Internalization and Recycling of the CB1 Cannabinoid Receptor

Tolerance develops rapidly to cannabis, cannabinoids, and related drugs acting at the CB1 cannabinoid receptor. However, little is known about what happens to the receptor as tolerance is developing. In this study, we have found that CB1 receptors are rapidly internalized following agonist binding and receptor activation. Efficacious cannabinoid agonists (WIN 55,212-2, CP 55,940, and HU 210) caused rapid internalization. Methanandamide (an analogue of an endogenous cannabinoid, anandamide) was less effective, causing internalization only at high concentration, whereas delta9-tetrahydrocannabinol caused little internalization, even at 3 microM. CB1 internalized via clathrin-coated pits as sequestration was inhibited by hypertonic sucrose. Internalization did not require activated G protein alpha(i), alpha(o), or alpha(s) subunits. A region of the extreme carboxy terminus of the receptor was necessary for internalization, as a mutant CB1 receptor lacking the last 14 residues did not internalize, whereas a mutant lacking the last 10 residues did. Steps involved in the recycling of sequestered receptor were also investigated. Recovery of CB1 to the cell surface after short (20 min) but not long (90 min) agonist treatment was independent of new protein synthesis. Recycling also required endosomal acidification and dephosphorylation. These results show that CB1 receptor trafficking is dynamically regulated by cannabimimetic drugs.     

Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB₁ receptor activity. Here we investigated the possibility of a negative regulation by CB₁ receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2′-chloroethylamide (ACEA) in a CB₁-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL^−/− mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB₁ activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.     

Imaging the Brain Marijuana Receptor: Development of a Radioligand that Binds to Cannabinoid CB1 Receptors In Vivo

Abstract: The major active ingredient of marijuana, (−)-Δ⁹-tetrahydrocannabinol, exerts its psychoactive effects via binding to cannabinoid CB1 receptors, which are widely distributed in the brain. Radionuclide imaging of CB1 receptors in living human subjects would help explore the presently unknown physiological roles of this receptor system, as well as the neurochemical consequences of marijuana dependence. Currently available cannabinoid receptor radioligands are exceedingly lipophilic and unsuitable for in vivo use. We report the development of a novel radioligand, [¹²³I]AM281{ N -(morpholin-4-yl)-5-(4-[¹²³I]iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide}, that is structurally related to the CB1-selective antagonist SR141716A [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide]. Baboon single photon emission computed tomography studies, mouse brain dissection studies, and ex vivo autoradiography in rat brain demonstrated rapid passage of [¹²³I]AM281 into the brain after intravenous injection, appropriate regional brain specificity of binding, and reduction of binding after treatment with SR141716A. AM281 has an affinity in the low nanomolar range for cerebellar binding sites labeled with [³H]SR141716A in vitro, and binding of [¹²³I]AM281 is inhibited by several structurally distinct cannabinoid receptor ligands. We conclude that [¹²³I]AM281 has appropriate properties for in vivo studies of cannabinoid CB1 receptors and is suitable for imaging these receptors in the living human brain.     

Relationships Between Ligand Affinities for the Cerebellar Cannabinoid Receptor CB1 and the Induction of GDP/GTP Exchange

The hypothesis of these studies is that ligand efficacy at the neuronal CB1 receptor is dependent on the ratio of ligand affinities for the active and inactive states of the receptor. Agonist efficacy was determined in rat cerebellar membranes using agonist-induced guanosine 5'-O-(3-[35S]thiotriphosphate) binding; efficacy was variable among the CB1 agonists examined. Ligand affinities for the active and inactive state of the CB1 receptor were determined by competition with [3H]CP55940 and [3H]SR141716A in the presence of 5'-guanylylimidodiphosphate, respectively. All of the agonists investigated had a higher affinity for the active state than the inactive state. The fraction of CB1 receptors in the active state at a maximally effective concentration was calculated for each agonist and was found to correlate significantly with agonist efficacy. These studies demonstrate that the CB1 receptor of the cerebellum can assume an active conformation in the absence of agonist and that the variability in efficacy among CB1 receptor agonists can be explained by the relative affinities of these ligands for the CB1 receptor in the active and inactive states.     

