Evaluation of the goat cellular immune response to rBtuB-Hia-FlgK peptides from Brucella melitensis.

Brucellosis is a zoonosis caused by Brucella; B. melitensis is the most prevalent species in goats and humans. Previously, three B. melitensis peptides, rBtuB-Hia-FlgK showed antigen-specific immune responses in rodent models. The goal of this study was to evaluate the goat Th1/Th2 immune response to B. melitensis peptides. Twenty-eight animals were separated into four groups and were immunized with the rBtuB-Hia-FlgK peptides cocktail, adjuvant, PBS and Rev-1 vaccine, respectively. Peripheral blood samples were collected on days 0, 15, and 80 post-inoculation. The CD4+ and CD8+ T cells proliferation, and cytokine production of the Th-1 (IL-2, IL-12, TNF-α, and IFN-γ) and Th-2 profiles (IL-4, IL-5, and IL-10) were evaluated. An increase of CD4+/CD8+ at 15 days post-vaccination was observed and continued until the 80th. In addition, the IFN-γ, TNF-α, and IL-2 mRNA expression were typically induced by the 15th day, but only IFN-γ levels were observed at day 80 post-immunization. Brucella pathogenesis is distinguished by the presence of a large amount of Th-1 cytokines. Although a reduced amount of IFN-γ in the culture supernatant was accurately detected compared with Rev-1 after 15 days, it could be influenced by the sampling schedule, as a higher cytokine production might be induced as early as the first-week post-vaccination. The results indicate that rBtuB-Hia-FlgK induced an immune response similar to the Rev-1 vaccine. The possible use of inert molecules with the unique ability to typically induce cellular response similar to attenuated vaccine represents an attractive option that should not be ruled out.

Experimental analyses of synergistic combinations of antibiotics with a recently recognised antibacterial agent, lacidipine.

The cardiovascular drug lacidipine (Lc) is known to possess antibacterial activity. Further potentiation of action is possible by synergism between Lc and an antibiotic or a non-antibiotic. The minimum inhibitory concentration (MIC) of antibiotics, Lc and other non-antibiotics were detected by the agar dilution technique in different bacteria. Synergism was determined by disc diffusion assay, the fractional inhibitory concentration (FIC) index through checkerboard assessment and, also, the protective capacity of the combination by administering the drugs along with 50 x LD(50) challenge dose of virulent Salmonella typhimurium in animal experiments. Synergism between Lc and penicillin was found to be statistically significant (P

Release of copper from CuT 380A co-incubated with human semen and its effect on sperm function in vitro.

Release of copper and its effect on functional integrity of human sperms in vitro were assessed following co-incubation of semen with CuT 380A. After 30 min of incubation with semen, release of copper ions from CuT 380A was found to be 9.2 to 40 times higher compared to control incubations with PBS. Sperm function tests, when simultaneously performed following loss of motility in sperms (> 95%) after 120 min of copper exposure, depicted a significant (P < 0.001) reduction in sperm viability and hypo-osmotic swelling (HOS) response. However, the affected sperm populations revealed no significant alterations in other functional tests like acrosomal status or nuclear chromatin decondensation. It is therefore concluded that the high release of copper from CuT 380A drastically lowers sperm motility, viability and HOS response but only marginally affects the acrosome status or nuclear chromatin condensation in short term incubations.

Germ cell death and their removal during initial stages of testicular ischemia and cryptorchidism: a comparative analysis.

