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Positron emission tomography studies in major depression show reduced serotonin (5-HT)1A receptor antagonist-binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5-HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III-R criteria. Measurements of [35S]GTPgammaS binding to Galphai/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5-HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Galphai/o, Galphaq/11 or Galphas proteins, or in the activity of cAMP-dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5-HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3-kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5-triphosphate. Finally, the activation of extracellular signal-regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist-stimulated 5-HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5-HT1A receptor transduction pathways that may have implications for cell survival.

Purpose: Serotonin regulates colonic motility receptors expressed on neural fibers and smooth muscle. Colonic inertia is characterized by delayed colonic transit. Abnormalities in serotonin receptor protein, as judged by immunoreactivity levels, could contribute to the origin of colonic inertia. The aim of this study was to investigate the expression of serotonin receptor(s) immunoreactivity in the left colon of patients with colonic inertia compared with controls., Methods: Sixteen patients who underwent subtotal colectomy for colonic inertia were assessed. Colonic transit time was measured with the radiopaque marker technique and presented as the number of retained markers in the colon on Day 5. The control group consisted of 18 patients who underwent left hemicolectomy for colonic carcinoma; histologically normal tissues from the left colon were used. Immunohistochemical staining for serotonin receptor was performed with a rabbit anti-idiotypic antibody. The average positive area (square pixels) in the mucosa, muscularis mucosa, submucosa, and circular and longitudinal muscles per microscopic field (63x) was calculated based on measurement of the positively stained area in 20 randomly chosen microscopic fields in each related structure. The Scion Image computer analysis system was used., Results: Serotonin receptor(s) immunoreactivity was mainly detected in the muscular mucosa, circular muscles, and longitudinal muscles and rarely in the mucosa and submucosa. In muscularis mucosa and circular muscle, the positive areas were significantly less in the colonic inertia group than in controls (muscularis mucosa: 29.1 +/- 10.8 vs 109.7 +/- 28.2, P < 0.05; circular muscle: 25.6 +/- 6.2 vs 90.2 +/- 19.1, P < 0.01). There were significantly positive correlations in the control group in serotonin receptor(s) immunoreactivity levels between circular muscle and longitudinal muscle (r = 0.54, P < 0.05) and between muscular mucosa and longitudinal muscle (r = 0.57, P < 0.05) but not in colonic inertia patients. In addition, the positive areas in the circular muscle were positively correlated to the colonic transit time (Spearman's rank correlation, 0.83; P < 0.01)., Conclusion: In colonic inertia patients, the serotonin receptor(s) immunoreactivity level is lower in muscular mucosa and circular muscle. The absence of a correlation of serotonin receptor(s) immunoreactivity in the muscular mucosa and muscularis propria in the patient group implies that an uncoordinated expression of serotonin receptors may also contribute to colonic inertia. However, the positive correlation between serotonin receptor(s) immunoreactivity levels in the circular muscle and the transit time observed in colonic inertia patients suggests a decrease in stimulatory subtypes and at the same time an increase in inhibitory subtypes of serotonin receptors in this tissue.

Five adenosines within the coding sequence of the serotonin 2C receptor (5-HT2C) pre-mRNA are converted to inosines by RNA editing (named A, B, C' (E), C, and D sites). In human prefrontal cortex (PFC), the most abundant 5-HT2C mRNA sequences result from editing at the A site, or from the editing combinations AC'C, ABCD, and ABD. In suicide victims with a history of major depression, C' site editing is significantly increased, D site editing is significantly decreased, and the C site shows a trend toward increased editing. Treatment of mice with the antidepressant drug fluoxetine (Prozac) causes changes in C', C, and D site editing that are exactly opposite to those seen in suicide victims. Thus, one outcome of fluoxetine treatment may be to reverse the abnormalities in 5-HT2C pre-mRNA editing seen in depressed suicide victims.

