Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells.

1. A cloned cDNA encoding a human 5-hydroxytryptamine3 receptor type A subunit (h5-HT3R-As) was transfected into human embryonic kidney (HEK 293) cells maintained in cell culture and a stable cell line expressing a high density of the recombinant receptor was selected. 2. Membrane homogenates prepared from transfected, but not untransfected, cells exhibited a homogeneous and saturable population (Bmax = 4.49 +/- 0.46 pmol mg-1 protein) of sites that bound the radiolabelled 5-HT3 receptor antagonist, [3H]-granisetron with high affinity (pKD = 8.87 +/- 0.08). Kinetic studies (at 37 degrees C) revealed rapid association (kappa +1 4.76 +/- 0.3 x 10(8) M-1 min-1) and dissociation (kappa -1 = 0.21 +/- 0.003 min-1) of the radioligand. 3. Selective and non-selective 5-HT3 receptor ligands competed for [3H]-granisetron binding with a rank order of potency (granisetron > ondansetron > meta-chlorophenylbiguanide > 5-HT > 2-methyl-5-HT > metoclopramide > > phenylbiguanide > cocaine > (+)-tubocurarine) identical to that established for 5-HT3 receptors endogenous to the human CNS. 4. In electrophysiological recordings performed on transfected cells, voltage-clamped at a holding potential of -60 mV, locally applied 5-HT (10 microM) evoked transient inward current responses that reversed in sign at a potential of -1.0 +/- 1.1 mV. Such responses were antagonized in a reversible manner by granisetron (1 nM). 5. The construction of a stable cell line expressing a high density of recombinant human 5-HT3 receptors which display appropriate pharmacology and function will assist in the further characterization of this receptor subtype and the exploration of species differences in 5-HT3 receptor pharmacology.     

Trichloroethanol potentiation of gamma-aminobutyric acid-activated chloride current in mouse hippocampal neurones.

1. The action of 2,2,2-trichloroethanol on gamma-aminobutyric acid (GABA)-activated Cl- current was studied in mouse hippocampal neurones in tissue culture by use of whole-cell patch-clamp recording. 2. Trichloroethanol increased the amplitude of currents activated by 1 microM GABA or 0.1 microM muscimol. Trichloroethanol, 1-25 mM, potentiated current activated by 1 microM GABA in a concentration-dependent manner with an EC50 of 3.0 +/- 1.4 mM and a maximal response (Emax) of 576 +/- 72% of control. 3. Trichloroethanol potentiated currents activated by GABA concentrations < 10 microM, but did not increase the amplitude of currents activated by concentrations of GABA > or = 10 microM. Despite marked potentiation of currents activated by low concentrations of GABA, trichloroethanol did not significantly alter the EC50, slope, or Emax of the GABA concentration-response curve. 4. Trichloroethanol, 5 mM, potentiated GABA-activated current in neurones in which ethanol, 10-500 mM, did not. The effect of trichloroethanol was not altered by the putative ethanol antagonist, Ro 15-4513. Trichloroethanol did not potentiate currents activated by pentobarbitone. 5. In the absence of exogenous GABA, trichloroethanol at concentrations > or = 2.5 mM activated a current that appeared to be carried by Cl- as its reversal potential changed with changes in the Cl- gradient and as it was inhibited by the GABAA antagonists, bicuculline methiodide and picrotoxin. 6. Since trichloroethanol is thought to be the active metabolite of chloral hydrate and other chloral derivative anaesthetics, potentiation of the GABA-activated current in central nervous system neurones by trichloroethanol may contribute to the sedative/hypnotic effects of these agents.     

Agonist interactions with 5-HT3 receptor recognition sites in the rat entorhinal cortex labelled by structurally diverse radioligands.

1. The pharmacological properties of 5-HT3 receptor recognition sites labelled with [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5-HT3 receptor complex. 2. In rat entorhinal cortex homogenates, [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 microM) with high affinity (Bmax = 75 +/- 5, 53 +/- 5, 92 +/- 6 and 79 +/- 6 fmol mg-1 protein, respectively; pKd = 9.41 +/- 0.04, 8.69 +/- 0.14, 8.81 +/- 0.06 and 10.14 +/- 0.04 for [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330, respectively, n = 3-8). 3. Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38-8.51, granisetron 8.62-8.03), whilst the agonists, 5-hydroxytryptamine (5-HT), phenylbiguanide (PBG) and 2-methyl-5-HT competed at sub-micromolar concentrations (pIC50; 5-HT 7.16-6.42, PBG 7.52-6.40, 2-methyl-5-HT 7.38-6.09). 4. Competition curves generated with increasing concentrations of quipazine, PBG, 5-HT and 2-methyl-5-HT displayed Hill coefficients greater than unity when the 5-HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for the binding of [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 by granisetron generated Hill coefficients around unity.(ABSTRACT TRUNCATED AT 250 WORDS)     

An electrophysiological investigation of the properties of a murine recombinant 5-HT3 receptor stably expressed in HEK 293 cells.

