Temperature-dependent interaction of the bovine hippocampal serotonin(1A) receptor with G-proteins

The ligand binding and G-protein coupling of the bovine hippocampal 5-HT1A receptor as a function of temperature was monitored. There is an almost complete and irreversible loss in agonist binding at 50 degrees C. However, the antagonist binding is reduced only by 50%, and this could be reversed if the temperature is lowered to 25 degrees C. Interestingly, the agonist binding of the 5-HT1A receptor in membranes exposed to 50 degrees C is inhibited to a much lesser extent by GTP-gamma-S, a non-hydrolysable analogue of GTP, indicating uncoupling of the 5-HT1A receptor to G-proteins at 50 degrees C. We propose that high temperature selectively and irreversibly inactivates G-proteins thereby affecting G-protein-receptor interaction and agonist binding of the 5-HT1A receptor.     

Cholesterol modulates ligand binding and G-protein coupling to serotonin(1A) receptors from bovine hippocampus

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven-transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding activity and G-protein coupling of the bovine hippocampal 5-HT(1A) receptor by depleting cholesterol from native membranes using methyl-beta-cyclodextrin (MbetaCD). Removal of cholesterol from bovine hippocampal membranes using varying concentrations of MbetaCD results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT to 5-HT(1A) receptors. This is accompanied by alterations in binding affinity and sites obtained from analysis of binding data. Importantly, cholesterol depletion affected G-protein-coupling of the receptor as monitored by the GTP-gamma-S assay. The concomitant changes in membrane order were reported by changes in fluorescence polarization of membrane probes such as DPH and TMA-DPH, which are incorporated at different locations (depths) in the membrane. Replenishment of membranes with cholesterol led to recovery of ligand binding activity as well as membrane order to a considerable extent. Our results provide evidence, for the first time, that cholesterol is necessary for ligand binding and G-protein coupling of this important neurotransmitter receptor. These results could have significant implications in understanding the influence of the membrane lipid environment on the activity and signal transduction of other G-protein-coupled transmembrane receptors.     

Interaction of Serotonin1A Receptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin1A (5-HT1A) receptor, an important member of the G-protein-coupled receptor superfamily. 2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics. 3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane. 4. The local anesthetics showed inhibition of agonist binding to the 5-HT1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT1A receptor brought about by local anesthetics is independent of the membrane cholesterol content. 5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.     

The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin 1A receptor

The serotonin(1A) (5-HT(1A)) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding of the bovine hippocampal 5-HT(1A) receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT(1A) receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT(1A) receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT(1A) receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT(1A) receptor function.     

Mutagenesis of the Human 5-HT1B Receptor: Differences from the Closely Related 5-HT1A Receptor and the Role of Residue F331 in Signal Transduction

Abstract

We have used a combination of sequence comparisons, computer-based modeling and site-directed mutagenesis to investigate the molecular interactions involved in ligand binding and signal transduction of the human 5-HT_1B receptor. Two amino acid residues, S212 in transmembrane region (TM) V and F331 in TM VI, were replaced by alanines. These amino acids are conserved in many G protein-coupled receptors and therefore likely to be important for receptor function. The mutant receptors were expressed in Chinese hamster ovary cells. The 5-HT-like agonist 5-carboxamido-tryptamine (5-CT) bound with 15-fold lower affinity to the S212A mutant as compared to wild-type receptor and the antagonist methiothepin bound with 17-fold lower affinity to the F331A mutant. No reduction in the affinity of 5-HT was seen for the S212A mutant, although an equivalent mutation in the 5-HT_1A receptor resulted in a 100-fold reduction of 5-HT binding. The inhibition of forskolin-stimulated cyclic AMP production by 5-HT was significantly reduced in cells expressing the F331A mutant, even though the endogenous ligand 5-HT bound with somewhat increased affinity. Methiothepin acted as an inverse agonist and increased the forskolin-stimulated cyclic AMP production at both the wild-type receptor and the mutants, and the effect was stronger on the F331A mutant. These results suggest that F331 is involved in the conformational changes necessary for signal transduction.     