Germ cell death and their removal from the seminiferous epithelium are common in the affected testis in conditions of unilateral ischemia or cryptorchidism; the similarities and differences, however, have not been studied between these two conditions. The present study was designed to examine the severity of the effect on testicular germ cells during the initial stages of both ischemia and cryptorchidism, which have significant implications on the restoration of fertility following surgical repair. Complete absence of spermatids was observed following 12 hr of ischemia as compared to 7 days of cryptorchidism. Germ cell removal in either case was in the direction of lumen to basement membrane leaving only a single layer of cells by 24 hr of unilateral ischemia as compared to 15 days of cryptorchidism. Levels of intratesticular testosterone was found lower in cryptorchidism (7 days) but not in ischemia till 24 hrs. Giant cells frequently observed in cryptorchid testis were absent in the ischemic seminiferous epithelium. There was a gradual increase in the number of apoptotic and non-viable cells; the latter was more than 95% by 24 hr of ischemia. In contrast, approximately 85% testicular cells were nonviable till 15 days of cryptorchidism. The 1c peak representing the population of haploid cells was significantly reduced in cryptorchidism (7 days), while the peak was completely abolished by 24 hr of ischemia. Rise in the levels of oxidative stress in the affected testis was observed identically during the initial stages. These findings indicate that coupled with the rise in tissue oxidative stress, the number of apoptotic/nonviable germ cells was alarmingly high (> 80%) by 15 days of cryptochidism or 24 hr of ischemia. Restoration of complete spermatogenesis following surgical repair may not be possible in such cases because of these acute adverse effects.

Alteration of metabolism of acetylcholine induced by 2-deoxy-D-glucose in the gastroduodenum of the rat.

2-Deoxy-D-glucose (2-DG) administered intraperitoneally, dose-dependently increased the secretion of gastric acid, and the changes were comparable with those on the activity of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) in the stomach. Double-reciprocal plot analysis of the increased activity of CAT and AChE, induced by 2-DG, showed that the changes were due to the increase of Vmax, with no change in the Km-value for the substrates. The uptake of [3H]choline and subsequent synthesis of [3H]ACh was observed in the forestomach, corpus and antrum of the stomach and in the duodenum. 2-Deoxy-D-glucose significantly increased the uptake of [3H]choline and synthesis of [3H]ACh in every region of the stomach and in the duodenum, in a dose-dependent manner. The increase of secretion of gastric acid, induced by 2-DG paralleled that of uptake of [3H]choline and synthesis of [3H]ACh at an early stage. The conversion of [3H]choline taken up to [3H]ACh was negligibly influenced by 2-DG. Neither the content of ACh and choline, nor the turnover rate of ACh, were changed by administration of 2-DG. 2-Buten-4-olide (2-B4O), which inhibits the activity of the vagus nerve through the central nervous system, prevented 2-DG-induced uptake of [3H]choline and subsequent synthesis of [3H]ACh, as well as the increase in secretion of gastric acid. These results suggest that the uptake of [3H]choline and subsequent synthesis of [3H]ACh are closely related to the neuronal activity of the vagus nerve, and that cholinergic neuronal activity is dependent upon quantitative changes of metabolism of ACh in the gastroduodenum.

In vivo receptor occupancy of NRA0045, a putative atypical antipsychotic, in rats.

We have previously reported that (R)-(+)-2-amino-4-(4-fluorophenyl)-5-[1-[4-(4-fluorophenyl)-4-oxobutyl]+ ++pyrrolidin-3-yl]thiazole (NRA0045) is a novel antipsychotic agent with affinities for dopamine D4, 5-hydroxytryptamine 2A (5-HT2A) and alpha1 receptors. In the present study, in vivo receptor occupancy of 5-HT2A, alpha1, dopamine D2 and D3 receptors by NRA0045 was assessed, based on in vivo and ex vivo receptor binding, and findings were compared to reference antipsychotic drugs (haloperidol, risperidone, clozapine). Intraperitoneal administration of haloperidol highly occupied the dopamine D2 receptor in the striatum and nucleus accumbens, and alpha1 adrenoceptors in the frontal cortex. Occupation of the 5-HT2A receptor in the frontal cortex and the dopamine D3 receptor in the nucleus accumbens and islands of Cajella was moderate. By contrast, atypical antipsychotics such as risperidone and clozapine dose-dependently occupied the 5-HT2A receptor in the frontal cortex, with moderate to negligible occupancy of the D2 receptor in the striatum and the nucleus accumbens. Clozapine and risperidone also occupied the alpha1 adrenoceptor in the frontal cortex, and clozapine did not occupy the dopamine D3 receptor. As seen with other atypical antipsychotics, intraperitoneal administration of NRA0045 dose-dependently occupied the 5-HT2A receptor and the alpha1 adrenoceptor in the frontal cortex, while it was without effect on dopamine D2 and D3 receptors in the striatum, nucleus accumbens and islands of Cajella. Thus, the strong occupancy of 5-HT2A and alpha1 receptors is involved in the pharmacological action of NRA0045.

