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We used Y(1)R/LacZ transgenic mice to investigate the interaction between NPY, GABA and Y(1) receptors in the amygdala. Immunolabeling of GABA and NPY positive neurons and histochemical staining of beta-galactosidase revealed NPY and GABA colocalization and close contacts of NPY-positive fibers with GABAergic neurons also expressing the Y(1)R/LacZ transgene.

The neurosteroid allopregnanolone, a reduced metabolite of progesterone, induces anxiolytic effects by enhancing GABA(A) receptor function. Neuropeptide Y (NPY) and GABA are thought to interact functionally in the amygdala, and this interaction may be important in the regulation of anxiety. By using Y(1)R/LacZ transgenic mice, which harbour a fusion construct comprising the promoter of the mouse gene for the Y(1) receptor for NPY linked to the lacZ gene, we previously showed that long-term treatment with benzodiazepine receptor ligands modulates Y(1) receptor gene expression in the medial amygdala. We have now investigated the effects of prolonged treatment with progesterone or allopregnanolone on Y(1)R/LacZ transgene expression, as determined by quantitative histochemical analysis of beta-galactosidase activity. Progesterone increased both the cerebrocortical concentration of allopregnanolone and beta-galactosidase expression in the medial amygdala. Finasteride, a 5alpha-reductase inhibitor, prevented both of these effects. Long-term administration of allopregnanolone also increased both the cortical concentration of this neurosteroid and transgene expression in the medial amygdala. Treatment with neither progesterone nor allopregnanolone affected beta-galactosidase activity in the medial habenula. These data suggest that allopregnanolone regulates Y(1) receptor gene expression through modulation of GABA(A) receptor function, and they provide further support for a functional interaction between GABA and neuropeptide Y in the amygdala.

NPY is a potent orexigenic signal and represents a key component of targets through which leptin exerts a regulatory restraint on body adiposity. Part of the orexigenic effects of NPY are mediated by hypothalamic NPY-Y(1) receptors. Here we studied the effect of fasting, leptin, and glucose administration on Y(1) receptor gene expression using a transgenic mouse model carrying a mouse Y(1) receptor/LacZ fusion gene. Transgene expression was determined by quantitative analysis of beta-galactosidase histochemical staining in the paraventricular, arcuate, ventromedial, and dorsomedial hypothalamic nuclei and in the medial amygdala, as a control region. Food deprivation for 72 h decreased transgene expression in the paraventricular nucleus but not in the arcuate nucleus. Leptin treatment, that was per se ineffective, counteracted the decrease of transgene expression induced in the paraventricular nucleus by 72 h fasting. Supplementing the drinking water with 10% glucose increased beta-galactosidase expression both in the paraventricular nucleus and arcuate nucleus of control mice. Finally, none of the treatments altered transgene expression in the dorsomedial hyphothalamic, ventromedial, and amygdaloid nuclei. Results suggest that changes in energetic balance affect Y(1) receptor expression in the paraventricular and arcuate nuclei and that leptin regulates the NPY-Y(1) system in the paraventricular nucleus. Different regulatory signals might modulate the NPY-Y(1) transmission in the dorsomedial hyphothalamic and ventromedial hyphothalamic nuclei.

