Elevated 5-HT 2A receptors in postmortem prefrontal cortex in major depression is associated with reduced activity of protein kinase A.

Previous human postmortem brain tissue research has implicated abnormalities of 5-HT receptor availability in depression and suicide. Although altered abundance of 5-HT 1A, 5-HT 2A, and 5-HT 2C receptors (5-HT(1A), 5-HT(2A), and 5-HT(2C)) has been reported, the causes remain obscure. This study evaluated the availability of these three receptor subtypes in postmortem brain tissue specimens from persons with a history of major depression (MDD) and normal controls and tested the relationships to protein kinases A and C (PKA, PKC). Samples were obtained from postmortem brain tissue (Brodmann area 10) from 20 persons with a history of MDD and 20 matched controls as determined by a retrospective diagnostic evaluation obtained from family members. Levels of 5-HT(1A), 5-HT(2A), and 5-HT(2C) receptor were quantitated via Western blot analyses. Basal and stimulated PKA and PKC activity were also determined. The depressed samples showed significantly increased 5-HT(2A) receptor abundance relative to controls, but no differences in 5-HT(1A) or 5-HT(2C) receptors. Basal and cyclic AMP-stimulated PKA activity was also reduced in the depressed sample; PKC activity was not different between groups. 5-HT(2A) receptor availability was significantly inversely correlated with PKC activity in controls, but with PKA activity in the depressed sample. Increased 5-HT(2A) receptor abundance and decreased PKA activity in the depressed sample are consistent with prior reports. The correlation of 5-HT(2A) receptor levels with PKA activity in the depressed group suggests that abnormalities of 5-HT(2A) receptor abundance may depend on receptor uncoupling and heterologous regulation by PKA.

Metabotropic glutamate receptor 5-induced phosphorylation of extracellular signal-regulated kinase in astrocytes depends on transactivation of the epidermal growth factor receptor.

G-protein-coupled receptors (GPCRs) induce the phosphorylation of mitogen-activated protein (MAP) kinase by actions on any of a number of signal transduction systems. Previous studies have revealed that activation of the G(q)-coupled metabotropic glutamate receptor 5 (mGluR5) induces phosphorylation of the MAP kinase extracellular signal-regulated kinase 2 (ERK2) in cultured rat cortical astrocytes. We performed a series of studies to determine the mechanisms underlying mGluR5-induced phosphorylation of MAP kinase in these cells. Interestingly, our studies suggest that mGluR5-mediated ERK2 phosphorylation is dependent on the activation of G(alphaq) but is not mediated by the activation of phospholipase Cbeta1, activation of protein kinase C, or increases in intracellular calcium. Studies with peptide inhibitors suggest that this response is not dependent on G(betagamma) subunits. However, the activation of ERK2 was dependent on activation of the epidermal growth factor (EGF) receptor and activation of a Src family tyrosine kinase. Furthermore, activation of mGluR5 induced an association of this receptor and the EGF receptor, suggesting the formation of a signaling complex involved in the activation of ERK2. These data suggest that mGluR5 increases ERK2 phosphorylation in astrocytes by a novel mechanism involving the activation of G(alphaq) and both receptor and nonreceptor tyrosine kinases but that is independent of the activation of phospholipase Cbeta1.

From neurotransmitter to gene: identifying the missing links.

Survival demands adaptation to changes in the environment, and some of the most important adaptations are accomplished by the nervous system. Adaptive processes such as learning and memory are mediated by complex intracellular adjustments that result, at least in part, from changes in the levels of certain proteins. The involvement of neurotransmitters in the regulation of protein biosynthesis has recently become the focus of much investigation. Questions that are beginning to be explored include: how do neurotransmitters regulate the expression of specific genes in various cell populations? How can a gene product be regulated in opposing or coordinated fashion by different stimuli? How does a neurotransmitter exert differential regulatory influences in the same cell type under different growth conditions? Teresa Esterle and Elaine Sanders-Bush examine these questions with an emphasis on the studies that explore the ability of neurotransmitters to have diverse actions on the same target gene in different tissues or on different genes in the same cell.

Dissecting intracellular signaling pathways with membrane-permeable peptides.

