Receptors In Guinea Pig Brain Research Articles

Guinea pig hippocampal 5‐HT1E receptors: a tool for selective drug development

Recent studies have indicated that the serotonin [5-hydroxytryptamine (5-HT)] 1E receptor, originally discovered in human brain tissue, is not expressed in rat or mouse brain. Thus, there have been few reports on 5-HT(1E) receptor drug development. However, expression of 5-HT(1E) receptor mRNA has been shown in guinea pig brain. To establish this species as an animal model for 5-HT(1E) drug development, we identified brain regions that exhibit 5-carboxyamidotryptamine, ritanserin, and LY344864 - insensitive [(3)H]5-HT binding (characteristic of the 5-HT(1E) receptor). In hippocampal homogenates, where 5-HT(1E) receptor density was sufficiently high for radioligand binding analysis, 100 nM 5-carboxyamidotryptamine, 30 nM ritanserin, and 100 nM LY344864 were used to mask [(3)H]5-HT binding at non-5-HT(1E) receptors. The K(d) of [(3)H]5-HT was 5.7 +/- 0.7 nM and is indistinguishable from the cloned receptor K(d) of 6.5 +/- 0.6 nM. The affinities of 16 drugs for the cloned and hippocampal-expressed guinea pig 5-HT(1E) receptors are essentially identical (R(2) = 0.97). These findings indicate that using these conditions autoradiographical distribution and signal transduction studies of the 5-HT(1E) receptor in guinea pig brain are feasible. Using the guinea pig as an animal model should provide important insights into possible functions of this receptor and the therapeutic potential of selective human 5-HT(1E) drugs.

Irreversible blockade of sigma‐1 receptors by haloperidol and its metabolites in guinea pig brain and SH‐SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [(3)H](+)-pentazocine binding to sigma(1) receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P(1), P(2) and P(3) subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other sigma(1) antagonists or (-)-sulpiride), [(3)H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of sigma(1) receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-alpha-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked sigma(1) receptors in guinea pig brain homogenate and P(2) fraction in vitro. We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated sigma(1) receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P(2) fraction membranes, which suggests that HP is metabolized to inactivate sigma(1) receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of sigma(1) receptors in brain homogenates. These results suggest that HP may irreversibly inactivate sigma(1) receptors in guinea pig and human cells, probably after metabolism to reduced HP.

SB-699551-A (3-cyclopentyl- N-[2-(dimethylamino)ethyl]- N-[(4′-{[(2-phenylethyl)amino]methyl}-4-biphenylyl)methyl]propanamide dihydrochloride), a novel 5-ht 5A receptor-selective antagonist, enhances 5-HT neuronal function: Evidence for an autoreceptor role for the 5-ht 5A receptor in guinea pig brain

This study utilised the selective 5-ht 5A receptor antagonist, SB-699551-A (3-cyclopentyl- N-[2-(dimethylamino)ethyl]- N-[(4′-{[(2-phenylethyl)amino]methyl}-4-biphenylyl)methyl]propanamide dihydrochloride), to investigate 5-ht 5A receptor function in guinea pig brain. SB-699551-A competitively antagonised 5-HT-stimulated [ 35S]GTPγS binding to membranes from human embryonic kidney (HEK293) cells transiently expressing the guinea pig 5-ht 5A receptor (p A 2 8.1 ± 0.1) and displayed 100-fold selectivity versus the serotonin transporter and those 5-HT receptor subtypes (5-HT 1A/B/D, 5-HT 2A/C and 5-HT 7) reported to modulate central 5-HT neurotransmission in the guinea pig. In guinea pig dorsal raphe slices, SB-699551-A (1 μM) did not alter neuronal firing per se but attenuated the 5-CT-induced depression in serotonergic neuronal firing in a subpopulation of cells insensitive to the 5-HT 1A receptor-selective antagonist WAY-100635 (100 nM). In contrast, SB-699551-A (100 or 300 nM) failed to affect both electrically-evoked 5-HT release and 5-CT-induced inhibition of evoked release measured using fast cyclic voltammetry in vitro. SB-699551-A (0.3, 1 and 3 mg/kg s.c.) did not modulate extracellular levels of 5-HT in the guinea pig frontal cortex in vivo. However, when administered in combination with WAY-100635 (0.3 mg/kg s.c.), SB-699551-A (0.3, 1 or 3 mg/kg s.c.) produced a significant increase in extracellular 5-HT levels. These studies provide evidence for an autoreceptor role for the 5-ht 5A receptor in guinea pig brain.