1. The pharmacological and biophysical properties of a recombinant 5-HT3 receptor have been studied by use of patch-clamp techniques applied to HEK 293 cells stably transfected with the murine 5-HT3 R-A cDNA. 2. At a holding potential of -60 mV, 77% of cells investigated responded to ionophoretically applied 5-HT with an inward current. Such currents were unaffected by methysergide (1 microM), or ketanserin (1 microM), but were antagonized in a concentration-dependent and reversible manner by the selective 5-HT3 receptor antagonist, ondansetron (IC50 = 440 pM) and the non-selective antagonists (+)-tubocurarine (IC50 = 1.8 nM) and metoclopramide (IC50 50 nM). 3. The 5-HT-induced current reversed in sign (E5-HT) at approximately -2mV and exhibited inward rectification. The influence of extra- and intracellular ion substitutions upon E5-HT indicates the 5-HT-evoked current to be mainly mediated by a mixed monovalent cation conductance. 4. Calcium and magnesium (0.1-10 nM) produced a concentration-dependent, voltage-independent, inhibition of the 5-HT-induced response. Zinc (0.3-300 microM) exerted a biphasic effect with low concentrations enhancing, and high concentrations depressing, the 5-HT-evoked current. 5. Fluctuation analysis of inward currents evoked by a low (1 microM) concentration of 5-HT suggests the current to be mediated by the opening of channels with a conductance of 420 fS. 6. The pharmacological and biophysical properties of the 5-HT3 R-A are similar to those previously described for 5-HT3 receptors native to murine neuroblastoma cell lines, with the exception that the function of the recombinant receptor was enhanced by low concentrations of zinc. This observation suggests that the properties of the native receptor are not completely represented by the 5-HT3 R-A subunit alone.     

Zolmitriptan stimulates a Ca(2+)-dependent K(+) current in C6 glioma cells stably expressing recombinant human 5-HT(1B) receptors

Stimulation of a Ca(2+)-dependent K(+) current by zolmitriptan, a 5-HT(1B/1D) receptor partial agonist, was investigated in C6 glioma cells stably expressing recombinant human 5-HT(1B) receptors. Outward K(+) currents (I(K)) were examined in non-transfected C6 glioma cells and in cells expressing cloned human 5-HT(1B) receptors using the patch-clamp technique in the whole-cell configuration. In C6 glioma cells expressing recombinant human 5-HT(1B) receptor, zolmitriptan increased I(K) in a concentration-dependent manner (maximum increase 16.3+/-7.8%, n=5, p<0.001) with a pD(2) value (geometric mean with 95% confidence intervals) of 7.03 (7.90-6.10). Zolmitriptan failed to elicit increases in I(K) in non-transfected C6 cells. In the presence of the mixed 5-HT(1B/1D) receptor antagonist, N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2(-methyl-4(5-methyl-1 ,2,4)-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamide 2HCl (GR 127935, 0. 1 mcM), zolmitriptan (1 mcM) failed to significantly increase I(K) in C6 cells expressing human 5-HT(1B) receptors confirming that zolmitriptan-evoked responses were indeed mediated by human 5-HT(1B) receptors. In C6 cells expressing cloned human 5-HT(1B) receptors, zolmitriptan-induced increases in I(K) were prevented by the calcium chelator, EGTA (5 mM) when included in the patch pipette (maximum increase -3.3+/-4.2%, n=4, P=NS). The Ca(2+)-dependent K(+) channel blockers, iberiotoxin (0.1 mcM) and tetraethylammonium (TEA, 1 mM), abolished zolmitriptan-induced increases in I(K) (4.5+/-7.3%, n=4 and -0.8+/-1.7%, n=4, respectively, P=NS in each case) in C6 cells expressing human 5-HT(1B) receptors, confirming the involvement of Ca(2+)-dependent K(+) channels. In conclusion, the 5-HT(1B/1D) receptor partial agonist, zolmitriptan, stimulates I(K/Ca) in C6 glioma cells stably transfected with human 5-HT(1B) receptors suggesting an increase of hyperpolarizing current.     

The effect of 5-HT and selective 5-HT receptor agonists and antagonists on rat dorsal vagal preganglionic neurones in vitro.

