Neuropeptide Y in the adrenal gland: characterization, distribution and drug effects.

The occurrence of neuropeptide Y-like immunoreactivity in the adrenal gland was investigated by means of radioimmunoassay, chromatography and immunohistochemistry. The adrenal levels of neuropeptide Y-like immunoreactivity varied considerably between species with lowest amounts in the rat and highest in the cow where immunoreactivity was observed in chromaffin cells and nerve fibres in the capsule and cortex. The distribution of neuropeptide Y-like immunoreactivity in the cow medulla overlapped that of enkephalin-like immunoreactivity. Chromatographic characterisation of rat and cow adrenal extracts showed that the majority of the neuropeptide Y-like immunoreactivity was similar in molecular weight and solubility properties to porcine neuropeptide Y. Rat adrenal contained additional material some of which may represent oxidised neuropeptide Y. The administration of insulin and reserpine to rats in vivo showed that the turnover of adrenal neuropeptide Y-like immunoreactivity is regulated by the splanchnic nerve. Splanchnic activation following insulin-induced hypoglycaemia elicited a 60% depletion of neuropeptide Y-like immunoreactivity 2 h post insulin injection. A smaller (maximum 40%) depletion of neuropeptide Y-like immunoreactivity was measured 24 h after reserpine injections which may be explained by splanchnic activation. Five days after reserpine injections the levels of adrenal neuropeptide Y-like immunoreactivity were increased to 200% of control levels and remained slightly elevated at 25 days. Adrenal enkephalin-like immunoreactivity showed similar but not identical changes following reserpine. The reserpine-induced elevation in neuropeptide Y-like immunoreactivity at the 5-day time point was abolished in rats with a chronic bilateral splanchnectomy. This evidence indicates that neuropeptide Y may be considered as a new adrenal medullary hormone.

Distribution of neuropeptide Y-like immunoreactivity in the rat central nervous system--I. Radioimmunoassay and chromatographic characterisation.

The distribution of neuropeptide Y-like immunoreactivity in the rat brain was investigated by means of immunochemical techniques. In the first part of the study (present paper) neuropeptide Y radioimmunoassays were characterised and the chromatographic properties and regional distribution of neuropeptide Y-like immunoreactivity was investigated. The second part of the study (accompanying paper) involved immunohistochemical techniques. Extracts from several regions of rat brain were found to contain immunoreactivity that behaved like synthetic porcine neuropeptide Y in three test systems: dilution in the radioimmunoassay (test of antigenic properties), gel chromatography (molecular weight), reverse phase high performance liquid chromatography (solubility properties). Experiments were conducted to optimise the extraction of neuropeptide Y. Boiling 0.1 M sodium phosphate buffer, pH 7.4, extracted at least two times as much immunoreactivity from whole brain pieces as other buffers. The nature of the extracted immunoreactivity was confirmed using chromatography. Experiments (using added iodinated or unlabelled neuropeptide Y standards) demonstrated that the differences between extraction media could not be explained by differential recovery of the peptide, although differences in recovery between media existed. Tissue sample weight was found to influence neuropeptide Y recovery. Evidence that rat neuropeptide Y-like immunoreactivity was not identical to the porcine peptide was obtained from experiments which demonstrated an early eluting peak of immunoreactivity in addition to the main peak on high performance liquid chromatograms. This material could be generated by oxidation of extracted rat neuropeptide Y, suggesting the presence in the rat peptide of a methionine residue. Some evidence of high molecular weight neuropeptide Y precursors was obtained from chromatography of hypothalamus extracts. Bovine pancreatic polypeptide-like material represented less than 1% of the amounts of neuropeptide Y in the brain. The distribution of neuropeptide Y-like immunoreactivity was non-uniform in the rat brain with highest concentrations observed in the hypothalamus, amygdaloid complex and periaqueductal central gray matter. Other regions of forebrain contained moderate to high concentrations including olfactory tubercle, striatum, nucleus accumbens, neocortex and hippocampus. Negligible amounts were detected in the cerebellum. In spinal cord immunoreactivity was concentrated in the dorsal horn, although measurable amounts were found in the ventral horn. The neurointermediate but not anterior lobe of the pituitary contained neuropeptide Y.

Distribution of neuropeptide Y-like immunoreactivity in the rat central nervous system--II. Immunohistochemical analysis.