Localization of NK 1 and NK 3 receptors in guinea-pig brain

In this study the localizations of tachykinin neurokinin-1 (NK 1) and neurokinin-3 (NK 3) receptors in the guinea-pig brain are described. In agreement with studies in rat and human brain, the neurons that exhibited the most marked NK 1 receptor immunoreactivity were found in the dorsomedial caudate putamen. NK 1 receptors were also widely distributed in diencephalic structures and in the mid and hind brain. NK 3 receptors were distributed in both superficial and deep layers of the cortex and many appeared to be located on cells with astrocyte-like morphology in the glia limitans. In several regions including the thalamus, hypothalamus, amygdala, periaqueductal gray, substantia nigra and area postrema, both NK 1 and NK 3 immunoreactivity were found. The present study revealed that tachykinin receptors are widely distributed in the guinea-pig central nervous system.

Autoradiographic quantitation and anatomical mapping of GTP sensitive-galanin receptors in the guinea pig central nervous system

Galanin is a 29-amino acid peptide widely distributed in the mammalian central nervous system. Galanin receptors in the guinea pig brain were visualized using [ 125I]galanin by in vitro receptor quantitative autoradiography. Scatchard analysis of [ 125I]galanin binding to slide-mounted sections revealed saturable binding to a single class of high affinity receptors with a K D of approximately 1 nM. Specific [ 125I]galanin binding sites were detected in a large number of brain areas (concentration range: from non detectable to 99.32 fmol/mg of tissular proteins). The anatomical mapping revealed high densities essentially in the telencephalon (e.g. lateral septal nuclei, amygdala, hippocampal dentate gyrus) and the diencephalon (e.g. the anterodorsal and medial habenular thalamic nuclei, the paraventricular, dorsomedian and median mammillary hypothalamic nuclei, the posterior lobe of the pituitary). Addition of Mg 2+ and GTP increased binding in some areas such as the zona incerta, the median eminence and the arcuate nucleus, and decreased it in other areas such as the amygdala, the hippocampus and the mammillary nuclei. This regional heterogeneity in the effect of Mg 2+ and GTP can be interpreted as: (1) different rates of galanin receptor occupancy by endogenous peptide; (2) a differential coupling of GTP binding proteins to galanin receptors in the brain structures; and (3) a different nature of receptors. At any rate, this study provides evidence for a specific GTP-sensitive galanin receptor in guinea pig brain with an extensive distribution suggesting various physiological implications. Comparison with studies performed in several mammals shows that the overall distribution of galanin receptors is well preserved among species. These data suggest that galanin may posses similar functional properties in the different species tested so far. Nevertheless, very distinct differences were found in some areas like the cortex, the hippocampus and the pituitary.

5-HT receptors in mammalian brain: receptor autoradiography and in situ hybridization studies of new ligands and newly identified receptors.