1. Whole-cell patch-clamp recordings were made from 142 visually identified rat dorsal vagal preganglionic neurones (DVMs). Applications of 5-hydroxytryptamine (5-HT, 20 microM, 2 min) elicited a slow depolarization (8.2 +/- 0.5 mV, n = 59) in 95% of the cells tested, accompanied by an increase in excitability. In (68%) of DVMs the depolarization was associated with an increase in apparent membrane resistance (Rmt 22.7 +/- 2.2%). These depolarizations and increases in Rm (14.3 +/- 2.6%, n = 8) were maintained in a medium which blocked synaptic transmission. 2. The response to 5-HT was associated with a reversal potential (Erev) of -91 +/- 1 mV at an extracellular K+ concentration (LK+]o) of 4.2 mM. This correlated well with the K+ equilibrium potential (Ek = -89 mV). 3. The depolarizing effect of 5-HT was attenuated by the 5-HT2A/2C receptor antagonists, ketanserin (1 microM), LY 53,857 (1 microM) and the 5-HT1A/2A receptor antagonist, spiperone (1 microM). The 5-HT1A receptor antagonist, pindobind 5-HT1A (5 microM), had no effect on the depolarizing response to 5-HT. 4. The effect of 5-HT was mimicked by the 5-HT2A/2C receptor agonist, alpha-methyl-5-HT (50 microM), the 5-HT1 receptor agonist, 5-carboxamidotryptamine (20 microM) and the putative 5-HT4 agonist, 5-methyoxytryptamine (5 microM). The selective 5-HT4 receptor antagonist, GR113808, had no effect on the depolarizing effect of 5-HT or 5-MEOT on DVMs. 5. The 5-HT3 antagonists, MDL 72222 (10 microM) and ICS-205-930 (1 and 10 microM), partially reduced the effect of 5-HT. The 5-HT3 receptor agonist, 2-methyl-5-HT (100-300 microM), excited a proportion of neurones tested (56%) by evoking a depolarizing and/or an increase in postsynaptic potentials (p.s.ps). 6. These results are consistent with direct, postsynaptic actions of 5-HT on DVMs via 5-HT2A receptors, being mediated, in part, by the reduction of K+ conductance.     

Evidence that the atypical 5-HT3 receptor ligand, [3H]-BRL46470, labels additional 5-HT3 binding sites compared to [3H]-granisetron.

1. The radioligand binding characteristics of the 3H-derivative of the novel 5-HT3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5-HT3 receptor radioligand [3H]-granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen. 2. In rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, [3H]-BRL46470 bound with high affinity (Kd (nM): 1.57 +/- 0.18, 2.49 +/- 0.30, 1.84 +/- 0.27, 3.46 +/- 0.36, respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 102 +/- 16, 44 +/- 4, 968 +/- 32 and 2055 +/- 105, respectively; mean +/- s.e. mean, n = 3-4) but failed to display specific binding in human putamen homogenates. 3. In the same homogenates of rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen as used for the [3H]-BRL46470 studies, [3H]-granisetron also bound with high affinity (Kd (nM): 1.55 +/- 0.61, 2.31 +/- 0.44, 1.89 +/- 0.36, 2.03 +/- 0.42 and 6.46 +/- 2.58 respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 39 +/- 4, 20 +/- 2, 521 +/- 47, 870 +/- 69 and 18 +/- 2, respectively; mean +/- s.e. mean, n = 3-4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ketanserin-sensitive depressant actions of 5-HT receptor agonists in the neonatal rat spinal cord.

1. The monosynaptic reflex (MSR), recorded in vitro from the neonatal rat spinal cord, was depressed by 5-hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), methysergide and R(+)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), and also by the selective 5-HT1D agonists, sumatriptan and N-methyl-3-(1-methyl-1-piperidinyl)-1H-indole-5-ethane sulphonamide (GR 85548). 2. Ketanserin (1 microM) and methiothepin (1 microM) reduced the duration of depressions elicited by 5-CT, but not those produced by 5-HT, sumatriptan, GR 85548, methysergide or 8-OH-DPAT. 3. The IC50 for MSR depression by 5-CT was 3.6, 2.1-6.2 nM (n = 4), by sumatriptan was 15.2, 12.9-18.0 nM (n = 32), by GR 85548 was 18.4, 11.7-29.1 nM (n = 12), by methysergide was 29.8, 10.2-87.1 nM (n = 4) and by 8-OH-DPAT was 0.21, 0.11-0.43 microM (n = 3) (geometric means and 95% confidence limits). 4. Ketanserin (0.1 or 1 microM) antagonized competitively responses to sumatriptan (apparent pA2 7.8 +/- 0.1, n = 5), GR 85548 (apparent pA2 7.6, unpaired data, n = 5), methysergide (apparent pA2 7.9 +/- 0.12, n = 4) and 8-OH-DPAT (apparent pA2 8.3 +/- 0.1, n = 3). Concentration-response curves to 5-CT showed a smaller, parallel shift to the right (apparent pA2 6.8 +/- 0.1, n = 4), but responses to 5-HT were unaffected by ketanserin (1 microM) (n = 4). 5. Methiothepin (1 microM) antagonized competitively responses to GR 85548 (apparent pA2 7.7, unpaired data, n = 5). 6. Mianserin (0.3 microM), a concentration sufficient to cause substantial block of 5-HT2C-mediated responses but have only a small effect on 5-HT1D-mediated actions, caused a small, non-parallel shift of the concentration-response curve to sumatriptan. 7. Depression of the MSR by sumatriptan was not blocked by (+/-)-cyanopindolol (0.1 microM), (+/-)-propranolol (0.5 or 1 microM) or spiroxatrine (0.1 microM), and depression of MSR by 8-OH-DPAT was not blocked by spiroxatrine (0.1 microM). (+/-)-Cyanopindolol (0.1 and 1 microM) itself induced a slow depression of the MSR. 8. The novel 5-HT1D antagonist, N-[4-methyl-1-piperazinyl) phenyl]2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide (GR 127935, 30 nM to 1 microM) caused a concentration-related depression of the reflex (up to 50%) usually slow in onset. Neither with these concentrations nor with concentrations in the range 1-3 nM was there any unequivocal blockade of responses to sumatriptan. 9. It is concluded that sumatriptan, GR 85548, methysergide and 8-OH-DPAT depress the MSR in the neonate rat spinal cord via ketanserin-sensitive receptors, which have some similarities to 5-HT1D alpha receptors but which are not blocked by GR 127935. 5-HT released by tryptaminergic pathways may act via the same receptors to depress the MSR. 5-HT applied to the cord probably acts via a different, possibly novel 5-HT receptor to depress the MSR.     

