Stephen M. Waters

Immunohistochemical localization of the neuropeptide Y Y1 receptor in rat central nervous system.

The diverse effects of neuropeptide Y (NPY) are mediated through interaction with G-protein coupled receptors. Pharmacological analysis suggests the Y1 receptor mediates several of NPYs central and peripheral actions. We sought to determine the distribution of Y1 protein throughout the rat central nervous system by means of indirect immunofluorescence using the tyramide signal amplification method and a novel, amino terminally-directed Y1 antisera. This antisera was verified as specific for Y1 by solution-phase competition ELISA, Western blot and in situ blocking experiments. High concentrations of Y1 immunoreactivity were found in the claustrum, piriform cortex (superficial layer), arcuate hypothalamic nucleus, interpeduncular nucleus, paratrigeminal nucleus, and lamina II of the spinal trigeminal nucleus and entire spinal cord. Moderate levels of Y1 immunoreactivity were found the in the main olfactory bulb, dorsomedial part of suprachiasmatic nucleus, paraventricular hypothalamic nucleus, ventral nucleus of lateral lemniscus, pontine nuclei, mesencephalic trigeminal nucleus, external cuneate nucleus, area postrema, and nucleus tractus solitarius. Low levels of Y1 immunostaining were distributed widely throughout layers II-III of the cerebral cortex (i.e., orbital, cingulate, frontal, parietal, insular, and temporal regions), nucleus accumbens core, amygdalohippocampal and amygdalopiriform areas, dentate gyrus, CA1 and CA2 fields of hippocampus, principal and oral divisions of the spinal trigeminal nucleus, islands of Calleja and presubiculum. These findings are discussed with reference to previously reported receptor autoradiography, immunohistochemistry and mRNA analyses to further support the role of Y1 in NPY-mediated biology.

C5a, But Not C3a, Increases VEGF Secretion in ARPE-19 Human Retinal Pigment Epithelial Cells

Purpose: We examined the potential for the pro-inflammatory complement proteins C5a and C3a to increase VEGF expression in ARPE-19 cells. Materials and Methods: Expression of complement receptors in ARPE-19 cells was evaluated by RT-PCR. VEGF secretion from ARPE-19 cells treated with C5a or C3a was determined by ELISA. Results: C5a and C3a receptor, but not C5L2, were detected in human eye tissue and ARPE-19 cells. C5a, but not C3a, treatment increased VEGF secretion from ARPE-19 cells, an effect inhibited by the C5aR antagonist, NDT 9513727. Conclusions: C5a receptor mediates increased VEGF secretion from ARPE-19 cells, suggesting a role for the C5a receptor in the pathogenesis of macular degeneration.

Molecular Characterization of the Gerbil C5a Receptor and Identification of a Transmembrane Domain V Amino Acid That Is Crucial for Small Molecule Antagonist Interaction

Anaphylatoxin C5a is a potent inflammatory mediator associated with pathogenesis and progression of several inflammation-associated disorders. Small molecule C5a receptor (C5aR) antagonist development is hampered by species-specific receptor biology and the associated inability to use standard rat and mouse in vivo models. Gerbil is one rodent species reportedly responsive to small molecule C5aR antagonists with human C5aR affinity. We report the identification of the gerbil C5aR cDNA using a degenerate primer PCR cloning strategy. The nucleotide sequence revealed an open reading frame encoding a 347-amino acid protein. The cloned receptor (expressed in Sf9 cells) bound recombinant human C5a with nanomolar affinity. Alignment of the gerbil C5aR sequence with those from other species showed that a Trp residue in transmembrane domain V is the only transmembrane domain amino acid unique to small molecule C5aR antagonist-responsive species (i.e. gerbil, human, and non-human primate). Site-directed mutagenesis was used to generate human and mouse C5aRs with a residue exchange of this Trp residue. Mutation of Trp to Leu in human C5aR completely eliminated small molecule antagonist-receptor interaction. In contrast, mutation of Leu to Trp in mouse C5aR enabled small molecule antagonist-receptor interaction. This crucial Trp residue is located deeper within transmembrane domain V than residues reportedly involved in C5a- and cyclic peptide C5a antagonist-receptor interaction, suggesting a novel interaction site(s) for small molecule antagonists. These data provide insight into the basis for small molecule antagonist species selectivity and further define sites critical for C5aR activation and function.