Synthesis and SAR of 1-alkyl-2-phenylethylamine derivatives designed from N,N-dipropyl-4-methoxy-3-(2-phenylethoxy)phenylethylamine to discover sigma(1) ligands.

The synthesis and structure-activity relationships (SAR) of 1-alkyl-2-phenylethylamine derivatives 5-8 designed from N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine hydrochloride (1, NE-100) are presented. The SAR between compound 1 and 1-alkyl-2-phenylethylamine derivatives suggested that the alkyl group on the 1-position carbon of 2-[4-methoxy-3-(2-phenylethyl)phenyl]ethylamine derivatives played the role of one of the propyl groups on the aminic nitrogen of compound 1. (-)-N-Propyl-1-butyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylam ine hydrochloride ((-)-6d, NE-537) and (-)-N-propyl-1-(3-methybutyl)-2-[4-methoxy-3-(2-phenylethoxy )phenyl]e thylamine hydrochloride ((-)-6i, NE-535), typical compounds in this series, have potent and selective sigma(1) affinity.

Design, synthesis, structure-activity relationships, and biological characterization of novel arylalkoxyphenylalkylamine sigma ligands as potential antipsychotic drugs.

sigma Receptor antagonists may be effective antipsychotic drugs that do not induce motor side effects caused by ingestion of classical drugs such as haloperidol. We obtained evidence that 1-(2-dipropylaminoethyl)-4-methoxy-6H-dibenzo[b,d]pyran hydrochloride 2a had selective affinity for sigma receptor over dopamine D2 receptor. This compound was designed to eliminate two bonds of apomorphine 1 to produce structural flexibility for the nitrogen atom and to bridge two benzene rings with a -CH2O- bond to maintain the planar structure. In light of the evidence, N, N-dipropyl-2-(4-methoxy-3-benzyloxylphenyl)ethylamine hydrochloride 10b was designed. Since compound 10b had eliminated a biphenyl bond of 6H-dibenzo[b,d]pyran derivative 2a, it might be more released from the rigid structure of apomorphine 1 than compound 2a. The chemical modification of compound 10b led to the discovery that N, N-dipropyl-2- [4-methoxy-3-(2-phenylethoxyl)phenyl]ethylamine hydrochloride 10g (NE- 100), the best compound among arylalkoxyphenylalkylamine derivatives 3, had a high and selective affinity for sigma receptor and had a potent activity in an animal model when the drug was given orally. We report here the design, synthesis, structure-activity relationships, and biological characterization of novel arylalkoxyphenylalkylamine derivatives 3.

Synthesis, SARs, and pharmacological characterization of 2-amino-3 or 6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as potent, selective, and orally active group II metabotropic glutamate receptor agonists.

