Introduction

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The ubiquitous neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) has so far been shown to interact with seven receptor classes, classified as 5-HT₁ to 5-HT₇ receptors. Three of them were defined as Gs-protein-coupled receptors: the 5-HT₄, 5-HT₆, and 5-HT₇ receptors. In this study, we focus on the functional properties of the 5-HT₄ receptor class, in which a new level of structural diversity has been highlighted in recent years.

The wide distribution of 5-HT₄ receptors, from the central nervous system to the peripheral tissues, suggests a wide range of functional roles. In the central nervous system, 5-HT₄ receptors have been localized in rat basal ganglia, hippocampus, and olfactory tubercule (Compan et al., 1996; Vilaro et al., 1996). In human brain, radioligand binding (Domenech et al., 1994; Reynolds et al., 1995) and in situ hybridization studies (Bonaventure et al., 2000) have shown predominant expression of 5-HT₄ receptors in basal ganglia and limbic structures. Functionally, central 5-HT₄ receptors have been implicated in diverse processes such as anxiety, memory, and cognition (Silvestre et al., 1996; Fontana et al., 1997). 5-HT₄ receptors have also been shown to be widely distributed in peripheral organs such as the gastrointestinal tract, the myocardium, the urinary bladder, and the adrenal glands (for review, see Ford and Clarke, 1993). Furthermore, pharmacological differences have been identified in various tissues and species, suggesting a heterogeneity of 5-HT₄ receptors (Ford and Clarke, 1993; Leung et al., 1996). Indeed, Gerald et al. (1995) have initially cloned two alternative splice variants coding for short (5-HT_4S) and long (5-HT_4L) isoforms of the receptor, which could be the structural basis for functional diversity. So far, seven receptor variants that differ in their C termini have been identified: 5-HT_4a (previously named 5-HT_4S), 5-HT_4b (previously named 5-HT_4L), 5-HT_4c, 5-HT_4d, 5-HT_4e, 5-HT_4f, and 5-HT_4g (Claeysen et al., 1997; Blondel et al., 1998; Bender et al., 2000). Moreover, by cloning the human 5-HT₄ receptor gene, our group has recently shown the existence of an additional site of alternative splicing (5-HT_4h), leading to an extended second extracellular loop that could theoretically combine with each C-terminal variant (Bender et al., 2000). Interestingly, the only experimentally isolated h5-HT_4hb-receptor variant showed an altered functional pharmacology because the prototypic 5-HT₄ receptor antagonist GR113808 exhibited partial agonistic properties. This finding supported the hypothesis of the existence of a functional diversity among 5-HT₄ receptor splice variants. In addition, we have recently shown a differential tissue distribution of the h5-HT₄ receptor isoforms (Bender et al., 2000), which may also contribute to tissue-specific functional differences. To date, all isoforms have been shown to activate adenylyl cyclase (AC) in vitro, and no difference in signal transduction between C-terminal 5-HT₄ receptor variants has been demonstrated. Although the functional significance for the existence of 5-HT₄ receptor splice variants is currently not well understood, studies on other receptors suggest that different C termini may discriminate between signal transduction pathways. G-protein selectivity was shown to be different for the C-terminal splice variants of the prostaglandin E receptor EP3 (Satoh et al., 1999), whereas somatostatin receptor isoforms have been found to differ in the coupling efficiency and desensitization events (Vanetti et al., 1993). To better understand the functional significance of 5-HT₄ receptor isoforms, we investigated the pharmacological profile and signal transduction of h5-HT_4a and h5-HT_4b receptors, two initially cloned C-terminal splice variants.

We have found major differences between the two variants, not only in the pharmacology of the cAMP response and the fractions of agonist high-affinity sites, but also in the selection of the G-proteins activated by the receptors and the second messengers triggered by agonists. Interestingly, this differential signaling was receptor variant and compound specific. These data provide further evidence for functional differences between 5-HT₄ receptor isoforms, underscoring a physiological relevance for the existence of 5-HT₄ receptor splice variants.