Peptides can be designed that mimic protein interaction motifs and thus, can be used to specifically and selectively block particular steps in signal transduction cascades where protein interactions have been previously identified. This protocol describes methods to synthesize peptides coupled to a membrane-permeable sequence (MPS), designed from the signal sequence of Kaposi fibroblast growth factor, which has been previously shown to translocate covalently attached cargo peptides across the cell membrane. To increase efficiency, yield, and versatility in the preparation of these membrane-permeable peptides, a modular synthesis strategy based on two unprotected peptide segments was designed. The modular synthesis strategy allows the MPS and functional peptides to be synthesized separately. In this manner, the functional domain of a peptide or protein, synthesized by traditional fluoroenylmethyloxy-carbonyl (Fmoc) chemistry or derived from recombinant expression, may be purchased commercially to expedite synthesis. Subsequently, the MPS domain may be attached to any functional domain using a one-step conjugation reaction. This protocol provides detailed methods for peptide synthesis, activation of the MPS, and the subsequent conjugation protocol.

Adaptive regulation of central serotonin receptors linked to phosphoinositide hydrolysis.

Central 5-HT2 and 5-HT1C receptors appear to be relatively resistant to up-regulation. 5-HT2 receptors are not upregulated after denervation of 5-HT neurons and chronic administration of antagonists elicits a paradoxical downregulation of both receptors. This article explores the possible explanations for these anomalous findings.

Agonist-directed signaling of serotonin 5-HT2C receptors: differences between serotonin and lysergic acid diethylamide (LSD).

For more than 40 years the hallucinogen lysergic acid diethylamide (LSD) has been known to modify serotonin neurotransmission. With the advent of molecular and cellular techniques, we are beginning to understand the complexity of LSD's actions at the serotonin 5-HT2 family of receptors. Here, we discuss evidence that signaling of LSD at 5-HT2C receptors differs from the endogenous agonist serotonin. In addition, RNA editing of the 5-HT2C receptor dramatically alters the ability of LSD to stimulate phosphatidylinositol signaling. These findings provide a unique opportunity to understand the mechanism(s) of partial agonism.

RNA editing of the human serotonin 5-HT2C receptor. alterations in suicide and implications for serotonergic pharmacotherapy.

RNA encoding the human serotonin 5-HT2C receptor (5-HT(2C)R) undergoes adenosine-to-inosine RNA editing events at five positions, resulting in an alteration of amino acids in the second intracellular loop. Several edited 5-HT(2C)Rs possess a reduced G-protein coupling efficiency compared to the completely non-edited isoform. The current studies show that the efficacy of the hallucinogenic drug lysergic acid diethylamide and of antipsychotic drugs is regulated by RNA editing, suggesting that alterations in editing efficiencies or patterns might result in the generation of a 5-HT(2C)R population differentially responsive to serotonergic drugs. An examination of the efficiencies of RNA editing of the 5-HT(2C)R in prefrontal cortex of control individuals vs. subjects diagnosed with schizophrenia or major depressive disorder revealed no significant differences in RNA editing among the three populations. However, subjects who had committed suicide (regardless of diagnosis) exhibited a statistically significant elevation of editing at the A-site, which is predicted to change the amino acid sequence in the second intracellular loop of the 5-HT(2C)R. These findings suggest that alterations in RNA editing may contribute to or complicate therapy in certain psychiatric disorders.

The serotonin autoreceptor: antagonism by quipazine.

