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CELLULAR AND MOLECULAR

Ca²⁺ Responses in Chinese Hamster Ovary-K1 Cells Demonstrate an Atypical Pattern of Ligand-Induced 5-HT_1A Receptor Activation

Centre de Recherche Pierre Fabre, Department of Cellular and Molecular Biology, Castres, France

Received for publication June 19, 2003
Accepted July 30, 2003.

	Abstract

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Little experimental evidence has been reported for diverse signaling via 5-hydroxytryptamine (5-HT)_1A receptors despite the fact that agonists seem to be more efficacious at dorsal raphe somatodendritic 5-HT_1A autoreceptors than at postsynaptic 5-HT_1A receptors. The present study investigated Ca²⁺ responses in Chinese hamster ovary (CHO)-K1 cells expressing a human 5-HT_1A receptor by 5-HT, prototypical 5-HT_1A agonists, N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methyl-6-; methylaminopyridin-2-yl)-methylaminomethyl]-piperidine (F 14679), and especially N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-; methylaminomethyl]piperidine (F 13640) as representative ligands of a new chemical class (methylamino-pyridine) that combines both high efficacy and selectivity for 5-HT_1A receptors. 5-HT (pEC₅₀ = 6.70 ± 0.02) induced a pertussis toxin-sensitive, transient high-magnitude Ca²⁺ response. High-magnitude Ca²⁺ responses (E_max, percentage versus 5-HT) were also found with F 13640 (107 ± 4), 5-carboxamidotryptamine (100 ± 3), and F 14679 (87 ± 3). In contrast, the prototypical 5-HT_1A receptor agonists buspirone, ipsapirone, and 8-(hydroxy-2-(di-n-propylamino)tetralin, and also flesinoxan and eptapirone, were virtually inactive (5). This atypical pattern of 5-HT_1A receptor activation contrasts with the broad spectrum of the ligands' partial agonist properties as observed by measuring guanosine 5'-O-(3-[³⁵ S]thio)triphosphate ([³⁵S]GTPS) binding responses with membranes of either CHO-K1 or C6-glial cells stably expressing a human 5-HT_1A receptor. Remarkably, differences between ligands that seem small in the [³⁵S]GTPS binding assay translate into huge differences in the magnitude of Ca²⁺ responses. Therefore, some of these 5-HT_1A ligands (i.e., F 13640) may in a selective way induce responses that may be not at all be achieved with other ligands (i.e., buspirone). In conclusion, the pharmacology of 5-HT_1A receptor ligands seems to be codetermined by the effector pathway.

In the present study, we measured 5-HT ligand-mediated Ca²⁺ responses in CHO-K1 cells transfected with a recombinant human 5-HT_1A receptor. Although 5-HT_1A receptors can activate phospholipase C (Raymond et al., 1999), this effect is considered to be host-specific and not as efficient as coupling to the inhibition of adenylate cyclase. Several 5-HT ligands were investigated here: prototypical 5-HT_1A agonists [i.e., 8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT), buspirone, and ipsapirone], flesinoxan and eptapirone, 5-carboxamidotryptamine (5-CT) as a high-efficacy but nonselective 5-HT_1A ligand (Pauwels et al., 1997), N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methyl-6-methylaminopyridin-2-yl)-methylaminomethyl]-piperidine (F 14679; Koek et al., 2001) and, especially N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-methylaminome-thyl]piperidine (F 13640; Colpaert et al., 2002) as representative ligands of a new chemical class (5-methyl-pyridin-2ylmethyl amine derivatives) that combines both high-efficacy and selectivity for 5-HT_1A receptors. We found that highly efficacious 5-HT_1A receptor agonists could induce a transient, high-magnitude Ca²⁺ response in CHO-K1 cells; the amplitude of the ligands' Ca²⁺ responses was similar to that of 5-HT. Prototypical 5-HT_1A ligands (i.e., 8-OH-DPAT), commonly considered as partial agonists (Koek et al., 2001), were no longer capable to induce a significant Ca²⁺ response. The Ca²⁺ response data were compared with 5'-O-(3-[³⁵S]thiotriphosphate ([³⁵S]GTPS) binding responses as a sensitive read-out for a wide spectrum of 5-HT_1A receptor ligand activities. The Ca²⁺ data are discussed with regard to agonist-selective 5-HT_1A receptor signaling.