In recent years the family of mammalian serotonin receptors has grown to 14 different subtypes, characterized by pharmacological or molecular biological techniques. In parallel, new ligand molecules have been developed for their study. However, selective ligands are not yet available to study every one of them. In addition the degree of selectivity of ligands, hitherto regarded as specific for a particular receptor subtype has been called in question by their affinities for newly discovered receptors. Consequently, a re-evaluation of past ligand receptor autoradiography work is necessary in view of the redefined receptor profiles of these ligands, and the introduction of newly developed ligands. A further difficulty for the characterization of these receptors is the absence of selective antagonist ligands which, for some of the subtypes, have become available only recently. In an attempt to overcome these difficulties we have combined in situ hybridization histochemistry and receptor ligand autoradiography to study the regional and cellular localization of several serotonin receptors in the rodent brain. In addition, for some receptors, we have expanded these studies to primates, including humans. We have found that the distribution of 5-HT1A receptors in monkey brain, labelled with the agonist 3H-8-OH-DPAT and the antagonist 3H-WAY 100635 was very similar at the levels examined, and corresponded well with that observed for the cells containing mRNA coding for this receptor, confirming the somatodendritic localization of 5-HT1A receptors in monkey brain. The labelling conditions to visualize 5-HT1F receptors in guinea pig brain, namely 3H-sumatriptan in the presence of 10(-8) M 5-CT to block 5-HT1D receptors, are suitable for visualizing this receptor, since the results agreed with those observed by in situ hybridization. By using 3H-ketanserin and 3H-mesulergine in parallel with in situ hybridization using the corresponding oligonucleotides, we were able to show that these ligands label respectively 5-HT2A and 5-HT2C binding sites in monkey brain. 5-HT4 receptors were localized in the brain of several species including humans by using 125I-SB 207710. In situ hybridization experiments performed in guinea pig confirmed that 5-HT4 receptors are localized on the terminals of the striatopallidal and striatonigral projections. 5-HT7 binding sites were labelled in rat and guinea pig brains by incubating with 3H-5-CT in the presence of 100 microM WAY 100135 and 250 microM GR 127935; the distribution obtained in both species agreed, in general, with that of the corresponding mRNA coding for them. These results are an illustration of the understanding of our current knowledge of the chemical neuroanatomy of the mammalian 5-HT system.

Characterization of the human brain putative A2B adenosine receptor expressed in Chinese hamster ovary (CHO.A2B4) cells.

1. An [3H]-adenine pre-labelling methodology was employed to assay cyclic AMP generation by adenosine analogues in Chinese hamster ovary (CHO.A2B4) cells, transfected with cDNA which has been proposed to code for the human brain A2B adenosine receptor, and in guinea-pig cerebral cortical slices. 2. Adenosine analogues showing the following rank order of potency in the CHO.A2B4 cells (pD2 value): 5'-N-ethylcarboxamidoadenosine (NECA, 5.91) > adenosine (5.69) > 2-chloroadenosine (5.27) > N6-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.06). The purportedly A2A-selective agonist, CGS 21680, failed to elicit a significant stimulation of cyclic AMP generation at concentrations up to 10 microM in CHO.A2B4 cells. In the guinea-pig cerebral cortex, NECA was more potent than APNEA with pD2 values of 5.91 and 4.60, respectively. 3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity in CHO.A2B4 cells (ca. 10 fold stimulation of cyclic AMP), while, in comparison, maximal responses to adenosine (32% NECA response), 2-chloroadenosine (61%), and APNEA (73%) were reduced. 4. Antagonists of NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA2 value in CHO.A2B4 cells: guinea-pig cerebral cortex): XAC (7.89: 7.46) > CGS 15943 (7.75: 7.33) > DPCPX (7.16: 6.91) > PD 115,199 (6.95: 6.39) > 8FB-PTP (6.52: 6.55) > 3-propylxanthine (4.63: 4.59). 5. We conclude that, using the agents tested, the A2B adenosine receptor cloned from human brain expressed in Chinese hamster ovary cells exhibits an identical pharmacological profile to native A2B receptors in guinea-pig brain.

3H]LY303870, a novel nonpeptide radioligand for the NK-1 receptor.