Several studies have shown that neuropeptide Y (NPY) is involved in the stimulation of gonadotropin hormone releasing hormone (GnRH) and luteinizing hormone (LH) secretion and that these effects are modulated by gonadal steroid feedback. The NPY regulation of GnRH release is probably mediated by the activation of the Y(1) receptor subtype. In this study we examined the regulation of the Y(1) receptor gene transcription by estrogens in transiently transfected NG108-15 neuroblastoma glioma cells. A chimeric plasmid containing the murine Y(1) receptor promoter fused to the firefly luciferase reporter gene was induced by approximately 2-fold in response to 17 beta-estradiol treatment. The estrogen-mediated enhancement of luciferase activity was dose-dependent, blocked by the estrogen receptor (ER) antagonist ICI 182,780, and was strictly dependent on the presence of ER alpha, since it occurred only in NG108-15 cells cotransfected with an expression vector for the human ER. Mutational analysis was performed to investigate whether the hemipalindromic estrogen-responsive elements (EREs) flanking the Y(1) receptor gene are responsible for conferring estradiol inducibility to the Y(1) receptor gene promoter. Mutation of the ERE1 half site at position -932, or mutation of the ERE2 half site at position -809, relative to the ATG, failed to affect the 17 beta-estradiol-mediated enhancement of luciferase activity. Conversely, mutation of both ERE1 and ERE2 half sites completely abolished activation of luciferase activity induced by estrogen. We also examined whether 17 beta-estradiol stimulates the transcriptional activity of the Y(1) receptor gene by binding to ER beta. Results demonstrated that luciferase activity was not modulated by estrogens when cells were transfected with the expression plasmid bearing the human ER beta. Moreover coexpression of both ER alpha and ER beta completely abolished the estrogen-induced activation of luciferase activity observed in the presence of ER alpha. Our data suggest that estrogens activate Y(1) receptor gene transcription possibly via a direct interaction of ER alpha with the hemipalindromic EREs flanking the Y(1) receptor gene., (Copyright 2000 S. Karger AG, Basel)

The relative content of peripheral benzodiazepine receptor (pBR) mRNA was examined by reverse transcriptase-polymerase chain reaction in lymphocytes of obsessive-compulsive disorder (OCD) patients, according to their clinical course of illness. pBR mRNA significantly decreased only in chronic OCD patients (n=8) as compared to controls (n=10), whereas no significant changes were observed in episodic OCD patients (n=7). We suggest that modulation of pBR gene expression might delineate a clinical heterogeneity in OCD.

NPY exerts anxiolytic effects, which are mediated by activation of Y1 receptors in the amygdala. It has been shown that diazepam counteracts the anxiogenic effect of Y1 receptor antagonists, suggesting that NPYergic and GABAergic systems are coupled in the regulation of anxiety. We used a transgenic mouse model, expressing a mouse Y1 receptor-beta-galactosidase fusion gene (Y1R/LacZ), to study the effect of positive or negative modulators of GABA(A) receptors on Y1 receptor gene expression. Mice were treated for 14 days with diazepam (4 or 20 mg/kg), the anxiolytic beta-carboline-derivative abecarnil (0.3 or 6 mg/kg) and the anxiogenic beta-carboline FG7142 (20 mg/kg). Transgene expression was determined by quantitative analysis of beta-galactosidase histochemical staining in the medial amygdala and in the medial habenula as a control region. Chronic treatment with 20 mg/kg diazepam or 6 mg/kg abecarnil significantly increased, whereas FG 7142 decreased, transgene expression in the medial amygdala. A transient decrease in transgene expression was observed in the medial amygdala six hours after the acute treatment with 20 mg/kg FG 7142 but not with diazepam or abecarnil. No significant changes were observed in the medial habenula. These data suggest that modulation of GABA(A) receptor function may regulate Y1 receptor gene expression in medial amygdala.

The Y1 receptor for neuropeptide Y (NPY) is highly expressed in mammalian CNS where it mediates the activation of several neurobiological functions. We have previously demonstrated that a 1.3-kb fragment upstream of the transcription initiation sites of the murine Y1 receptor gene is able to direct specific expression of reporter genes in neuronal cell cultures. In the present study transgenic mice harbouring this putative promoter region linked to the LacZ reporter gene were generated and analysed for temporal and spatial distribution. Ten transgenic lines expressed beta-galactosidase in the CNS but not in other organs such as heart, liver and kidney. Histochemical analysis of brain from adult transgenic mice showed specific expression of the transgene in specific brain regions with little variation. Four transgenic lines showed characteristic patterns of beta-galactosidase activity in the brain that are consistent with the expression of the endogenous gene. Prominent LacZ activity was present in several telencephalic and diencephalic structures, including deeper layers of cerebral cortex, amygdaloid complex, hippocampus, preoptic area, several thalamic and hypothalamic nuclei and habenula. The ontogeny analysis indicates that the LacZ expression agrees with the temporal expression pattern of rat Y1 receptor mRNA. These data demonstrate that the 1.3-kb upstream region of the murine Y1 receptor gene contains the cis acting elements required for establishing a CNS-restricted and developmental stage-specific pattern of expression in vivo. Moreover they provide further information on the distribution of this NPY subtype receptor in mammalian brain.