	Materials and Methods

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Transfection of CHO-K1 Cells with Human Recombinant 5-HT_1A Receptor. Subconfluent CHO-K1 cells were transiently transfected with 10 µg of a wild-type human 5-HT_1A receptor plasmid containing the entire receptor coding sequence (R.C.2.1.5HT.01.A, GenBank accession no. X57829 [GenBank] ) by electroporation (Wurch et al., 1996). Cells were plated in 96-well plates with 0.2 ml of nutrient mixture Ham's F12 plus 10% heat-inactivated fetal calf serum and 1% dimethyl sulfoxide at about 60,000 cells/well. Cells were assayed for intracellular Ca²⁺ responses between 24 and 48 h after transfection.

Measurement of Intracellular Ca²⁺ Responses. Intracellular Ca²⁺ responses were measured upon 1-h loading with 2 µM Fluo-3 fluorescent calcium indicator dye as described previously (Pauwels et al., 2000). Either 5-HT or other 5-HT ligands were assayed between 1 nM and 10 µM for their Ca²⁺ responses. Data for Ca²⁺ responses were obtained in arbitrary fluorescent units and were not translated into Ca²⁺ concentrations. Fluorescent readings were made every 2 s for the first 3 min by using a fluorometric imaging plate reader (Molecular Devices Corp., Sunnyvale, CA). E_max values were defined as the ligand's maximal high-magnitude Ca²⁺ response in percentage versus that obtained with 10 µM 5-HT. pEC₅₀ values correspond to a ligand concentration at which 50% of its own maximal high-magnitude Ca²⁺ response was measured. Antagonists were preincubated for 10 min before 5-HT and the Ca²⁺ response recorded for a further 3 min. Antagonist potency (pIC₅₀ value) was defined as the concentration required to antagonize 50% of the Ca²⁺ response induced by 1 µM 5-HT. This was calculated as the difference in surface area between the 5-HT and ligand conditions.

[³⁵S]GTPS Binding Responses. [³⁵S]GTPS binding responses were determined on membrane preparations of CHO-K1 or C6-glial cells stably transfected with a recombinant human 5-HT_1A receptor as described previously (Pauwels et al., 1997). Incubation mixtures were prepared in glass tubes and consisted of 0.4 ml of membrane preparation (20-40 µg of protein) and 0.05 ml of either 5-HT or another 5-HT ligand in the presence of 30 µM GDP. After an incubation of 30 min at 25°C, 0.05 ml [³⁵S]GTPS (0.5 nM) was added for an additional period of 30 min. The reactions were stopped by adding 3 ml of ice-cold 20 mM HEPES (pH 7.4) containing 3 mM MgCl₂ and rapid filtration as described previously (Pauwels et al., 1997). Maximal stimulation of [³⁵S]GTPS binding was defined in the presence of 10 µM 5-HT. E_max values were expressed as a percentage of the response obtained with 10 µM 5-HT.

Protein Content. Protein levels were estimated with the dye-binding assay using the Bio-Rad kit (Bradford, 1976). Bovine serum albumin was used as a standard.

Statistical Analysis. Statistical analysis was performed on E_max values using a two-tailed Student's t test.

Materials. CHO-K1 and C6-glial cells were obtained from American Type Culture Collection (Rockville, MD). Cell culture media, fetal calf serum, culture plates, and Bordetella pertussis toxin were obtained from Gibco Biocult (Paisley, UK). [³⁵S]GTPS (1100 Ci/mmol) was obtained from Amersham Biosciences Inc. (Les Ulis, France). Fluo-3 was obtained from Molecular Probes (Eugene, OR). 5-HT and probenicid acid were from Sigma-Aldrich (St. Louis, MO). 5-CT, 8-OH-DPAT, and buspirone were obtained from Sigma/RBI (Natick, MA). Flesinoxan, ipsapirone, N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100635), 1'-methyl-5-(2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-carbonyl)-2,3,6,7-tetrahydrospiro[furo[2,3-f]indole-3,4'-piperidine] (SB 224289), F 13640, and F 14679 were synthesized at the Centre de Recherche Pierre Fabre (Castres, France). Stock solutions of compounds were prepared at 10^-3 M. Serial dilutions were made in incubation buffer.