Sustained pharmacological blockade of NK1 substance P receptors causes functional desensitization of dorsal raphe 5-HT 1A autoreceptors in mice

Antagonists at NK1 substance P receptors have demonstrated similar antidepressant properties in both animal paradigms and in human as selective serotonin reuptake inhibitors (SSRIs) that induce desensitization of 5-HT 1A autoreceptors within the dorsal raphe nucleus (DRN). We investigated whether this receptor adaptation also occurs upon NK1 receptor blockade. C57B/L6J mice were treated for 21 days with the selective NK1 receptor antagonist GR 205171 (10 mg/kg daily) through subcutaneously implanted osmotic mini pumps, and DRN 5-HT 1A autoreceptor functioning was assessed using various approaches. Recording of DRN serotonergic neurons in brainstem slices showed that GR 205171 treatment reduced (by approximately 1.5 fold) the potency of the 5-HT 1A receptor agonist, ipsapirone, to inhibit cell firing. In parallel, the 5-HT 1A autoreceptor-mediated [35S]GTP-gamma-S binding induced by 5-carboxamidotryptamine onto the DRN in brainstem sections was significantly decreased in GR 205171-treated mice. In vivo microdialysis showed that the cortical 5-HT overflow caused by acute injection of the SSRI paroxetine (1 mg/kg) was twice as high in GR 205171-treated as in vehicle-treated controls. In the DRN, basal 5-HT outflow was significantly enhanced by GR 205171 treatment. These data supported the hypothesis that chronic NK1 receptor blockade induces a functional desensitization of 5-HT 1A autoreceptors similar to that observed with SSRIs.     

Design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT1A/5-HT3 ligands

A series of new benzimidazole-arylpiperazine derivatives III were designed, synthesized and evaluated for binding affinity at serotoninergic 5-HT(1A) and 5-HT(3) receptors. Compound IIIc was identified as a novel mixed 5-HT(1A)/5-HT(3) ligand with high affinity for both serotonin receptors and excellent selectivity over alpha(1)-adrenergic and dopamine D(2) receptors. This compound was characterized as a partial agonist at 5-HT(1A)Rs and a 5-HT(3)R antagonist, and was effective in preventing the cognitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test.     

Cloning, expression, and characterization of the unique bovine A1 adenosine receptor. Studies on the ligand binding site by site-directed mutagenesis.

The bovine brain A1 adenosine receptor (A1AR) is distinct from other A1ARs in that it displays the unique agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine and has a 5-10-fold higher affinity for both agonists and antagonists. The cDNA for this receptor has been cloned from a size-selected (2-4-kb) bovine brain library and sequenced. The 2.0-kb cDNA encodes a protein of 326 amino acid residues with a molecular mass of 36,570 daltons. The amino acid sequence fits well into the seven-transmembrane domain motif typical of G protein-coupled receptors. Northern analysis in bovine tissue using the full length cDNA demonstrates mRNAs of 3.4 and 5.7 kb with a tissue distribution consistent with A1AR binding. Subcloning of the cDNA in a pCMV5 expression vector with subsequent transfection into both COS7 and Chinese hamster ovary cells revealed a fully functional A1AR which could inhibit adenylylcyclase and retained the unique pharmacologic properties of the bovine brain A1AR. The A1AR was found to have a single histidine residue in each of transmembrane domains 6 and 7. Histidine residues have been postulated by biochemical studies to be important for ligand binding. Mutation of His-278 to Leu-278 (seventh transmembrane domain) dramatically decreased both agonist and antagonist binding by greater than 90%. In contrast, mutation of His-251 to Leu-251 decreased antagonist affinity and the number of receptors recognized by an antagonist radioligand. In contrast, agonist affinity was not perturbed but the number of receptors detected by an agonist radioligand was also reduced. These data suggest that both histidines are important for both agonist and antagonist binding, but His-278 appears critical for ligand binding to occur.     