Pyrazole antagonists of the CB1 receptor with reduced brain penetration

Type 1 cannabinoid receptor (CB1) antagonists might be useful for treating obesity, liver disease, metabolic syndrome, and dyslipidemias. Unfortunately, inhibition of CB1 in the central nervous system (CNS) produces adverse effects, including depression, anxiety and suicidal ideation in some patients, which led to withdrawal of the pyrazole inverse agonist rimonabant (SR141716A) from European markets. Efforts are underway to produce peripherally selective CB1 antagonists to circumvent CNS-associated adverse effects. In this study, novel analogs of rimonabant (1) were explored in which the 1-aminopiperidine group was switched to a 4-aminopiperidine, attached at the 4-amino position (5). The piperidine nitrogen was functionalized with carbamates, amides, and sulfonamides, providing compounds that are potent inverse agonists of hCB1 with good selectivity for hCB1 over hCB2. Select compounds were further studied using in vitro models of brain penetration, oral absorption and metabolic stability. Several compounds were identified with predicted minimal brain penetration and good metabolic stability. In vivo pharmacokinetic testing revealed that inverse agonist 8c is orally bioavailable and has vastly reduced brain penetration compared to rimonabant.     

Characterization of CB1 Receptors on Rat Neuronal Cell Cultures: Binding and Functional Studies Using the Selective Receptor Antagonist SR 141716A

Abstract: This study was undertaken to characterize further the central cannabinoid receptors in rat primary neuronal cell cultures from selected brain structures. By using [³H]SR 141716A, the specific CB1 receptor antagonist, we demonstrate in cortical neurons the presence of a high density of specific binding sites ( B _max = 139 ± 9 fmol/mg of protein) displaying a high affinity ( K _D = 0.76 ± 0.09 n M ). The two cannabinoid receptor agonists, CP 55940 and WIN 55212-2, inhibited in a concentration-dependent manner cyclic AMP production induced by either 1 µ M forskolin or isoproterenol with EC₅₀ values in the nanomolar range (4.6 and 65 n M with forskolin and 1.0 and 5.1 n M with isoproterenol for CP 55940 and WIN 55212-2, respectively). Moreover, in striatal neurons and cerebellar granule cells, CP 55940 was also able to reduce the cyclic AMP accumulation induced by 1 µ M forskolin with a potency similar to that observed in cortical neurons (EC₅₀ values of 3.5 and 1.9 n M in striatum and cerebellum, respectively). SR 141716A antagonized the CP 55940- and WIN 55212-2-induced inhibition of cyclic AMP accumulation, suggesting CB1 receptor-specific mediation of these effects on all primary cultures tested. Furthermore, CP 55940 was unable to induce mitogen-activated protein kinase activation in either cortical or striatal neurons. In conclusion, our results show nanomolar efficiencies for CP 55940 and WIN 55212-2 on adenylyl cyclase activity and no effect on any other signal transduction pathway investigated in primary neuronal cultures.     

线粒体大麻素受体1在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响

目的:探讨线粒体CB1受体(mitochondrial cannabinoid receptor1,mtCB1)在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响。方法:原代培养新生的Wistar大鼠海马神经元,将培养至第8天的海马神经元采用随机数字表分为5组(n=60):正常组(N组):正常培养,不做任何处理;缺氧复氧组(H/R组):采用氧糖剥夺法构建海马神经元缺氧复氧损伤模型,缺氧6h,复氧20 h;缺氧复氧组+ACEA+AM251组(H/R+ACEA+AM251组):缺氧6 h结束后立即加入ACEA和AM251,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+ACEA+Hemopressin(H/R+ACEA+Hemo组):缺氧6h结束后立即加入ACEA和Hemopressin,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+赋形剂组(H/R+V组):同样于缺氧6h结束后立即加入二甲基亚砜(DMSO),终浓度0.1%,复氧20 h。使用激光共聚焦显微镜检测细胞内Ca~(2+)的浓度,流式细胞仪检测细胞凋亡率,Western blot检测凋亡诱导因子(AIF)、线粒体分裂相关蛋白Drp1、Fis1,细胞凋亡相关蛋白细胞色素C(Cytc)和Rho相关的卷曲蛋白激酶1(ROCK1)的表达。结果:与N组相比,H/R组、H/R+ACEA+AM251组、H/R+ACEA+Hemo组和H/R+V组的细胞内Ca~(2+)浓度、细胞凋亡率、以及AIF、Drp1、Fis1、Cytc、ROCK1蛋白的表达水平均明显增加(P0.05);与H/R组相比,H/R+ACEA+Hem组上述各检测指标明显降低(P0.05),H/R+ACEA+AM251组和H/R+V组各指标比较差异无统计学意义(P0.05)。结论:线粒体CB1受体(mtCB1受体)可能通过降低细胞内ROS的含量来减少细胞内Ca~(2+)浓度和ROCK1的表达,进而抑制线粒体分裂,并最终减轻海马神经元缺氧复氧损伤。     