Background: The aim of this study was to assess whether the decrease of peripheral benzodiazepine receptor (pBR) number in peripheral blood mononuclear cells (PBMC), previously observed in patients with generalized anxiety disorder, is paralleled by changes in the relative content of messenger RNA (mRNA) encoding pBR., Methods: Eight patients with a DSM-III-R diagnosis of generalized anxiety disorder were examined before, during, and after 2'-chloro-N-desmethyl-diazepam treatment. Eight healthy subjects were analyzed in parallel. The relative content of pBR mRNA was determined by reverse-transcriptase-polymerase chain reaction, using beta-actin as internal standard. Kinetic binding properties of pBR were measured using 3H-PK11195 as a ligand., Results: pBR and pBR mRNA were significantly decreased in untreated generalized anxiety disorder patients as compared to controls (by 45% and 70%, respectively). Both pBR density and mRNA levels returned to control values during treatment or after withdrawal, which also coincided with recovery from anxiety., Conclusions: These results suggest that the turnover rate of pBR is reduced in PBMC of generalized anxiety disorder patients, and that this change occurs at the transcriptional level.

We previously isolated a 1.3-kb genomic fragment in the 5'-flanking region of the murine neuropeptide Y (NPY) Y1 receptor gene, which is able to drive the expression of LacZ reporter gene in neuronal cells. We determined the ability of deletion mutants of this region to modulate transcription of the heterologous luciferase gene in the Y1 receptor-expressing neuroblastoma/ glioma NG108-15 cells and the Y1 receptor-deficient 293 cells. Results suggest the presence of a cell type-specific core promoter (-399 to -218 from the initiator ATG) and, upstream, of two positive and two negative regulatory elements. Sequence analysis of the Y1 receptor promoter identified two decameric sequences corresponding to consensus binding sites for nuclear factor-kappa B/Rel proteins. Gel shift analysis indicated that a 29-bp oligonucleotide comprising the two putative kappa B sites, which we refer to as Y1-kappa B sequence, specifically binds kappa B-related complexes in nuclear extracts from rat brain areas, NG108-15 cells, and the murine T cell clone A.E7. In nuclear extracts from A.E7 and NG108-15 cells, the Y1-kappa B sequence specifically binds an additional complex whose molecular nature remains to be elucidated. Through transient transfection studies, we also demonstrated that the Y1-kappa B sequence acts as an enhancer element, inferring its potential role in regulation of the Y1 receptor gene expression.

The homologous regulation of the muscarinic m1 and m3 receptors mRNA was studied in rat corticostriatal neuronal cultures. In response to the full agonist carbachol the m1 and m3 receptor mRNA levels were modulated and showed different time-courses. The m1 receptor mRNA increased to 270% of prestimulation levels after 6 h, while the m3 receptor transcript transiently increased to 170% after 2 h. Conversely, the muscarinic receptor partial agonist oxotremorine caused a rapid and sustained increase in both mRNA species as soon as after 1 h of exposure. A comparable increase in both receptor mRNAs was induced by the partial agonist [4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethyl-ammonium chloride] (McN-A-343). The observation that 1 h exposure to the non selective antagonist N-methyl-scopolamine also enhanced the m1 and m3 receptor transcripts suggests that in corticostriatal neurons muscarinic receptor partial agonists exert an antagonist-like effect on the m1 and m3 receptor mRNAs.

The repeated intracerebroventricular administration of nerve growth factor (5 micrograms/2.5 microliters) to neonatal rats induced the activation of choline acetyltransferase in forebrain cholinergic neurons that was paralleled by a concomitant change in the density of muscarinic cholinergic receptors in the cerebral cortex. The administration of nerve growth factor altered muscarinic binding sites in a biphasic fashion during postnatal development. A significant stimulation of the developmental increase in the density of muscarinic binding sites occurred in nerve growth factor-treated animals at days 2 and 3 after birth. Conversely, nerve growth factor induced a significant decrease in the receptor number at postnatal days 8 and 14. Muscarinic receptor number returned to control values after treatment, suggesting that nerve growth factor-induced changes to muscarinic cholinergic receptors are reversible. Nerve growth factor administration did not affect muscarinic cholinergic receptor density in striatal membranes and did not alter the relative content of cortical messenger RNAs encoding m1 and m3 muscarinic cholinergic receptor subtypes at postnatal day 14, as determined by reverse transcriptase-polymerase chain reaction. The up- and down-regulation of muscarinic cholinergic receptors induced by nerve growth factor during postnatal development may be temporally related events associated with concomitant changes in the activity of choline acetyltransferase.