	Experimental Procedures

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Materials. [³H]GR113808 (specific activity of 3.7 TBq/mmol), 5-hydroxy[³H]tryptamine triacetate ([³H]5-HT, specific activity of 4.44 TBq/mmol), and [³⁵S]GTPS (specific activity of 37 MBq/ml) were obtained from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). The mammalian expression vector pcDNA3 was obtained from Invitrogen (Carlsbad, CA). Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), sodium butyrate, Geneticin (G-418), and calf serum were from Invitrogen. The Bradford protein assay was performed with the reagent supplied by Bio-Rad (Nazareth Eke, Belgium). The Adenylyl Cyclase Activation FlashPlate kit was supplied by PerkinElmer Life Sciences (Brussels, Belgium). The Bordatella pertussis toxin (PTX) was from Calbiochem (La Jolla, CA). The liquid scintillation spectrometer, the scintillation fluids Ultima Gold MV and Ultimaflo AF, and the Unifilter-96 GF/B plates were from Packard (Meriden, CT). GDP and dilithium salt were from Hoffman-La Roche (Basel, Switzerland). Fluo3-acetoxymethyl ester was purchased from Molecular Probes (Leiden, The Netherlands). Forskolin and probenecid were from Sigma (St Louis, MO). Cisapride and prucalopride are proprietary compounds from Janssen Pharmaceutica (Antwerp, Belgium). Renzapride, SB204070, GR113808, SDZ205070, and SDZ HTF919 were synthesized by Janssen Pharmaceutica for its own purposes. 5-HT and 5-MeOT were from Acros Organics (Geel, Belgium). For pharmacological testing, compounds were dissolved and diluted in dimethyl sulfoxide except for indoleamines, which were dissolved in water and protected from light throughout the experiment. The final dimethyl sulfoxide concentration never exceeded 0.5% (v/v). The GraphPad Prism program was from GraphPad Software, Inc. (San Diego, CA).

Stable Transfections and Selection of Monoclonal Cell Lines. The h5-HT_4a and h5-HT_4b receptors were stably expressed in HEK 293 cells. Expression was under the control of the constitutive cytomegalovirus promoter provided by the pcDNA3 vector. The calcium phosphate transfection method was used with modifications as described previously (Lesage et al., 1998). Stable monoclonal cell lines were created by limited dilution and selected by [³H]GR113808 radioligand binding after culture under Geneticin (G-418) selection (800 µg/ml). Receptor expression levels of a range of isolated monoclonal cell lines were investigated with the antagonist [³H]GR113808 in radioligand binding experiments; nontransfected HEK 293 cells did not show specific [³H]GR113808 binding (data not shown). Of the 39 monoclonal cell lines obtained after transfection with the h5-HT_4a receptor, six had expression levels of about 2000 fmol/mg of protein after treatment with sodium butyrate (see below). Clone 8 (maximal expression levels ~3100 fmol/mg) was used for further studies and characterization. Another clone (clone 1) with a lower expression level (maximal ~1500 fmol/mg) was used to investigate the contribution of different receptor expression levels in the functional tests performed in this study. Of the 33 monoclonal cell lines expressing the h5-HT_4b receptor, cell clone 9 had an expression level of about 7000 fmol/mg of protein. Clone 9 was used for further study and characterization, and as for the other receptor splice variant, a clone of lower expression level (clone 4, maximal ~2000 fmol/mg) was used to investigate the effect of different receptor expression levels in the functional tests performed in this study.

Cell Culture and Treatments. Cells were grown in DMEM containing 10% dialyzed calf serum, 10⁵ IU/l penicillin G, 0.1 g/l streptomycin, 0.1 g/l pyruvate, and 0.292 g/l L-glutamine, under 5% CO₂ at 37°C. Cells were grown under selection (800 µg/ml G418) for 48 h every 2 weeks. Culture plates were coated with PBS containing 10 µg/ml poly(L-lysine) for 45 min at 37°C and washed once with PBS before the cells were seeded.

Membrane Preparations and Radioligand Binding. Cells were cultured on 150-mm Petri dishes and washed twice with ice-cold PBS. The cells were then scraped from the plates with a cell scraper, suspended in 50 mM Tris-HCl buffer, pH 7.4, and harvested by centrifugation at 16,000g for 10 min. The pellet was re-suspended in 5 mM Tris-HCl, pH 7.4, and homogenized with an Ultra Turrax homogenizer (IKA Labortecnik, Staufen, Germany). The resulting membranes were collected by centrifugation at 25,000g for 20 min and stored at 70°C in 50 mM Tris-HCl buffer, pH 7.4, at a protein concentration of approximately 1 mg/ml. The Bradford protein assay was used for protein determination with bovine serum albumin as a standard.

