Douglas A. Schober

Autoradiographic localization of subtypes of angiotensin II antagonist binding in the rat brain

The non-peptide angiotensin II receptor compounds DuP 753 and WL 19 were utilized to detect subtypes of [125I]Sar1-Ile8-angiotensin II binding to angiotensin II receptors in the rat brain. In rat forebrain homogenates, DuP 753 and WL 19 produced a partial displacement of [125I]Sar1-Ile8-angiotensin II binding with DuP 753 displacing approximately 65% of the binding and WL 19 displacing approximately 35% of the binding. Using the techniques of quantitative receptor autoradiography, a distinct regional distribution of the subtypes of angiotensin II antagonist bind was detected. The angiotensin II-1 binding site (the receptor subtype preferentially displaced by DuP 753) appeared to predominate in the dipsogenic, cardiovascular and endocrine areas, including the subfornical organ, paraventricular and periventricular nuclei of the hypothalamus, anterior pituitary, dorsal motor nucleus of the vagus, nucleus of the solitary tract and the area postrema. Additional areas that contained predominantly the angiotensin II-1 receptor subtype were the ventral hippocampus, substantia gelatinosa of the trigeminal nucleus, nucleus of the lateral olfactory tract, piriform cortex and median preoptic nucleus. The angiotensin II-2 binding site (displaced by WL 19) was the predominant subtype in the thalamus, inferior olive, lateral septum, subthalamic nucleus, locus coeruleus, medial geniculate and medial amygdala. Several areas of the brain appeared to contain both receptor subtypes, including the superior and inferior colliculi, and the olfactory bulb. The angiotensin II-1 binding site was concentrated in areas of the brain involved in mediating angiotensin II effects on drinking, endocrine status and blood pressure. Localization of angiotensin II-2 sites in the thalamus and areas of the brain which process sensory information suggests a novel modulatory role for angiotensin II at this receptor subtype. These results indicate that DuP 753 and WL 19 are highly selective for angiotensin II binding site subtypes in the brain and that, in general these subtypes are compartmentalized in distinct brain regions. The non-peptide compounds used in these studies should provide excellent tools to discern the functional role of angiotensin II receptor subtypes in the brain.

Neuropeptide Y receptor subtypes in the basolateral nucleus of the amygdala modulate anxiogenic responses in rats

The behavioral effects induced by intra-amygdala stimulation of the neuropeptide Y (NPY) Y(2) and the NPY Y(5) receptor subtypes were assessed in the social interaction (SI) test. Microinjections of NPY(3-36), an NPY Y(2) preferring agonist, into the basolateral nucleus of the amygdala (BLA) produced bi-directional dose-response curve. At low doses NPY(3-36) has an anxiogenic effect while at higher doses it produced an anxiolytic effect. Pretreatment with the NPY Y(5) receptor antagonist Novartis 1(1 nmol), an analog of CGP71683A synthesized by Eli Lilly and Company, IN, blocked the anxiolytic effects of NPY(3-36) (80 pmol), while pretreatment with BIBO 3304 (200 pmol), a Y(1) antagonist, had no effect, suggesting that the Y(5), but not the Y(1) receptor was involved in the anxiolytic behavior produced following intra-amygdalar NPY(3-36) administration. In addition, the Y(5) antagonist had no behavioral effect when given alone at 1.0 nmol. These findings support the hypothesis that amygdalar Y(2) receptors may play a role in mediating anxiogenic effects, while Y(5) receptors may be involved in the anxiolytic behaviors of NPY.

Neuropeptide Y-Y2 receptors mediate anxiety in the amygdala.

The behavioral effects of direct injection of the neuropeptide Y (NPY) Y2 receptor agonist C2-NPY into the basolateral nucleus of the amygdala (BLA) was assessed in rats utilizing the social interaction test (SI). C2-NPY decreased SI time in a dose-dependent manner with a significant change observed at a dose of 80 pmol/100 nl. The anxiogenic effects produced by intra-amygdalar C2-NPY injections were reversed with intraperitoneal administration of alprazolam (1 mg/kg), a known anxiolytic. These findings support the hypothesis that Y2 receptors are involved in the regulation of the anxiety response.

Structure-activity relationship of a series of diaminoalkyl substituted benzimidazole as neuropeptide Y Y1 receptor antagonists

A series of benzimidazoles (4) was synthesized and evaluated in vitro as potent and selective NPY Y1 receptor antagonists. Substitution of the piperidine nitrogen of 4 with appropriate R groups resulted in compounds with more than 80-fold higher affinity at the Y receptor compared to the parent compound 5 (R = H). The most potent benzimidazole in this series was 21 (Ki = 0.052 nM).