Activation of the 5-HT1C receptor expressed in Xenopus oocytes by the benzazepines SCH 23390 and SKF 38393.

1. A cloned 5-HT1C receptor expressed in Xenopus laevis oocytes was used to characterize the action of four dopamine D1-selective benzazepines at the 5-HT1C receptor. Additionally, the apparent binding of the D1-selective benzazepines to 5-HT1C receptors was measured in the choroid plexus of the pig. 2. In voltage-clamped oocytes expressing the cloned 5-HT1C receptor, 5-hydroxytryptamine (5-HT) elicited a characteristic inward current response with an EC50 of 13 nM. SCH 23390 acted as a stereoselective agonist (or partial agonist) with an EC50 of about 550 nM. SKF 38393 (1 microM-1 mM), SKF 77434 (100 microM), and SKF 82958 (100 microM) also acted as agonists (or partial agonists) at the cloned 5-HT1C receptor. SKF 38393 was not stereoselective at the 5-HT1C receptor. 3. The response to SCH 23390 activated slowly and, although the response contained many oscillations characteristic of the activation of the phosphatidylinositol signal transduction system, SCH 23390 rarely elicited the rapid spike-like response seen routinely in response to 5-HT. However, the responses to SKF 38393, SKF 77434, and SKF 82958 were identical in appearance to the response to 5-HT, except that the responses to the benzazepines were smaller. These comparisons were made by applying both a benzazepine and 5-HT to each individual oocyte expressing the cloned 5-HT1C receptor. 4. Consistent with the responses measured in oocytes, SCH 23390 bound stereoselectively to 5-HT1C receptors in the choroid plexus of the pig (Ki = 6.3 nM), and SKF 38393 bound non-stereoselectively with lower affinity (Ki = 2.0-2.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation by calcineurin of 5-HT3 receptor function in NG108-15 neuroblastoma x glioma cells.

1. We have investigated the mechanism of regulation of 5-HT3 receptor channel sensitivity in voltage-clamped (-80 mV) NG108-15 neuroblastoma cells. 2. The 5-HT-induced inward current activated rapidly. The fast onset was followed by a biphasic decay which was characterized by two time constants, tau 1 (1.1 +/- 0.21s) and tau 2 (8.9 +/- 1.6s), respectively. Brief applications of 5-HT, applied at 2 min intervals, induced a decrease in the amplitude of the 5-HT3 receptor-mediated peak inward currents. 3. Buffering of intracellular calcium with the calcium chelator BAPTA (10 mM) instead of EGTA (10 mM) attenuated the 5-HT-induced loss of responsiveness of 5-HT3 receptors. Omission of calcium from the extracellular medium yielded a similar attenuation of loss of responsiveness. 4. Inclusion of the protein kinase inhibitor, staurosporine (1 microM) or of okadaic acid (1 microM), an inhibitor of protein phosphatases 1 and 2A, in the intracellular buffer solution did not affect 5-HT3 receptor sensitivity. 5. Injection of cyclosporin A-cyclophilin A complex (20 nM), which potently inhibits calcineurin, did not affect the time constants of the biphasic decay of the 5-HT response tau 1 (1.4 +/- 0.28s) and tau 2 (11.3 +/- 1.7s). The complex, however, prevented the loss of 5-HT3, receptor responsiveness upon repeated application of 5-HT. A similar, but weaker effect was observed after intracellular application of the autoinhibitory peptide domain of calcineurin (1 microM). 6. The recovery of desensitized 5-HT3 receptors upon a second application of 5-HT (1 microM) showed a half-life time (tau 1/2) of 2.6 +/- 0.12 min in control cells which was reduced to 1.6 +/- 0.09 min in cells treated with cyclosporin A-cyclophilin A (20 nM) complex. 7. We conclude that calcineurin does not affect the fast decay of the 5-HT3 receptor response but may be involved in a slower process which regulates channel activity.     

Ability of 5-HT4 receptor ligands to modulate rat striatal dopamine release in vitro and in vivo.