The purpose of this study was to attempt to reproduce previous findings regarding the antagonist specificity of the 5HT autoreceptor and to find additional antagonists of this receptor. Crude synaptosomal preparations of the rat hypothalamus were loaded with [3H]5HT, placed on glass microfiber filters and superfused with modified Krebs--Henseleit buffer at 37 degrees C. The release of [3H]5HT was stimulated by raising the buffer K+ concentration and was Ca2+-dependent. In the presence of 100 nM fluoxetine (a selective 5HT uptake inhibitor), exogenous 5HT inhibited the K+-induced release of [3H]5HT but did not affected basal [3H]5HT release. The K+-induced [3H]5HT release was maximally inhibited by 30 nM 5HT to a level of 66.4 +/- 4.0% of control. The concentration of 5HT required to inhibit half-maximally K+-induced [3H]5HT release was approx. 7 nM. Methiothepin and quipazine were found to block the inhibition of K+-induced [3H]5HT release by exogenous 5HT (30 nM). The IC50S for blockade of the effects of 5HT were approx. 3.8 and 670 nM for methiothepin and quipazine, respectively. Several other putative 5HT antagonists, the dopamine receptor antagonist, spiperone and the alpha receptor antagonist, phentolamine, were without effect. Thus, the 5HT autoreceptor appears to have a unique specificity for certain 5HT antagonists. In addition, blockade of 5HT autoreceptors may be one mechanism by which quipazine produces behavioral effects characteristic of a 5HT receptor agonist.

Selective 5HT-2 antagonists inhibit serotonin stimulated phosphatidylinositol metabolism in cerebral cortex.

Evidence suggests that the serotonin 5HT-1 receptor site is functionally linked to adenylate cyclase in the brain, but a biochemical effector system which is linked to the serotonin 5HT-2 receptor site has not been found. In the present paper we report an investigation of 5HT stimulated phosphatidylinositol (PI) hydrolysis in rat cerebral cortex and have found that selective 5HT-2 antagonists (pizotifen and ketanserin) block 5HT's effect upon PI metabolism. These data suggest that 5HT stimulated PI hydrolysis is mediated by the 5HT-2 binding site.

Adaptive changes in the 5-HT2 binding site after chronic administration of agonists and antagonists.

This study confirms and extends an earlier report that acute administration of the serotonin (5-HT) antagonist, mianserin, caused a marked decrease in the density of 5-HT2 binding sites in brain of the rat (Blackshear and Sanders-Bush, 1982). Using [3H]ketanserin, a selective ligand for the 5-HT2 site, the present study further investigated the mechanism of this effect. The effects of mianserin in vivo and in vitro were compared with those of trifluoromethylphenylpiperazine (TFPP), a directly acting 5-HT agonist. While TFPP, unlike mianserin, was not active in single doses, it caused a 58% decrease in the density of 5-HT2 sites after repeated doses. The adaptive change induced by chronic treatment with TFPP was qualitatively and quantitatively similar to that caused by chronic administration of mianserin. Furthermore, mianserin and TFPP had similar effects in vitro; preincubation of membranes from brain induced a concentration-dependent increase in the Kd value with no change in the Bmax value. Similar adaptive changes in 5-HT2 sites after administration of mianserin, a presumed 5-HT antagonist, and after TFPP, a presumed 5-HT agonist, suggests that adaptive mechanisms in the serotonergic system are different from those in other aminergic systems or that the drugs used to characterize 5-HT receptor systems need to be re-evaluated.

Stereoselective LSD-like activity in a series of d-lysergic acid amides of (R)- and (S)-2-aminoalkanes.

The 3-pentyl-, (R)- and (S)-2-pentyl-, 2-hexyl-, and 2-heptylamides of d-lysergic acid were synthesized and evaluated in biochemical and behavioral assays for LSD-like activity. In radioligand competition studies, the (R)-lysergamides were consistently more potent than the (S)-amides in displacing [3H]ketanserin from 5-HT2A receptors in rat cortical homogenate and in displacing [3H]-8-OH-DPAT ([3H]-8-hydroxy-2-(di-n- propylamino)tetralin) from rat hippocampal 5-HT1A receptors. As the amide alkyl was lengthened from pentyl to heptyl, the affinity of the (R)-isomers for 5-HT2A sites decreased, while affinity for 5-HT1A sites was maximal for the (R)-2-hexyllysergamide. In rats trained to discriminate 0.08 mg/kg LSD tartrate from saline, a similar stereoselective effect was noted in which the (R)-alkylamides were more potent than the (S)-isomers in producing the LSD-like discriminative stimulus effect. However, as the amide alkyl substituent was increased in length, LSD-like activity decreased, with only partial substitution for training drug being observed for the (R)-hexylamide. The (R)- and (S)-pentyllysergamides were also assayed for their ability to activate intracellular phosphoinositide hydrolysis. Consistent with the binding and behavioral studies, these assays showed that both isomers are potent agonists at the 5-HT2A receptor, but that the (R)-pentyllysergamide is approximately 20 times more active than the (S)-pentyllysergamide in stimulating phosphoinositide turnover.