Suicide and depression are associated with reduced serotonergic neurotransmission. In suicides, there is a reduction in serotonin transporter (SERT) sites and an increase in postsynaptic 5-HT(1A) receptors in localized regions of the prefrontal cortex. In depression, there is a diffuse decrease in SERT binding throughout the dorsoventral extent of the prefrontal cortex. Serotonergic innervation of the prefrontal cortex arises predominantly from neurons in the brainstem dorsal raphe nucleus (DRN). We, therefore, examined postmortem SERT binding and mRNA expression, as well as 5-HT(1A) autoreceptor binding in the DRN of 10 matched pairs of controls and depressed suicide victims. The concentration of SERT sites, SERT mRNA, and 5-HT(1A) binding was not different between controls and suicides (p >.05). In the DRN of suicides, the volume of tissue defined by 5-HT(1A) binding was 40% smaller than controls. An index of the total number of 5-HT(1A) receptors (receptor binding x volume of receptor distribution) was 43.3% lower in the DRN of suicides, compared with controls. The suicide group had 54% fewer DRN neurons expressing SERT mRNA compared with controls. In the serotonin neurons that expressed the SERT gene, expression per neuron was greater in suicides. Less total 5-HT(1A) and SERT binding is consistent with results of in vivo studies in depression. Less feedback inhibition of serotonin DRN firing via 5-HT(1A) autoreceptors and enhancement of serotonin action due to less uptake of serotonin, is consistent with compensatory changes in response to hypofunction in depressed suicides.

Agonist activation of G protein-coupled receptors induces an increase in the binding of guanosine 5'-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS); this increase in binding has been used as a tool to investigate receptor interaction with the heterotrimer guanine nucleotide-binding regulatory protein (G protein). The present study uses agonist-stimulated [(35)S]GTPgammaS binding to characterize serotonin 5-HT(2A/2C) receptors in rat brain membrane fractions and demonstrate the anatomical localization of the receptors by in vitro autoradiography on slide-mounted sections. The stimulatory effect of the agonist [1-(2,5-dimethoxy-4-iodophenyl)]-2 aminopropane (DOI) is compared to that of serotonin (5-HT). Autoradiography revealed a similar localization of DOI- and 5-HT-stimulated binding of [(35)S]GTPgammaS in distinct areas of prefrontal and parietal cortex, consistent with previously reported 5-HT(2A) receptor distribution. Specific binding was demonstrated in the frontal and parietal cortex, medial prefrontal, and cingular and orbital-insular areas as well as in the hippocampal formation, septal areas, the nucleus accumbens, and the choroid plexus. MDL 100105, a specific 5-HT(2A) antagonist, and ketanserin, an antagonist of 5-HT(2A/2C) receptors, blocked DOI stimulation in all labeled areas, whereas 5-HT stimulation was only partially blocked (70-80%). A small but significant inhibition was observed with the specific antagonist of 5-HT(2C/2B), SB 206553. This autoradiographic technique provides a useful tool for measuring in situ changes in specific receptor-Gq protein coupling in anatomically discrete brain regions, under physiological and pathological conditions., (Copyright 2000 Wiley-Liss, Inc.)

Mechanisms underlying the 5-HT2A receptor induction of intracellular Ca2+ mobilization and Ca2+ influx in type I astroglial cells in primary culture from newborn rat cerebral cortex were evaluated. The 5-HT-evoked Ca(2+)-transients, inhibited by the 5-HT2A antagonists ketanserin or 4-(4-fluorobenzoyl)-1-(4-phenylbutyl) piperidine oxalate, consisted of an initial peak caused by inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from internal stores, and a second sustained part which was due to Ca2+ transport over the plasma membrane. The responses were pertussis toxin-insensitive, suppressed by the phospholipase C inhibitor neomycin and were inhibited by the Ca(2+)-ATPase inhibitor thapsigargin. Furthermore, the responses were inhibited by the IP3 receptor antagonist heparin. When the second sustained part of the 5-HT-evoked response was studied, it was concluded that Ca2+ influx was not a result of opening of voltage operated calcium channels of either L, N or T-type. Instead it appeared that Ca2+ entered the cells through specialized voltage independent Ca2+ channels which were dependent of the IP3 production and subsequent Ca2+ release from internal stores. From this, we conclude that 5-HT opens Ca2+ channels in astrocytes which closely resemble depletion-operated Ca2+ channels (DOCCs).