The role of endogenous cannabinoids in the hypothalamo-pituitary-adrenal axis regulation: in vivo and in vitro studies in CB1 receptor knockout mice

Exogenous cannabinoids affect multiple hormonal systems including the hypothalamo-pituitary-adrenocortical (HPA) axis. These data suggest that endogenous cannabinoids are also involved in the HPA control; however, the mechanisms underlying this control are poorly understood. We assessed the role of endogenous cannabinoids in the regulation of the HPA-axis by studying CB1 receptor knockout (KO) and wild type (WT) mice. Basal and novelty stress-induced plasma levels of adrenocorticotropin (ACTH) and corticosterone were higher in CB1-KO than in WT mice. We investigated the involvement of the pituitary in the hormonal effects of CB1 gene disruption by studying the in vitro release of ACTH from anterior pituitary fragments using a perifusion system. Both the basal and corticotropin releasing hormone (CRH)-induced ACTH secretion were similar in CB1-KO and WT mice. The synthetic glucocorticoid, dexamethasone suppressed the CRH-induced ACTH secretion in both genotypes; thus, the negative feedback of ACTH secretion was not affected by CB1 gene disruption. The cannabinoid agonist, WIN 55,212-2 had no effects on basal and CRH-stimulated ACTH secretion by anterior pituitary slices. In our hands, the disruption of the CB1 gene lead to HPA axis hyperactivity, but the pituitary seems not to be involved in this effect. Our data are consistent with the assumption that endogenous cannabinoids inhibit the HPA-axis via centrally located CB1 receptors, however the understanding of the exact underlying mechanism needs further investigation.     

Cannabinoid CB1 receptor knockout mice exhibit markedly reduced voluntary alcohol consumption and lack alcohol-induced dopamine release in the nucleus accumbens

The mechanisms underlying predisposition to alcohol abuse and alcoholism are poorly understood. In this study, we evaluated the role of cannabinoid (CB1) receptors in (i) voluntary alcohol consumption, and (ii) acute alcohol-induced dopamine (DA) release in the nucleus accumbens, using mice that lack the CB1 receptor gene (CB1-/-). CB1-/- mice exhibited dramatically reduced voluntary alcohol consumption, and completely lacked alcohol-induced DA release in the nucleus accumbens, as compared to wild-type mice. The gender difference, with female mice consuming significantly more alcohol than wild-type male mice, was observed in wild-type mice, whereas this gender difference was nonexistent in CB1 mutant male and female mice. There was also a significant gender difference, with the wild-type, heterozygous, and mutant females consuming significantly more liquid and food than wild-type, heterozygous and mutant males. However, the total volume of fluid consumption and food intake did not differ between wild-type, heterozygous, and mutant mice. These results strongly suggest that the CB1 receptor system plays an important role in regulating the positive reinforcing properties of alcohol.     

Distribution and Ontogeny of 1S,3R-1-Aminocyclopentane-1,3-Dicarboxylic Acid-Sensitive and Quisqualate-Insensitive [3H]Glutamate Binding Sites in the Rat Brain