Dihydrobenzofuran analogues of hallucinogens. 4. Mescaline derivatives.

Dihydrobenzofuran and tetrahydrobenzodifuran functionalities were employed as conformationally restricted bioisosteres of the aromatic methoxy groups in the prototypical hallucinogen, mescaline (1). Thus, 4-(2-aminoethyl)-6,7-dimethoxy-2,3-dihydrobenzofuran hydrochloride (8) and 1-(8-methoxy-2,3,5,6-tetrahydrobenzo[1,2-b:5,4-b']difuran-4-yl)-2- aminoethane hydrochloride (9) were prepared and evaluated along with 1 for activity in the two-lever drug discrimination (DD) paradigm in rats trained to discriminate saline from LSD tartrate (0.08 mg/kg). Also, 1, 8, and 9 were assayed for their ability to displace [3H]ketanserin from rat cortical homogenate 5-HT2A receptors and [3H]8-OH-DPAT from rat hippocampal homogenate 5-HT1A receptors. In addition, these compounds were evaluated for their ability to compete for agonist and antagonist binding to cells expressing cloned human 5-HT2A, 5-HT2B, and 5-HT2C receptors. Finally, agonist efficacy was assessed by measurement of phosphoinositide hydrolysis in NIH 3T3 cells expressing the rat 5-HT2A or 5-HT2C receptors. Although 1 fully substituted for LSD in the DD assays (ED50 = 33.5 mumol/kg), neither 8 nor 9 substituted for LSD, with just 50% of the rats administered 8 selecting the drug lever, and only 29% of the rats administered 9 selecting the drug lever. All of the test compounds had micromolar affinity for the 5-HT1A and 5-HT2A receptors in rat brain homogenate. Curiously, the rank order of affinities of the compounds at 5-HT2A sites was opposite their order of potency in the behavioral assay. An evaluation for ability to stimulate phosphoinositide turnover as a measure of functional efficacy revealed that all the compounds were of approximately equal efficacy to serotonin in 5-HT2C receptors. At 5-HT2A receptors, however, 8 and 9 were significantly less efficacious, eliciting only 61 and 45%, respectively, of the maximal response. These results are consistent with the proposed mechanism of action for phenethylamine hallucinogens, that such compounds must be full agonists at the 5-HT2A receptor subtype. In contrast to the 2,5-dimethoxy-substituted phenethylamines, where rigidification of the methoxy groups had no deleterious effect on activity, the loss of activity in the 3,4,5-trioxygenated mescaline analogues may suggest that the 3 and 5 methoxy groups must remain conformationally mobile to enable receptor activation.

Lysergic acid diethylamide-induced Fos expression in rat brain: role of serotonin-2A receptors.

Lysergic acid diethylamide (LSD) produces altered mood and hallucinations in humans and binds with high affinity to serotonin-2A (5-HT(2A)) receptors. Although LSD interacts with other receptors, the activation of 5-HT(2A) receptors is thought to mediate the hallucinogenic properties of LSD. The goal of this study was to identify the brain sites activated by LSD and to determine the influence of 5-HT(2A) receptors in this activation. Rats were pretreated with the 5-HT(2A) receptor antagonist MDL 100907 (0.3 mg/kg, i.p.) or vehicle 30 min prior to LSD (500 microg/kg, i.p.) administration and killed 3 h later. Brain tissue was examined for Fos protein expression by immunohistochemistry. LSD administration produced a five- to eight-fold increase in Fos-like immunoreactivity in medial prefrontal cortex, anterior cingulate cortex, and central nucleus of amygdala. However, in dorsal striatum and nucleus accumbens no increase in Fos-like immunoreactivity was observed. Pretreatment with MDL 100907 completely blocked LSD-induced Fos-like immunoreactivity in medial prefrontal cortex and anterior cingulate cortex, but only partially blocked LSD-induced Fos-like immunoreactivity in amygdala. Double-labeled immunohistochemistry revealed that LSD did not induce Fos-like immunoreactivity in cortical cells expressing 5-HT(2A) receptors, suggesting an indirect activation of cortical neurons. These results indicate that the LSD activation of medial prefrontal cortex and anterior cingulate cortex is mediated by 5-HT(2A) receptors, whereas in amygdala 5-HT(2A) receptor activation is a component of the response. These findings support the hypothesis that the medial prefrontal cortex, anterior cingulate cortex, and perhaps the amygdala, are important regions involved in the production of hallucinations.