We have previously reported that Schwann cells cultured from rat sciatic nerves express 5-HT2A receptors. In this study we extend these in vitro observations to Schwann cells in situ. Since the serotonin (5-HT) levels in rat sciatic nerve are elevated following nerve injury, we examined Schwann cells in healthy and injured adult rat sciatic nerves. These nerves were double-labeled immunohistochemically with an anti-idiotypic antibody that recognizes 5-HT1B, 5-HT2A, and 5-HT2C receptors and an antibody against S100beta, a Schwann cell marker. 5-HT receptor labeling was observed in Schwann cells of healthy and regenerating nerves, but not of degenerating nerves, while S100beta labeling was observed in the Schwann cells of all nerves examined. The 5-HT receptor immunolabeling was cytoplasmic, as with the cultured Schwann cells. While staining was observed at the nodes of Ranvier, it was not restricted to these locations. These results suggest that myelinating rat Schwann cells normally express 5-HT receptors in vivo, and that receptor expression is reduced during times when 5-HT levels are elevated in the sciatic endoneurium.

Intracellular calcium responses of cultured rat Schwann cells to 5-hydroxytryptamine (5-HT) were examined using the calcium indicator dye fluo-3. Consistent changes in [Ca2+]i were observed with bath application of 5-HT and the basis of these responses was characterized. Application of 5-HT elicited a transient increase in intracellular calcium in a subpopulation of cultured Schwann cells. In many responding cells, the response recurred at approximately regular intervals following the initial transient. In some cases, these oscillations lasted for hours following removal of 5-HT from the bath. The increase in intracellular calcium evoked by 5-HT still occurred in the absence of extracellular calcium, suggesting that 5-HT induces calcium release from intracellular stores. Consistent with this hypothesis, the response to 5-HT was prevented by depletion of inositol trisphosphate-sensitive intracellular calcium stores with thapsigargin. Bath application of caffeine, known to activate Ca2+ release from ryanodine receptor-mediated stores, did not elicit an increase in [Ca2+]i. These results also suggested that 5-HT acted by stimulating a member of the 5-HT2 receptor family since this family employs inositol trisphosphate as a second messenger. In agreement with this interpretation, it was found that the 5-HT-induced intracellular calcium transients could be reversibly blocked by both ketanserin and spiperone, suggesting that the transients are mediated by 5-HT2A receptors. Additional support for this conclusion was obtained by immunocytochemistry using an anti-idiotypic antibody that recognizes a subset of 5-HT receptors.

In the present study, we performed the immunodetection of serotonergic (5-HT) receptor subtypes in the spinal cord by using an anti-idiotypic antiserum (TH8) at light and electron microscopic levels. This antibody has been shown to recognize 5-HT1B, 5-HT1C, and 5-HT2 receptor subtypes (Tamir et al.: J Neurochem 57:930-942, 1991). The TH8 immunoreactivity was observed in the dorsal and ventral horns of the gray matter. Light microscopy revealed that small cell bodies located in laminae I-III of the dorsal horn were intensely immunolabeled. A more homogenous and discrete staining was also observed throughout the entire dorsal horn. In the ventral horn, motoneurons were also immunoreactive (IR). Peroxidase deposits were observed as numerous patches covering the motoneuronal surface. Numerous interneurons were moderately and homogeneously immunostained. With the electron microscope, most of the labeled structures were identified as neurons (dendrites and perikarya) in both the dorsal and ventral horns. In the dorsal horn, immunoreactivity was present in dendrites and neuronal perikarya. A large majority of the immunoreactivity found in dendrites was not associated with synaptic differentiations. Indeed, the dendrites, in which peroxidase deposit was seen, were not locally involved in synapses. Very scarce synaptic varicosities were observed in close apposition with IR dendrites. In the ventral horn, TH8 immunoreactivity was present in dendrites, with an accumulation of peroxidase deposit on the active zone of synapses, facing presynaptic membranes. Both the postsynaptic membrane and the submembrane area were IR. In addition, a few astroglial fine processes were immunostained; most of them were observed in the dorsal horn. Scarce IR astroglial profiles were observed in the ventral horn. These observations show that such an antiserum constitutes a useful tool for the ultrastructural analysis of 5-HT receptor distribution. Finally, correlation between the immunocytochemical localization of 5-HT receptor subtypes and the modes of 5-HT transmission in the spinal cord (wiring and volume transmissions) is discussed in the present report.

