Subcellular fractionation of bovine ganglion stellatum: co-storage of noradrenaline, Met-enkephalin and neuropeptide Y in large 'dense-cored' vesicles

The subcellular localization of noradrenaline, Met-enkephalin and neuropeptide Y was studied in homogenates of bovine ganglion stellatum. After differential centrifugation most of the noradrenaline (70%) was found soluble, while both neuropeptide Y and Met-enkephalin were sedimented for more than 65%. However, the 3 substances co-sedimented mainly in the microsomal fraction. The microsomal fraction was further analyzed by differential and equilibrium density gradient centrifugation. In both types of gradient, Met-enkephalin and neuropeptide Y were found in the more dense region of the gradient, coinciding with the main peak of noradrenaline. In this fraction, the molar ratio of Met-enkephalin to noradrenaline was 1:95. The corresponding molar ratio for neuropeptide Y to noradrenaline was 1:253. These results indicate that neuropeptide Y and Met-enkephalin are stored with noradrenaline in 'heavy' or large 'dense cored' vesicles in the cell bodies of sympathetic neurons of bovine ganglion stellatum. We present here for the first time biochemical evidence for the co-localization of neuropeptides and a classical transmitter in a neuronal cell body.