Membrane cholesterol oxidation inhibits ligand binding function of hippocampal serotonin(1A) receptors

We have monitored the ligand binding function of the bovine hippocampal 5-HT(1A) receptor following treatment of native membranes with cholesterol oxidase. Cholesterol oxidase is a water soluble enzyme that acts on the membrane interface to catalyze the conversion of cholesterol to cholestenone. Oxidation of membrane cholesterol significantly inhibits the specific binding of the agonist and antagonist to 5-HT(1A) receptors. Fluorescence polarization measurements of membrane probes incorporated at different locations in the membrane revealed no appreciable effect on membrane order due to the oxidation of cholesterol to cholestenone. These results therefore suggest that the ligand binding function of the 5-HT(1A) receptor is a cholesterol-dependent phenomenon that is not related to the ability of cholesterol to modulate membrane order. Importantly, these results represent the first report on the effect of a cholesterol-modifying agent on the ligand binding function of this important neurotransmitter receptor.     

Serotonergic modulation of retinal input to the mouse suprachiasmatic nucleus mediated by 5-HT1B and 5-HT7 receptors

Serotonin (5-HT) and 5-HT receptor agonists can modify the response of the mammalian suprachiasmatic nucleus (SCN) to light. It remains uncertain which 5-HT receptor subtypes mediate these effects. The effects of 5-HT receptor activation on optic nerve-mediated input to SCN neurons were examined using whole-cell patch-clamp recordings in horizontal slices of ventral hypothalamus from the male mouse. The hypothesis that 5-HT reduces the effect of retinohypothalamic tract (RHT) input to the SCN by acting at 5-HT1B receptors was tested first. As previously described in the hamster, a mixed 5-HT(1A/1B) receptor agonist, 1-[3-(trifluoromethyl)phenyl]-piperazine hydrochloride (TFMPP), reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) evoked by selectively stimulating the optic nerve of wild-type mice. The agonist was negligibly effective in a 5-HT1B receptor knockout mouse, suggesting minimal contribution of 5-HT1A receptors to the TFMPP-induced reduction in the amplitude of the optic nerve-evoked EPSC. We next tested the hypothesis that 5-HT also reduces RHT input to the SCN via activation of 5-HT7 receptors. The mixed 5-HT(1A/7) receptor agonist, R(+)-8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), reduced the evoked EPSC amplitude in both wild-type and 5-HT1B receptor knockout mice. This effect of 8-OH-DPAT was minimally attenuated by the selective 5-HT1A receptor antagonist WAY 100635 but was reversibly and significantly reduced in the presence of ritanserin, a mixed 5-HT(2/7) receptor antagonist. Taken together with the authors' previous ultrastructural studies of 5-HT1B receptors in the mouse SCN, these results indicate that in the mouse, 5-HT reduces RHT input to the SCN by acting at 5-HT1B receptors located on RHT terminals. Moreover, activation of 5-HT7 receptors in the mouse SCN, but not 5-HT1A receptors, also results in a reduction in the amplitude of the optic nerve-evoked EPSC. The findings indicate that 5-HT may modulate RHT glutamatergic input to the SCN through 2 or more 5-HT receptors. The likely mechanism of altered RHT glutamatergic input to SCN neurons is an alteration of photic effects on the SCN circadian oscillator.     

Serotonergic regulation of blood glucose levels in the crayfish, Procambarus clarkii: site of action and receptor characterization

The present study investigated the site of action of 5-hydroxytryptamine (5-HT) and pharmacologically characterized the receptors involved in regulating blood glucose levels in the crayfish, Procambarus clarkii. Injection of 5-HT into intact animals increased glucose levels in a dose-dependent manner. In contrast, 5-HT failed to elicit a hyperglycemic response in eyestalk-ablated animals. Effects of several 5-HT receptor agonists and antagonists were examined. 5-CT, oxymetazoline (both 5-HT(1) receptor agonists) and alpha-methyl-5-HT (a 5-HT(2) receptor agonist), but not 1-phenylbiguanide, m-CPBG (both 5-HT(3) receptor agonists), or RS 67333 (a 5-HT(4) receptor agonist), induced hyperglycemic responses in a dose-dependent manner. In addition, 8-OH-DPAT (a 5-HT(1A) receptor agonist), L-694,247 (a 5-HT(1B/1D) receptor agonist), and DOI (a 5-HT(2A) receptor agonist) were effective in significantly increasing the glucose levels, whereas both BW 723C86 (a 5-HT(2B) receptor agonist) and m-CPP (a 5-HT(2C) receptor agonist) were ineffective. Finally, ketanserin (a 5-HT(2A) receptor antagonist), but not p-MPPF (a 5-HT(1A) receptor antagonist), GR 55562 (a 5-HT(1B/1D) receptor antagonist), SB 206553 (a 5-HT(2B/2C) receptor antagonist), or tropisetron (a 5-HT(3) receptor antagonist), was able to block 5-HT-induced hyperglycemia. The combined results support the hypothesis that 5-HT exerts its hyperglycemic effect by enhancing the release of hyperglycemic factor(s) from the eyestalks, and suggest that 5 HT-induced hyperglycemia is mediated by 5-HT(1)- and 5-HT(2)-like receptors.     

