Anoxic LTP sheds light on the multiple facets of NMDA receptors.

Hippocampal neurones in the CA1 region have become a model system to study the mechanisms of long-term potentiation (LTP) and memory processes. The CA1 region is also highly vulnerable to ischaemic or anoxic episodes which induce a selective and delayed degeneration of pyramidal neurones. In CA1 neurones, anoxic episodes generate a novel form of LTP to which we refer as anoxic LTP. In common with tetanic LTP, the induction of anoxic LTP is voltage- and NMDA receptor-dependent. However, in contrast with tetanic LTP, the expression of anoxic LTP is mediated exclusively by NMDA receptors. These observations suggest that anoxic-ischaemic episodes trigger a switch in favour of NMDA receptor-operated synaptic transmission. We suggest that the multiple forms of NMDA receptor-dependent LTPs are determined by extracellular and intracellular modulatory sites of this receptor.

NMDA receptor redox sites: are they targets for selective neuronal protection?

NMDA receptors play a central role in neuronal plasticity and in several pathological situations. Transient activation of this receptor triggers long-term potentiation, whereas sustained activation leads to cell death. Evidence for control of this activity by a redox site in cell cultures, brain tissues and in recombinant NMDA receptors are discussed by Henri Gozlan and Yehezkel Ben-Ari. The characteristics of this modulation and the consequences of redox state modifications on NMDA-mediated events are examined in vitro under physiological and pathological conditions. Since metabolic disorders enhance NMDA receptor function, the redox site could constitute a new target for selectively preventing in vivo the deleterious consequences of overactivation without blocking neuronal plasticity mediated by NMDA receptors.

Altered balance between thyrotropin-releasing hormone and dopamine in prolactinomas and other pituitary tumors compared to normal pituitaries.

We measured TRH and dopamine (DA) concentrations in prolactinomas and other pituitary tumors in order to further understand the roles of these two factors in the hormone hypersecretion and growth of these tumors. The mean TRH concentration (by RIA) in 16 prolactinomas was 247 +/- 92 (+/- SE) fmol/mg cell protein (range, 10-1297), near that found in normal pituitary tissue. The prolactinoma TRH content did not correlate with the patient's tumor size or plasma PRL level. By contrast, DA assayed by high pressure liquid chromatography was present in normal pituitary tissue (7.3 +/- 3.5 pmol/mg cell protein), but was very low or undetectable in the prolactinomas (23 fmol/mg cell protein or less). 3,4-Dihydroxyphenylacetic acid, also assayed by high pressure liquid chromatography, was undetectable in both normal pituitary tissue and prolactinomas. This imbalance between TRH and DA content also was found in GH-secreting and nonsecreting adenomas. The TRH content in 18 GH-secreting tumors (24 +/- 6 fmol/mg) was considerably lower than that in the prolactinomas (P less than 0.001). In 8 nonsecreting adenomas, the mean TRH concentration was 109 +/- 28 fmol/mg, about half of that in the prolactinomas. In those 2 types of adenomas, DA also was nearly undetectable (less than or equal to 73 fmol/mg cell protein). We conclude that the imbalance between TRH and DA contents in prolactinomas compared to those in normal pituitary tissue might participate in the mechanisms leading to hypersecretion of PRL and the growth of all types of pituitary adenomas.

Aging associated changes in serotoninergic and dopaminergic pre- and postsynaptic neurochemical markers in the rat brain.

Measurements of endogenous levels of serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), dopamine (DA) and dihydroxyphenyl acetic acid (DOPAC), and biochemical and autoradiographic investigations on 5-HT and DA receptors were made in various brain regions in male rats at three different ages: 3 months, 10 months and 22 months. Age-dependent decreases in 5-HT levels associated with parallel increases in 5-HIAA/5-HT ratio were observed in the hypothalamus, striatum, hippocampus and cerebral cortex, suggesting an accelerated 5-HT turnover in aged rats. Similarly, DA levels were lower, and DOPAC/DA ratio was higher in the striatum of 22-month-old compared to 3-month-old or 10-month-old rats. Of the three different classes of 5-HT receptors which were examined, 5-HT1B sites exhibited the largest age-dependent decrease in density, followed by 5-HT2 sites, while 5-HT1A sites remained practically unchanged during aging. By comparison, the loss of striatal D2 receptors in 22-month-old rats compared to young adults was much greater than that of any 5-HT receptor subtype. Such differential age-dependent alterations of the various classes of 5-HT receptors and of dopaminergic versus serotoninergic synaptic markers might be responsible for at least some of the functional deficits in aged animals.