[Leu31-Pro34] neuropeptide Y identifies a subtype of 125I-labeled peptide YY binding sites in the rat brain

Subtypes of the neuropeptide Y (NPY) receptor in the rat brain were identified by the use of the selective Y-1 analog, [Leu34-Pro34] NPY. In rat brain homogenate binding studies, [Leu31-Pro34] NPY was found to produce a partial inhibition of 100 pM 125I-labeled peptide YY (PYY) binding with a plateau at 50-1000 nM [Leu31-Pro34] NPY resulting in a 70% inhibition of binding. The C-terminal fragment NPY 13-36, a putative Y-2 agonist, exhibited very little selectivity in rat brain homogenates. Scatchard analysis of 125I-labeled PYY binding to rat brain homogenate yielded biphasic plots with Kd values of 40 and 610 pM. Inclusion of 100 nM [Leu31-Pro34] NPY was found to eliminate the low affinity component of 125I-labeled PYY binding leaving a single, high affinity binding site with a Kd of 68 pM. In autoradiographic studies, displacement curves indicated that [Leu31-Pro34] NPY completely inhibited binding in the cerebral cortex with little effect on the binding in the hypothalamus. On the other hand NPY 13-36 inhibited binding in the hypothalamus at low concentrations but required higher concentrations to inhibit binding in the cerebral cortex. Other brain regions such as the hippocampus, appeared to contain both subtypes. Subsequent to these studies, a quantitative autoradiographic map was conducted using 50-100 pM 125I-labeled PYY in the presence and absence of [Leu31-Pro34] NPY which produced a selective displacement of binding in certain distinct brain regions. These areas included the cerebral cortex, certain thalamic nuclei and brainstem while ligand binding was retained in other brain regions including the zona lateralis of the substantia nigra, lateral septum, nucleus of the solitary tract and the hippocampus. Numerous brain regions appeared to contain both receptor subtypes. Therefore, the Y-1 and Y-2 receptor subtypes exhibited a somewhat distinct distribution in the brain. In addition, 125I-labeled PYY appears to label the Y-2 receptor with relatively higher affinity when compared to the Y-1 receptor.

The Neuropeptide Y Y1 Antagonist, 1229U91, A Potent Agonist for the Human Pancreatic Polypeptide-Preferring (NPY Y4) Receptor

Recently, a novel high-affinity peptide antagonist, 1229U91, was published as a selective neuropeptide Y Y1 antagonist. The selectivity of 1229U91 was evaluated in the human NPY Y1 receptor containing cell line, SK-N-MC, and cells containing the cloned human NPY Y2, the pancreatic polypeptide-preferring (NPY Y4), and the NPY Y5 receptors. 1229U91 potently displaced [125I]-peptide YY (PYY) binding to human NPY Y1 receptors (IC50 = 0.245+/-0.004 nM, n = 4). but displayed little affinity for the human NPY Y2 and Y5 receptors (IC50 > 1000 nM). Interestingly, 1229U91 displaced [125I]-PYY with even greater affinity at the human NPY Y4 receptor (IC50 = 0.081+/-0.009 nM, n = 4). Using a cyclic AMP accumulation assay, 1229U91 blocked NPY inhibition of forskolin-induced adenylate cyclase activity in NPY Y1 receptor containing SK-N-MC cells. In the human NPY Y4 receptor expressing cell line, 1229U91 did not block pancreatic polypeptide (PP) inhibition of forskolin stimulated adenylate cyclase. However, in the absence of PP, 1229U91 was able to inhibit forskolin stimulated cyclic AMP accumulation (IC50 = 7.16+/-2.8 nM, n = 4). We conclude that 1229U91 binds non-selectively with high affinity to both human NPY Y1 and Y4 receptors. Furthermore, 1229U91 displays antagonist activity at the NPY Y1 receptor, while having agonist activity at the NPY Y4 receptor.

[125I]Leu31, Pro34-PYY is a High Affinity Radioligand for Rat PP1/Y4 and Y1 Receptors: Evidence for Heterogeneity in Pancreatic Polypeptide Receptors

Cloned receptors for the PP-fold peptides are subdivided into Y1, Y2, PP1/Y4, Y5 and Y6. NPY and PYY have similar affinity for Y1, Y2, Y5 and Y6 receptors while PP has highest affinity for PP1. Pro34-substituted analogs of NPY and PYY have selectivity for Y1 and Y1-like receptors over Y2 receptors. In the present study, we found the putative Y1-selective radioligand, [125I]Leu31, Pro34-PYY, also binds with high affinity to the rat PP1 receptor in cell lines expressing the receptor. However, in rat brain sections, [125I]Leu31, Pro34-PYY does not appear to bind to the interpeduncular nucleus, a brain region containing a high density of [125I]-bPP binding sites. Therefore, it appears there is additional heterogeneity in receptors recognizing PP.