The effects of anti-idiotypic antibodies (alpha-id) that recognize serotonin [5-hydroxytryptamine (5-HT)] receptors on myenteric neurons of the guinea pig small intestine were characterized electrophysiologically, and alpha-id binding sites were located immunocytochemically. Initial applications of the alpha-id mimicked each of three actions of 5-HT: a rapid depolarization, associated with a fall in input resistance (Rin), which was inhibited by the 5-HT3 antagonists tropisetron (> or = 1 microM) and renzapride (100 microM); a slow membrane depolarization, associated with increased Rin, that was inhibited by the 5-HT1P antagonist renzapride but was unaffected by a 5-HT4 blocking concentration of tropisetron (10 microM); and a hyperpolarization, associated with decreased Rin, that was antagonized by the 5-HT1A inhibitor NAN-190. Cross-desensitization was observed between responses to 5-HT and the alpha-id. After exposure to the alpha-id, subsequent responses to the alpha-id, 5-HT, and stimulus-evoked slow excitatory postsynaptic potentials were antagonized; however, responses to carbachol and substance P were unaffected. The alpha-id thus specifically inhibits the effects of endogenously released and exogenously applied 5-HT. The alpha-id bound to sites on myenteric and submucosal neurons and a subepithelial nerve plexus. Binding of the alpha-id was blocked by 5-HT1P-, 5-HT3-, and 5-HT4-specific antagonists. We concluded that the alpha-id binds selectively to all known subtypes of 5-HT receptor in the enteric nervous system and is thus useful for investigating the gastrointestinal function of 5-HT.

Parafollicular (PF) cells of the thyroid gland are neural crest derivatives, which costore the neurotransmitter, 5-hydroxytryptamine (5-HT) with calcitonin. PF cells are located adjacent to follicular (F) cells within the basement membrane of thyroid follicles. It has been proposed that 5-HT serves an intercellular signalling function in the thyroid and that F cells are its target. This proposal was tested by using cell lines derived from PF (medullary thyroid carcinoma [MTC]) and F (FRTL-5) cells to study the mechanisms that mediate the secretion and action of 5-HT. Secretion of 5-HT by MTC cells was evoked by thyroid stimulating hormone, thyrotropin (TSH), elevated extracellular calcium (increases [Ca2+]e), or by agents that increase intracellular cAMP (increases [cAMP]i). When protein kinase C (PKC) was down-regulated by prolonged treatment of MTC cells with phorbol 12-myristate 13-acetate (PMA), or PKC was inhibited by staurosporin, the TSH- or PMA-evoked secretion of 5-HT was blocked; however, interference with PKC function did not affect 5-HT secretion evoked by increases [Ca2+]e or increases [cAMP]i. In the putative targets, FRTL-5 cells, 5-HT increased the turnover of phosphoinositides (PI), cytosolic calcium (increases [Ca2+]i), increases [cAMP]i, and biphasically modified the effect of TSH on cAMP. All of these 5-HT effects were inhibited by 5-HT2 receptor antagonists (spiperone and ketanserin) and by pertussis toxin (PTx), suggesting that the actions of 5-HT are mediated by 5-HT2 receptors, which are coupled to a G protein. This suggestion was supported by the following additional observations: FRTL-5 membranes bound the 5-HT2 agonist, [125I]2,5-dimethoxy-4-iodophenylisopropylamine ([125I]-DOI), and anti-idiotypic antibodies, which recognize 5-HT2 receptors. [125I]-DOI binding was inhibited by guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) and the antibodies were displaced by spiperone. Data are consistent with the hypothesis that 5-HT serves as a PF to F cell messenger.