1. The ability of 5-HT4 (5-hydroxytryptamine4) receptor ligands to modify dopamine release from rat striatal slices in vitro and in the striatum of freely moving rats was assessed by the microdialysis technique. 2. The release of dopamine from slices of rat striatum continually perfused with Krebs buffer was enhanced by 5-HT4 receptor agonists; 5-HT (10 microM), 5-methoxytryptamine (5-MeOT; 10 microM), renzapride (10 microM) and (S)-zacopride (10 microM) maximally increased dopamine release by 133 +/- 5, 214 +/- 25, 232 +/- 29 and 264 +/- 69%, respectively (mean +/- s.e.mean, n = 3-8). The drug-induced responses were maximal within the first 2 min of drug application, and subsequently declined. The non-selective 5-HT3/5-HT4 receptor antagonist, SDZ205-557 (10 microM), failed to modify basal dopamine release from striatal slices but completely antagonized the (S)-zacopride (10 microM)-induced increase in dopamine release. 3. To allow faster drug application, the modulation of dopamine release from rat striatal slices in a static release preparation was also investigated. The 5-HT4 receptor agonist, renzapride (10 microM) also enhanced dopamine release in this preparation (maximal increase = 214 +/- 35%, mean +/- s.e.mean, n = 14), whilst a lower concentration of renzapride (3 microM) was less effective. The renzapride-induced response was maximal within the first 2 min of drug application, before declining. In this preparation, the stimulation of dopamine release by renzapride (10 microM), was completely antagonized by the selective 5-HT4 receptor antagonist, GR113808 (100 nM). In addition, both the Na+ channel blocker, tetrodotoxin (100 nM) and the non-selective protein kinase A inhibitor, H7 (100 nM) completely prevented the stimulation of dopamine release induced by renzapride (10 microM). 4. In vivo microdialysis studies demonstrated that the 5-HT4 receptor agonists, 5-MeOT (10 microM), renzapride (100 microM) and (S)-zacopride (100 microM) maximally elevated extracellular levels of dopamine in the striatum by 220 +/- 20, 161 +/- 10 and 189 +/- 53%, respectively (mean +/- s.e.mean, n = 5-9). A lower concentration of renzapride (10 microM) was less effective. The elevation of extracellular striatal dopamine levels induced by either renzapride (100 microM) or (S)-zacopride (100 microM) were completely antagonized by the non-selective 5-HT4 receptor antagonist, SDZ205-557 (100 microM). In addition, the elevation of extracellular levels of dopamine induced by either 5-MeOT (10 microM) or renzapride (100 microM) was completely prevented by the selective 5-HT4 receptor antagonist, GR113808 (1 microM) and the renzapride (100 microM)-induced response was also completely prevented by the non-selective protein kinase A inhibitor, H7 (1 microM). In this in vivo preparation, both GR113808 (1 microM) and H7 (1 microM), when perfused alone, reduced extracellular levels of dopamine. 5. In conclusion, the present study provides evidence that the 5-HT4 receptor facilitates rat striatal dopamine release in vitro and in vivo.     

The interaction of antidepressant drugs with central and peripheral (enteric) 5-HT3 and 5-HT4 receptors.

1. A combined study of receptor binding in central neuronal cell membranes and functional responses in isolated segments of guinea-pig small intestine allowed characterization of the interaction of four antidepressant drugs with central and peripheral 5-HT3 and 5-HT4 receptors. 2. Clomipramine, paroxetine and fluoxetine inhibited [3H]-DAU 6215 binding to 5-HT3 recognition sites in NG 108-15 cells with IC50 values in the range 1.3-4 microM. Litoxetine had an IC50 of 0.3 microM. The specific binding of [3H]-GR 113808 to 5-HT4 recognition sites in pig striatal membranes was inhibited by all four antidepressants with negligible potency (IC50 values > or = 20 microM). 3. In whole ileal segments, concentration-response curves to 5-HT were biphasic, with the high- and low-potency phases involving 5-HT4 and 5-HT3 receptors, respectively. Curves to 2-methyl-5-hydroxytryptamine (2-methyl-5-HT: a 5-HT3 receptor agonist) and 5-methoxytryptamine (5-MeOT: a 5-HT4 receptor agonist) were monophasic. All antidepressants were used at concentrations lacking anticholinoceptor properties, as demonstrated in both electrically stimulated longitudinal muscle-myenteric plexus preparations (LMMPs) and in unstimulated LMMPs following addition of acetylcholine (100 nM). 4. Fluoxetine (0.1-1 microM) and litoxetine (0.3-3 microM) antagonized both the high- and low-potency phases of the 5-HT curve. Schild analysis for the low-potency phase yielded pA2 estimates of 6.6 +/- 0.3 (Schild slope of 1.1) and of 6.6 +/- 0.1 (Schild slope of 1.1), respectively. At higher concentrations (3 microM), fluoxetine markedly inhibited the 5-HT response maximum. Clomipramine (10-300 nM) inhibited, by a mechanism independent of concentration, both phases of the 5-HT curve with a reduction of the maximum response. Paroxetine (1 microM) was ineffective on the high-potency phase, but caused a rightward shift of the low-potency phase (pKB: 6.1 +/- 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Blockade by ONO-NT-012, a unique prostanoid analogue, of prostaglandin E2-induced allodynia in conscious mice.