Irreversible inactivation of beta-adrenergic receptors of C6 glioma cells. Synthesis and study of a thiol derivative of propranolol.

The beta-adrenergic receptor of C6 glioma cells contains a disulfide bridge which can be reduced by dithiothreitol (DTT). On intact cells, N-ethylmaleimide (NEM) (5 mM) does not change the affinity of [3H] H2-alprenolol ([3H] DHA) but reduces the total number of beta-adrenergic cell receptors by 21 +/- 3 per cent ; (N = 3). After receptor reduction by DTT, NEM irreversibly blocks the accessibility of the beta-adrenergic receptors to [3H]DHA. On isolated membranes, incubation in the presence of either NEM (5 mM) or isoproterenol (5.10(-7) M) does not significantly modify the total number of beta-adrenergic receptors accessible to [3H]DHA. Incubation of membranes with both NEM and isoproterenol reduces the number of binding sites by 33 +/- 2 per cent ; (N = 3). A thiol derivative of propranolol was synthetized. Its affinity is 10 times lower than that of propranolol. This sulfur derivative reduces the total number of beta-adrenergic receptors by 22 +/- 3 per cent (N = 3) when incubated with the native receptor and by 55 +/- 4 per cent (N = 4) when incubated with the reduced receptor. DTT does not significantly reverse the blockade induced by propranolol-SH. A model is proposed for explaining these results.

The main features of central 5-HT1 receptors.

The 5-HT1 receptor family comprises five different pharmacologic subtypes, designated 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, and 5-HT1E, whose common property is to bind 5-HT with nanomolar affinity. Recent investigations with molecular biology approaches led to the cloning and sequencing of 5-HT1A receptors in the rat and in the human, and of the 5-HT1C receptor in the rat. Although the 5-HT1A and 5-HT1C protein binding subunits exhibit the same structure with seven hydrophobic transmembrane domains, an extracellular N terminal and an intracellular C tail, their respective amino-acid sequences are markedly different. Indeed, a higher degree of sequence homology is found between the 5-HT1C and 5-HT2 receptors than between the former and 5-HT1A receptors, suggesting that the 5-HT1C subtype in fact belongs to the 5-HT2 class of central 5-HT receptors. All other 5-HT1 receptor subtypes are negatively coupled to adenylyl cyclase, whereas the 5-HT1C subtype, like 5-HT2 receptors, is positively coupled to phospholipase C. The respective regional distributions and regulatory properties, as well as pending questions regarding the ultrastructural localization, synthesis, mutual interactions, and axonal flow of 5-HT1 receptor subtypes, are also discussed.

Differential effects of d-fenfluramine and p-chloroamphetamine on H75/12-induced depletion of 5-hydroxytryptamine and dopamine in the rat brain.