(+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (4, LY354740), a highly selective and orally active group II metabotropic glutamate receptor (mGluR) agonist, has increased interest in the study of group II mGluRs. Our interest focused on a conformationally constrained form of compound 4, because it appeared that the rigid form resulted in not only selectivity for group II mGluR but was orally active. Therefore, we introduced a fluorine atom to compound 4, based on the molecular size (close resemblance to hydrogen atom) and electronegativity (effects on the electron distribution in the molecule) of this atom and carbon-fluorine bond energy. Compound (+)-7 (MGS0008), the best compound among 3-fluoro derivatives 7-10, retained the agonist activity of compound 4 for mGluR2 and mGluR3 ((+)-7: EC(50) = 29.4 +/- 3.3 nM and 45.4 +/- 8.4 nM for mGluR2 and mGluR3, respectively; 4: EC(50) = 18.3 +/- 1.6 nM and 62.8 +/- 12 nM for mGluR2 and mGluR3, respectively) and increased the oral activity of compound 4 ((+)-7: ED(50) = 5.1 mg/kg and 0.26 mg/kg for phencyclidine (PCP)-induced hyperactivity and PCP-induced head-weaving behavior, respectively; 4: ED(50) = >100 mg/kg and 3.0 mg/kg for PCP-induced hyperactivity and PCP-induced head-weaving behavior, respectively). In addition, a compound [(3)H]-(+)-7 binding study using mGluR2 or 3 expressed in CHO cells was successful ((+)-7: K(i) = 47.7 +/- 17 nM and 65.9 +/- 7.1 nM for mGluR2 and mGluR3, respectively; 4: K(i) = 23.4 +/- 7.1 nM and 53.5 +/- 13 nM for mGluR2 and mGluR3, respectively). On the basis of a successful result of compound 7, we focused on the introduction of a fluorine atom on the C6 position of compound 4. (1R,2S,5R, 6R)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid ((-)-11) exhibited a high degree of agonist activity for group II mGluRs equal to that of compound 4 or 7 ((-)-11: K(i) = 16.6 +/- 5.6 and 80.9 +/- 31 nM for mGluR2 and mGluR3, respectively). Our interest shifted to modification on CH(2) at C4 position of compound 11, since replacement of the CH(2) group with either an oxygen atom or sulfur atom yielded compound 5 or 6, resulting in increased agonist activity. We selected a carbonyl group instead of CH(2) at the C4 position of compound 11. The carbonyl group might slightly change the relative conformation of three functional groups, the amino group and two carboxylic acids, which have important roles in mediating the interaction between group II mGluRs and their ligand, compared with the CH(2) group of 4, oxygen atom of 5, and sulfur atom of 6. (1R,2S,5S,6S)-2-Amino-6-fluoro-4-oxobicyclo[3.1. 0]hexane-2,6-dicarboxylic acid monohydrate ((+)-14, MGS0028) exhibited a remarkably high degree of agonist activity for mGluR2 (K(i) = 0.570 +/- 0.10 nM) and mGluR3 (K(i) = 2.07 +/- 0.40 nM) expressed in CHO cells but not mGluR4, 6, 7, 1a, or 5 expressed in CHO cells (K(i) = >100 000 nM). Furthermore, compound (+)-14 strongly inhibited phencyclidine (PCP)-induced head-weaving behavior (ED(50) = 0.090 microg/kg) and hyperactivity (ED(50) = 0.30 mg/kg) in rats. Thus, (+)-7 and (+)-14 are potent, selective, and orally active group II mGluR agonists and might be useful not only for exploring the functions of mGluRs but in the treatment of schizophrenia.

The atypical antipsychotic profile of NRA0045, a novel dopamine D4 and 5-hydroxytryptamine2A receptor antagonist, in rats.