1. Physiological and molecular evidence for the presence and functional role of M1 muscarinic cholinergic receptors (mAChRs) in adult guinea-pig ventricular cells is presented. 2. Whole-cell clamp measurements of the L-type calcium current (ICa) in isolated myocytes were performed. Caesium was used to suppress potassium currents. ICa was increased by the muscarinic agonist carbachol in cells pretreated with pertussis toxin which blocked the M2 mAChR-triggered cascade of intracellular signalling, while it was not changed in untreated cells. 3. If the M2-mediated regulation of ICa was blocked by directly saturating the cell with cyclic adenosine monophosphate (cAMP) through the patch pipette, application of carbachol induced a further small increase of the current above the level reached after cAMP perfusion. This increase was more pronounced in cells pretreated with pertussis toxin. 4. The carbachol-induced increase of ICa was blocked by the selective M1 mAChR antagonist pirenzepine. 5. The application of high concentrations of carbachol increased the accumulation of [3H]inositol monophosphate up to 240% above control levels. This increase was reduced by application of pirenzepine. 6. The expression of M1 receptor mRNA in ventricular cardiocytes was shown by reverse transcriptase-polymerase chain reaction. 7. These results suggest that M1 mAChR regulation of ICa can be a component of the paradoxical positive inotropism induced by high concentrations of muscarinic agonists.

A putative novel G protein-coupled receptor was isolated using the polymerase chain reaction (PCR) and conventional library screening. Degenerate primers corresponding to the thyroid and the sixth transmembrane regions that are conserved among the G protein-coupled receptors were used to amplify selectively gene fragments of new members of this receptor family from a neuroblastoma-glioma cell line cDNA. One of the PCR amplification products that by sequence analysis appeared to contain a partial fragment of a novel G protein-coupled receptor gene was used as a probe to screen a rat forebrain cDNA library to isolate a full-length clone. Sequence comparison indicates that the new gene belongs to the G protein-coupled receptor family and displays the highest structural similarity with the dog RDC1 orphan receptor and several hormone peptide receptors. The mRNA for this putative receptor is not expressed in any specific region of the brain but seems to be localized in a restricted number of peripheral tissues, including lung, testes, and kidney. We suggest that the 4-24 cDNA clone encodes for a novel G protein-coupled receptor that may belong to hormone peptide receptor family.

The murine gene for the NPY-1 receptor subtype for neuropeptide Y was characterized by DNA sequencing and expression studies. It comprises three exons with a 6,400 bp 5'-untranslated and a 80 bp internal intronic sequence. The 5'-flanking region of this gene lacks TATA or CCAAT consensus sequences in the proximity to the multiple transcription initiation sites. A 1,300 bp genomic fragment of the 5'-flanking region drives the expression of the lacZ reporter gene in NG108-15 cells and primary cultured neurons but not in glial and human embryonic kidney cells. In addition, it contains consensus sequences for various transcription factors including cAMP- and glucocorticoid-responsive elements.

The effect of nerve growth factor (NGF) on muscarinic receptor subtypes was investigated in a primary culture of telencephalic neurons prepared from neonatal rats. The treatment with 100 ng/ml of NGF significantly enhanced choline acetyltransferase (ChAT) activity and intracellular acetylcholine (ACh) content during cultivation. The same treatment induced an early transient increase of the number of muscarinic cholinergic receptors (mAChR), as measured by [3H]quinuclidinyl benzilate binding to cell homogenate, that was followed by a dramatic decrease of the receptor density from the 9th day of culture. Atropine completely prevented the decrease of the maximal number of muscarinic recognition sites induced by NGF. Prolonged exposure of telencephalic neurons to NGF also induced a significant reduction of the relative content of the messenger RNA (mRNA) encoding m1 and m3 receptors, while the m4 transcript was increased by the treatment. We suggest that the prolonged stimulation of cholinergic neurons by NGF induces a downregulation of m1 and m3 mAChR and their mRNAs on the postsynaptic site, while it increases the synthesis of the functionally distinct m4 receptor subtype, which might be presynaptically localized on cholinergic neurons. The transient increase of the receptor number that occurs at the first days of culture was not paralleled by changes in the relative content of mAChR mRNAs and might be associated with the trophic activity of NGF on cholinergic synapses during early development.