Hallucinogens and Drosophila: linking serotonin receptor activation to behavior.

The similarity of mode of action, behavior, and gene response between Drosophila melanogaster and mammalian systems, combined with the power of genetics, have recently made the fly an attractive system to study underlying mechanisms of drug abuse, addiction, and mental disorders. The present studies define the behavioral and molecular effects of the powerful hallucinogen lysergic acid diethylamide in Drosophila. Pharmacological activation of serotonin receptors in the fly by lysergic acid diethylamide induces behaviors not unlike those observed in mammalian systems. These include alterations in visual processing abilities, reduced locomotor activity, and altered gene expression within the brain. Many of these effects are due to activation of the same serotonin receptor subtypes that are thought to be the primary mediators of hallucinogenic drug effects in humans as well as the acute symptoms of schizophrenia.We suggest that Drosophila can be used as a genetically tractable model system to define the molecular events leading from serotonin receptor activation to behavior, possibly revealing new targets for hallucinogenic agents and for the treatment of neuropsychiatric disorders such as schizophrenia.

Ultrastructural localization of serotonin2A receptors in the middle layers of the rat prelimbic prefrontal cortex.

Cortical serotonin(2A) receptors are hypothesized to be involved in the pathology and treatment of schizophrenia. Light microscopic studies in the rat prefrontal cortex have localized serotonin(2A) receptors to the dendritic shafts of pyramidal and local circuit neurons. Electrophysiological studies have predicted that these receptors are also located on glutamate terminals, whereas neurochemical studies have hypothesized that they are located on dopamine terminals in this area. The present study sought to determine the ultrastructural localization of immunoperoxidase labeling for serotonin(2A) receptors in the middle layers of the prelimbic portion of the rat prefrontal cortex. Serotonin(2A) receptor immunoreactivity was observed in 325 identifiable structures. Of these, 73% were postsynaptic profiles that were composed of either dendritic shafts (58%) or dendritic spine heads and necks (42%). Twenty-four percent of the labeled profiles were presynaptic axons and varicosities; most of these had morphological features that were characteristic of monoamine axons: thin diameter, lack of myelination, occasional content of dense-cored vesicles, and infrequent formation of synapses in single sections. The remainder of the labeled profiles (4%) were glial processes. These findings suggest that serotonin(2A) receptor-mediated effects within the rat prelimbic prefrontal cortex are primarily postsynaptic in nature, affecting both the spines of pyramidal cells and the dendrites of pyramidal and local circuit neurons in this area. The results further suggest that serotonin acts presynaptically via this receptor subtype, most likely at receptors on monoamine fibers, and only rarely directly on glutamate axons.

Clozapine and other 5-hydroxytryptamine-2A receptor antagonists alter the subcellular distribution of 5-hydroxytryptamine-2A receptors in vitro and in vivo.