The effects of the two 5-HT-releasing drugs, p-chloroamphetamine and d-fenfluramine, on central serotoninergic and dopaminergic systems were compared in adult rats. Both drugs (0.5-5.0 mg/kg i.p., 2 hr before death) produced a dose-dependent reduction in levels of 5-HT, but only p-chloroamphetamine decreased the levels of 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus, striatum and cerebral cortex. Within the dose range tested, d-fenfluramine did not affect the levels of DA and of its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in brain. By contrast, p-chloroamphetamine significantly increased the levels of DA and HVA and decreased the levels of DOPAC, notably in the striatum. As expected of a 5-HT uptake inhibitor, d-fenfluramine at small doses (0.2-0.5 mg/kg) prevented the depletion of 5-HT due to 4-methyl-alpha-ethyl-meta-tyramine (H75/12, 40 mg/kg i.p.), whereas at large doses (1.0-5.0 mg/kg) d-fenfluramine, like p-chloroamphetamine (0.2-1.0 mg/kg), slightly enhanced the effect of H75/12. Neither d-fenfluramine (0.5 mg/kg) nor p-chloroamphetamine (0.5 mg/kg) affected the depletion of DA due to H75/12. These data indicate that p-chloroamphetamine is a 5-HT-releasing drug, at any dose between 0.2 and 5.0 mg/kg, whereas d-fenfluramine acts as a 5-HT uptake inhibitor at 0.2-0.5 mg/kg and as a 5-HT releasing drug at larger doses. On account of the potential neurotoxicity of 5-HT-releasing drugs but not 5-HT uptake inhibitors, it can be inferred that d-fenfluramine is very probably devoid of any neurotoxic action in the dose range (less than 1.0 mg/kg) required for its anorectic action.

Tianeptine stimulates uptake of 5-hydroxytryptamine in vivo in the rat brain.

The neurochemical effects of the atypical tricyclic antidepressant, tianeptine, were further assessed on central serotoninergic and dopaminergic systems in the rat. Acute treatment with tianeptine (10 mg/kg i.p.) significantly enhanced the levels of metabolites of 5-HT and DA, 5-hydroxyindole acetic acid and dihydroxyphenylacetic acid respectively, in the brain stem, striatum and cerebral cortex. These effects could be prevented by the administration of drugs acting selectively (or preferentially) on serotoninergic systems such as d,l-fenfluramine and 4-methyl-alpha-ethyl-metatyramine (H75/12), suggesting that the increased metabolism of DA was secondary to a modification of serotoninergic systems in tianeptine-treated rats. In contrast to that found with inhibitors of the uptake of 5-HT, treatment with tianeptine markedly enhanced depletion of 5-HT due to administration of H75/12. However, depletion of DA induced by H75/12, was not altered by tianeptine. In vitro measurement of the uptake of [3H]5-HT also confirmed that tianeptine exerted opposite effects to those of classical tricyclic antidepressants, since the in vivo administration of tianeptine (2 x 10 mg/kg i.p.) induced a significant increase in the uptake of [3H]5-HT in cortical synaptosomes. The fact that both inhibitors of the uptake of 5-HT and tianeptine which, in contrast, enhanced the in vivo uptake of 5-HT, are potent antidepressants, challenges the current hypothesis on the central mechanisms of action of these drugs.

Differential effects of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on various 5-HT receptor binding sites in the rat brain.

The effects of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an alkylating agent producing irreversible blockade of various membrane bound receptors in brain, were investigated on four different types of serotonin receptors, 5-HT1A, 5-HT1B, 5-HT2A and 5-HT3, in various brain regions in the rat. In addition, the fate of central benzodiazepine- and "R"-zacopride-specific binding sites was also examined in rats treated with EEDQ. Membrane binding assays and/or quantitative autoradiography with appropriate radioligands indicated that EEDQ inactivated 5-HT1A, 5-HT1B and 5-HT2A sites, but was poorly active on 5-HT3, benzodiazepine and "R" sites. Among the receptors affected by EEDQ, hippocampal 5-HT1A sites were the most sensitive to the alkylating agent (ID50 approximately 1 mg/kg i.p.), followed by the cortical 5-HT2A (ID50 approximately 3 mg/kg i.p.) and the striatal 5-HT1B (ID50 approximately 6 mg/kg i.p.) sites. Pretreatment by selective ligands partially protected hippocampal 5-HT1A sites from irreversible inactivation by EEDQ (10 mg/kg i.p.) with the following order of efficacy: WAY 100635 > spiperone > BMY 7378 > ipsapirone. Similarly, pretreatment by spiperone (5 mg/kg i.p.) also reduced the ability of EEDQ to inactivated cortical 5-HT2A receptors. Analyses of the time-course recovery of respective binding sites after EEDQ administration showed that the turnover rate of 5-HT1A sites did not significantly differ in the dorsal raphe nucleus and in various forebrain areas (hippocampus, septum, cerebral cortex; half-life: approximately 4 days), but was lower than that of cortical 5-HT2A sites (half-life: 2.9 days).