Neuropeptide Y (NPY) is an important central and peripheral modulator of neural and endocrine functions. This neuropeptide interacts with at least two pharmacologically distinct receptors, termed Y1 and Y2. At Y1 receptors, the NPY analog [Leu31,Pro34] NPY, but not the carboxyl-terminal fragment NPY-(18-36), displaces radiolabeled NPY and the sequence-related peptide YY, whereas Y2 receptors exhibit the opposite selectivity. We have used cultured mammalian 293 cells for the high level transient expression of a previously cloned putative neuropeptide receptor of rat brain. We report that this receptor displays the ligand binding properties and selectivity of a Y1 receptor, with a single high affinity site for 125I-NPY (Kd, 0.7 +/- 0.2 nM). The functionality of the recombinantly expressed receptor was demonstrated by an inhibition of adenylyl cyclase and a concomitant mobilization of intracellular Ca2+.

Muscarinic cholinergic receptors were analysed in lymphocyte membranes from 35 patients with early (n = 20) and late onset (n = 15) Alzheimer's disease (AD), 86 patients with other neurological disorders and 60 normal controls by the specific binding of 3H-N-methyl-scopolamine (3H-NMS). The number of binding sites of 3H-NMS (Bmax) was significantly decreased both in early and late onset AD groups as compared with age-matched controls, by 54% and 40%, respectively, whereas the apparent binding affinity (Kd) was the same in all disease and control groups. In addition, the average Bmax in early AD was significantly lower than in late AD. The density of the binding of 3H-NMS was also significantly lower in a subgroup of old subjects with Down's syndrome (DS), whereas no changes were found in younger individuals with DS or in patients with Parkinson's disease, whether they were demented or not, multi-infarct dementia, myasthenia gravis or epilepsy. In the AD group, the difference in binding sites was unrelated either to the severity of dementia or disease duration. Treatment of the patients with cholinergic agents did not alter the binding values in any of the examined group. We conclude that the alteration of lymphocyte muscarinic receptors is highly associated with AD, but whether this reflects the central cholinergic deficit in these patients is uncertain.

A novel cDNA encoding a putative G protein-coupled receptor was isolated from a rat forebrain cDNA library by homology screening. Sequence comparison demonstrates that the encoded polypeptide containing seven transmembrane domains shows highest homology with tachykinin receptors, particularly in the transmembrane domains and the first intracellular loop. The mRNA for this new receptor seems specifically expressed in brain and is prominently localized in specific nuclei of the rat brain, including thalamus, cerebral cortex and hippocampus. We speculate that a neuropeptide may be the natural ligand of this novel G protein-coupled receptor.

Homologous desensitization of muscarinic acetylcholine receptors (mAChR) was studied using primary cultures of corticostriatal neurons from neonatal rats. Prolonged incubation with carbachol attenuated phospholipase C responsiveness to muscarinic agonists and decreased the number of cell surface mAChR, as measured by binding of N-[3H] methylscopolamine to neuronal monolayers. When neurons were exposed to carbachol for 15 min, 40% of the mAChR lost from the membrane domain was recovered in the cytosol; a decrease of the total neuronal receptors was detected following an incubation with the agonist lasting longer than 15 min. Both 8-Br-cyclic AMP and forskolin neither affected N-[3H]methylscopolamine binding to cell monolayers or did they prevent the agonist-mediated mAChR desensitization. 8-Br-cyclic GMP also failed to decrease mAChR number. Pertussis toxin failed to prevent the homologous desensitization of mAChR under conditions that blocked the agonist-mediated inhibition of forskolin-stimulated cyclic AMP formation. The phorbol ester 12-O-tetradecanoyl-phorbol-12, 13-acetate induced a concentration-dependent decrease of N-[3H]methylscopolamine binding to neuronal monolayers. However, the protein kinase C inhibitors sphingosine and the ganglioside monosialosyl-gangliotetraglicosylceramide inhibited the 12-O-tetradecanoyl-phorbol-12,13-acetate-induced but not the agonist-induced desensitization of mAChRs. Furthermore, incubation with muscarinic agonists failed to translocate protein kinase C from cytosol to plasma membranes, as measured by binding of the phorbol ester [3H]-4-beta-phorbol-12,13-dibutyrate to neuronal monolayers. In corticostriatal neurons the agonist-induced desensitization and internalization of mAChR involves neither protein kinase C and protein kinase A activation nor changes in cyclic GMP and cyclic AMP content.