In this study, we demonstrate that clozapine and other atypical antipsychotic drugs induce a paradoxical internalization of 5-hydroxytryptamine-2A receptors in vitro and a redistribution of 5-hydroxytryptamine-2A receptors in vivo. We discovered that clozapine, olanzapine, risperidone and the putative atypical antipsychotic drug MDL 100,907 all induced 5-hydroxytryptamine-2A receptor internalization in fibroblasts stably expressing the 5-hydroxytryptamine-2A receptor in vitro. Two 5-hydroxytryptamine-2A antagonists (mianserin and ritanserin), which have been demonstrated to reduce negative symptoms in schizophrenia, also caused 5-hydroxytryptamine-2A receptor internalization. Four different drugs, each devoid of 5-hydroxytryptamine-2A antagonist activity, had no effect on the subcellular distribution of 5-hydroxytryptamine-2A receptors in vitro. Treatment of rats for seven days with clozapine induced an increase in intracellular 5-hydroxytryptamine-2A receptor-like immunoreactivity in pyramidal neurons, while causing a decrease in labeling of apical dendrites in the medial prefrontal cortex. This redistribution of 5-hydroxytryptamine-2A receptors in pyramidal neurons was also seen when rats were chronically treated with another atypical antipsychotic drug, olanzapine. The typical antipsychotic drug haloperidol, however, did not induce a redistribution of 5-hydroxytryptamine-2A receptors in pyramidal neurons in the medial prefrontal cortex. Taken together, these results demonstrate that several atypical antipsychotic drugs with high 5-hydroxytryptamine-2A receptor affinities induce a redistribution of 5-hydroxytryptamine-2A receptors both in vivo and in vitro. It is conceivable that the loss of 5-hydroxytryptamine-2A receptors from the apical dendrites of pyramidal neurons is important for the beneficial effects of atypical antipsychotic drugs and other 5-hydroxytryptamine-2A antagonists in schizophrenia.

5-Hydroxytryptamine2C receptors on spinal neurons controlling penile erection in the rat.

The localization of 5-hydroxytryptamine2C receptors in the lumbosacral spinal cord of the rat was investigated using selective antibodies raised against the carboxyl-terminal part of the rat receptor. The distribution of immunoperoxidase labelling at the light microscope level revealed numerous labelled neurons in the gray matter, with a higher intensity in the sacral parasympathetic nucleus, the dorsal gray commissure and particularly the motoneurons of the ventral horn. Confocal microscope analysis showed that immunostaining was mainly intracellular (motoneurons), but could also be associated with the membrane of cell bodies and dendrites. Actually, electron microscope immunogold experiments demonstrated an exclusive staining of the cis-Golgi apparatus. Following pseudo-rabies virus transsynaptic retrograde labelling from the corpus cavernosum, labelled neurons were found in the sacral parasympathetic nucleus and the dorsal gray commissure of the L6-S1 segments. All virus-labelled neurons exhibited 5-hydroxytryptamine2C receptor immunoreactivity. These results indicate that all parasympathetic preganglionic neurons and their related interneurons which contribute to the innervation of cavernosal tissue bear 5-hydroxytryptamine2C receptors. In the sacral parasympathetic nucleus, most neurons which were retrogradely-labelled from the pelvic ganglion with Fast Blue also showed 5-hydroxytryptamine2C receptor immunoreactivity. In the ventral horn, motoneurons retrogradely labelled from the ischiocavernosus muscle and the bulbospongiosus muscle, both of which are involved in erection and ejaculation, were also 5-hydroxytryptamine2C receptor-immunopositive. The supraspinal serotoninergic control of erection at the lumbosacral level therefore appears to be strongly associated with the activation of 5-hydroxytryptamine2C receptors, consistent with the proerectile properties of 5-hydroxytryptamine2C agonists.

RNA-editing of the 5-HT(2C) receptor alters agonist-receptor-effector coupling specificity.

1. The serotonin(2C) (5-HT(2C)) receptor couples to both phospholipase C (PLC)-inositol phosphate (IP) and phospholipase A(2) (PLA(2))-arachidonic acid (AA) signalling cascades. Agonists can differentially activate these effectors (i.e. agonist-directed trafficking of receptor stimulus) perhaps due to agonist-specific receptor conformations which differentially couple to/activate transducer molecules (e.g. G proteins). Since editing of RNA transcripts of the human 5-HT(2C) receptor leads to substitution of amino acids at positions 156, 158 and 160 of the putative second intracellular loop, a region important for G protein coupling, we examined the capacity of agonists to activate both the PLC-IP and PLA(2)-AA pathways in CHO cells stably expressing two major, fully RNA-edited isoforms (5-HT(2C-VSV), 5-HT(2C-VGV)) of the h5-HT(2C) receptor. 2. 5-HT increased AA release and IP accumulation in both 5-HT(2C-VSV) and 5-HT(2C-VGV) expressing cells. As expected, the potency of 5-HT for both RNA-edited isoforms for both responses was 10 fold lower relative to that of the non-edited receptor (5-HT(2C-INI)) when receptors were expressed at similar levels. 3. Consistent with our previous report, the efficacy order of two 5-HT receptor agonists (TFMPP and bufotenin) was reversed for AA release and IP accumulation at the non-edited receptor thus demonstrating agonist trafficking of receptor stimulus. However, with the RNA-edited receptor isoforms there was no difference in the relative efficacies of TFMPP or bufotenin for AA release and IP accumulation suggesting that the capacity for 5-HT(2C) agonists to traffic receptor stimulus is lost as a result of RNA editing. 4. These results suggest an important role for the second intracellular loop in transmitting agonist-specific information to signalling molecules.