Opioid receptors and neuropeptides in the CNS in rats treated chronically with amoxapine or amitriptyline.

The central mechanism responsible for the potentiation by antidepressant drugs of analgesia induced by morphine, was explored by measuring the levels of various neuropeptides (met-enkephalin, leu-enkephalin, dynorphin, substance P and cholecystokinin-like materials) and the density of delta and mu opioid binding sites in the spinal cord of rats treated for 14 days with amoxapine (10 mg/kg i.p., daily) or amitriptyline (10 mg/kg i.p., daily). Similar measurements were made in the hypothalamus and cerebral cortex for comparison. Chronic treatment with amoxapine or amitriptyline did not affect the levels of dynorphin, substance P and cholecystokinin, but markedly enhanced the levels of leu-enkephalin in the three structures examined. The levels of met-enkephalin were also increased after treatment with amitriptyline but only in the spinal cord and hypothalamus. No changes in opioid receptors were found in the cerebral cortex, but the densities of delta and mu opioid binding sites were increased in the spinal cord, and decreased in the hypothalamus of rats treated with amoxapine or amitriptyline. These changes induced by antidepressants in opioidergic markers at the spinal level might account for the potentiation of the action of morphine in amoxapine- or amitriptyline-treated rats. In addition, the observed alterations in the same markers in the hypothalamus could be associated with changes induced by antidepressants in neuroendocrine regulation.

Quantification of 5-hydroxytryptamine1A receptors in the cerebellum of normal and X-irradiated rats during postnatal development.

5-Hydroxytryptamine1A receptors were studied in rats during the first postnatal month in the normal cerebellum and in the granule cell-deprived cerebellum produced by X-irradiation at postnatal day 5. Quantitative autoradiographic studies on sagittal sections of cerebellar vermis, using [1251]BH-8-MeO-N-PAT as radioligand or specific anti-receptor antibodies, revealed that 5-hydroxytryptamine1A receptors existed in the molecular/Purkinje cell layer but at variable density from one lobule to another. Thus, in both normal and X-irradiated rats, the posterior lobules were more heavily labelled than the anterior ones, and the density of 5-hydroxytryptamine1A sites decreased progressively in all the cerebellar folia down to hardly detectable levels at postnatal day 21. However, the intensity of labelling remained higher at postnatal day 8 and postnatal day 12 in X-irradiated rats than in age-paired controls. Measurements of [3H]8-OH-DPAT specific binding to membranes from whole cerebellum confirmed that the density of 5-hydroxytryptamine1A sites per mg membrane protein (Bmax) was higher in X-irradiated animals than in age-paired controls. However, on a "per cerebellum" basis, no significant difference could be detected between the total number of 5-hydroxytryptamine1A sites, which progressively increased in both control and X-irradiated animals during the first postnatal month. These results therefore show that 5-hydroxytryptamine1A receptors are not located on developing granule cells. The progressive decrease in 5-hydroxytryptamine1A receptor density during the first postnatal month did not reflect a transient expression of 5-hydroxytryptamine1A receptors in the cerebellum of newborn rats, but resulted from the progressive "dilution" of these sites in this growing structure. The higher density of 5-hydroxytryptamine1A sites in X-irradiated rats simply reflected a lower "dilution" due to the delayed growth of the cerebellum in these animals.

Baroreceptor reflex inhibition induced by the stimulation of serotonin3 receptors in the nucleus tractus solitarius of the rat.