Functional characterization of the novel antipsychotic iloperidone at human D2, D3, alpha 2C, 5-HT6, and 5-HT1A receptors

Iloperidone has demonstrated an interesting monoamine receptor profile in radioligand binding studies, with nanomolar affinity for certain noradrenaline, dopamine, and serotonin receptors. In this study, the agonist/antagonist activity of iloperidone was determined in cell lines expressing recombinant human D(2A), D(3), alpha(2C), 5-HT(1A), or 5-HT(6) receptors. With the exception of 5-HT(6) receptors, these receptors are negatively coupled to cyclase. Thus, after stimulation with forskolin, the agonists dopamine (at D(2A) and D(3)), noradrenaline (at alpha(2C)), or 8-OH-DPAT (at 5-HT(1A)) induced a reduction in cAMP accumulation. Conversely, activation of the 5-HT(6) receptor by 5-HT led to an increase in cAMP accumulation. Iloperidone alone was devoid of significant agonist activity but inhibited the agonist response in all 5 cell lines in a surmountable and concentration-dependent fashion. Iloperidone was most potent at D(3) receptors (pK(B) 8.59 +/- 0.20; n = 6), followed by alpha(2C) (pK(B) 7.83 +/- 0.06; n = 15), 5-HT(1A) (pK(B) 7.69 +/- 0.18; n = 10), D(2A) (pK(B) 7.53 +/- 0.04; n = 11) and 5-HT(6) (pK(B) 7.11 +/- 0.08; n = 11) receptors.     

Serotonin 5-HT1D Receptors in Human Prefrontal Cortex and Caudate: Interaction with a GTP Binding Protein

Radioligand binding studies were performed to characterize serotonin 5-HT1D receptors in postmortem human prefrontal cortex and caudate homogenates. [3H]5-HT binding, in the presence of pindolol (to block 5-HT1A and 5-HT1B receptors) and mesulergine (to block 5-HT1C receptors), was specific, saturable, reversible, and of high affinity. Scatchard analyses of [3H]5-HT-labeled 5-HT1D sites in human prefrontal cortex produced a KD value of 4.2 nM and Bmax of 126 fmol/mg protein. In competition experiments, 8-hydroxydipropylaminotetralin, trifluoromethylphenylpiperazine, mesulergine, 4-bromo-2,5-dimethoxyphenylisopropylamine, and ICS 205-930 had low affinity for [3H]5-HT-labeled 5-HT1D sites, indicating that the pharmacology of the 5-HT1D site is distinct from that of previously identified 5-HT1A, 5-HT1B, 5-HT1C, 5-HT2, and 5-HT3 sites. 5-HT1D sites in human brain have a similar pharmacology to the 5-HT1D sites previously identified in rat, porcine and bovine brains. Guanyl nucleotides, guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and guanosine 5'-(beta, gamma-imido)-triphosphate (Gpp(NH)p), modulated the binding of [3H]5-HT to 5-HT1D sites, whereas adenyl nucleotides had no effect. These findings are supportive of the presence of serotonin 5-HT1D receptors in human prefrontal cortex and caudate which appear to be coupled to a GTP binding protein.     