In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes increasing amounts of acetylcholine (ACh) from the third day in vitro (DIV), which reaches a plateau around the 10 day of culture. Muscarinic cholinergic receptors (mAChR), measured by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB), are detectable from the fifth DIV and increase linearly during the time of culture. At the twelfth DIV, the density of mAChRs (approximately 600 fmol/mg protein) is comparable to the density of mAChR in adult rat cortex. These receptors are coupled to second messenger systems, since muscarinic agonists inhibit adenylate cyclase activity and stimulate phosphoinositide breakdown with efficacies and potencies similar to those found in adult rat cortex. Moreover, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, we were able to demonstrate the presence of the m1, m3, and m4 mAChR subtype mRNAs in this neuronal culture at 12 DIV. Our data suggest that corticostriatal neuronal cultures develop in vitro ACh-synthesizing neurons and functionally active cholinergic receptors. This therefore makes them ideally suited to study the development and properties of brain mAChR subtypes.

Enzymes can be used in high-performance liquid chromatography post-column reactors to improve sensitivity and specificity of detection for some compounds by converting the compounds to easily detectable products. The enzymes can be covalently bound to a post-column reactor, but a simpler approach is to bind them by adsorption to an ion exchanger or a hydrophobic interaction support. This technique has been applied to electrochemical detection of acetylcholine by using adsorption of choline oxidase and cholinesterase to a 3-cm long commercially available weak anion-exchange cartridge. Conversion of acetylcholine to peroxide is quantitative during the 10-sec residence time in the cartridge. Enzyme elution from the cartridge is negligible when low ionic strength mobile phases are used. Fresh enzyme needs to be added to the cartridge at only 1-2 week intervals.

In hippocampal slices, somatostatin 14 and its stable analog L363 [cyclo(Phe-Pro-Phe-D-Trp-Lys-Thr)] fail to modify muscarinic signal transduction mediated by stimulation of phosphoinositide breakdown, whereas somatostatin 14 mimics oxotremorine in inhibiting adenylate cyclase activity of hippocampal membranes. The simultaneous addition of somatostatin 14 and oxotremorine elicits a nonadditive convergent inhibition of adenylate cyclase activity. Both L363 and oxotremorine nonadditively stimulate a high-affinity guanosine 5'-triphosphatase activity of hippocampal membranes. This stimulation could be operative in mediating the convergent inhibition of adenylate cyclase activity elicited by the binding of specific ligands to somatostatin and muscarinic recognition sites present in hippocampal membranes. Because L363 competitively displaces muscarinic agonists fand antagonists from their specific recognition sites, one might infer that the two recognition sites interact functionally; that is, somatostatin reduces the efficacy of oxotremorine and/or vice versa.

The properties of the binding of the muscarinic receptor ligands, [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]NMS) in human mononuclear cells were compared. The binding of [3H]QNB showed a high, non-specific component and lack of saturability in both intact mononuclear cells and preparations of lysed mononuclear cell membranes. Conversely the specific binding of [3H]NMS had a high affinity and was saturable at concentrations greater than 30 nM in both intact and broken cells. Classical muscarinic receptor antagonists displaced specific binding of [3H]NMS binding according to the law of mass action, while displacement curves for pirenzepine and muscarinic agonists were very shallow (nH less than 1), suggesting the presence of more than one subtype of muscarinic receptor on mononuclear cell membranes. Binding studies with [3H]NMS to purified mononuclear cell subpopulations demonstrated that muscarinic binding sites were mainly localized on thymus-derived (T) lymphocytes and large granule lymphocytes. Moreover evidence is presented of an age-dependent increase of the density of muscarinic binding sites on T-lymphocytes. The results are discussed in terms of the usefulness of the binding of [3H]NMS in studying the physiological function of muscarinic receptors on human T-lymphocytes and their possible changes in patients with neurological diseases.