We synthesized a potent and selective antagonist radioligand for the neurokinin (NK)-1 receptor and characterized its binding to guinea pig striatal membranes. (R)-N-[2-[Acetyl[3H3][(2-methoxyphenyl)-methyl]amino]- 1-(1H-indol-3-ylmethyl) ethyl][1,4'-bipiperidine]- 1'-acetamide ([3H]LY303870) binds to a single class of sites with an equilibrium KD of 0.22 nM and a Bmax of 723 fmol/mg of protein. Unlabeled LY303870 potently inhibited the binding with an IC50 of 0.56 nM, whereas the less active (S)-enantiomer (LY306155) was substantially less potent. The nonpeptide NK-1 antagonists (+/-)-CP96,345 and (+/-)-RP 67580 had IC50 values of 0.74 and 49 nM, respectively. Substance P (SP) was also a potent inhibitor with with an IC50 of 3.1 nM. The inhibition by SP could be separated into two components: a high-affinity component with a Ki of 0.53 nM and a lower-affinity component with a Ki of 155 nM. Addition of 100 microM guanylyl 5'-imidodiphosphate [Gpp(NH)p] in the incubation increased the relative amount of the low-affinity agonist state of the receptor. Consistent with the antagonist properties of LY303870, the dissociation rate of [3H]-LY303870 was not changed by the presence of 100 microM Gpp(NH)p. The distribution of [3H]LY303870 binding sites in the guinea pig brain closely matched the distribution of NK-1 receptors labeled by [3H]SP. Therefore, [3H]LY303870 is a potent and selective antagonist radioligand for NK-1 receptors in guinea pig brain. In addition, regulation of NK-1 agonist affinity by guanine nucleotides is similar to that seen for monoaminergic receptors.

Autoradiographic characterization of binding sites for [ 3H]NE-100 in guinea pig brain

The receptor binding specificity and neuroanatomical distribution of [ 3H]NE-100 (N, N- dipropyl-2- [4- methoxy-3- (2- phenylethoxy) phenyl] ethylamine monohydrochloride)-labeled sigma receptor in guinea pig brain were examined using quantitative autoradiography. NE-100 potently inhibited [ 3H]NE-100 binding to slide-mounted sections of guinea pig brain with the IC 50 value of 1.09 nM, therefore, NE-100 apparently has high affinity binding sites. Competition studies, under conditions similar to those used to visualize the receptor, yielded the following rank order of potency: NE-100 > haloperidol > DuP734 > (+)pentazocine ⪢ (−)pentazocine. Non-sigma ligands such as phencyclidine (PCP), MK-801 and (−)sulpiride had negligible affinities for [ 3H]NE-100 binding sites. High densities of [ 3H]NE-100 binding sites displaceable by haloperidol were present in the granule layer of the cerebellum, the cingulate cortex, the CA3 region of the hippocampus, the hypothalamus and the pons. The distribution of [ 3H]NE-100 binding sites was consistent with that of [ 3H](+)pentazocine, a sigma 1 ligand. These sigma sites may possibly be related to various aspects of schizophrenia.

Synthesis using a Fmoc-based strategy and biological activities of some reduced peptide bond pseudopeptide analogues of dynorphin A1.

Eight analogues of Dyn A(1-11)-NH2 incorporating the enzymatically stable psi(CH2-NH) isosteric peptide bond replacement were synthesized and tested for binding affinity at the central opioid mu, delta, and kappa receptors in guinea pig brain (GPB) homogenates and for activity at the peripheral kappa (and mu) receptors in the guinea pig ileum (GPI). The peptidic analogues were synthesized by solid phase techniques using a Fmoc/tert-butyl strategy, and the psi(CH2-NH) bond, or reduced bond, was introduced via reductive alkylation of the N-terminal amino group of the growing peptide with a Fmoc-N(alpha)-protected amino aldehyde. The synthesis of Fmoc-N(alpha)-protected amino aldehydes also is described. Several other peptides have been previously synthesized incorporating this modification and showed for instance increased enzymatic stability and antagonist properties. Results obtained in the GPB show that modifications of the peptide bond in the address site (analogues 4-9) do not affect the binding at the kappa receptor and, with a few exceptions, at the mu and delta receptors. On the other hand, analogues 2 and 3, modified in the message segment of Dyn A(1-11)-NH2, show a decrease in binding affinity at all three receptors. In the GPI, the results are more varied as the influence of the peptide bond modification seems to be more important than in the GPB. Finally, selected analogues were tested with no indication for antagonist activity at the kappa peripheral receptor.