M1 and M2 Muscarinic Receptor Subtypes Regulate Antidepressant-Like Effects of the Rapidly Acting Antidepressant Scopolamine

Scopolamine produces rapid and significant symptom improvement in patients with depression, and most notably in patients who do not respond to current antidepressant treatments. Scopolamine is a nonselective muscarinic acetylcholine receptor antagonist, and it is not known which one or more of the five receptor subtypes in the muscarinic family are mediating these therapeutic effects. We used the mouse forced-swim test, an antidepressant detecting assay, in wild-type and transgenic mice in which each muscarinic receptor subtype had been genetically deleted to define the relevant receptor subtypes. Only the M1 and M2 knockout (KO) mice had a blunted response to scopolamine in the forced-swim assay. In contrast, the effects of the tricyclic antidepressant imipramine were not significantly altered by gene deletion of any of the five muscarinic receptors. The muscarinic antagonists biperiden, pirenzepine, and VU0255035 (N-[3-oxo-3-[4-(4-pyridinyl)-1-piper azinyl]propyl]-2,1,3-benzothiadiazole-4-sulfonamide) with selectivity for M1 over M2 receptors also demonstrated activity in the forced-swim test, which was attenuated in M1 but not M2 receptor KO mice. An antagonist with selectivity of M2 over M1 receptors (SCH226206 [(2-amino-3-methyl-phenyl)-[4-[4-[[4-(3 chlorophenyl)sulfonylphenyl]methyl]-1-piperidyl]-1-piperidyl]methanone]) was also active in the forced-swim assay, and the effects were deleted in M2−/− mice. Brain exposure and locomotor activity in the KO mice demonstrated that these behavioral effects of scopolamine are pharmacodynamic in nature. These data establish muscarinic M1 and M2 receptors as sufficient to generate behavioral effects consistent with an antidepressant phenotype and therefore as potential targets in the antidepressant effects of scopolamine.

Characterization of the neuropeptide Y5 receptor in the human hypothalamus: a lack of correlation between Y5 mRNA levels and binding sites

Neuropeptide Y (NPY) is a 36-amino-acid peptide that appears to play a central role in the control of feeding behavior. Recently, a cDNA encoding a novel NPY receptor subtype (Y5) was cloned from the rat and human hypothalamus, and shown to have a pharmacology consistent with NPY-induced feeding. We have subsequently cloned this cDNA from human hypothalamus and stably expressed it in CHO cells. Consistent with earlier reports, hY5 has a high affinity for NPY, [Leu31, Pro34]NPY, and NPY(3-36), but low affinity for larger C-terminal deletions of NPY and BIBP3226. High levels of hY5 mRNA were found in the human testis, brain, spleen and pancreas, with lower levels in several other tissues. In the human brain, hY5 mRNA levels were typically higher than hY2, but lower in comparison to hY1 receptor mRNA. To quantify the relative amounts of hY1, hY2 and hY5 mRNA in the human hypothalamus, we employed competitive RT-PCR. Interestingly, the relative amount of hY5 mRNA was substantially higher than either hY1 or hY2. However, pharmacological characterization of NPY binding sites in human hypothalamus membranes revealed predominantly the hY2 subtype. These data establish that while hY5 mRNA levels are very high in the human hypothalamus, conventional radioligand binding techniques do not detect hY5-like binding site. Whether hY5-like binding sites exist in the other human tissues that express hY5 mRNA (and what function hY5 has in those tissues) awaits future investigation.

Comparison of (R)‐[3H] Tomoxetine and (R/S)‐[3H] Nisoxetine Binding in Rat Brain

Abstract: (R)‐[3H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower‐affinity site. The goal of the present study was to identify the nature of this low‐affinity site by comparing the binding properties of (R)‐[3H]tomoxetine with those of (R/S)‐[3H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas (R)‐[3H]tomoxetine also bound to a second, lower‐affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE‐containing neurons. However, low levels of (R)‐[3H]‐tomoxetine binding were seen in the caudate‐putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where (R/S)‐[3H]nisoxetine binding was almost absent. In homogenates of the caudate‐putamen, the NE uptake inhibitors desipramine and (R)‐nisoxetine and the serotonin (5‐HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 nM (R)‐nisoxetine to mask the binding of (R)‐[3H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [3H]citalopram. Therefore, (R)‐[3H]tomoxetine binds to the NE uptake site with high affinity and the 5‐HT uptake site with somewhat lower affinity.