Measurements of cAMP Formation. After 2 days of growth, cells were detached from the Petri dishes with 3 ml of EDTA (0.04% w/v in PBS) and resuspended in PBS without Ca²⁺ and Mg²⁺. The cells were centrifuged at 500g for 5 min. The pellet was resuspended in the "stimulation buffer" provided with the PerkinElmer kit and diluted to a concentration of 10⁶ cells/ml, and 50 µl was added per well of the Flashplate (50,000 cells/well). Compounds were diluted in PBS without Ca²⁺ and Mg²⁺ containing 1 µM pargyline and 1 µM paroxetine. Fifty microliters of the compound solution was added per well, followed by an incubation for 20 min at 37°C. After the incubation period, a direct radioimmunoassay using ¹²⁵I-cAMP was performed by the addition of 100 µl of detection mix per well according to the supplier's instructions. After incubation for 18 h at room temperature, counting was performed in a Topcount HTS9912V counter (Packard).

[³⁵S]GTPS Binding Assays. For further functional testing, the [³⁵S]GTPS binding assay was performed on membrane preparations. Membranes were incubated (105 µg/ml) in 20 mM HEPES-NaOH, pH 7.4, containing 10 mM MgCl₂, 1 µM GDP, 1 µM pargyline, and 1 µM paroxetine, in the presence of 0.25 nM [³⁵S]GTPS and compounds. The final volume was 250 µl and the incubation was performed in polypropylene tubes (PPN-tube-96; Micronic bv, Lelystad, The Netherlands) at 37°C for 20 min. The incubation was stopped by filtration on Unifilter-96 GF/B plates with ice-cold phosphate buffer, 10 mM Na₂HPO₄, adjusted to pH 7.4 with a solution of 10 mM NaH₂PO₄. The filter plates were dried at room temperature, 30 µl of Microscint was added per well, and plates were counted in a Topcount HTS9912V counter (Packard).

Measurements of Intracellular Calcium Concentration. The intracellular [Ca²⁺]_i was measured with a fluorometric imaging plate reader (FLIPR; Molecular Devices, Crawley, England). Cells were seeded at 40,000 cells/well in 96-well plates (Cluster plates, black with clear bottom; Costar; Merck, Overijse, Belgium) for 2 days until they reached confluence. Cells were loaded with 2 µM Fluo3-acetoxymethyl ester in DMEM supplemented with 20 mM HEPES, pH 7.4, and 2.5 mM probenecid, for 1 h at 37°C under 5% CO₂. The plates were washed three times with 5 mM HEPES, pH 7.4, 1.25 mM CaCl₂, 140 mM NaCl, 1 mM MgCl₂, 5 mM KCl, and 10 mM glucose, containing 2.5 mM probenecid (to prevent extrusion of Fluo-3), 1 µM pargyline, and 1 µM paroxetine. [Ca²⁺]_i was measured by FLIPR, and the peak of the calcium transient was considered as the relevant signal. Treatment of the cells with 10 µM ionomycin (Ca²⁺-inophore) was performed as a control for dye loading and cell viability.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Characterization of Cell Lines in Ligand Concentration Binding. The two human 5-HT₄ receptor isoforms investigated in this study, h5-HT_4a and h5-HT_4b, were stably transfected into HEK 293 cells. In this study, we have primarily used a high-level expressing clone of each splice variant, the h5-HT_4a clone 8 and the h5-HT_4b clone 9. In addition, clones with lower expression levels, h5-HT_4a clone 1 and h5-HT_4b clone 4, were employed in certain experiments to investigate the effect of receptor expression on the functional properties of each receptor variant. The isolated monoclonal cell lines were characterized by saturation binding, using two radioligands, an antagonist, [³H]GR113808, and the natural agonist, [³H]5-HT. The experiments were performed on membrane preparations from cells treated with sodium butyrate and from untreated cells. Independent of expression levels, the concentration binding isotherms of the antagonist [³H]GR113808 to h5-HT_4b and h5-HT_4a receptors expressed in different clones showed rectangular hyperbolae. The resulting linear Scatchard plots revealed a single high-affinity binding site (K_D values ranging between 0.05 and 0.1 nM for both 5-HT₄ receptor isoforms). Furthermore, whereas treatment with sodium butyrate increased all B_max values by almost 70%, it did not alter mean K_D values. The calculated K_D and B_max values are presented in Table 1. For comparison, the B_max values of the two lower level expressing clones, treated with sodium butyrate, were 1500 (±160) and 2087 (±513) fmol/mg of protein for the h5-HT_4a clone 1 and the h5-HT_4b clone 4, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Scatchard plots derived from [3H]5-HT saturation binding curves performed on membrane preparations from HEK 293 cells stably transfected with h5-HT4 receptor isoforms. Saturation binding was performed with [3H]5-HT concentrations ranging from 0.2 to 40 nM to visualize the low-affinity sites. The membranes were prepared from h5-HT4a (A) or h5HT4b (B) expressing cells under sodium butyrate-treated () or nontreated conditions (). The parabolic Scatchard plots show the two affinity sites for both h5-HT4 receptor isoforms. The data shown represent a typical example out of three independent experiments performed in duplicate.