Enteropancreatic 5-hydroxytryptamine (5-HT)-immunoreactive nerve fibers have recently been described. To test the hypothesis that pancreas contains 5-HT receptors similar to those that characterize the enteric nervous system, binding of 5-[3H]HT to pancreatic membranes was investigated. Membranes were prepared by differential centrifugation from guinea pig pancreas, and binding of 5-[3H]HT was measured by use of a rapid filtration assay. Alternatively, 5-[3H]HT-binding sites were analyzed and located by radioautography on frozen sections of unfixed pancreas. The 5-HT1P receptor-specific antagonist, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), was used to define nonspecific binding in all assays. High-affinity reversible binding of 5-[3H]HT to pancreatic membranes was found. Good agreement was observed when Kd was determined from saturation isotherms (Kd = 2.1 +/- 0.4 nM; Bmax = 0.6 +/- 0.07 pmol/mg protein) or from the ratio of association and dissociation constants (2.7 +/- 0.04 nM). As is true of 5-[3H]HT binding to enteric 5-HT1P receptors, binding of 5-[3H]HT to pancreatic membranes was inhibited by 5-HTP-DP, 5- and 6-hydroxyindalpine (agonists at 5-HT1P receptors), and guanine nucleotides. In contrast, binding of 5-[3H]HT to pancreatic membranes was not inhibited by a variety of compounds known to bind to other types of 5-HT receptor, to the neuronal 5-HT transporter, to alpha- or beta-adrenoceptors, or to dopamine receptors. We concluded that pancreas contains 5-HT1P receptors with properties similar to those of bowel. Radioautography and immunocytochemistry with an anti-idiotypic antibody that recognizes 5-HT receptors revealed that pancreatic 5-HT1P receptors (identified with appropriate inhibitors of 5-[3H]HT or antibody binding) were concentrated in ganglia, nerves, and unidentified cells in both exocrine and endocrine pancreas. These observations suggest that future studies of 5-HT's role in pancreatic physiology should concentrate on enteropancreatic innervation and 5-HT1P receptors.

Anti-idiotypic antibodies were generated by immunizing rabbits with affinity-purified antibodies to serotonin (5-hydroxytryptamine; 5-HT). Anti-5-HT activity was removed from the resulting antisera by chromatography through a 5-HT affinity column. The anti-idiotypic antibodies were demonstrated by enzyme-linked immunosorbent assay to bind to affinity-purified whole anti-5-HT antibodies and their Fab fragments. Anti-idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5-HT were coupled, competed with 5-HT (covalently bound to protein) for the binding sites on anti-5-HT antibodies and serotonin binding protein. The anti-idiotypic antibodies antagonized the binding of [3H]5-HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti-idiotypic antibodies also blocked the binding of the 5-HT1B-selective ligand (-)-[125I]iodocyanopindolol (in the presence of 30 microM isoproterenol) to cortical membranes. In contrast, anti-idiotypic antibodies failed to inhibit binding of the 5-HT1A-selective ligand 8-hydroxy-2-(di-n-[3H]propylamino)-tetralin [( 3H]8-OH-DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti-idiotypic antibodies may recognize some 5-HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti-idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5-HT1C or 5-HT2 receptor or with a genomic clone encoding the 5-HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5-HT1C and 5-HT2 receptors, but not on the surfaces of HeLa cells that expressed 5-HT1A receptors. Immunostaining of cells by the anti-idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5-HT1C receptors was blocked by mesulergine (but not ketanserin, 8-OH-DPAT, or spiperone), whereas that of cells expressing 5-HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8-OH-DPAT). The anti-idiotypic antibodies failed to inhibit the uptake of [3H]5-HT by serotonergic neurons. It is concluded that the anti-idiotypic antibodies generated with anti-5-HT serum recognize the 5-HT1B, 5-HT1C, and 5-HT2 receptor subtypes; however, neither 5-HT1A receptors nor 5-HT uptake sites appear to react with these antibodies.