	Results

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In contrast to nontransfected CHO-K1 cells, 5-HT (10 µM) induced a Ca²⁺ response in CHO-K1 cells transiently transfected with a human 5-HT_1A receptor (Fig. 1). The amplitude of this Ca²⁺ response (3432 ± 244 arbitrary fluorescence units) was smaller than that obtained at the endogenously expressed bradykinin receptor upon stimulation with 10 µM bradykinin (6519 ± 113 arbitrary fluorescence units). The kinetic properties of these two Ca²⁺ responses differed. The onset time of maximal activation was faster with bradykinin (16 ± 1 s) than with 5-HT (30 ± 1 s), whereas the attenuation of the Ca²⁺ signal upon maximal activation was slightly greater for bradykinin than for 5-HT. In contrast to the bradykinin-mediated Ca²⁺ response, pertussis toxin-treatment prevented the 5-HT-mediated Ca²⁺ response (Fig. 1C).

View larger version (15K): [in this window] [in a new window]   Fig. 1. 5-HT and bradykinin-induced Ca2+ responses in nontransfected and 5-HT1A receptor-transfected CHO-K1 cells. CHO-K1 cells were transiently transfected with the human 5-HT1A receptor (10 µg of plasmid). Basal, bradykinin-(10 µM), and 5-HT (10 µM)-induced Ca2+ responses were measured every 2 s for 3 min. Curves illustrate a representative experiment. A, nontransfected CHO-K1 cells; B, 5-HT1A receptor-transfected CHO-K1 cells (0.83 ± 0.18 pmol/mg protein binding sites on intact cells as estimated by [3H] WAY 100635, 1 nM); and C, 5-HT1A receptor-transfected CHO-K1 cells upon overnight treatment with pertussis toxin (20 ng/ml).