1. The atypical antipsychotic profile of (R)-(+)-2-amino-4-(4-fluorophenyl)-5-[1-[4-(4-fluorophenyl)-4-oxobutyl] pyrrolidin-3-yl] thiazole (NRA0045), a potent dopamine D4 and 5-hydroxytryptamine (5-HT)2A receptor antagonist, was examined in rats. 2. Spontaneous locomotor activity was decreased dose-dependently with i.p. administration of clozapine (ED50 3.7 mg kg-1), haloperidol (ED50 0.1 mg kg-1) and chlorpromazine (ED50 0.9 mg kg-1), whereas inhibition of this type of behaviour induced by i.p. administration of NRA0045, at doses up to 10 mg kg-1, did not exceed 50%. 3. Locomotor hyperactivity induced by methamphetamine (MAP, 2 mg kg-1, i.p.) in rats (a model of antipsychotic activity) was dose-dependently antagonized by NRA0045 (ED50 0.4 mg kg-1, i.p., and 0.3 mg kg-1, p.o., respectively), clozapine (ED50 0.3 mg kg-1, i.p. and 0.8 mg kg-1, p.o., respectively), haloperidol (ED50 0.02 mg kg-1, i.p. and 0.1 mg kg-1, p.o., respectively), chlorpromazine (ED50 0.3 mg kg-1, i.p. and 3.3 mg kg-1, p.o., respectively). In contrast, the MAP (3 mg kg-1, i.v.)-induced stereotyped behaviour in rats (a model of extrapyramidal symptoms) was not affected by NRA0045 or clozapine, at the highest dose given (30 mg kg-1, i.p.). Haloperidol (ED50 0.3 mg kg-1, i.p.) and chlorpromazine (ED50 4.8 mg kg-1, i.p.) strongly blocked the MAP-induced stereotyped behaviour. NRA0045 and clozapine selectively blocked behaviour associated with activation of the mesolimbic/mesocortical dopamine neurones rather than nigrostriatal dopamine neurones. 4. Extracellular single-unit recording studies demonstrated that MAP (1 mg kg-1, i.v.) decreased the firing rate in the substantia nigra (A9) and ventral tegmental area (A10) dopamine neurones in anaesthetized rats. NRA0045 completely reversed the inhibitory effects of MAP on A10 dopamine neurones (ED50 0.1 mg kg-1, i.v.), whereas the inhibitory effects of MAP on A9 dopamine neurones were not affected by NRA0045, in doses up to 1 mg kg-1 (i.v.). Clozapine completely reversed the inhibitory effects of MAP on A10 dopamine neurones (ED50 1.9 mg kg-1, i.v.) and on A9 dopamine neurones (ED50 2.5 mg kg-1, i.v.). Haloperidol completely reversed the inhibitory effects of MAP on A10 (ED50 0.03 mg kg-1, i.v.) and on A9 dopamine neurones (0.02 mg kg-1, i.v.). NRA0045, like clozapine, was more potent in reversing the effects of MAP on A10 than A9 dopamine neurones. 5. Prepulse inhibition (PPI) is impaired markedly in humans with schizophrenia. The disruption of PPI in rats by apomorphine (0.5 mg kg-1, s.c.) was reversed significantly by NRA0045 (3 mg kg-1, i.p.), clozapine (3 mg kg-1, i.p.) and haloperidol (0.3 mg kg-1, i.p.). 6. Phencyclidine (PCP) elicits predominantly psychotic symptoms in normal humans and in schizophrenics. NRA0045 (0.03-0.3 mg kg-1, i.p.) and clozapine (0.1-1 mg kg-1, i.p.) significantly and dose-dependently shortened the PCP(1.25 mg kg-1, i.p.)-induced prolonged swimming latency in rats in a water maze task, whereas haloperidol (0.01-0.1 mg kg-1, i.p.) did not significantly alter swimming latency. 7. These findings suggest that NRA0045 may have unique antipsychotic activities without the liability of motor side effects typical of classical antipsychotics.

Involvement of protein kinase C activation in regulation of acetylcholine release from rat hippocampal slices by minaprine.