Acetylcholine (ACh) and vasoactive intestinal peptide (VIP) probably coexist in cholinergic neurons of rodent salivary glands. In this tissue, cholinergic drugs regulate release of both ACh and VIP from postganglionic cholinergic neurons. In the present study we investigated whether VIP could modulate the metabolism of ACh in mouse submandibular gland cholinergic neurons using ACh turnover rate (TRACh) as a parameter. The TRACh was estimated via measurement of the formation of [3H]ACh during constant rate infusion of [3H]choline. Choline and ACh were separated by reverse phase high-performance liquid chromatography and were detected electrochemically after enzymatic postcolumn reaction. We calculated that the TRACh was about 3 nmol/mg of protein per hr. Pilocarpine, a muscarinic agonist decreased the TRACh about 5-fold whereas atropine methyl Br, a muscarinic antagonist, caused a large increase in turnover. Turnover, therefore, appears to be regulated by a feedback mechanism triggered by occupancy of postsynaptic receptors. VIP infused i.v. (40 micrograms/kg/min) decreased the TRACh by about 50%. Atropine completely prevented the inhibition of the TRACh induced by VIP. These results suggest that, by changing postsynaptic or presynaptic muscarinic receptor function, VIP may participate in the control of ACh metabolism. Parasympathetic decentralization of salivary glands did not prevent the effect of either atropine or VIP on TRACh. This finding suggests that the central afferent input to the ganglionic cells is not required for the regulation of ACh metabolism and, therefore, the feedback loop probably acts via a postganglionic mechanism which is not elucidated by present experiments.

The binding of [3H]spiperone to striatal membranes from rabbit brain was evaluated in untreated male and female and in female rabbits after treatment with ovine prolactin (1 mg/kg, i.v.) or with saline. Unsubstantial differences were found between male and female striatal [3H]spiperone binding sites, both in affinity and density. Ovine prolactin treatment induced an increased binding site density and a lower affinity than occurred in the female controls. These results appear similar to those obtained after chronic neuroleptic treatment and confirm that prolactin is able to affect the dopaminergic neurotransmission system.

Choline oxidase and cholinesterase were found to retain their activity for 1-2 weeks at room temperature while adsorbed to a commercially available anion-exchange cartridge. These enzymes convert acetylcholine to H2O2. Acetylcholine can be measured in tissue extracts by separation at pH 7 on a polymeric reverse-phase high-performance liquid chromatography column, conversion of acetylcholine to H2O2 on a postcolumn enzyme-loaded anion-exchange cartridge, and electrochemical detection of the H2O2 formed.

In GH3 pituitary cell homogenates, acetylcholine (ACh) (IC50 200 nM) inhibits adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in a concentration- and GTP-dependent manner. Maximal inhibition was obtained with 10 microM ACh and corresponded to approximately a 50% decrease in basal enzyme activity. ACh inhibition is antagonized by atropine and is mimicked by muscarinic receptor agonists, but not by nicotine. ACh reduces the adenylate cyclase stimulation by vasoactive intestinal peptide (VIP), without changing its EC50. In intact GH3 cells, ACh decreases the cyclic AMP content and the rate of prolactin release in a concentration-dependent manner. When the cells are simultaneously exposed to VIP and ACh, the VIP-induced increases in cyclic AMP accumulation and prolactin release are reduced by 80% and 40%, respectively. The potency of VIP is not significantly changed by the presence of ACh, and vice versa.