The distribution of neuropeptide Y-like immunoreactivity in the rat brain and spinal cord was investigated by means of the peroxidase-antiperoxidase procedure of Sternberger using a rabbit anti-neuropeptide Y serum. A widespread distribution of immunostained cells and fibres was detected with moderate to large numbers of cells in the following regions: olfactory bulb, anterior olfactory nucleus, olfactory tubercle, striatum, nucleus accumbens, all parts of the neocortex and the corpus callosum, septum including the anterior hippocampal rudiment, ventral pallidum, horizontal limb of the diagonal band, amygdaloid complex. Ammon's horn, dentate gyrus, subiculum, pre- and parasubiculum, lateral thalamic nucleus (intergeniculate leaflet), bed nucleus of the stria terminalis, medial preoptic area, lateral hypothalamus, mediobasal hypothalamus, supramammillary nucleus, pericentral and external nuclei of the inferior colliculus, interpeduncular nucleus, periaqueductal central gray, locus coeruleus, dorsal tegmental nucleus of Gudden, lateral superior olive, lateral reticular nucleus, medial longitudinal fasciculus, prepositus hypoglossal nucleus, nucleus of the solitary tract and spinal nucleus of the trigeminal nerve. In the spinal cord cells were found in the substantia gelatinosa at all levels, the dorsolateral funiculus and dorsal gray commissure in lumbosacral cord. The pattern of staining was found to be similar to that observed with antisera to avian and bovine pancreatic polypeptide, but to differ in some respects from that observed with antisera to molluscan cardioexcitatory peptide. The presence of neuropeptide Y immunoreactive fibres in tracts such as the corpus callosum, anterior commissure, lateral olfactory tract, fimbria, medial corticohypothalamic tract, medial forebrain bundle, stria terminalis, dorsal periventricular bundle and other periventricular areas, indicated that in addition to the localisation of neuropeptide Y-like peptide(s) in interneurons in the forebrain, neuropeptide Y may be found in long neuronal pathways throughout the brain.

Neurotensin facilitates dopamine release in vitro from rat striatal slices.

Neurotensin, (0.1-10 microM) stimulated the release of [3H]dopamine from rat striatal slices in a calcium-dependent manner, and potentiated the K+-evoked release of [3H]dopamine and endogenous dopamine. This effect was dose-dependent, (1 nM-1 microM) with an EC50 of approximately 10 nM, and was mediated by means of a receptor of similar structure-activity profile to those described in other tissues.

Survival of basal ganglia neuropeptide Y-somatostatin neurones in Huntington's disease.

The content of somatostatin-like immunoreactivity (SRIF-LI) and neuropeptide Y-like immunoreactivity (NPY-LI) has been measured in both control post-mortem human brains and in Huntington's disease brains. The content of both SRIF-LI and NPY-LI was found to be significantly increased in the basal ganglia of Huntington's disease brains compared with a control group. The nature of the SRIF-LI and NPY-LI in both control and Huntington's disease brains was investigated after separation on Sephadex G25 and G50 columns. Using a C-terminal-directed SRIF radioimmunoassay (RIA), 3 peaks of immunoreactivity were measured, whilst an N-terminal-directed SRIF RIA detected two peaks of immunoreactivity. In each case, the elution profile did not differ between control and Huntington's disease caudate nucleus. The content of immunoreactivity in each peak was found to be increased in Huntington's disease brains compared with controls. Only one peak of NPY-LI was detected in both control and Huntington's disease caudate after separation on Sephadex G25 and G50 columns. Immunohistochemical staining of the caudate and putamen of control and Huntington's disease brains revealed a population of neurones containing NPY-LI. The number of NPY-positive neurones was found to be increased in both the caudate and putamen of Huntington's disease brains compared to control caudate and putamen.

Subcellular distribution of neuropeptide Y-like immunoreactivity in guinea pig neocortex.

Neuropeptide Y-like immunoreactivity (NPY-LI) was enriched in synaptosomal fractions of neocortex, which on lysis yielded vesicle-rich fractions. The distribution of NPY-LI on a sucrose density gradient was similar to that of somatostatin, with a concentration in heavy vesicles. The peptides were not found in light vesicles in contrast to the distribution of noradrenaline. Both homogenate and vesicular NPY-LI coeluted with synthetic NPY on reverse-phase HPLC.