Involvement of protein kinase C (PKC) activation in the regulation of acetylcholine (ACh) release from rat hippocampal slices by minaprine, [3(2-morpholinoethylamino)-4-methyl-6-phenylpyridazine] was investigated. Phorbol esters such as 4 beta-phorbol 12,13-dibutylate (PDBu) and 12-o-tetradecanoylphorbol 13-acetate (TPA) enhanced high K(+)-evoked [3H]ACh release from rat hippocampal slices. The enhancing effect of phorbol ester was attenuated by PKC inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). However, the inactive phorbol analogue, 4 alpha-phorbol 12,13-didecanoate (PDD) had no effect on [3H]ACh release. Minaprine and 4-aminopyridine (4-AP) attenuated the inhibitory effect of 5-hydroxytryptamine (5-HT) on high K(+)-evoked [3H]ACh release from rat hippocampal slices. The attenuating effect of minaprine on the inhibition of [3H]ACh release by 5-HT was prevented by staurosporine, while that of 4-AP was not influenced. Furthermore, PDBu blocked voltage-dependent, rapidly inactivating 42K efflux from rat brain synaptosomes. These results suggest that minaprine attenuates the inhibitory effect of 5-HT on ACh release via PKC activation in rat hippocampus, and that the blockade of the K+ channel with PKC activation might be involved in the action of minaprine.

Regulation of NMDA-induced [3H]dopamine release from rat hippocampal slices through sigma-1 binding sites.

To examine the interaction between ionotropic glutamate receptors and sigma binding sites, we made use of [3H]dopamine release from rat hippocampal slices. Agonists for ionotropic glutamate receptors such as N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate evoked release of [3H]dopamine from rat hippocampal slices, in a dose-dependent manner. (+)-Pentazocine, a prototype sigma1 agonist, attenuated the NMDA-induced [3H]dopamine release dose-dependently and significantly as did non-competitive NMDA antagonists such as 5-methyl-10,11-dihydro-5H-dibenzo(a,b)cyclohepten-5,10-imine maleate (MK-801) and phencyclidine. In contrast, (+)-pentazocine had no effect on AMPA- or on kainate-induced [3H]dopamine release. Sigma-1 receptor antagonists including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl] ethylamine monohydrochloride (NE-100), 1(cyclopropylmethyl)-4-(2'-(4"-fluorophenyl)-2'-oxoethylpiperidine (DuP734) and 1-(cyclopropylmethyl)-4-(2',4"-cianophenyl)-2'-oxoethyl)-pip eridine hydrobromide (XJ448) prevented significantly the inhibitory effect of (+)-pentazocine on NMDA-induced [3H]dopamine release, without affecting the release of [3H]dopamine evoked by NMDA. The inhibitory effect of (+)-pentazocine on [3H]dopamine release was preserved even in the presence of tetrodotoxin. These results suggest that sigma1 binding sites selectively interact with the NMDA receptor channel complex among ionotropic glutamate receptors, and that sigma1 binding sites may be involved in modulating the release of dopamine in the rat hippocampus by interacting with the NMDA receptor on dopaminergic nerve terminal.

Purification and partial characterization of K+ channel blockers from the venom of Dendroaspis angusticeps.

Two polypeptides (designated DTX-A and DTX-B) were purified from crude snake venom of Dendroaspis angusticeps using gel filtration, cation exchange column chromatography and cation exchange high performance liquid chromatography, and their blocking actions of K+ channels were investigated in rat brain synaptosomes. Both DTX-A and DTX-B inhibited the voltage-dependent 42K efflux from the synaptosomes. DTX-A blocked 42K efflux of both the rapidly inactivating phase (component T) and the slowly inactivating phase (component S). The inhibitory effect of DTX-A on component T was pronounced compared with that on component S. However, DTX-B selectively blocked 42K efflux of component S. The molecular weights of DTX-A and DTX-B were estimated to be ca 10,000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition of these toxins is different from that of polypeptide purified from the venom of D. angusticeps (alpha-, beta-, gamma- and delta-DTX). These results suggest that DTX-A and DTX-B are new polypeptides which block voltage-dependent K+ channels selectively, and that they are useful tools for investigating the K+ channel.