The stimulation of inositol phospholipid (PI) hydrolysis by excitatory amino acids was measured in the rat hippocampus or striatum after 3 different chemical or surgical lesions of putative glutamatergic pathways. Intrahippocampal infusions of kainate preferentially destroyed neurons in the CA3-4 areas, denervating the CA1 area of the ipsilateral and contralateral hippocampus. Infusions of colchicine selectively destroyed granule cells of fascia dentata, denervating the CA3 area of the ipsilateral hippocampus. Ablation of the frontal cortex selectively reduced the glutamatergic afferents to the striatum. These 3 lesions potentiated the ibotenate, glutamate and quisqualate stimulation of PI hydrolysis, while N-methyl-D-aspartate remained ineffective. This stimulation was inhibited by 2-amino-4-phosphonobutyric acid but not by phencyclidine. These lesions also increased the stimulation of PI hydrolysis elicited by norepinephrine, but failed to enhance the stimulation by carbamylcholine. These results support the hypothesis that signal transduction in a subclass of excitatory amino acid receptors present in rat brain may undergo plastic modifications following denervation.

Bilateral (3 nmol/side) i.c.v. infusion of ethylcholine aziridinium ion (AF64A) causes a 70% decrease of hippocampal acetylcholine content lasting for longer than 30 days without changing the density of hippocampal recognition sites for muscarinic ligands. In hippocampal slices prepared from rats receiving i.c.v. AF64A, the activation of phosphoinositide turnover or the inhibition of cyclic AMP accumulation elicited by muscarinic receptor agonists is facilitated. This AF64A treatment also causes a long-lasting decrease of hippocampal norepinephrine and serotonin (5-HT) content. Even a smaller dose of AF64A (1.5 nmol/side) reduces the hippocampal 5-HT content. The number of alpha-1 adrenoceptor recognition sites is slightly increased by 3 nmol/side of AF64A and the stimulation of phosphoinositide turnover by norepinephrine is facilitated. In contrast the decrease of hippocampal 5-HT concentration elicited by AF64A fails to change the 5-HT receptor indices that were measured. These results indicate that in rat hippocampus muscarinic receptors and alpha-1 adrenoceptors are denervated by AF64A and that this denervation promotes a receptor supersensitivity. These results also suggest that we could not find appropriate conditions to express a complete specificity of AF64A in destroying cholinergic axons and therefore this drug cannot be used readily to induce a selective deficiency of central cholinergic transmission.

Chronic administration of dihydroergotoxine, at the two doses of 2.5 mg/kg and 5 mg/kg decreases the binding of dopamine 3H-agonists to striatal membranes. By contrast the binding of dopamine 3H-antagonists is decreased in the animals treated with the higher dose and increased in those treated with the lower one. In old rats, in which a partial loss of both 3H-antagonist and 3H-agonist binding sites is observed, the DHT treatment confirms to increase the binding of 3H-antagonists, without affecting that of 3H-agonists. Thus, aging and ergot alkaloids seem to discrimate between DA-agonist and DA-antagonist receptor sites suggesting that this receptors are separate entityes.

In rat hippocampal slices kept in Krebs-Henseleit medium, an increase of K+ ions to 12 mM potentiates the stimulation of phosphoinositide turnover elicited by carbachol and (+/-)-cis-methyldioxolane. Oxotremorine is inactive if tested in Krebs-Henseleit medium but it stimulates by 220% the phosphoinositide turnover when K+ is increased to 12 mM. The K+ facilitation of the carbachol stimulation of phosphoinositide turnover was blocked by pirenzepine, a muscarinic antagonist. This drug was equally potent in inhibiting the carbachol stimulation of phosphoinositide turnover both in normal and 12 mM K+ Krebs medium. This facilitatory effect of K+ appears to be preferential for muscarinic receptors, since it failed to increase the activation of phosphoinositide breakdown induced by norepinephrine and histamine. The K+ potentiation of the muscarinic stimulation of phosphoinositide turnover is not mediated by a release of one of the endogenous neurotransmitters stored in these slices because such a facilitation occurs in Ca2+-deprived Krebs-Henseleit medium and failed to occur following a depolarizing dose of veratrine. Our experiments excluded that K+ facilitates carbachol stimulation of phosphoinositide turnover because it modifies the binding characteristics of muscarinic receptors; however, they cannot exclude that K+ acts at the receptor transducer coupling.