Adenylyl Cyclase Activation. The activity of AC after h5-HT₄ receptor stimulation with various reference ligands was estimated on living cells, which were not treated with sodium butyrate to elevate receptor expression levels. An increase of basal cAMP levels, found in the high expressing clones compared with wild-type HEK 293 cells, revealed the constitutive activity of both 5-HT₄ receptors, which is consistent with previous reports (Claeysen et al., 1999, 2000). Compared with the basal values measured in the wild-type HEK 293 cells, the measured constitutive activity was 482% (±135) and 881% (±156) (mean ± S.D., n = 3) in 5-HT_4a and 5-HT_4b receptor-expressing cells, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   cAMP formation investigated in HEK 293 cells stably transfected with h5-HT4 receptor isoforms. Concentration-response curves of cAMP accumulation were performed on whole cells expressing h5-HT4a (A) or h5-HT4b (B) receptors stimulated with 5-HT (), prucalopride (), cisapride (), SDZ-HTF919 (), renzapride (), 5-MeOT (), SB204070 (), and SDZ205,557 (). The maximal response for each compound was normalized to the maximal stimulation obtained with 5-HT (% of 5-HTmax). Results are expressed as fitted curves to the mean (±S.D.) of independent experiments performed in duplicate, as summarized in Table 2.

Changes in Intracellular Calcium Concentration. To investigate other potential signal transduction pathways, we measured the modulations of [Ca²⁺]_i after h5-HT_4a and h5-HT_4b receptor activation. The same reference compounds as described for the cAMP experiments were employed. The h5-HT_4a receptor showed fast increase in [Ca²⁺]_i after exposure to all tested agonists, independent of the expression level found in both investigated clones. Interestingly, the Ca²⁺ response induced by the h5-HT_4a receptor was dependent on the chemical nature of the agonists. For the two agonistic benzamide-like compounds, cisapride and prucalopride, the peaks of the calcium transients were two and three times higher than for the natural agonist 5-HT and showed a different time course (Fig. 3). Both cisapride- and prucalopride-stimulation needed a longer time (>100 s) to reach maximal [Ca²⁺]_i than was observed with indoleamines (<50 s).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Transients of [Ca2+]i on HEK 293 cells stably transfected with h5-HT4 receptor isoforms. Time course of changes in [Ca2+]i triggered via the h5-HT4b () or the h5-HT4a () receptor were studied by the calcium-sensitive fluorescence of Fluo3 measured with FLIPR on sodium butyrate-treated cells. Ordinates represent the increase of RFU measured at the peak of the Ca2+ transient. The receptors were stimulated by 106 M 5-HT (A) or 106 M prucalopride (B). The arrows indicate the time when the compound was added to cells. The results were normalized to the basal fluorescence levels of the cells before compound addition.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   [Ca2+]i measurements on HEK 293 cells stably transfected with h5-HT4 receptor isoforms. Changes in [Ca2+]i in cells expressing the h5-HT4b (A and B) or the h5-HT4a (C and D) receptor under either sodium butyrate-treated (A and C) or nontreated (B and D) conditions measured with FLIPR as indicated in Fig. 3. The compounds used for receptor stimulation were 5-HT (), prucalopride (), cisapride (), renzapride (), and 5-MeOT (). Data are expressed as fitted curves to the mean (±S.D.) peak values (in RFU) of triplicate determinations and represent a typical experiment out of independent experiments as summarized in Table 3. Note that sodium butyrate treatment is not necessary to obtain a Ca2+ response.