Alterations of peptide metabolism and neuropeptidase activity in senile dementia of the Alzheimer's type.

Work in our laboratory has shown that in addition to previously characterized changes in the level of neuropeptides in SDAT brain, the activity of degradative enzymes responsible for peptide metabolism is also affected. In addition to other reported alterations in peptide metabolism, we have observed that SS-28 degradation is increased in Brodmann area 22 whereas substance P degradation is increased in temporal cortex. Changes in the degradation of these neuropeptides known to be affected in SDAT correlate well with alterations in the activity of specific neuropeptidases. Trypsin-like serine protease activity is increased in SDAT Brodmann area 22 which parallels the increased degradation of SS-28. The activity of MEP 24.15 is decreased in temporal cortex which corresponds to the decreased degradation of substance P. Changes in the activity of these degradative enzymes in SDAT brain can potentially affect the action of other neuropeptide substrates because the neuropeptidases discussed here terminate the action of several neuropeptides. As more neuropeptide and degradative peptidase alterations are discovered in SDAT, greater emphasis may be placed on the role that peptides and neuropeptidases play in the progression of SDAT.

Subchronic haloperidol administration decreases aminopeptidase N activity and [Met5]enkephalin metabolism in rat striatum and cortex

Previously we have shown that subchronic intraperitoneal (i.p.) administration of haloperidol decreases the degradation of [Met5]enkephalin by regional brain slices (Waters et al., 1995, J. Pharmacol. Exp. Ther. 274, 783). In the present study, subchronic (7-day i.p.) administration of haloperidol (1 mg/kg) decreased the accumulation of aminopeptidase-derived fragments Tyr and Gly-Gly-Phe-Met on cortical and striatal slices. The accumulation of Tyr-Gly-Gly, however, was not altered by haloperidol treatment on slices from either region. Further, aminopeptidase N activity was decreased in P2 membranes isolated from either the cortex or striatum of haloperidol-treated animals. These data suggest that the haloperidol-induced decrease in [Met5]enkephalin metabolism results, at least in part, from a reduction in the activity of aminopeptidase N.

Effect of dopaminergic drugs on processing and degradative neuropeptidase mRNA in rat frontal cortex and caudate-putamen

Drugs which act upon central dopamine receptors alter the level, mRNA expression and in vitro degradation of neuropeptides associated with dopamine neuron regulation. Changes in the degradation of certain neuropeptides are correlated with significant alterations in the activity of specific neuropeptidases, namely aminopeptidase N (APN) and neutral endopeptidase 24.11 (NEP 24.11). In the present study, we sought to examine the molecular mechanism of neuropeptidase activity changes in response to dopaminergic drug treatment. The effects of dopaminergic drugs on the mRNA level of APN and NEP 24.11 were determined by RNase protection assays of RNA extracted from rat frontal cortex and caudate-putamen. Additionally, the effects of dopaminergic drugs on the mRNA expression for the neuropeptide processing enzymes, prohormone convertase 1 (PC1) and PC2, were determined. After 7-day administration of the dopamine receptor antagonist, haloperidol (1 mg/kg), no effect on the mRNA expression of APN, NEP 24.11, PC1 or PC2 was observed in either of the rat brain regions studied. Administration of the dopamine receptor agonist, apomorphine (5 mg/kg, bid), altered only the expression of APN mRNA in rat caudate-putamen, where the greatest effect on APN activity has been previously observed. These results suggest that alterations in other post-transcriptional events, such as mRNA translation or insertion of neuropeptidase protein into the membrane, likely play a larger role than changes in mRNA expression in the modulation of neuropeptidase activity.