1. In the presence of atropine (0.2 microM) and indomethacin (2 microM), the effects of 5-hydroxytryptamine (5-HT) have been studied on electrically-evoked, neurogenic contractions of the guinea-pig proximal colon in vitro. 2. 5-HT, at higher concentrations than 1 nM, caused an increase in electrically (1 Hz, 0.3 ms, 160 mA)-evoked, atropine-resistant contractions in a concentration-dependent manner and at 30 nM produced a maximal effect (pEC50 value of 8.20 +/- 0.11, n = 6). The enhancing effects of 5-HT on the electrically evoked contractions were mimicked by alpha-methyl-5-HT (pEC50 value of 6.59 +/- 0.05, n = 6). 3. Both hexamethonium (100 microM) and spantide (10 microM), selective antagonists for nicotinic and tachykinin receptors respectively, significantly reduced the enhancement of the electrically evoked contractions by 5-HT (30 nM). 4. DAU 6285 (3 microM), a 5-HT4 receptor antagonist, abolished the enhancing action of 5-HT (30 nM), but metitepine (0.03 microM), a 5-HT1/5-HT2 receptor antagonist, ketanserin (0.01 microM), a 5-HT2 receptor antagonist, and ondansetron (1 microM), a 5-HT3 receptor antagonist, had no effect on the enhancement. The enhancing effects of alpha-methyl-5-HT (1 microM) were also abolished by DAU 6285 (3 microM). 5. Both 5-HT (30 nM) and alpha-methyl-5-HT (1 microM) had no effect on contractions to exogenous substance P (0.15-0.3 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Coupling of an endogenous 5-HT1B-like receptor to increases in intracellular calcium through a pertussis toxin-sensitive mechanism in CHO-K1 cells.

1. Chinese hamster ovary cells (CHO-K1) express an endogenous 5-hydroxytryptamine (5-HT)1B-like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)-sensitive mechanism. Furthermore, the human adenosine A1 receptor when expressed in CHO-K1 cells (CHO-A1) has been shown to mobilize intracellular Ca2+ through a PTX-sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5-HT1B-like receptor was able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO-A1 cells. 2. In agreement with previous studies using CHO cells, 5-hydroxytryptamine (5-HT) elicited a concentration-dependent inhibition of forskolin-stimulated [3H]-cyclic AMP production in CHO-A1 cells (p[EC50] = 7.73 +/- 0.13). 5-HT (1 microM) inhibited 47 +/- 5% of the [3H]-cyclic AMP accumulation induced by 3 microM forskolin. Forskolin stimulated [3H]-cyclic AMP accumulation was also inhibited by the 5-HT1 receptor agonists (p[EC50] values) 5-carboxyamidotryptamine (5-CT; 8.07 +/- 0.08), RU 24969 (8.12 +/- 0.33) and sumatriptan (5.80 +/- 0.31). 3. 5-HT elicited a concentration-dependent increase in [Ca2+]i in CHO-A1 cells (p[EC50] = 8.07 +/- 0.05). In the presence of 2 mM extracellular Ca2+, 5-HT (1 microM) increased [Ca2+]i from 174 +/- 17 nM to 376 +/- 22 nM. The 5-HT1 receptor agonists (p[EC50] values), 5-carboxyamidotryptamine (5-CT; 7.9 +/- 0.02), RU 24969 (8.1 +/- 0.07) and sumatriptan (5.9 +/- 0.11) all elicited concentration-dependent increases in [Ca2+]i. Similar maximal increases in [Ca2+]i were obtained with each agonist. The selective 5-HT1A receptor agonist, 8-OH-DPAT (10 microM) did not stimulate increases in [Ca2+]i. 5-HT (100 microM) and 5-CT (10 microM) did not stimulate a measurable increase in [3H]-inositol phosphate accumulation in CHO-A1 cells. 4. 5-HT (1 microM)-mediated increases in [Ca2+]i were insensitive to the 5-HT receptor antagonist, ritanserin (5-HT2; 100 nM), ketanserin (5-HT2; 100 nM), LY-278,584 (5-HT3; 1 microM) and WAY 100635 (5-HT1A; 1 microM). The response to 5-HT (100 nM) was antagonized by the non-selective 5-HT1 antagonist, methiothepin (pKb = 8.90 +/- 0.09) and the 5-HT1D antagonist GR 127935 (pKb = 10.44 +/- 0.06). 5. Pretreatment with PTX (200 ng ml-1 for 4 h) completely attenuated the Ca2+ response to 100 microM 5-HT. 6. In untransfected CHO-K1 cells, 5-HT (1 microM), RU 24969 (1 microM), and 5-CT (1 microM) elicited increases in [Ca2+]i similar to those observed in CHO-A1 cells. 7. These data demonstrate that in CHO-K1 cells the endogenously expressed 5-HT1B-like receptor couples to the phospholipase C/Ca2+ signalling pathway through a PTX-sensitive pathway, suggesting the involvement of Gi/Go protein(s).     