Attenuation of serotonin-induced suppression of [(3)H]acetylcholine release from rat cerebral cortex by minaprine: Possible involvement of the serotonin-2 receptor and K(+) channel.

5-Hydroxytryptamine (serotonin, 5-HT) and 5-HT(2) receptor agonist 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) inhibited K(+)-induced [(3)H]acetylcholine ([(3)H]ACh) release from slices and crude synaptosomes of rat cerebral cortex. Minaprine [3(2-morpholinoethylamino)-4-methyl-6-phenylpyridazine] and the 5-HT(2) receptor antagonists, ketanserin and methysergide, had no effect on [(3)H]ACh release alone. However, they attenuated the inhibition of [(3)H]ACh release by 5-HT and DOB. The attenuating effect of 5-HT was not mimicked by the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), neither was it prevented by the 5-HT(1A) and 5-HT(1B) mixed receptor antagonist, propranolol, or by the 5-HT(3) receptor antagonist, cocaine. Minaprine inhibited [(3)H]ketanserin binding more strongly than [(3)H]5-HT binding in the cerebral cortex. The inhibitory effect of minaprine on [(3)H]ketanserin binding was noncompetitive in Scatchard plot analysis. The inhibitory effect of 5-HT on [(3)H]ACh release was also suppressed by the voltage-dependent K(+) channel blockers, phencyclidine, tetraethylammonium (TEA) and 4-aminopyridine, but was not suppressed by the Ca(2+)-dependent K(+) channel blocker, apamin. These results suggest that minaprine suppresses the inhibitory effect of 5-HT on ACh release in the cerebral cortex by blocking the 5-HT(2) receptor and the voltage-dependent K(+) channel on the nerve endings of cholinergic neurons.

Anxiety-like behavior in mice lacking the angiotensin II type-2 receptor.

The main biological role of angiotensin II type 2 receptor (AT2) has not been established. We made use of targeted disruption of the mouse AT2 gene to examine the role of the AT2 receptor in the central nervous system (CNS). AT2-deficient mice displayed anxiety-like behavior compared with wild-type mice. However, AT2-deficient mice showed no depressant-like activity and no change in hexobarbital-induced sleeping time as compared with findings in wild-type mice. Both noradrenergic and corticotropin-releasing factor (CRF) neuronal systems appear to be involved in this anxiety-like behavior. Diazepam, captopril (angiotensin I converting enzyme inhibitor), prazosin (alpha1 antagonist) reversed the anxiety-like behavior in these AT2-deficient mice, whereas yohimbine (alpha2 antagonist), phenylephrine (alpha1 agonist), clonidine (alpha2 agonist), isoproterenol (beta1/beta2 agonist), propranolol (beta1/beta2 antagonist) and alpha-helical CRF9-41 (CRF receptor antagonist) has no apparent effects on anxiety-like behavior in AT2-deficient mice. In addition, concentrations of plasma adrenocorticotropic hormone (ACTH) and corticosterone in AT2-deficient mice did not differ from these in wild-type mice, hence, there are probably no endocrine abnormalities involving the hypothalamic-pituitary-adrenal axis (HPA). The amygdala appears to play an important role in many of the responses to fear and anxiety. The number of [3H]prazosin but not [125I]CRF binding sites in the amygdala was significantly reduced in AT2-deficient mice. These findings indicate that the noradrenergic system is involved in mediating the anxiety-like behavior in AT2-deficient mice.

A newly found angiotensin II receptor subtype mediates cyclic GMP formation in differentiated Neuro-2A cells.

In search of the functional role of the newly found angiotensin II (Ang II) binding site which is expressed in differentiated Neuro-2A cells, we found that Ang II causes a marked stimulation of cGMP formation dose-dependently. The stimulation was blocked by the nonselective Ang II receptor antagonist [Sar1,Ile8]Ang II but not by the AT1 antagonist DuP 753 or the AT2 antagonist PD 123319. These results suggest that Ang II increased cGMP level via a new Ang II receptor subtype in differentiated Neuro-2A cells.

