000:12:1. This biochemical study supports immunohistochemical studies which described co-localization of neuropeptide Y in adrenaline cells in the rat, mouse, cat, guinea-pig and man and co-localization of neuropeptide Y with enkephalins in bovine adrenal chromaffin cells. Our results are however in contrast with the report of other immunohistochemical work which claimed co-localization of neuropeptide Y in noradrenaline cells of rat, cat, dog, horse and cow.