Bicuculline antagonizes 5-HT(3A) and alpha2 glycine receptors expressed in Xenopus oocytes

The present study examined the effects of bicuculline on the mouse 5-hydroxytryptamine(3A) receptor (5-HT(3A) receptor and the human alpha2 subunit of the glycine receptor. Bicuculline antagonized both the 5-HT(3A) receptor (IC(50)=20.12+/-0.39 microM) and the alpha2 glycine receptor (IC(50)=169.40+/-1.73 microM). A competitive form of antagonism by bicuculline was suggested by experiments in which the EC(50)s for 5-HT and glycine were increased in the 5-HT(3A) and alpha2 glycine receptors, respectively, as bicuculline concentrations were increased. A competitive nature of antagonism by bicuculline at the 5-HT(3A) receptor was also suggested by displacement of the competitive antagonist, [3H]GR65630 in SF21 insect cells expressing the 5-HT(3A) receptor (K(i)=19.01+/-0.71 microM). Our data and that of others reveal that bicuculline, a purported selective antagonist of the GABA(A) receptor, antagonizes at least one receptor subclass in every member of the superfamily of ligand-gated ion channels.     

Investigation into the 5-hydroxytryptamine-induced atropine-resistant neurogenic contraction of guinea-pig proximal colon.

1. The aim of this study was to characterize the receptors mediating the atropine-resistant neurogenic contraction to 5-hydroxytryptamine (5-HT) in the longitudinal muscle of the guinea-pig proximal colon and to determine the type of tachykinin receptors involved in the contractile response to 5-HT by the use of selective antagonists. 2. In the presence of atropine (0.3 microM), guanethidine (5 microM), hexamethonium (100 microM), ketanserin (0.1 microM) and indomethacin (3 microM), 5-HT (0.01-3 microM) produced concentration-dependent neurogenic contractions of colonic strips and at 0.3 microM produced a maximal effect (pEC50 = 7.39 +/- 0.09, n = 18). The 5-HT4 receptor stimulant, 5-methoxytryptamine (5-MeOT, 0.03-10 microM) also produced neurogenic contractions with similar maximum effect to those of 5-HT (pEC50 = 6.89 +/- 0.16). 3. The 5-HT4 receptor antagonist, DAU 6285 (3 microM) shifted the concentration-response curves to both 5-HT and 5-MeOT to the right without significant depression of the maximum, but the 5-HT1/5-HT2 receptor antagonist, metitepine (0.1 microM) and the 5-HT3 receptor antagonist, ondansetron (0.3 microM) had no effect on the control curves to 5-HT and 5-MeOT. 4. The selective NK1 receptor antagonist, FK 888 (1 microM) markedly attenuated the contractions to 5-HT and 5-MeOT. In contrast, the selective NK2 receptor antagonist, SR 48968 (10 nM) and the selective NK3 receptor antagonist, SR 142801 (10 nM) had no effect on the contractions to 5-HT and 5-MeOT. 5. These results indicate that the 5-HT-induced atropine-resistant neurogenic contraction of guinea-pig proximal colon is due to activation of 5-HT4 receptors, presumably located on excitatory motor neurones, innervating the longitudinal muscle. The contraction evoked by activation of the 5-HT4 receptors is mediated primarily via NK1 receptors but not NK2 or NK3, suggesting that the 5-HT4 receptor-mediated contraction is evoked indirectly via tachykinin release from tachykinin-releasing excitatory neurones.     

GLC-3: a novel fipronil and BIDN-sensitive, but picrotoxinin-insensitive, L-glutamate-gated chloride channel subunit from Caenorhabditis elegans

1. We report the cloning and expression of a novel Caenorhabditis elegans polypeptide, GLC-3, with high sequence identity to previously cloned L-glutamate-gated chloride channel subunits from nematodes and insects. 2. Expression of glc-3 cRNA in XENOPUS oocytes resulted in the formation of homo-oligomeric L-glutamate-gated chloride channels with robust and rapidly desensitizing currents, an EC(50) of 1.9+/-0.03 mM and a Hill coefficient of 1.5+/-0.1. GABA, glycine, histamine and NMDA all failed to activate the GLC-3 homo-oligomer at concentrations of 1 mM. The anthelminthic, ivermectin, directly and irreversibly activated the L-glutamate-gated channel with an EC(50) of 0.4+/-0.02 microM. 3. The GLC-3 channels were selective for chloride ions, as shown by the shift in the reversal potential for L-glutamate-gated currents after the reduction of external Cl(-) from 107.6 to 62.5 mM. 4. Picrotoxinin failed to inhibit L-glutamate agonist responses at concentrations up to 1 mM. The polycyclic dinitrile, 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile (BIDN), completely blocked L-glutamate-induced chloride currents recorded from oocytes expressing GLC-3 with an IC(50) of 0.2+/-0.07 microM. The phenylpyrazole insecticide, fipronil, reversibly inhibited L-glutamate-gated currents recorded from the GLC-3 receptor with an IC(50) of 11.5+/-0.11 microM. 5. In this study, we detail the unusual antagonist pharmacology of a new GluCl subunit from C. elegans. Unlike all other native and recombinant nematode GluCl reported to date, the GLC-3 receptor is insensitive to picrotoxinin, but is sensitive to two other channel blockers, BIDN and fipronil. Further study of this receptor may provide insights into the molecular basis of non-competitive antagonism by these compounds.     