NE-100, a novel potent sigma ligand, preferentially binds to sigma 1 binding sites in guinea pig brain.

The selectivity of N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) for sigma 1 and sigma 2 binding sites was studied by means of binding of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine ([3H]DTG) in guinea pig brain. NE-100 inhibited [3H](+)-pentazocine binding to sigma 1 binding sites potently with an IC50 value of 1.54 +/- 0.26 nM while it had a weak effect on [3H]DTG binding to sigma 2 binding sites. The inhibitory effect of NE-100 on [3H](+)-pentazocine was 55 times more potent than that on [3H]DTG binding. These results suggest that NE-100 is a potent and selective ligand for sigma 1 binding sites.

Discrimination of (+)-3-PPP sites from DTG sites by FH-510, a novel potent sigma ligand, in rat brain.

The effect of 5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol monohydrochloride (FH-510) on the binding of sigma ligands such as [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([3H](+)-3-PPP) and [3H]1,3-di-o-tolylguanidine ([3H]DTG) to rat brain membranes was studied. The inhibitory effect of FH-510 on [3H](+)-3-PPP binding to membranes of rat brain was 260 times more potent than that on [3H]DTG binding. Scatchard plot analysis showed that FH-510 inhibited [3H](+)-3-PPP binding in a competitive manner, while the inhibitory effect of FH-510 on [3H]DTG binding was noncompetitive. These results suggest that (+)-3-PPP sites could be discriminated from DTG sites, and that FH-510 binds preferentially to (+)-3-PPP recognition sites in rat brain.

FH-510, a potent and selective ligand for rat brain sigma recognition sites.

FH-510 (5,8-dimethyl-4-(2-di-n-propylaminoethyl)carbazol) is a potent ligand for sigma ligand binding sites in rat brain membranes and has an IC50 value of 4.8 +/- 1.0 nM, which is comparable to that of haloperidol (2.2 +/- 0.2 nM). The high selectivity of FH-510 for sigma binding sites was evaluated by its lack of significant affinity for other binding sites, including those for dopamine D2 and phencyclidine. When administered to mice orally, FH-510 suppressed (+)-N-allylnormetazocine ((+)-SKF10,047)-induced stereotyped behavior with an ED25 value of 0.54 mg/kg, which is 14-fold more potent than that for (+)-alpha-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine butanol ((+)-BMY14802) (7.6 mg/kg). These results suggest that FH-510 may be a potent and selective sigma ligand.

Neuropharmacological profiles of a novel atypical antipsychotic, NRA0562, in rats.

Neuropharmacological profiles of 5-(2-[4-(6-fluoro-1H-indole-3-yl) piperidine-1-yl] ethyl)-4-(4-fluorophenyl) thiazole-2-carboxylic acid amide (NRA0562) in rats were examined using electrophysiological and immunohistochemical methods. The firing rates of the substantia nigra pars compacta (A9) and the ventral tegmental area (A10) dopamine neurons were inhibited by methamphetamine (1 mg/kg, i.v.). NRA0562 dose-dependently reversed the inhibitory effects of methamphetamine on A9 and on A10 dopamine neurons. NRA0562 was more potent to reverse the inhibitory effects of methamphetamine on A10 (ED(50)=0.3 mg/kg) than on A9 (ED(50)=0.9 mg/kg) dopamine neurons. NRA0562 produced significant increases in Fos-like immunoreactivity in both the nucleus accumbens and the dorsolateral striatum. The difference between the number of Fos-like immunoreactivity produced by NRA0562 in the nucleus accumbens vs. dorsolateral striatum, referred to as the atypical index, was positive. Similar results could be observed with risperidone, an atypical antipsychotic. These results suggest that NRA0562 may have the atypical antipsychotic activities seen with risperidone, but without the liability of motor side effects typical of classical antipsychotics.