Characterization and distribution of putative 5‐ht7 receptors in guinea‐pig brain

1. In the presence of (-)-cyanopindolol (1.0 microM) and sumatriptan (1.0 microM), 0.5 nM [3H]-carboxamidotrytamine ([3H]-5-CT) labelled a single population of receptors in guinea-pig cerebral cortex membranes. 2. 5-HT-displaceable binding was rapid, saturable and reversible. A high affinity binding site was characterized both by equilibrium saturation (Kd = 0.76 +/- 0.28 nM; Bmax = 68.1 +/- 26.7 fmol mg-1 protein) and kinetic (Kd = 0.18 +/- 0.05 nM) analysis. The pharmacological profile of this site was similar to the profile obtained in transfected CHO-K1 cells expressing guinea-pig 5-ht7 receptors. 3. Autoradiographic analysis revealed a discrete localization of binding sites in guinea-pig brain, with the highest density of sites in the medial thalamic nuclei and related limbic and cortical regions. Moderate levels of binding were detected in sensory relay nuclei, substantia nigra, hypothalamus, central grey and dorsal raphe nuclei. This distribution corresponded to that observed using in situ hybridization with [35S]-UTP labelled riboprobes complementary to mRNA encoding the guinea-pig 5-ht7 receptor. 4. In conclusion, under appropriate conditions, [3H]-5-CT labelled a single population of saturable binding sites that corresponded to an endogenous 5-ht7 receptor in guinea-pig brain. The distribution of 5-ht7 receptors in thalamocortical and limbic brain regions suggests a role for these receptors in sensory and affective behaviours.

Opioid peptide receptor studies. 3. Interaction of opioid peptides and other drugs with four subtypes of the kappa 2 receptor in guinea pig brain.

Using guinea pig, rat, and human brain membranes depleted of mu and delta receptors by pretreatment with the site-directed acylating agents BIT (mu selective) and FIT (delta selective), previous studies from our laboratory resolved two subtypes of the kappa 2 binding site, termed kappa 2a and kappa 2b. In more recent studies, we used 6 beta-[125Iodo]-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([125I]IOXY) to characterize multiple kappa 2 binding sites in rat brain. The results indicated that [125I]IOXY, like [3H]bremazocine, selectively labels kappa 2 binding sites in rat brain membranes pretreated with BIT and FIT. In the rat brain, using 100 nM [D-Ala2-MePhe4,Gly-ol5]enkephalin to block [125I]IOXY binding to the kappa 2b site, we resolved two subtypes of the kappa 2a binding site. In the present study we examined the binding of [125I]IOXY to the kappa 2 receptors of guinea pig brain. As observed in rat brain, [125I]IOXY, under appropriate assay conditions, selectively labels kappa 2 binding sites. Quantitative binding studies readily demonstrated the presence of kappa 2a and kappa 2b binding sites. The kappa 2a binding sites were selectively assayed using 5 microM [Leu5]enkephalin to block [125I]IOXY binding to the kappa 2b sites, and kappa 2b sites were selectively assayed using 5 microM (-)-(1S,2S)-U50,488 to block [125I]IOXY binding to the kappa 2a sites. Under these conditions, two subtypes of the kappa 2a site were resolved with high (kappa 2a-1) and low (kappa 2a-2) affinity for nor-BNI (Ki values = 0.88 and 476 nM) and CI977 (Ki values = 17.5 and 95,098 nM). Similarly, two subtypes of the kappa 2b site were observed with high (kappa 2b-1) and low (kappa 2b-2) affinity for [D-Ala2-MePhe4,Gly-ol5]enkephalin (DAMGO) (Ki values = 97 and 12,321 nM) and alpha-neoendorphin (Ki values = 33 and 5308 nM). Two-site models were also resolved in the presence of 100 microM 5'-guanylyimidodiphosphate (GppNHp). We carried out detailed ligand selectivity analysis of the multiple kappa 2 binding sites. Most test agents were either nonselective or selective for the kappa 2a-1 site. Nalbuphine was moderately selective for the kappa 2a-2 site. Similarly, although most test agents were either nonselective or selective for the kappa 2b-1 site, butorphanol, and the delta antagonists naltrindole, naltriben, and 7-benzylidene-7-dehydronaltrexone were moderately selective for the kappa 2b-2 site.(ABSTRACT TRUNCATED AT 400 WORDS)

Dihydrocodeinone-hydrazone, dihydrocodeinone-oxime, naloxone-3-OMe-oxime, and clocinnamox fail to irreversibly inhibit opioid kappa receptor binding.