Characterization of putative 5-ht7 receptors mediating direct relaxation in Cynomolgus monkey isolated jugular vein.

1. 5-Hydroxytryptamine (5-HT) receptors mediating contraction and relaxation are present in Cynomolgus monkey isolated jugular vein denuded of endothelium. 2. In the absence of spasmogen, alpha-methyl-5-HT and sumatriptan contracted the tissues with potency values (pEC50) of 6.8 (n = 2) and 6.4 +/- 0.1 (mean +/- s.e. mean, n = 3), respectively. In contrast, 5-HT caused an initial contraction (10 nM - 1 microM), followed by relaxation (1 microM - 32 microM). The contractile effect of alpha-methyl-5-HT was antagonized by ketanserin with a pKB value of 8.1 (n = 2). 5-Carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-OH-DPAT did not contract or relax the tissues in the absence of spasmogen. 3. In tissues precontracted with U46619 (10 nM) and in the presence of 5-HT1A, 5-HT1B, 5-HT2A, 5-HT3, and 5-HT4 receptor blockade, 5-CT and 5-MeOT caused endothelium-independent relaxation with potency values of 7.5 +/- 0.1 (n = 21) and 5.7 +/- 0.1 (n = 4), respectively. The potency of 5-HT was 7.2 (n = 2) while alpha-methyl-5-HT did not start to relax the tissues below a concentration of 10 microM. 4. Relaxations elicited by 5-CT were antagonized by the following compounds (with pKB values in parentheses): methiothepin (9.7), mesulergine (8.1), metergoline (8.0), clozapine (7.8), mianserin (7.7), spiperone (7.3), ritanserin (7.1), methysergide (7.0) and ketanserin (5.7). 5. It is concluded that the 5-HT receptor mediating endothelium-independent relaxation may be a functional correlate of the putative 5-ht7 receptor.     

Ethanol and trichloroethanol alter gating of 5-HT3 receptor-channels in NCB-20 neuroblastoma cells

Alcohol potentiation of 5-HT3 receptors was examined in NCB-20 neuroblastoma cells using whole-cell patch-clamp electrophysiological techniques. Activation of the receptor with the weak partial agonist dopamine (DA) was used to examine alcohol effects under conditions of full agonist occupancy, but low probability of channel opening. Dopamine activation of the receptor increased in a concentration-dependent manner (EC50=0.28 mM), and on average maximal responses to DA were 8.0+/-0.8% of the maximal response to 5-HT. Ethanol (EtOH) and trichloroethanol (TCEt) potentiated DA-activated ion current mediated by 5-HT3 receptors. Potentiation of responses to a maximally effective dopamine concentration averaged 52.0+/-8.0% for EtOH and 567+/-43% for TCEt, which was comparable to the potentiation observed when receptors were activated by a low concentration of 5-HT. The alcohols increased both the potency and efficacy with which dopamine activated the receptor. The observation that alcohols increase the maximal efficacy of dopamine activation of the receptor indicates that one action of alcohols on the 5-HT3 receptor is to increase the probability of channel opening independent of any effect on agonist affinity.     

Characterization of 5-HT3 and ''atypical'' 5-HT receptors mediating guinea-pig ileal contractions in vitro.

1. Neuronal 5-hydroxytryptamine (5-HT) receptors mediating contraction of guinea-pig ileal segments have been characterized in vitro by the use of methysergide to block 5-HT1-like and 5-HT2 receptors. Concentration-response curves to 5-HT were biphasic (first phase, defined as those responses occurring between 1 nM and 0.32 microM 5-HT, -log EC50 = 7.15 +/- 0.08; second phase, defined as these responses occurring between 0.32 microM and 32 microM 5-HT, -log EC50 = 5.32 +/- 0.03) but monophasic to 5-methoxytryptamine (-log EC50 = 7.0 +/- 0.08) and 2 methyl 5-HT (-log EC50 = 5.2 +/- 0.13). The maximal response of the first phase to 5-HT and the maximal response to 5-methoxytryptamine were 30 +/- 4% and 35 +/- 5% respectively of the maximum response to the second phase of the 5-HT concentration-effect curve (set at 100%). In contrast, the maximal response to 2-methyl-5-HT equalled that obtained with 5-HT (second phase). 2. The responses comprising the second phase of the concentration-effect curve to 5-HT were antagonized by 1 microM ICS 205-930, ondansetron, granisetron, quipazine, N-methyl-quipazine and (R,S)-zacopride and the following pKB values, with 5-HT as the agonist, were obtained at the 5-HT3 receptor: ICS 205-930 7.61 +/- 0.05, ondansetron 6.90 +/- 0.04, granisetron 7.90 +/- 0.04, (S)-zacopride 8.11 +/- 0.06, (R,S)-zacopride 7.64 +/- 0.11, and (R)-zacopride 7.27 +/- 0.06.(ABSTRACT TRUNCATED AT 250 WORDS)