Results on antagonism of the 5-HT-induced Ca²⁺ response are reported in Fig. 2. WAY 100635, methiothepin, and buspirone fully antagonized the 5-HT response. The 5-HT_1B receptor antagonist SB 224289 (1 µM) exerted little, if any, effect (Fig. 2B). Prototypical 5-HT_1A receptor agonists (i.e., buspirone, 8-OH-DPAT, and ipsapirone) did not induce a significant Ca²⁺ response despite the fact that their maximal [³⁵S]GTPS binding responses as obtained with membranes of 5-HT_1A receptor-transfected CHO-K1 cells were between 56 and 79% compared with 5-HT (Table 1); similar findings were obtained with flesinoxan and eptapirone. Even a comparison with [³⁵S]GTPS binding data as obtained with membranes of C6-glial cells stably transfected with a 5-HT_1A receptor (Pauwels et al., 1997) indicated lower but still significant activity for each of these compounds (Table 1). Similarly, these compounds behaved as either partial (i.e., buspirone) or efficacious agonists (i.e., eptapirone) by monitoring their cAMP responses in transfected HeLa cells (Table 1). In contrast, F 13640, 5-CT, and F 14679 induced large Ca²⁺ responses with a magnitude that was similar or apparently identical to that of 5-HT (Table 1). F 13640 and F14679 [GenBank] , in contrast to 5-CT, are highly selective for the 5-HT_1A receptor. Table 2 compares binding affinities between 5-HT_1A and two receptor subtypes (5-HT_1B and 5-HT_2A) that have been postulated to be endogenously expressed in CHO-K1 cells and C6-glial cells (Giles et al., 1996; Pauwels et al., 1996). F 13640 and F 14679 do not significantly bind to 5-HT_1B and 5-HT_2A receptors. Moreover, we could not measure a 5-HT-mediated Ca²⁺ response in nontransfected CHO-K1 cells (Fig. 1A). Therefore, the observed Ca²⁺ responses with F 13640 and F 14679 can be considered to be due to activation by 5-HT_1A receptors. A comparison with the [³⁵S]GTPS binding responses in transfected C6-glial membranes (Fig. 3; Table 1) indicated a significantly lower maximal [³⁵S]GTPS binding response for F 13640 with an unmodified potency (p > 0.05), and a significantly lower maximal [³⁵S]GTPS binding response for 5-CT and F 14679 accompanied with an enhanced potency (p 0.01). A comparison with the [³⁵S]GTPS binding responses in transfected CHO-K1 membranes did not reveal attenuated maximal responses, whereas pEC₅₀ values were significantly enhanced (p 0.005), although less for F 13640 (4 times) than 5-HT (11 times), 5-CT (14 times), and F 14679 (76 times) (Table 1). Figure 4 illustrates the atypical pattern of 5-HT ligand-mediated maximal Ca²⁺ responses in CHO-K1 cells versus their spectrum of maximal [³⁵S]GTPS binding responses for both transfected CHO-K1 and C6-glial membranes. The magnitude of Ca²⁺ responses produced by the 5-HT_1A receptor ligands examined here in CHO-K1 cells correlated weakly with their [³⁵S]GTPS binding response at 5-HT_1A receptors in CHO-K1 (Spearman's rank correlation, r² = 0.61, p = 0.009, n = 9) and C6-glial cells (Spearman's rank correlation, r² = 0.56, p = 0.016, n = 9) (Fig. 5).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Antagonism of the 5-HT-induced Ca2+ response by buspirone, methiothepin, and WAY 100635, but not SB 224289, in CHO-K1 cells transiently transfected with a 5-HT1A receptor. CHO-K1 cells were transiently transfected with a human 5-HT1A receptor plasmid and assayed for a 5-HT (1 µM)-induced Ca2+ response. A, WAY 100635, methiothepin, and buspirone were given at indicated concentrations 10 min before 5-HT (1 µM). Ca2+ data are expressed in percentage of the maximal Ca2+ response as obtained with 10 µM 5-HT. Curves were constructed using mean values ± S.E.M. obtained in five to seven independent transfection experiments. pIC50 values are 7.96 ± 0.13 (WAY 100635), 7.16 ± 0.08 (methiothepin), and 6.34 ± 0.09 (buspirone). B, WAY 100635 (1 µM) and SB 224289 (1 µM) were given at 10 min before 5-HT was assayed at the indicated concentrations. Ca2+ data are expressed in percentage of the maximal Ca2+ response as obtained with 10 µM 5-HT. Curves were constructed using mean values ± S.E.M. (5-HT alone) and mean values (5-HT + WAY 100635 and 5-HT + SB 224289) obtained in five and two independent transfection experiments.

View this table: [in this window] [in a new window]   TABLE 1 Emax and pEC50 values of 5-HT ligands Ca2+, [35S]GTPS binding, and cAMP responses. by recombinant human 5-HT1A receptor Ca2+ responses were determined in 5-HT1A receptor-transfected CHO-K1 cells and expressed as a percentage of the Ca2+ response induced by 10 µM 5-HT. [35S]GTPS binding responses were determined on membranes of stably 5-HT1A receptor transfected CHO-K1 or C6-glial cells in the presence of 30 µM GDP and expressed versus 10 µM 5-HT-mediated response. Mean values ± S.E.M. are given for a minimum of three independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Concentration-dependent Ca2+ and [35S]GTPS binding responses of 5-HT ligands in either CHO-K1 or C6-glial cells transfected with a 5-HT1A receptor. Ca2+ responses in transiently 5-HT1A receptor-transfected CHO-K1 cells were expressed as a percentage of the maximal Ca2+ response induced by 10 µM 5-HT. [35S]GTPS binding responses at membranes of stably transfected C6-glial cells were measured with 30 µM GDP. Data are expressed as a percentage of the 10 µM 5-HT-induced [35S]GTPS binding response. Curves were constructed using mean values ± S.E.M. for a minimum of three independent transfection experiments. The 5-CT-mediated [35S]GTPS binding response data are from Pauwels et al. (1997). Mean Emax and pEC50 values are summarized in Table 1.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Emax values of 5-HT ligand's Ca2+ and [35S]GTPS binding responses. Emax values for Ca2+ and [35S]GTPS binding responses were obtained with 5-HT1A receptor transfected CHO-K1 cells and membranes of stably 5-HT1A receptor-transfected CHO-K1 (A) or C6-glial cells (B), respectively (Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Relationship between the magnitudes of 5-HT ligand's Ca2+ and [35S]GTPS binding responses (higher rank numbers represent a higher amplitude, and are based on the Emax values described in Table 1), r2 represents Spearman's rank correlation coefficient. A, [35S]GTPS binding responses in membranes of stably 5-HT1A receptor-transfected CHO-K1 cells. B, [35S]GTPS binding responses in membranes of stably 5-HT1A receptor transfected C6-glial cells.