Inositol Phosphates or Extracellular Calcium Influx. Mobilization of calcium from intracellular stores would classically involve the activation of phospholipase C and was investigated by means of IP measurements. Assays were performed after h5-HT_4a receptors had been stimulated with the natural agonist 5-HT and the benzofuran prucalopride. No increase of IP concentrations could be detected (data not shown) in contrast to a parallel control performed with CHO cells expressing h5-HT_2A receptors. Our results, therefore, indicate that the h5-HT_4a-induced calcium mobilization does not involve the phospholipase C/IP₃ pathway.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Characterization of the h5-HT4a receptor-mediated Ca2+ response. h5-HT4a cells were stimulated by vehicle (basal), 106 M 5-HT, 106 M prucalopride, or 106 M forskolin. A, the experiments were performed using a modification of the standard buffer containing 1.25 mM extracellular calcium (black bars) by replacing calcium with 2 mM EGTA () or in the standard buffer with a 24-h preincubation with 100 ng/ml PTX () or vehicle (). The ordinate represents the mean RFU measured at the peak of the Ca2+ transient normalized to the mean RFU obtained before the stimulation (% of basal). Data are expressed as the mean (±S.D.) of three independent experiments performed in triplicate. B, the experiments were performed in standard buffer containing 1.25 mM extracellular calcium after a 15-min preincubation with 200 µM bepridil () or vehicle (). The ordinate represents the mean RFU measured at the peak of the Ca2+ transient. Data are expressed as the mean (±S.D.) of nine determinations and represent a typical experiment out of three independent experiments.

[³⁵S]GTPS Binding. G-protein coupling to receptors was investigated by measuring [³⁵S]GTPS binding to membrane preparations from cells expressing either h5-HT₄ receptor isoform. Results were expressed as percentage increase in [³⁵S]GTPS binding over basal levels (Fig. 6). The levels of stimulation were found to differ between h5-HT_4a and h5-HT_4b receptors, and were in the range of 50 and 100% of stimulation, respectively. To investigate potential differences in pharmacology, the same six agonists and three putative antagonists were used as described above. The pEC₅₀ values derived from fitted concentration-response curves are presented in Table 4. We were not able to detect any stimulatory effects on [³⁵S]GTPS binding with the two antagonists GR113808 and SDZ205,557. However, SB204070 triggered a weak response via h5-HT_4b receptors. Despite the higher levels of stimulation triggered via the h5-HT_4b receptor, no significant differences in the potency of the agonistic compounds were detected between the both receptor variants.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effects of PTX pretreatment on [35S]GTPS binding on membranes from HEK 293 cell line stably transfected with h 5-HT4 receptor isoforms. The h5-HT4b (A and B) and h5-HT4a (C and D) receptor-expressing membranes were employed in [35S]GTPS binding using a series of agonistic compounds. The membranes in B and D are treated with 100 ng/ml PTX, whereas A and C show membranes from vehicle-treated cells. Results are expressed as percent stimulation over basal levels. The nonstimulated (basal) [35S]GTPS binding levels were 4150 ± 274 and 3711± 977 dpm for the 5-HT4a and 5-HT4b receptor preparations, respectively. The compounds used for receptor stimulation were 5-HT (), prucalopride (), cisapride (), SDZ-HTF919 (), renzapride (), 5-MeOT (), SB204070 (), and SDZ205,557 (). Data are expressed as fitted curves to the mean (±S.D.) of three independent experiments performed in duplicate.

View this table: [in this window] [in a new window]   TABLE 4 Effects of PTX-pretreatment on [35S]GTPS binding on membranes from HEK 293 stably transfected with h5-HT4a and h5-HT4b receptors PTX stands for membrane preparations from cells treated with 100 ng/ml PTX. Presented results are the means of three independent experiments performed in duplicate. The pEC50 values were calculated by nonlinear regression analysis of each concentration-response curve.

View this table: [in this window] [in a new window]   TABLE 5 Inhibition of maximal [35S]GTPS binding by 100 ng/ml PTX on membranes from HEK 293 stably transfected with h5-HT4b receptors Experiments were performed as described for Table 4. Results are means of three independent experiments performed in duplicate and are based on fitted concentration-response curves as presented in Fig. 6. Only the calculated differences between the fitted maximal responses from nontreated and PTX-treated membranes are presented.

