Neuropeptide Y (NPY) Y1 and Y5 receptor subtypes mediate many of NPY's diverse actions in the central nervous system. The present studies use polyclonal antibodies directed against the Y1 and Y5 receptors to map and compare the relative distribution of these NPY receptor subtypes within the rat brain. Antibody specificity was assessed by using Western analysis, preadsorption of the antibody with peptide, and preimmune serum controls. Immunostaining for the Y1 and Y5 receptor subtypes was present throughout the rostral-caudal aspect of the brain with many regions expressing both subtypes: cerebral cortex, hippocampus, hypothalamus, thalamus, amygdala, and brainstem. Further studies using double-label immunocytochemistry indicate that Y1R immunoreactivity (-ir) and Y5R-ir are colocalized in the cerebral cortex and caudate putamen. Y1 receptor ir was evident in the central amygdala, whereas both Y1- and Y5-immunoreactive cells and fibers were present in the basolateral amygdala. Corresponding with the physiology of NPY in the hypothalamus, both Y1R- and Y5R-ir was present within the paraventricular (PVN), supraoptic, arcuate nuclei, and lateral hypothalamus. In the PVN, Y5R-ir and Y1R-ir were detected in cells and fibers of the parvo- and magnocellular divisions. Intense immunostaining for these receptors was observed within the locus coeruleus, A1-5 and C1-3 nuclei, subnuclei of the trigeminal nerve and nucleus tractus solitarius. These data provide a detailed and comparative mapping of Y1 and Y5 receptor subtypes within cell bodies and nerve fibers in the brain which, together with physiological and electrophysiological studies, provide a better understanding of NPY neural circuitries.

Overproduction of Apo CIII causes elevated plasma triglyceride levels in transgenic animals and is associated with hypertriglyceridemia in humans. The regulation of apo CIII production is likely to play an important role in controlling plasma triglyceride levels. As an initial step in determining the role of transcriptional regulation in the production of apo CIII and in triglyceride metabolism, we have begun to characterize the activity of specific transcriptional regulatory elements in the CIII promoter. In the current study, we have identified and characterized an NF-kappa B regulatory element located 150 nucleotides upstream from the transcriptional start site of the apo CIII gene. Purified NF-kappa B, as well as an NF-kappa B protein in HepG2 cell nuclear extracts, bound specifically to this sequence element. The hepatic protein was induced by phorbol ester (PMA), and reacted with antibodies to the p50 and p65 subunits of NF-kappa B. The NF-kappa B element conferred PMA and IL1-beta inducible transcriptional activity to a heterologous promoter/reporter construct when transfected into HepG2 cells. Analysis of the full length CIII promoter demonstrated that the inducible activity of the NF-kappa B element was suppressed by sequences in the apo CIII enhancer element located approximately 500 nucleotides upstream of the NF-kappa B binding site. A deletion removing the enhancer restored the PMA inducible activity of the NF-kappa B binding site. These results indicate that apo CIII gene expression is regulated by NF-kappa B, and suggest that apo CIII production may be modulated by cellular signals, like inflammatory cytokines, that activate NF-kB.

Regulation of vasopressin (VP) and oxytocin (OT) secretion involves integration of neural signals from hypothalamic osmoreceptors, ascending catecholaminergic and peptidergic cell groups in the brain stem, and local and autoregulatory afferents. Neuropeptide Y (NPY) is one factor that stimulates the release of VP and OT from the supraoptic (SON) and paraventricular nuclei of the hypothalamus via activation of Y1 receptors (Y1R). The current studies were designed to assess the regulation and distribution of NPY Y1R expression in the SON of male rats that were either given 2% NaCl drinking water (24-72 h) or water deprived (48 h). Subjecting male rats to these conditions resulted in significant increases in both the number of cells expressing Y1R immunoreactivity (ir) and the amount of Y1R protein per cell within the SON. Y1R immunoreactivity was increased in the magnocellular but not medial parvocellular paraventricular nuclei, and Y1R mRNA levels were increased in the SON of salt-loaded rats. Subpopulations of both VP and OT cells in the hypothalamus express Y1R immunoreactivity and a greater percentage of VP-ir cells express Y1R after salt loading. To control for potential effects of dehydration-induced anorexia, a group of euhydrate animals was pair fed with animals consuming 2% NaCl. No detectable change in Y1R expression was observed in the SON of pair-fed animals, even though body weights were significantly lower than controls. These data demonstrate that NPY Y1R gene and protein expression are increased in the SON of salt-loaded and water-deprived animals and provide a mechanism whereby NPY can support VP/OT release during prolonged challenges to fluid homeostasis.

In atrial cells, the open probability of G protein-activated ACh-sensitive K+(KACh) channels can be increased approximately fivefold by intracellular ATP (ATPi). Using inside-out patches, we examined how proteases, changes in intracellular pH, and different anions affect G protein-mediated activation and ATP-induced stimulation of the KACh channel. Treatment with trypsin (0.5 mg/ml) removed the GTP dependence of the KACh channel and abolished the ATP-induced stimulation. Intracellular GTP activated KACh channels at all intracellular pH values tested (6.0–8.0), with the concentration at which half-maximal activation ( K ½) occurred ranging from 0.3 (pH 8.0) to 6.7 (pH 6.0) μM. However, the ATPi-induced increase in KACh channel activity was inhibited at pH 8.0 ( K ½ = pH 7.4). All anions tested except sulfate, phosphate, fluoride, and iodide supported GTP-induced activation. Of the anions that supported GTP-induced activation, only citrate blocked the ATP-induced stimulation of the KACh channel. These results indicate that the GTP- and ATP-mediated effects on the KACh channel use separate signaling pathways. The ATP-mediated effect involves a trypsin- and pH-sensitive mechanism.