Previous studies suggested that in the nucleus tractus solitarius, cardiovascular responses to serotonin may involve the simultaneous activation of more than one receptor subtype. In the present study, the cardiovascular effects of the local application of serotonin and different serotonin3 agonists and antagonists into the nucleus tractus solitarius were analysed in intact and unilaterally ganglionectomized rats. Unilateral injections of serotonin (5-15 nmol) produced a dose-dependent increase in blood pressure and partially antagonized the arterial baroreflex responses evoked by an i.v. injection of phenylephrine. Similar blood pressures response were obtained after unilateral microinjections of phenylbiguanide (5 nmol) and 2-methyl-serotonin (5 nmol), two serotonin3 receptor agonists. Bilateral microinjections of serotonin or phenylbiguanide produced more pronounced blood pressure effects and antagonized completely the baroreflex responses. Both blood pressure and baroreflex effects were antagonized by prior injections of specific serotonin3 antagonists such as zacopride (100 pmol) and ondansetron (100 pmol). Concomitant autoradiographic studies performed in intact and ganglionectomized rats, using [125I]iodozacopride, confirmed that serotonin3 receptors in the nucleus tractus solitarius are mainly located on vagal afferent fibers. In addition, serotonin microinjections made in the nucleus tractus solitarius ipsilateral to the ganglionectomy revealed a significant reduction in cardiovascular responses compared to intact animals. These results suggest that in the nucleus tractus solitarius of the rat, serotonin is involved in the reflex regulation of blood pressure through the stimulation of serotonin3 receptors presumably located on vagal afferent fibers. Since bicuculline antagonized the serotonin-mediated pressor responses, a serotonin3-dependent activation of an inhibitory GABAergic system within the nucleus tractus solitarius might be involved in blood pressure regulatory mechanisms.

Production and characterization of polyclonal antibodies recognizing the intracytoplasmic third loop of the 5-hydroxytryptamine1A receptor.

The portion of the complementary DNA encoding the third intracellular loop of the rat 5-hydroxytryptamine1A (serotonin) receptor was subcloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein coupled with the glutathione S-transferase of Schistosoma japonicum. The fusion protein was purified on a glutathione-agarose affinity column and used to immunize rabbits for the production of polyclonal anti-5-hydroxytryptamine1A receptor antibodies. Enzyme-linked immunosorbent assay revealed that antibodies were produced as early as one month after the first injection of the fusion protein, and immune response plateaued at a maximum after the third (monthly) booster injection. These antibodies only marginally affected the specific binding of [3H]8-hydroxy-2-(di-n-propyl-amino) tetralin to solubilized and membrane bound 5-hydroxytryptamine1A receptors, and did not interfere with serotonin-induced inhibition of forskolin-stimulated adenylate cyclase negatively coupled to 5-hydroxytryptamine1A receptors in rat hippocampal membranes. However, antibodies were able to immunoprecipitate 5-hydroxytryptamine1A receptor binding sites solubilized from rat hippocampal membranes. The distribution of immunoautoradiographic labelling and immunohistochemical staining of rat brain sections exposed to the antibodies raised against the fusion protein superimposed to that of 5-hydroxytryptamine1A receptor binding sites labelled by specific radioligands, with marked enrichment in the limbic areas (dentate gyrus and CA1 area in the hippocampus, lateral septum, entorhinal cortex) and the anterior raphe nuclei. The differential cellular location of immunoreactivity within the hippocampus (where dendritic fields but not pyramidal cell somas were immunostained) and the median raphe nucleus (where the plasmic membrane of somas was strongly immunoreactive) suggests that the addressing of 5-hydroxytryptamine1A receptors might differ from one neuronal cell type to another.

Anpirtoline, a novel, highly potent 5-HT1B receptor agonist with antinociceptive/antidepressant-like actions in rodents.