Abstract: Displacement of [³H]glutamate by 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid and quisqualate (in the presence of saturating concentrations of ionotropic glutamate receptor agonists) was used to characterize optimal ionic conditions, distribution, and the ontogeny of glutamate receptor binding sites in rat brain. Using rat forebrain membranes or receptor autoradiography, optimal 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive [³H]glutamate binding was found in the presence of 100 m M bromide ions and in the absence of calcium ions. Under these conditions, [³H]glutamate binding was relatively quisqualate insensitive. In regions of the neonatal (11-day-old) and adult rat brain, this [³H]glutamate binding was highest in forebrain (striatum, cerebral cortex, and hippocampus) and hypothalamus/midbrain but was lower in the cerebellum, olfactory bulb, and pons/medulla regions. 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [³H]glutamate binding was present in the rat forebrain at 1 day of age and gradually increased more than twofold by day 50 (adult). Thus, in the presence of bromide ions and in the absence of calcium ions, [³H]glutamate labels a subpopulation of metabotropic glutamate receptors that are sensitive to 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid but insensitive to quisqualate. Expression of [³H]glutamate binding under these conditions was both regionally and developmentally regulated in rat brain, suggesting that [³H]glutamate is labeling a distinct population of metabotropic glutamate receptors.     

Pharmacological and Physicochemical Properties of Pre-Versus Postsynaptic 5-Hydroxytryptamine1A Receptor Binding Sites in the Rat Brain: A Quantitative Autoradiographic Study

Numerous data suggested that the pharmacological and biochemical properties of 5-hydroxytryptamine1A (5-HT1A) receptors exhibit some regional differences in the CNS, notably within the raphe nuclei compared with various forebrain areas (such as the hippocampus). This possibility has been further investigated in the dorsal raphe nucleus and two areas within the hippocampus, the dentate gyrus and the CA1 area, using the quantitative autoradiographic technique. The potencies of 5'-guanylylimidodiphosphate to inhibit the specific binding of 125I-Bolton-Hunter-8-methoxy-2-(N-propyl-N-propylamino)tetralin (125I-BH-8-MeO-N-PAT) to 5-HT1A sites and of N-ethylmaleimide to block these sites irreversibly were identical in the dorsal raphe nucleus and the hippocampal areas in rat brain sections. In contrast, slight but significant differences were noted in the pH dependence and pharmacological properties of 5-HT1A sites labeled by 125I-BH-8-MeO-N-PAT in these three regions. Similarly, heat denaturation experiments and tissue exposure to either phospholipase A2 or the alkylating agent 8-methoxy-2-(N-2'-chloropropyl,N-propyl)aminotetraline revealed regional differences in the properties of 5-HT1A sites. However, in most cases, the observed variations were of greater amplitude between the CA1 area and the dentate gyrus, where 5-HT1A sites are located postsynaptically, than between any one of these areas and the dorsal raphe nucleus where they act as (presynaptic) somatodendritic autoreceptors. These data further support that subtypes of 5-HT1A receptors probably exist in the rat brain, but this heterogeneity seems unrelated to the pre- or post-synaptic location of these receptors.     

Prolonged Treatment with Ligands Affects Ligand Binding to the Human Serotonin1A Receptor in Chinese Hamster Ovary Cells

SUMMARY 1. The serotonin_1A receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins, and appear to be involved in several behavioral and cognitive functions.2. We monitored the effect of prolonged treatment of the human serotonin_1A receptor expressed in Chinese hamster ovary (CHO) cells with pharmacologically well-characterized ligands on its binding to the agonist 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT) and antagonist 4-(2′-methoxy)-phenyl-1-[2′-(N-2″-pyridinyl)-p-fluorodobenzamido]ethyl-piperazine (p-MPPF).3. Our results indicate that prolonged treatment with the specific agonist (8-OH-DPAT) differentially affects subsequent binding of the agonist and antagonist to the receptor in a manner independent of receptor-G-protein coupling. Importantly, our results show that prolonged treatment with the commonly used antagonist p-MPPF, and its iodinated analogue 4-(2′-methoxy)-phenyl-1-[2′-(N-2″-pyridinyl)-p-iodobenzamido]ethyl-piperazine (p-MPPI), which have earlier been reported to display similar binding properties to serotonin_1A receptors, induces significantly different effects on the ligand binding function of serotonin_1A receptors.     