5-Hydroxytryptamine (5-HT) receptors have been analyzed and related to potential roles played by 5-HT in the physiology of the enteric nervous system (ENS). Three subtypes of 5-HT receptor--5-HT1P, 5-HT3, and 5-HT1A--have been found on enteric neurons. Receptors have been identified by intracellularly recording the electrical activity of enteric neurons and by studying the binding of radioligands and polyclonal anti-idiotypic antibodies raised against antibodies to 5-HT. Radioligand binding has been assessed by rapid filtration and by radioautography. 5-HT1P receptors mediate slow depolarizations of myenteric neurons that are associated with a closure of K+ channels. These responses can be inhibited by N-acetyl-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP) and by the substituted benzamide, BRL 24924. 5-HT1P-like responses can be mimicked by 5- and 6-hydroxyindalpine, by another substituted benzamide, the S stereoisomer of zacopride, and by anti-idiotypic antibodies. 5-HT1P receptors can be labeled by 3H-5-HT and 3H-5-hydroxyindalpine with high affinity and are located on neurons of both enteric plexuses and on processes of intrinsic neurons in the gastrointestinal mucosa. A similar distribution of binding sites for anti-idiotypic antibodies is revealed by immunocytochemistry. Excitatory postsynaptic potentials (EPSPs) mediated by 5-HT are abolished by 5-HT1P antagonists. Blockade of 5-HT1P receptors is accompanied by acceleration of the rate of gastric emptying. Mucosal application of cholera toxin activates enteric neurons in both plexuses; this action is blocked by 5-HT1P or 5-HT3 antagonists and by anti-idiotypic antibodies. 5-HT3 receptors are responsible for fast depolarizations associated with increased membrane conductance. These responses are antagonized by ICS 205-930 and mimicked by 2-methyl-5-HT and anti-idiotypic antibodies. 5-HT1A receptors have been reported to mediate hyperpolarizing responses associated with a rise in membrane conductance. Hyperpolarizing responses are also elicited by the 5-HT1A agonists, 8-hydroxy-di-n-propylaminotetralin (8-OH-DPAT) and 5-carboxyamidotryptamine. It is proposed that 5-HT1P receptors and perhaps 5-HT3 receptors are involved in initiating the peristaltic reflex and in regulating gastric emptying. No physiologic role has yet been identified for 5-HT1A receptors in the ENS.

The release of [3H]5-HT and its binding protein, SBP, from the guinea pig enteric nervous system was analyzed. Release of both [3H]5-HT and [3H]NE from strips of longitudinal muscle with adherent myenteric plexus preloaded with the respective radioactive amine was evoked by high K+ and the ionophore X537A. However, Ca2+-dependence could not be shown for [3H]5-HT release by either agent or for [3H]NE release by X537A. However, Ca2+-dependence (as well as inhibition of release by high Mg2+ and tetrodotoxin) could be demonstrated for the release of radioactivity evoked by electrical field stimulation of everted segments of ileum preloaded by perfusion through the serosal lumen with [3H]5-HT. Light and electron microscopic radioautography revealed that the sources of released radioactivity were axons, especially axonal varicosities containing a mixture of small clear and large dense-cored vesicles. SPB, but not the cytosol marker protein, lactic dehydrogenase, was spontaneously released from the perfused everted ileum. A marked increase in SBP (but not LDH) release was provoked by electrical field stimulation at 10 Hz, and this increased release (but not the spontaneous release) was Ca2+-dependent. It is concluded that SBP and 5-HT are probably stored together, at least in part in vesicles, and that both can be released by exocytosis from depolarized axon terminals.

A protein fraction that binds serotonin was obtained from astrocytes in primary cultures. It was found to differ in some of its physical, chemical, and pharmacological properties from neuronal serotonin binding protein.

This study was designed to pinpoint the site at which N-hexanoyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP-hex) exerts its previously reported effect on thalamic neurons in rats. The animals were prepared under chloralose-urethane anesthesia for a stereotaxic approach to either the nucleus ventralis posterolateralis (nVPL) or the centrum medianum-parafascicularis complex (CM-Pf) of the thalamus. Individual neurons in these nuclei were separately activated by single-pulse stimulation of the sciatic nerve or the thalamic fibers that form reciprocal connections between the CM-Pf and the second somatosensory (SII) region of the nVPL. Poststimulus time histograms were constructed from computer readouts of the stimulus-evoked responses of a neuron during a 500-ms period accumulated in a digital computer 100X. In addition, the number of spikes accumulated in each histogram was compared to the number of spikes accumulated under identical conditions on the same neuron after intracarotid infusion of 5-HTP-DP-hex. The effect of the drug was reversed by the infusion of 5-HTP. Statistical evaluation of the accumulated spike counts indicated that 5-HTP-DP-hex suppressed only the excitation of CM-Pf neurons from the SII of the nVPL; the input of the sciatic nerve into the CM-Pf remained unaltered. Furthermore, no effect was exerted by this dipeptide on the afferent excitation of neurons in the SII of the nVPL.(ABSTRACT TRUNCATED AT 250 WORDS)

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.