1. The purpose of the present study was to relate the effects of the novel drug, anpirtoline, on 5-hydroxytryptamine (5-HT) receptor subtypes to its antinociceptive and antidepressant-like actions in rodents. 2. Binding assays with rat brain membranes have shown that anpirtoline bound with a much higher affinity to 5-HT1B receptor (Ki = 28 nM) than to 5-HT1A (Ki = 150 nM) and 5-HT2 (Ki = 1.49 microM) receptors. 3. Like 5-HT, anpirtoline concentration-dependently inhibited forskolin-stimulated adenylate cyclase activity in homogenates from the rat substantia nigra. Both effects were not additive, and could be prevented by 5-HT1B receptor antagonists such as propranolol and penbutolol. 4. In superfused rat and pig brain cortex slices preincubated with [3H]-5-HT, the electrically evoked tritium overflow was inhibited by anpirtoline and 5-HT. Whereas 5-HT was equipotent in both tissues (EC50 = 69 nM), anpirtoline was markedly less potent in pig brain cortex slices (EC50 = 1190 nM) than in rat brain cortex slices (EC50 = 55 nM). The concentration-response curve for anpirtoline was shifted to the right by metitepine in both preparations. 5. In the social behaviour deficit test, anpirtoline and trifluoromethylphenyl-piperazine were effective in reversing the isolation-induced impairments in mice, an effect shown only by compounds with agonist properties at the 5-HT1B receptor. 6. In the electrostimulated pain test using mice, anpirtoline dose-dependently increased the pain threshold with an ED50 of 0.52 mg kg-1, i.p. The antinociceptive activity of anpirtoline was abolished by pretreatment with cyproheptadine or propranolol.7. In the forced swimming test in rats, anpirtoline induced a dose-related increase in swimming activity. With an ED50 value of 4.6mgkg-1, i.p., anpirtoline was 4 times more potent than the two standard compounds imipramine and desipramine. The decrease of immobility time or the increase of active periods in this model of behavioural despair is suggested to be characteristic of antidepressant drugs.8. Anpirtoline exhibits both antinociceptive and antidepressant-like activities in animals. It is probable that anpirtoline elicits these pharmacological effects via its agonist effect on 5-HT1B and 5-HT1A receptors.

5-HT3 receptor antagonism by anpirtoline, a mixed 5-HT1 receptor agonist/5-HT3 receptor antagonist.

1. The aim of this study was to provide evidence that anpirtoline, which is an agonist at 5-HT1B and 5-HT1D receptors and also displays submicromolar affinity for 5-HT1A recognition sites, in addition, acts as an antagonist at 5-HT3 receptors. 2. In radioligand binding studies on rat brain cortical membranes, anpirtoline inhibited specific binding of [3H]-(S)-zacopride to 5-HT3 receptor recognition sites (pKi: 7.53). 3. In N1E-115 neuroblastoma cells in which [14C]-guanidinium was used as a tool to measure cation influx through the 5-HT3 receptor channel, the 5-HT-induced influx was concentration-dependently inhibited by anpirtoline. In this respect, anpirtoline mimicked other 5-HT3 receptor antagonists; the rank order of potency was ondansetron > anpirtoline > metoclopramide. 4. The concentration-response curve for 5-HT as a stimulator of [14C]-guanidinium influx was shifted to the right by anpirtoline (apparent pA2: 7.78). 5. In urethane-anaesthetized rats, anpirtoline inhibited (at lower potency than zacopride and tropisetron) the 5-HT- or phenylbiguanide-induced bradycardia (Bezold-Jarisch reflex), but did not induce this reflex by itself. 6. Intravenous infusion of cisplatin in the domestic pig caused a consistent emetic response which was antagonized by anpirtoline. 7. It is concluded that anpirtoline, which was previously characterized as a 5-HT1 receptor agonist also proved to be a 5-HT3 receptor antagonist in several experimental models and, hence, exhibits a unique pattern of properties at different 5-HT receptors.

Involvement of tryptophan residue(s) in the specific binding of agonists/antagonists to 5-HT3 receptors in NG108-15 clonal cells.