Previous work from our lab identified two subtypes of the opioid kappa receptor. Whereas the kappa1 receptor can be labeled by [3H]U69,593 (5 alpha,7 alpha,8 beta-(-)- N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-phenyl- benzeneacetamide), the kappa2 receptor can be labeled by [125I]OXY (6 beta-125iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan). Other data demonstrate that [125I]IOXY, like [3H]bremazocine, labels two populations of kappa2 receptors in guinea pig brain: kappa2a and kappa2b binding sites. In the present study, we tested the hypothesis that certain dihydrocodeinone and oxicodone derivatives, which have been shown to irreversibly block low affinity [3H]naloxone binding sites, would also bind irreversibly to opioid kappa receptor subtypes. We also tested the novel irreversible mu receptor antagonist, clocinnamox (14 beta-(p-chlorocinnamoylamino)-7,8-dihydro-N-cyclopropylmethylno rmorphinone mesylate). Wash-resistant inhibition (WRI) assays were conducted to detect apparent irreversible inhibition. The proportion of WRI attributable to inhibition of receptor binding, termed receptor inhibition (RI), was calculated by the equation: RI = WRI (wash-resistant inhibition) - SI (supernatant inhibition or inhibition attributable to residual drug.) Dihydrocodeinone-hydrazone, dihydrocodeinone-oxime and naloxone-3-OMe-oxime failed to produce any wash-resistant inhibition of kappa receptor binding. In contrast, preincubating guinea pig membranes with 1 microM clocinnamox produced a substantial degree of wash-resistant inhibition (greater than 90%) at kappa1 and kappa2 binding sites. However, as indicated by supernatant inhibition values of 70% to 90%, there was a large amount of residual clocinnamox which remained despite the use of an extensive washing procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor binding profiles of KB-5492, a novel anti-ulcer agent, at σ receptors in guinea-pig brain

We studied the receptor binding profile of 4-methoxyphenyl 4-(3,4,5-trimethoxybenzyl)-1-piperazine acetate monofumarate monohydrate (KB-5492), a novel anti-ulcer agent, for the σ receptor in guinea-pig brain membranes. KB-5492 selectively inhibited specific [ 3H]1,3-di(2-tolyl)guanidine (DTG) binding to the σ receptor ( IC 50 = 3.15 μ M ) with a pseudo-Hill coefficient of 0.3. Computer-assisted analysis revealed that KB-5492 bound to high- and low-affinity sites. Although KB-5492 had weak affinity for α 2- adrenoceptors at 10 μM, it was almost inactive at a concentration of 10 μM in 33 other binding assays for receptors, second messenger system and ion channels. σ Receptor ligands such as haloperidol, DTG, (+)-3-(3-hydroxyphenol)- N-(1-propyl)piperidine (3-PPP), rimcazole and (−)-3-PPP inhibited specific [ 3H]DTG binding and their IC 50 values were 0.003, 0.044, 0.33, 0.67 and 1.03 μM, respectively. On the other hand, various anti-ulcer agents such as cetraxate, cimetidine, omeprazole, sofalcone, sucralfate, teprenone and troxipide could hardly displace specific [ 3H]DTG binding at 100 μM. Scatchard-Rosenthal analysis indicated that [ 3H]DTG bound to a single site, and K D and B max values for [ 3H]DTG were 87 .3 nM and 679.3 fmol/mg protein, respectively. KB-5492 significantly decreased the B max value, but did not affect the K D value . In contrast, haloperidol and DTG significantly increased the K D values, but did not affect the B max values. These findings indicate that KB-5492 selectively bound to the [ 3H]DTG-labeled σ receptor and that other anti-ulcer agents had little affinity for the σ receptor. Furthermore, KB-5492 inhibited specific [ 3H]DTG binding in a non-competitive manner.