Adenylyl Cyclase Activation after PTX Pretreatment. AC stimulation was investigated on h5-HT_4b- and, as a control, on h5-HT_4a receptors on cells pretreated with PTX and on untreated cells. Because the basal levels of cAMP were increased after PTX pretreatment the results were expressed as a percentage of the maximal stimulation obtained on nontreated cells. Consistent with the findings in [³⁵S]GTPS binding experiments, cAMP formation induced by 5-HT or prucalopride was found to be increased by PTX pretreatment in the h5-HT_4b-variant indicating a des-inhibition of the cAMP-pathway (Fig. 7). The increase in maximally accumulated cAMP was 27% (±5) for 5-HT and 29% (±6) for prucalopride stimulation. In contrast, no significant modulation of the AC response by PTX treatment was found with the h5-HT_4a receptor variant (Fig. 7).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Effects of PTX pretreatment on cAMP formation in HEK 293 cell lines stably transfected with h5-HT4 receptor isoforms. The h5-HT4a (A) and the h5-HT4b (B) receptor-expressing cells were stimulated by 5-HT ( and ) or prucalopride ( and ). Cells were treated with 100 ng/ml PTX (open symbols) or vehicle (solid symbols) 24 h before the experiment. Concentration-response curves of cAMP formation were derived from cells as mentioned in Fig. 2. The maximal response of a compound was normalized to the maximal stimulation obtained with 5-HT in nontreated cells (% of 5-HTmax). Results are expressed as fitted curve to the mean (±S.D.) of three independent experiments performed in duplicate.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

This study uses the initially cloned and widely distributed 5-HT_4a and 5-HT_4b receptors to investigate the pharmacological and functional properties of these receptor splice variants in stably transfected HEK 293 cells. Analysis of saturation binding performed with [³H]GR113808 revealed K_D values that were consistent with previous findings on transiently transfected cells (Ansanay et al., 1996; Van den Wyngaert et al., 1997) and different brain tissues (Grossman et al., 1993; Waeber et al., 1994; Arranz et al., 1998). We confirm that C-terminal variations of h5-HT_4a and h5-HT_4b receptors did not modify binding K_D values, consistent with the findings on EP3 and somatostatin receptors (Tsunehisa et al., 1993; Vanetti et al., 1993) and very recently also 5-HT₄ receptors (Bach et al., 2001). Detailed saturation binding performed with [³H]5-HT, revealed the presence of two affinity sites for both h5-HT_4a and h5-HT_4b receptors. Previous studies could only show a single affinity site for [³H]5-HT of 20 (±7) nM for the rat 5-HT_4b receptor expressed in COS7 cells, although the shallow competition binding curves and partial GTPS sensitivity indicated the existence of two affinity sites (Adham et al., 1996). The high concentrations of [³H]5-HT used in that study (5-100 nM) may have precluded the detection of the high-affinity site described in this report (K_D values ranging from 1.3 to 3.2 nM).

Comparison of saturation binding B_max data using [³H]GR113808 and [³H]5-HT allowed us to estimate the fraction of receptors present in the agonist high-affinity state. This fraction of high-affinity receptors was not dependent on expression levels, either modified by sodium butyrate pretreatment of cells or using different monoclonal cell lines, but characteristic for a given splice variant. The h5-HT_4b receptors showed a consistently higher proportion of non-G-protein-coupled or inactive receptors than the h5-HT_4a splice variant, based on the assumption that the natural agonist [³H]5-HT labels only active receptors in R*- and R*G-state with high affinity. The independence of the fraction of [³H]5-HT high-affinity sites from expression levels is analogous to the equilibrium J constant calculated in the absence of ligand as recently shown by Claeysen and colleagues (2000). They noticed that in COS-7 cells transfected with human or mouse 5-HT_4a receptors, the expression levels had no effect on the equilibrium J constant.

The pharmacological profile obtained for cAMP formation was partially divergent compared with previously reported results. We have shown that benzamide-like compounds in our system were full agonists on both variants, in contrast to reports classifying renzapride and cisapride as partial agonists (Ouadid et al., 1992; Blondel et al., 1998). Moreover, SB204070 and SDZ205,557, described as antagonists, were found to be partial agonists on both h5-HT_4a and h5-HT_4b receptors. In addition, small but statistically significant differences in efficacy and/or potency of agonists were found between the two splice variants. Direct extrapolation of these results to tissue-based studies should bear in mind that functional responses found in tissues may reflect not only different expression levels but also be the result of the stimulation of several 5-HT₄ receptor splice variants expressed in a given tissue.