	Discussion

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The Ca²⁺ response data as obtained with the 5-HT_1A receptor in transfected CHO-K1 cells indicate an atypical pharmacological 5-HT_1A receptor profile. Either a highly efficacious Ca²⁺ response or almost no Ca²⁺ response was found with the series of investigated 5-HT ligands. It seemed that the ligands induce either a "yes" or "no" response. The Ca²⁺ response induced by the native ligand 5-HT is likely to occur via activation of the recombinant 5-HT_1A receptor; no Ca²⁺ signal could be detected in nontransfected CHO-K1 cells, although the Ca²⁺ pathway was adequately responsive to bradykinin. CHO cells have previously been reported to express endogenous 5-HT_1B receptors, which are negatively coupled to adenylyl cyclase and positively coupled to increases in intracellular Ca²⁺ formation (Dickenson and Hill, 1995; Giles et al., 1996). In the present study, the selective 5-HT_1B receptor antagonist SB 224289 (Gaster et al., 1998) affected the 5-HT-mediated Ca²⁺ response little, if at all, whereas the selective 5-HT_1A receptor antagonist WAY 100635 (Fletcher et al., 1996) fully blocked the response. The 5-HT-mediated Ca²⁺ response was also sensitive to inhibition by pertussis toxin treatment. Thus, like the [³⁵S]GTPS binding responses, the Ca²⁺ effect in CHO-K1 cells seem to be mediated by endogenous G_i/o proteins. The [³⁵S]GTPS binding response monitors G protein activation of endogenous G_i/o proteins, whereas the Ca²⁺ response is probably mediated by endogenous G subunits of activated G_i/o proteins in CHO-K1 cells. Although dual coupling of the cloned 5-HT_1A receptor to both adenylyl cyclase and phospholipase C in HeLa cells is apparently mediated via the same G_i3 protein (Fargin et al., 1991), this may be different for CHO-K1 and C6-glial cells.

The Ca²⁺ response data strongly suggest that these 5-HT_1A receptor ligands can be divided in two different classes. A first class of ligands (F 13640, 5-CT, and F 14679) seem to demonstrate a maximal effect that is similar to that induced by 5-HT. These compounds also acted with a significantly higher efficacy in the Ca²⁺ response compared with their [³⁵S]GTPS binding responses in C6-glial cells. A second class of ligands (buspirone, flesinoxan, 8-OH-DPAT, ipsapirone, and eptapirone), with definite partial agonist properties in the [³⁵S]GTPS binding responses, were inactive or almost inactive in the Ca²⁺ response. It is possible that both classes of 5-HT receptor ligands recognize a distinct population of 5-HT_1A receptor conformations that may affect in a different manner the downstream cascade of effector proteins. Activation of both populations of 5-HT_1A receptor conformations would result in [³⁵S]GTPS binding responses with a broad spectrum of partial agonist properties. Activation of only a single population of receptor conformations would result in an efficacious Ca²⁺ response, whereas the other population of receptor conformations would result in very low efficacy. For instance, buspirone was almost free of intrinsic activity in the Ca²⁺ response and fully antagonized the 5-HT-induced Ca²⁺ response. Similar observations for buspirone have previously been obtained using HeLa cells and Ca²⁺ mobilization (Hoyer et al., 1991); this contrasts with buspirone's partial or efficacious agonist activity in [³⁵S]GTPS binding (e.g., Pauwels et al., 1997) and cAMP responses (Pauwels et al., 1993). Molecular dynamics simulations, considering the 5-HT_2A receptor (Shapiro et al., 2000), produced ligand-bound structures using substantially different binding interactions even among structurally similar ligands (differing by as little as one methyl group). Relatively minor changes in either receptor or ligand structure can produce drastic and unpredictable changes in both binding interactions and 5-HT_2A receptor activation. Differences in receptor reserve have often been invoked to explain why partial agonists may demonstrate either agonist (i.e., high receptor reserve) or antagonist (i.e., low receptor reserve) behaviors. In spite of prior evidence that dorsal raphe somatodendritic 5-HT_1A autoreceptors exhibit high receptor/effector coupling efficiency (receptor reserve) compared with postsynaptic receptors in hippocampus (Meller et al., 1990), there is no clear evidence of a difference at the level of receptor/G protein coupling (Meller et al., 2000). Alternatively, the 5-HT_1A receptor is able to couple to different Gi/o/z proteins (i.e., Butkerait et al., 1995), one of which may act preferentially on phospholipase C.