3H]SNF8702 autoradiography of CCK-B receptors in guinea pig brain and studies with a cloned rat CCK-B receptor.

3H]SNF8702 autoradiography of CCK-B receptors in guinea pig brain and studies with a cloned rat CCK-B receptor.

Inhibitory effects of NaCl and guanyl-5'yl-imidodiphosphate (GppNHp) on [3H]naloxone binding to kappa-opioid receptors in guinea pig cerebellum.

Studies were performed to characterize the opioid receptors in guinea pig brain using the radiolabeled opioid antagonists, [3H]naloxone and [3H]diprenorphine and the kappa-agonist [3H]U-69593. The binding of [3H]U-69593 to guinea pig cerebellar membranes was reduced by NaCl, guanyl-5'yl-imidodiphosphate (GppNHp) and NaCl+GppNHp, and [3H]naloxone binding to cerebellar membranes was also reduced by NaCl and GppNHp. In the guinea pig cerebral cortex and striatum and the rat cerebellum, [3H]naloxone binding was not affected significantly by GppNHp in the presence or absence of 100 nM [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) and [D-Ala2, D-Leu5]enkephalin (DADLE). Guinea pig cerebellar [3H]diprenorphine binding was not affected by NaCl, GppNHp or NaCl+GppNHp. Furthermore, [3H]naloxone binding was reduced after pretreating cerebellar membranes with N-ethylmaleimide (NEM), which also attenuated GppNHp-induced inhibition of cerebellar [3H]naloxone binding. These results suggest that the properties of [3H]naloxone binding in guinea pig cerebellum differ from those in other brain regions and rat cerebellum, and that the interaction of [3H]naloxone and [3H]U-69593, but not [3H]diprenorphine, with guinea pig cerebellar opioid receptors is associated with a G-protein.

Autoradiographic identification and characterization of σ receptors in guinea pig brain using [ 3H]1(cyclopropylmethyl)-4-(2′-(4≈-fluorophenyl)-2′-oxoethyl) piperidine ([ 3H]DuP 734), a novel σ receptor ligand

The psychotomimetric effects of certain cycloalkyls and benzomorphans that interact with σ receptors has led to the hypothesis that these sites may be important in the etiology of schizophrenia. DuP 734 [1-(cyclopropylmethyl)-4-(2′-(4≈-fluoro-phenyl)-2′-oxoethyl) piperidine HBr] is a novel σ receptor ligand. The receptor binding specificity and neuroanatomical distribution of [ 3H]DuP 734-labeled σ receptors in guinea pig brain were examined using quantitative autoradiography. [ 3H]DuP 734 binding (10 μM haloperidol displaceable) to slide-mounted sections of guinea pig brain was saturable and of high affinity ( K i = 3.9nM). Competition studies, under conditions identical to those used to visualize the receptor, yielded the following rank order of potency: DuP 734 > haloperidol > (+)-pentazocine > (−)-butaclamol > DTG > (+)-SKF 10,047 > (+)-3-PPP > (−)-pentazocine > (+)-butaclamol > U50,488H > (−)-SKF 10,047 > cinanserin > PCP ≫ MK801, sulpiride. High densities of [ 3H]DuP 734 binding sites displaceable by haloperidol were present in the limbic system, in particular the dorsal and ventral bands of Broca as well as the ventral pallidum. Within the hippocampus, the pyramidal layers were sparsely labeled, while higher densities of binding sites were evident in the dentate gyrus. The frontal cortex, the mammillary complex of the hypothalamus, the central gray and red nucleus of the midbrain, the pontine reticular nucleus, the Purkinje cell layer of the cerebellum and dorsal and ventral horns, as well as the central gray matter of the spinal cord, all showed enrichments of [ 3H]DuP 734 binding sites. Lower levels of binding were present in the other regions of the cerebral cortex including parietal, pyriform, occipital, cingulate cortex, as well as the basal ganglia, and negligible specific binding was present in the white matter tracts. The kinetic and pharmacological characteristics and distribution of [ 3H]DuP 734 binding sites in brain are similar to those previously reported for σ receptors.