Chemical modification of the 5-HT3 receptors in membranes from NG108-15 hybridoma cells was achieved using protein modifying reagents specific for various amino acid residues: N-bromosuccinimide for tryptophan, dithiothreitol for cystine, sodium tetrathionate for cysteine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline for aspartic and glutamic acids, diethylpyrocarbonate for histidine, tetranitromethane for tyrosine and 2,3-butanedione for arginine. Among all the reagents tested, N-bromosuccinimide produced the largest alteration in the specific binding of [3H]zacopride onto 5-HT3 receptors. A significant reduction in Bmax (approximately 50%) with no change in Kd were noted on [3H]zacopride specific binding to membranes which were incubated with 40 microM N-bromosuccinimide for 60 min at 25 degrees. The occupancy of 5-HT3 receptor binding sites by various 5-HT3 agonists and antagonists (phenylbiguanide, ondansetron, granisetron, MDL 72222) prevented, at least partially, any subsequent reduction in [3H]zacopride specific binding by N-bromosuccinimide treatment. However, neither m-chloro-phenylbiguanide, among the agonists, nor zacopride, among the antagonists, were able to prevent the effect of N-bromosuccinimide, suggesting that variations might exist in the molecular mechanisms implicated in the binding of 5-HT3 ligands to the recognition site on 5-HT3 receptors. Nevertheless, these data support the suggestion that tryptophan residue(s) are probably involved in the binding of agonists and antagonists onto 5-HT3 receptors in NG108-15 cell membranes.

Irreversible blockade of central 5-HT binding sites by 8-methoxy-2'-chloro-PAT.

We have synthesized 8-methoxy-2-(N-2'-chloropropyl, N-propyl) aminotetralin (8-methoxy-2'-chloro-PAT), an alkylating agent derived from the potent 5-HT agonist, 8-hydroxy-2-(N,N-dipropyl)-aminotetralin (PAT). As expected for an irreversible ligand, the blockade of 3H-PAT or 3H-5-HT binding to post-synaptic 5-HT1 (A and B) sites in rat hippocampal membranes pretreated with 8-methoxy-2'-chloro-PAT could not be prevented by extensive washing of membranes. Prior occupancy of 5-HT1 sites by 5-HT or PAT prevented any subsequent irreversible blockade by the alkylating agent. Similar irreversible blockade by 8-methoxy-2'-chloro-PAT was found on 3H-PAT binding to striatal membranes suggesting that presynaptic 5-HT binding sites (see Gozlan et al., Nature, Lond. 305, 140, 1983) were sensitive also to the alkylating agent. In contrast, the modifying agent N-ethylmaleimide (NEM) reduced markedly 3H-PAT binding to postsynaptic hippocampal 5-HT1 sites, but did not alter 3H-PAT binding to striatal presynaptic 5-HT sites. Although 8-methoxy-2'-chloro-PAT bound irreversibly to different classes of 5-HT binding sites (5-HT1A, 5-HT1B, presynaptic sites), it can be considered a selective alkylating agent, since it exerted no action on 3H-spiperone binding to 5-HT2 sites, 3H-muscimol binding to GABA sites, or 3H-flunitrazepam binding to benzodiazepine sites.

Physical evidence of the coupling of solubilized 5-HT1A binding sites with G regulatory proteins.

Previous investigations (El Mestikawy et al., J Neurochem 51: 1031-1040, 1988) have shown that 5-HT1A binding sites (R[5-HT1A]) solubilized by CHAPS from rat hippocampal membranes can be modulated by guanine nucleotides, as expected from their solubilization together with associated G regulatory proteins (G). Studies of the hydrodynamic properties of solubilized R[5-HT1A] have been presently carried out in order to assess in a more direct way the presence of R[5-HT1A]-G complexes in the soluble extract. Under control conditions, the sedimentation of a CHAPS extract from hippocampal membranes through a 5-30% sucrose gradient (200,000 g, 17 hr, 4 degrees) gave two maxima of [3H]8-OH-DPAT binding activity corresponding to sedimentation coefficients of 8.0 S and 10.0 S, respectively. Running the gradient in the presence of 1 microM GTP revealed a significant reduction of the 10.0 S peak, as expected from the loss of material (probably a G protein) normally associated with R[5-HT1A]. Conversely, attempts to prevent the dissociation of R[5-HT1A]-G by treatment of CHAPS soluble hippocampal extracts with the cross-linking reagent disuccinimidyl suberate (0.1 mM) resulted in a significant increase (+70%) in [3H]8-OH-DPAT binding activity associated with the appearance of a new sedimenting material with a higher coefficient (16.5 S). Furthermore, [3H]8-OH-DPAT binding became almost completely insensitive to guanine nucleotides as expected from the irreversible coupling by disuccinimidyl suberate of R[5-HT1A] with G protein(s). WGA-agarose chromatography of CHAPS soluble hippocampal extract supplemented with GTP allowed the physical separation of R[5-HT1A] from the bulk of G proteins, and a concomitant decrease of [3H]8-OH-DPAT high affinity binding capacity. Partial recovery of the latter could be achieved by reconstituting R[5-HT1A]-G complexes upon the addition of a mixture of pure bovine Gi + Go to G-deprived soluble extracts. Finally in vivo treatment with Pertussis toxin (5 micrograms intracerebroventricularly, 48 hr before killing) resulted in a significant reduction of the specific binding of [3H]8-OH-DPAT (-36%) to hippocampal membranes and corresponding CHAPS soluble extracts, and a marked decrease in the inhibitory effect of GppNHp. Accordingly the G protein associated with R[5-HT1A] belongs probably to the Gi or Go families.