Strikingly, F 14679 and flesinoxan displayed a small difference (up to 16%) in their maximal [³⁵S]GTPS binding response, whereas they showed an 82% difference in their maximal Ca²⁺ response. Therefore, it is unlikely that the herein observed Ca²⁺ results can be explained by the assumption that efficacy in the Ca²⁺ response is observable from a certain threshold of 5-HT_1A receptor activation as estimated by the [³⁵S]GTPS binding response. This would also suggest that the apparent efficacy of each 5-HT ligand in the Ca²⁺ response would be enhanced under conditions of more efficient coupling or attenuated when coupling efficacy would be lower. The present study clearly demonstrates some 5-HT ligands are more efficacious, whereas others are less or not at all efficacious in the Ca²⁺ response. This opposite observation on ligand efficacy suggests the pharmacology of the Ca²⁺ response is different from that observed with the [³⁵S]GTPS binding response, although both are effected via the 5-HT_1A receptor. This would suggest that 5-HT_1A ligands inducing a highly efficacious Ca²⁺ response may result in downstream effects that cannot at all be achieved with such prototypical ligands as buspirone. In confirmation of this, F 13640 produces a complete inhibition of formalin-induced pain behaviors in conditions where buspirone exerted no detectable effect (Colpaert et al., 2002).

In conclusion, the pharmacological observations with the Ca²⁺ response indicate two classes of 5-HT_1A receptor ligands. The Ca²⁺ response in CHO-K1 cells constitutes a useful tool to identify highly selective 5-HT_1A receptor ligands that are distinct from prototypical 5-HT_1A ligands. This study further illustrates that the pharmacology of 5-HT_1A receptors may be diverse and be codetermined by the effector pathway. The complex area of 5-HT_1A receptor-coupled effector systems requires further research to analyze the activation of these effector systems and characterizes this activation by effector-selective ligands.

	Acknowledgements

 
We thank Dr. T. Wurch for critical reading of the manuscript. We also thank S. Bernois and F. Finana for excellent technical assistance and S. Brignatz for expert secretarial assistance.

	Footnotes

 
DOI: 10.1124/jpet.103.055871.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine, serotonin; 8-OH-DPAT, 8-(hydroxy-2-(di-n-propylamino)tetralin; 5-CT, 5-carboxamidotryptamine; F 14679, N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methyl-6-; methylaminopyridin-2-yl)-methylaminomethyl]-piperidine; F 13640, N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-; methylaminomethyl]piperidine; CHO, Chinese hamster ovary; GTPS, 5'-O-(3-[³⁵ S]thio) triphosphate; WAY 100635, N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide; SB 224289 1'-methyl-5-(2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-; carbonyl)-2,3,6,7-tetrahydrospiro[furo[2,3-f]indole-3,4'-piperidine].