Inhibition of Forskolin‐Stimulated Cyclic AMP Formation by 1‐Aminocyclopentane‐trans‐1,3‐Dicarboxylate in Guinea‐Pig Cerebral Cortical Slices

The effects of the selective metabotropic glutamate receptor agonist 1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) on forskolin-stimulated cyclic AMP formation in guinea-pig cerebral cortex slices were determined. t-ACPD inhibited the accumulation of [3H]cyclic AMP by approximately 80%, with an IC50 value of 35 +/- 4 microM. The effect was reversible and stereoselective, with the 1S,3R isomer being approximately 400-fold more potent than the 1R,3S isomer. L-Glutamate (over a restricted concentration range) also partially inhibited the response to forskolin, but quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and N-methyl-D-aspartate (NMDA) were ineffective. The effect of t-ACPD was not blocked by antagonists of the phospholipase C-linked metabotropic glutamate receptor, the AMPA ionotropic glutamate receptor, or the NMDA receptor. In summary, our results indicate the presence of a glutamate receptor in guinea-pig brain that is activated selectively by t-ACPD and that is negatively linked to adenylyl cyclase.

Specific Binding of [3H]Pyrilamine to Histamine H1 Receptors in Guinea Pig Brain In Vivo: Determination of Binding Parameters by a Kinetic Four‐Compartment Model

The binding of [3H]pyrilamine, a selective ligand of histamine H1 receptors, to guinea pig brain in vivo was compared with its binding to a brain homogenate. The pharmacological properties (regional distribution, saturability, and stereoselectivity) of the [3H]pyrilamine binding in vivo were similar to those of the in vitro binding to brain homogenate. A dynamic four-compartment model was proposed for the analysis of the kinetics of [3H]pyrilamine binding in vivo. The receptor constants in vivo were determined by a computer-fitting method after correcting the radioactivity of arterial plasma and brain for the presence of radioactive metabolites. The in vivo association and dissociation were 213 and 42 times, respectively, slower than those of in vitro binding at 37 degrees C. A possible mechanism for slow association and dissociation in vivo is discussed.

Down‐regulation of progestin receptors in guinea pig brain: New findings using an immunocytochemical technique

Progesterone injection in estradiol-primed, ovariectomized guinea pigs results in down-regulation of hypothalamic progestin receptors determined by in vitro binding assays. In order to determine if progesterone also decreases immunostaining of progestin receptors and if progestin receptors are down-regulated preferentially in particular neuroanatomical areas, ovariectomized guinea pigs were injected with doses of estradiol benzoate (10 micrograms at 42 h before progesterone injection) and progesterone (500 micrograms at 4, 12, or 24 h before perfusion) that reliably induce the expression of lordosis and subsequent behavioral refractoriness to progesterone. Progestin receptor-immunoreactive cells were counted in sections from discrete parts of the preoptic area and hypothalamus. As expected, estradiol dramatically increased cell nuclear, and, to a lesser extent, cytoplasmic, immunostaining in defined regions of the preoptic area and hypothalamus. By 12 h after progesterone injection, the number of progestin receptor-immunoreactive cells had decreased in some areas, but not others. The rostral and caudal aspects of the ventrolateral hypothalamus were particularly responsive showing a substantial decrease in progestin receptor-immunoreactivity by 12 h after injection. No decreases in the progestin receptor-immunoreactive cell number were observed in any of the preoptic regions examined, although obvious decreases in immunostaining intensity were seen. The results of these immunocytochemical experiments extend earlier findings from in vitro progestin binding experiments and demonstrate that as with progestin binding, progestin receptor-immunoreactivity decreases when progesterone is injected in a behavioral desensitization procedure. Furthermore, they point to the ventrolateral hypothalamus as one site in which the down-regulation of progestin receptors may be particularly responsive to progesterone.