In situ molecular sizes of the various types of 5-HT binding sites in the rat brain.

The radiation inactivation technique has been used to estimate the molecular weights of 5-HT binding sites in various regions of the rat brain. Using 3H-5-HT or 3H-8-OH-DPAT as the ligand, the same molecular weight of 55,000-60,000 daltons was calculated for the postsynaptic 5-HT1A and 5-HT1B sites in the hippocampus and cerebral cortex. Studies with 3H-ketanserin as the selective ligand indicated a molecular weight in the same range for the post-synaptic 5-HT2 binding site in the cerebral cortex. In contrast, a higher value (67,000 daltons) was found for the presynaptic 5-HT3 site selectively labelled by 3H-8-OH-DPAT in the striatum and cerebral cortex. The curvilinear pattern of the radiation-induced inactivation of 5-HT1A and 5-HT1B binding sites suggested that both sites belong to complex polymeric structures. In contrast, the 5-HT2 and 5-HT3 sites may correspond to less cooperative structures since simple monoexponential inactivation curves were observed upon irradiation.

Common pharmacological and physico-chemical properties of 5-HT3 binding sites in the rat cerebral cortex and NG 108-15 clonal cells.

On account of the postulated existence of 5-HT3 receptor subtypes, the respective physico-chemical and pharmacological properties of specific binding sites for the potent 5-HT3 antagonist [3H]zacopride were compared using membranes from the rat posterior cortex or neuroblastoma-glioma NG 108-15 clonal cells. In both membrane preparations, [3H]zacopride bound to a single class of specific sites with a Kd close to 0.5 nM. However, the Bmax value in NG 108-15 cell membranes (970 +/- 194 fmol/mg protein) was approximately 50 times larger than that in cortical membranes (19 +/- 2 fmol/mg protein). The specific binding of [3H]zacopride was equally affected by temperature, pH and molarity of the assay medium, and equally insensitive to thiol- and disulfide-reagents (N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, dithiothreitol) and GTP in cortical as well as NG 108-15 cell membranes. Determination of the molecular size of [3H]zacopride specific binding sites by radiation inactivation yielded values close to 35 kDa for both membrane preparations. Finally, a highly significant positive correlation (r = 0.979) was found between the respective pKi values of 34 different drugs for their inhibition of [3H]zacopride specific binding to cortical or NG 108-15 cell membranes. Among them, the most potent was S(-)zacopride (pKi = 9.55), followed by BRL 43964, ICS 205-930, quipazine, R(+)zacopride, GR 38032F and MDL 72222. Atypical antidepressants (mianserin, amoxapine) and neuroleptics (clotiapine, loxapine and clozapine) were active in rather low concentrations (pKi less than 6.5), suggesting that recognition of 5-HT3 sites might be relevant to part of the in vivo effects of these drugs. Such identical physico-chemical and pharmacological properties of [3H]zacopride specific binding in cortical and NG 108-15 cell membranes strongly suggest that the same 5-HT3 receptor (subtype?) exists in these two preparations.