Address correspondence to: Dr. Petrus J. Pauwels, Centre d'Immunologie Pierre Fabre, 5, avenue Napoléon III-BP 497, F 74164 Saint-Julien-en-Genevois Cedex, France. E-mail: peter.pauwels{at}pierre-fabre.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Ariëns EJ (1964) The mode of action of biologically active compounds, in Molecular Pharmacology, vol 1, Academic Press, New York, NY.
Berg KA, Maayani S, Goldfarb J, Scaramellini C, Leff P, and Clarke WP (1998) Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: evidence for agonist-directed trafficking of receptor stimulus. Mol Pharmacol 54: 94-104.[Abstract/Free Full Text]
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.[CrossRef][Medline]
Butkerait P, Zheng Y, Hallak H, Graham TE, Miller HA, Burris KD, Molinoff PB, and Manning DR (1995) Expression of the human 5-hydroxytryptamine_1A receptor in Sf₉ cells. J Biol Chem 270: 18691-18699.[Abstract/Free Full Text]
Colpaert FC, Tarayre JP, Koek W, Pauwels PJ, Bardin L, Xu ZJ, Wiesenfeld-Hallin Z, Cosi C, Carilla-Durand E, Assié MB, et al. (2002) Large-amplitude 5-HT_1A receptor activation: a new mechanism of profound central analgesia. Neuropharmacology 43: 945-958.[CrossRef][Medline]
Colquhoun D (1998) Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 125: 924-947.[Medline]
Dickenson JM and Hill SJ (1995) Coupling of an endogenous 5-HT_1B-like receptor to increases in intracellular calcium through a pertussis toxin-sensitive mechanism in CHO-K1 cells Br J Pharmacol 116: 2889-2896.[Medline]
Fargin A, Yamamoto K, Cotecchia S, Goldsmith PK, Spiegel AM, Lapetina EG, Caron MG, and Lefkowitz RJ (1991) Dual coupling of the cloned 5-HT_1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same G_i protein. Cell Signal 3: 547-557.[CrossRef][Medline]
Fletcher A, Forster EA, Bill DJ, Brown G, Ciffe IA, Hartley JE, Jones DE, McLenachan A, Stanhope KJ, Critchely DJP, et al. (1996) Electrophysiological, biochemical, neurohormonal and behavioral studies with WAY-100635, a potent, selective and silent 5-HT_1A receptor antagonist. Behav Brain Res 73: 337-353.[CrossRef][Medline]
Gaster LM, Blaney FE, Davies S, Duckworth DM, Ham P, Jenkins S, Jennings AJ, Joiner GF, King FD, Mulholland KR, et al. (1998) The selective 5-HT_1B receptor inverse agonist 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl) biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydro-spiro[furo[2,3-f]indole-3, 4'-piperidine] (SB 224289) potency blocks terminal 5-HT autoreceptor function both in vitro and in vivo. J Med Chem 41: 1218-1235.[CrossRef][Medline]
Gether U, Liu S, and Kobilka BK (1995) Fluorescent labeling of purified ₂ adrenergic receptor: evidence for ligand-specific conformational changes. J Biol Chem 270: 28268-28275.[Abstract/Free Full Text]
Gettys TW, Fields TA, and Raymond JR (1994) Selective activation of inhibitory G protein -subunits by partial agonists of the human 5-HT_1A receptor. Biochemistry 33: 4283-4290.[CrossRef][Medline]
Giles H, Lansdell SJ, Bolofo ML, Wilson HL, and Martin GR (1996) Characterization of a 5-HT_1B receptor on CHO cells: functional responses in the absence of radio-ligand binding. Br J Pharmacol 117: 1119-1126.[Medline]
Hoyer D, Boddeke H, and Schoeffter P (1991) Second messengers in the definition of 5-HT receptors, in Serotonin: Molecular Biology, Receptors and Functional Effects, (Fozard JR and Saxena PR eds) pp 117-132, Basel, Birkhäuser.
Kenakin T (1997) Drug receptor theory, in Pharmacologic Analysis of Drug-Receptor Interaction, 3rd ed., pp. 1-42, Lippincott-Raven Press, New York, NY.