Modulation of cannabinoid agonist binding by 5-HT in the rat cerebellum

Cross-talk between cannabinoid CB1 and serotonin 5-HT receptors in rat cerebellar membranes was investigated using radioligand binding. In competition against the CB1 antagonist, [3 H]SR141716A, the agonist, WIN 55,212-2 yielded a biphasic isotherm. The majority of binding was to a high-affinity state that was significantly reduced by the GTP analogue, Gpp(NH)p. Interestingly, 5-HT enhanced the high-affinity binding constant of WIN 55,212-2 while attenuating the proportion of high-affinity binding. 5-HT also significantly reduced the proportion of high-affinity binding of the cannabinoid agonist, HU 210, but had no effect on the agonist, CP 55,940. The effect of 5-HT on WIN 55,212-2 binding was inhibited by the 5-HT2 receptor antagonist ritanserin as well as Gpp(NH)p, suggesting a dependence on the 5-HT2 receptor and on G protein-receptor interactions, respectively. Subsequent [3 H]WIN 55,212-2 dissociation kinetic experiments revealed that 5-HT promoted a slower-dissociating species of radiolabelled agonist-receptor complex. Our findings support a membrane-delimited cross-talk between two G protein-coupled receptors that are co-localized in certain cells of the central nervous system. Intriguingly, the cannabinoid agonist dependence of the 5-HT modulatory effect suggests that agonist-specific conformations of the CB1 receptor may also be important in determining the extent of this cross-talk.     

The sterol-binding antibiotic nystatin differentially modulates ligand binding of the bovine hippocampal serotonin1A receptor

We have monitored the ligand binding of the bovine hippocampal 5-HT1A receptor following treatment with the sterol-binding antifungal antibiotic nystatin. Nystatin considerably inhibits the specific binding of the antagonist to 5-HT1A receptors in a concentration-dependent manner. However, the specific agonist binding does not show significant changes. Fluorescence polarization measurements of membrane probes incorporated at different locations in the membrane revealed a substantial decrease in the membrane order in the interior of the bilayer. Experiments with cholesterol-depleted membranes indicate that the action of nystatin is mediated through membrane cholesterol. These results represent the first report on the effect of a cholesterol-perturbing agent on the ligand-binding activity of this important neurotransmitter receptor.     

GABA(B) receptors in 5-HT transporter- and 5-HT1A receptor-knock-out mice: further evidence of a transduction pathway shared with 5-HT1A receptors

The functional properties of GABA(B) receptors were examined in the dorsal raphe nucleus (DRN) and the hippocampus of knock-out mice devoid of the 5-HT transporter (5-HTT-/-) or the 5-HT(1A) receptor (5-HT(1A)-/-). Electrophysiological recordings in brain slices showed that the GABA(B) receptor agonist baclofen caused a lower hyperpolarization and neuronal firing inhibition of DRN 5-HT cells in 5-HTT-/- versus 5-HTT+/+ mice. In addition, [(35)S]GTP-gamma-S binding induced by GABA(B) receptor stimulation in the DRN was approximately 40% less in these mutants compared with wild-type mice. In contrast, GABA(B) receptors appeared functionally intact in the hippocampus of 5-HTT-/-, and in both this area and the DRN of 5-HT(1A)-knock-out mice. The unique functional changes of DRN GABA(B) receptors closely resembled those of 5-HT(1A) autoreceptors in 5-HTT-/- mice, further supporting the idea that both receptor types are coupled to a common pool of G-proteins in serotoninergic neurons.     

Dopamine D1 receptors characterized with [3H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D1 receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D1 receptors, as assessed by the binding of the selective antagonist [3H]SCH 23390. The binding of [3H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [3H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D1 receptors. Moreover, agonist high affinity binding to D1 receptors and its sensitivity to guanine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D1 receptors labeled with [3H] dopamine (agonist) or [3H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [3H]dopamine but not [3H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D1 receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D2 receptor, and the molecular mechanism responsible for this difference remains to be elucidated.     