Identification of rat serotonin 5-HT2C receptors as glycoproteins containing N-linked oligosaccharides.

Antibodies against a portion of the rat 5-HT2C receptor third intracellular loop were generated and used to identify receptors solubilized from cell lines and rat brain. Western blots of CHAPS-soluble proteins were probed with affinity-purified anti-2C antibodies. The specificity of anti-2C was demonstrated with extracts prepared from NIH/3T3 fibroblasts which stably express functional rat 5-HT2C or 5-HT2A receptors. Extracts from the 5-HT2C cell line, but not the 5-HT2A cell line, contained immunoreactive proteins with masses of 51-52 kDa and 58-68 kDa. In the brain, immunoreactive proteins were identified from choroid plexus extracts with masses of 51 kDa and 58-62 kDa. The major 58-62 kDa and minor 51 kDa proteins were not detected in extracts prepared from the hippocampus, striatum, or frontal cortex using the same amount of CHAPS-soluble protein. These results are consistent with previous studies demonstrating that 5-HT2C receptor binding sites and mRNA are most abundant in choroid plexus. The association of asparagine-linked (N-linked) oligosaccharides with the receptors was examined next. The 5-HT2C receptor cell line (3T3/2C) was grown in the presence of tunicamycin to metabolically inhibit N-linked glycosylation. Proteins from the cell extracts were detected with masses of 40 and 41 kDa. Extracts prepared from 3T3/2C cells (grown in the absence of tunicamycin) and from choroid plexus were incubated with N-glycosidase F to enzymatically remove available N-linked sugars. Immunoreactive proteins were detected with masses of 41 and 42 kDa from 3T3/2C cells and 41 kDa from choroid plexus. Neuraminidase, which cleaves sialic acid (N-acetylneuraminic acid) residues from glycoproteins, reduced the mass of the 51 and 58-62 kDa proteins from the choroid plexus to 50 and 54-58 kDa. In contrast, the 51-52 and 58-68 kDa proteins from 3T3/2C cells were not affected by treatment with neuraminidase. These results demonstrate that 5-HT2C receptors contain N-linked sugars and suggest that sialic acid residues associate with 5-HT2C receptors in the choroid plexus. The oligosaccharide moieties, which contribute up to approximately 30% of the relative mass as judged by SDS-polyacrylamide gel electrophoresis, may impart functional properties to 5-HT2C receptors.

Central serotonin receptors: effector systems, physiological roles and regulation.

Radioligand binding studies have revealed four distinct serotonin (5HT) binding sites in rat brain that are thought to function as 5HT receptors. These include the 5HT-1a, 5HT-1b, 5HT-1c, and 5HT-2 binding sites. Studies have shown that the 5HT-2 binding site mediates a number of effects of 5HT agonists and serves as a 5HT receptor in neuronal and non-neuronal tissues. The 5HT-2 site employs phosphoinositide hydrolysis for signal transduction. The 5HT-1c binding site is also a functional receptor that is linked to phosphoinositide hydrolysis. However, the physiological role of the 5HT-1c receptor is not yet known. Lack of appropriate pharmacological tools for probing the 5HT-1a and 5HT-1b binding sites has made it difficult to definitively determine whether these binding sites are coupled to biochemical effector systems or mediate any of the physiological responses to 5HT agonists. However, there is some evidence that the 5HT-1a site is coupled to adenylate cyclase, and a number of functional roles for the 5HT-1a and 5HT-1b sites have been proposed.