Koek W, Patoiseau JF, Assie MB, Cosi C, Kleven MS, Dupont-Passelaigue E, Carilla-Durand E, Palmier C, Valentin JP, John G, et al. (1998) F 11440, a potent, selective, high efficacy 5-HT_1A receptor agonist with marked anxiolytic and anti-depressant potential. J Pharmacol Exp Ther 287: 266-283.[Abstract/Free Full Text]
Koek W, Vacher B, Cosi C, Assie MB, Patoiseau JF, Pauwels PJ, and Colpaert FC (2001) 5-HT_1A receptor activation and antidepressant-like effects: F 13714 has high efficacy and marked antidepressant potential. Eur J Pharmacol 420: 103-112.[CrossRef][Medline]
Krumins AJ and Barber R (1997) The stability of the agonist ₂-adrenergic receptor-G_s complex: evidence for agonist-specific receptors states. Mol Pharmacol 52: 144-154.[Abstract/Free Full Text]
Leff P, Scaramellini C, Law C, and McKechnie K (1997) A three-state model of agonist action. Trends Pharmacol Sci 18: 355-362.[Medline]
Meller E, Goldstein M, and Bohmaker K (1990) Receptor reserve for 5-hydroxytryptamine_1A-mediated inhibition of serotonin synthesis: possible relationship to anxiolytic properties of 5-hydroxytryptamine_1A agonists. Mol Pharmacol 37: 231-237.[Abstract]
Meller E, Li H, Carr KD, and Hiller JM (2000) 5-Hydroxytryptamine_1A receptor-stimulated [³⁵S]GTPS binding in rat brain: absence of regional differences in coupling efficiency. J Pharmacol Exp Ther 292: 684-691.[Abstract/Free Full Text]
Pauwels PJ, Tardif S, Finana F, Wurch T, and Colpaert FC (2000) Ligand-receptor interactions as controlled by wild-type and mutant Thr(370)Lys 2B-adrenoceptor-G15 fusion proteins. J Neurochem 74: 375-384.[CrossRef][Medline]
Pauwels PJ, Tardif S, Wurch T, and Colpaert FC (1997) Stimulated [³⁵S]GTP S binding by 5-HT_1A receptor agonists in recombinant cell lines. Modulation of apparent efficacy by G-protein activation state. Naunyn-Schmiedeberg's Arch Pharmacol 356: 551-561.[CrossRef][Medline]
Pauwels PJ, Van Gompel P, and Leysen JE (1993) Activity of 5-HT receptor agonists, partial agonists and antagonists at cloned human 5-HT_1A receptors that are negatively coupled to adenylate cyclase in permanently transfected HeLa cells. Biochem Pharmacol 45: 375-383.[CrossRef][Medline]
Pauwels PJ, Wurch T, Amoureux MC, Palmier C, and Colpaert FC (1996) Stimulation of cloned human serotonin 5-HT_1D receptor sites in stably transfected C6 glial cells promotes cell growth. J Neurochem 66: 65-73.[Medline]
Raymond JR, Mukhin YV, Gettys TW, and Garnovskaya MN (1999) The recombinant 5-HT_1A receptor: G protein coupling and signaling pathways. Br J Pharmacol 127: 1751-1764.[CrossRef][Medline]
Shapiro DA, Kristiansen K, Kroeze WK, and Roth BL (2000) Differential model of agonist binding to 5-hydroxytryptamine_2A serotonin receptors revealed by mutation and molecular modeling of conserved residues in transmembrane region 5. Mol Pharmacol 58: 877-886.[Abstract/Free Full Text]
Watson C, Chen G, Irving P, Way J, Chen WJ, and Kenakin T (2000) The use of stimulus-biased assay systems to detect agonist-specific receptor active states: implications for the trafficking of receptor stimulus by agonists. Mol Pharmacol 58: 1230-1238.
Wurch T, Chastagnier C, Palmier C, Colpaert FC, and Pauwels PJ (1996) A 413 bp region upstream the human 5-HT_1A receptor gene is sufficient for its in vitro expression. Neurosci Res Commun 19: 75-82.[CrossRef]
Yang Q and Lanier SM (1999) Influence of G protein type on agonist efficacy. Mol Pharmacol 56: 651-656.[Abstract/Free Full Text]
Zhang D and Weinstein H (1993) Signal transduction by a 5-HT₂ receptor: a mechanistic hypothesis from molecular dynamics stimulations of the three-dimensional model of the receptor complexed to ligands. J Med Chem 36: 934-938.[CrossRef][Medline]

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