Benzimidazole derivatives. Part 5: design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT1A/5-HT3 ligands

A series of new mixed benzimidazole-arylpiperazine derivatives were designed by incorporating in general structure III the pharmacophoric elements of 5-HT(1A) and 5-HT(3) receptors. Compounds 1-11 were synthesized and evaluated for binding affinity at both serotoninergic receptors, all of them exhibiting high 5-HT(3)R affinity (K(i)=10-62nM), and derivatives with an o-alkoxy group in the arylpiperazine ring showing nanomolar affinity for the 5-HT(1A)R (K(i)=18-150nM). Additionally, all the synthesized compounds were selective over alpha(1)-adrenergic and dopamine D(2) receptors (K(i)>1000-10,000nM). Compound 3 was selected for further pharmacological characterization due to its interesting binding profile as mixed 5-HT(1A)/5-HT(3) ligand with high affinity for both receptors (5-HT(1A): K(i)=18.0nM, 5-HT(3): K(i)=27.2nM). In vitro and in vivo findings suggest that this compound acts as a partial agonist at 5-HT(1A)Rs and as a 5-HT(3)R antagonist. This novel mixed 5-HT(1A)/5-HT(3) ligand was also effective in preventing the cognitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test, suggesting a potential interest in the treatment of cognitive dysfunction.     

5-hydroxytryptamine evokes endothelial nitric oxide synthase activation in bovine aortic endothelial cell cultures.

Activation of endothelial nitric oxide synthase (eNOS) results in the production of nitric oxide (NO) that mediates the vasorelaxing properties of endothelial cells. The goal of this project was to address the possibility that 5-hydroxytryptamine (5-HT) stimulates eNOS activity in bovine aortic endothelial cell (BAEC) cultures. Here, we tested the hypothesis that 5-HT receptors mediate eNOS activation by measuring agonist-stimulated [3H]L-citrulline ([3H]L-Cit) formation in BAEC cultures. We found that 5-HT stimulated the conversion of [3H]L-arginine ([3H]L-Arg) to [3H]L-Cit, indicating eNOS activation. The high affinity 5-HT1B receptor agonist, 5-nonyloxytryptamine (5-NOT)-stimulated [3H]L-Cit turnover responses were concentration-(0.01 nM to 100 microM) and time-dependent. Maximal responses were observed within 10 min following agonist exposures. These responses were effectively blocked by the 5-HT1B receptor antagonist, isamoltane, the 5-HT1B/5-HT2 receptor antagonist, methiothepin, and the eNOS selective antagonists (0.01-10 microM): L-Nomega -monomethyl-L-arginine (L-NMMA) and L-N omega-iminoethyl-L-ornithine (L-NIO). Pretreatment of BAEC cultures with pertussis toxin (PTX; 1-100 ng/ml) for 16 hr resulted in significant inhibition of the agonist-stimulated eNOS activity, indicating the involvement of Gi proteins. These findings lend evidence of a 5-HT1B receptor/eNOS pathway, accounting in part for the activation of eNOS by 5-HT. Further investigation is needed to determine the role of other vascular 5-HT receptors in the stimulation of eNOS activity.     

Peripheral 5-carboxamidotryptamine induces hindlimb scratching by stimulating 5-HT1A receptors in rats.

Treatment of rats with 5-carboxamidotryptamine (5-CT) or 5-methoxy-tryptamine (5-MeOT) induces a hindlimb scratch response. These compounds have high affinity for 5-HT1A and 5-HT1D receptors. The selective 5-HT1A receptor agonist N,N-dipropyl-5-CT (DP-5-CT) also induced hindlimb scratching while the selective 5-HT1D receptor agonist, sumatriptan, did not. 5-CT-induced hindlimb scratching was inhibited dose-dependently by several 5-HT1A antagonists (BMY 7378, NAN-190, MDL 73005EF and pindobind-5-HT1A) as well as the non-selective 5-HT antagonist, methiothepin. Pretreatment of rats with the serotonin (5-HT) synthesis inhibitor, p-chlorophenylalanine (PCPA) or the 5-HT depleting agent, reserpine, markedly attenuated 5-CT-induced hindlimb scratching. These data suggest that hindlimb scratching induced by 5-HT agonists may not be centrally mediated but rather may be mediated by a neuronal 5-HT1A receptor localized outside the blood-brain barrier.