Farnesoid X Receptor, through the Binding with Steroidogenic Factor 1-responsive Element, Inhibits Aromatase Expression in Tumor Leydig Cells

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that regulates bile acid homeostasis. It is expressed in the liver and the gastrointestinal tract, but also in several non-enterohepatic tissues including testis. Recently, FXR was identified as a negative modulator of the androgen-estrogen-converting aromatase enzyme in human breast cancer cells. In the present study we detected the expression of FXR in Leydig normal and tumor cell lines and in rat testes tissue. We found, in rat Leydig tumor cells, R2C, that FXR activation by the primary bile acid chenodeoxycholic acid (CDCA) or a synthetic agonist GW4064, through a SHP-independent mechanism, down-regulates aromatase expression in terms of mRNA, protein levels, and its enzymatic activity. Transient transfection experiments, using vector containing rat aromatase promoter PII, evidenced that CDCA reduces basal aromatase promoter activity. Mutagenesis studies, electrophoretic mobility shift, and chromatin immunoprecipitation analysis reveal that FXR is able to compete with steroidogenic factor 1 in binding to a common sequence present in the aromatase promoter region interfering negatively with its activity. Finally, the FXR-mediated anti-proliferative effects exerted by CDCA on tumor Leydig cells are at least in part due to an inhibition of estrogen-dependent cell growth. In conclusion our findings identify for the first time the activators of FXR as negative modulators of the aromatase enzyme in Leydig tumor cell lines.     

Characterization of [3H]CP-96,501 as a Selective Radioligand for the Serotonin 5-HT1B Receptor: Binding Studies in Rat Brain Membranes

Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT_1B receptor than the commonly used 5-HT_1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [³H]CP-96,501, was found to bind with a high affinity ( K _D, 0.21 n M ) to a single binding site ( n _H, 1.0). The receptor density of this site ( B _max, 72 fmol/mg of protein) matched that of the 5-HT_1B receptor determined with [³H]5-HT. Competition curves of 16 serotonergic compounds in [³H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT_1B receptor determined with [³H]5-HT or [¹²⁵I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [³H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [³H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [¹²⁵I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [³H]CP-96,501 is a useful agonist radioligand for the 5-HT_1B receptor.     

The GTP-Insensitive Component of High-Affinity [3H]8-Hydroxy-2-(Di-n-Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5-Hydroxytryptamine1A Receptor

Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]-G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]-G protein complex.     

Hippocampal 8-[3H]Hydroxy-2-(Di-n-Propylamino)Tetralin Binding Site Densities, Serotonin Receptor (5-HT1A) Messenger Ribonucleic Acid Abundance, and Serotonin Levels Parallel the Activity of the Hypothalamopituitary-Adrenal Axis in Rat

We have previously demonstrated that susceptibility of the Lewis rat to inflammatory disease, compared with the relatively resistant Fischer F344/N rat, is related to a hyporesponsive hypothalamopituitary-adrenal axis to inflammatory and other stress mediators. Because serotonin (5-HT) and the 5-HT1A receptor are important stimulators of this axis, we have investigated the levels of 8-[3H]-hydroxy-2,3-(di-n-propylamino)tetralin binding sites, 5-HT1A mRNA, 5-HT, and 5-hydroxyindoleacetic acid in various brain regions of Lewis, outbred Harlan Sprague Dawley, and Fischer F344/N rats. Lewis rats expressed significantly fewer hippocampal and frontal cortical 8-[3H]-hydroxy-2,3-(di-n-propylamino)tetralin binding sites and less 5-HT1A mRNA than Harlan Sprague Dawley and Fischer F344/N rats. Adrenalectomy increased the number of 8-[3H]hydroxy-2,3-(di-n-propylamino)tetralin binding sites and 5-HT1A mRNA expression in the hippocampus of all three strains. Levels of hippocampal 5-HT in Fischer F344/N rats were significantly greater than levels detected in the same regions from Lewis and Harlan Sprague Dawley rats. Hypothalamic 5-HT and 5-hydroxyindoleacetic acid levels in Harlan Sprague Dawley rats were higher than the same area from the other two strains. Adrenalectomy increased the levels of 5-hydroxyindoleacetic acid in the hypothalamus of all three strains. We conclude that hippocampal 5-HT1A receptor densities and 5-HT levels in the rat parallel the activity and responsiveness of the hypothalamopituitary-adrenal axis.     

Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin

The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3'-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3'-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI. As expected of a possible correspondence between PI and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline- and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of PI were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).