A very recent study of Bach et al. (2001) investigating the cAMP response in h5-HT_4a and h5-HT_4b receptors could also not match the pharmacological pattern found on cells to measurement on right atrium membranes, although the 5-HT_4a isoform is thought to be predominant in that tissue (Blondel et al., 1997). In contrast to our study, they have found partial agonism of renzapride and cisapride. However, their pharmacological comparison is focused on antagonists, and the study uses [-³²P]ATP-loaded membranes for measurements of the cAMP, whereas our study is performed on intact cells. At similar expression levels, our EC₅₀ for 5-HT is approximately 10-fold lower than the ones reported by Bach et al. (2001). Therefore, the membrane based assay used by Bach et al., which allows a direct comparison to measurements performed on tissue samples, may suffer from impaired coupling and may not be able to reveal the subtle differences seen in this study on intact cells.

In addition to AC activation, 5-HT₄ receptors have also been shown to trigger other signal transduction pathways, such as reduction of potassium currents in mouse colliculi neurons (Fagni et al., 1992), calcium influx in adrenocortical cells (Contesse et al., 1996), and atrial myocytes (Ouadid et al., 1992). Our results revealed a clear functional difference between the two h5-HT₄ receptor isoforms in their ability and pharmacology to increase [Ca²⁺]_i. The h5-HT_4a receptor was able to trigger strong [Ca²⁺]_i increase, in contrast to h5-HT_4b receptors, which responded with a weaker signal and only to indoleamines. This signal transduction pathway was dependent on receptor expression levels, however, also detected the lowest expression level of the h5-HT_4a receptor [i.e., clone 1 expressing 1500 (±160) fmol/mg]. Interestingly, the calcium response was not only variant-specific but also dependent on compound structure, because benzamide-like compounds triggered higher responses than indoleamines.

The absence of IP pathway activation, the elimination of the response after removal of extracellular calcium, and the inhibition of the response by bepridil suggest that h5-HT_4a receptors were able to activate calcium channels. Nevertheless, the question of the cascade events triggering the calcium channel activation has yet to be assessed. The phosphodiesterase inhibitor IBMX and forskolin, a direct AC activator did not enhance the increase of [Ca²⁺]_i (data not shown), suggesting that, in contrast to the h5-HT₄-induced [Ca²⁺]_i response in adrenocortical cells (Contesse et al., 1996), the h5-HT_4a calcium response in HEK 293 cells was not dependent on the cAMP pathway. Moreover, PTX treatment did not modify the calcium response, which excluded the hypothesis of Gi/o-protein involvement in the mechanism. However, because it has been shown that after activation of G-protein heterotrimers, the -subunits are able to activate calcium channels (for review, see Holler et al., 1999), such a pathway has to be considered for the 5-HT_4a receptor. In addition, opening of Ca²⁺-channels subsequent to a -subunit-mediated activation of potassium channels could be another mechanism.

In regard to the G-protein coupling to h5-HT_4a and h5-HT_4b receptors, we have found a difference between the level of G-protein stimulation induced by h5-HT_4a and h5-HT_4b receptors. PTX pretreatment induced a reduction in h5-HT_4b receptor-mediated [³⁵S]GTPS binding to the level found for the h5-HT_4a receptor, suggesting the existence of an additional Gi/o component in the h5-HT_4b receptor G-protein coupling. This was confirmed by an enhanced increase of cAMP formation induced via the h5-HT_4b variant after a PTX pretreatment, which was not found for the h5-HT_4a receptor. Taken together, these results indicate that the h5-HT_4b receptor is able to activate both Gi/o- and Gs-proteins, in contrast to the h5-HT_4a-receptor, which couples only to Gs-proteins. The pEC₅₀ values of both PTX-pretreated and nontreated cells in [³⁵S]GTPS binding and cAMP formation were similar, suggesting that unlike for the D₂ receptor, 5-HT_4b receptor dual coupling was not dependent on the agonist concentration (Chang et al., 1997). Until now, dual G-protein coupling is hypothesized to have two main functions. On the one hand, it has been shown for other receptors that these were sequential events contributing to desensitization mechanisms (Lefkowitz, 1998). On the other hand, Gs- and Gi/o-proteins have been shown to couple simultaneously to the ₂ adrenergic receptor (Eason et al., 1992), the functional net result of cAMP production representing the integration of the two G-protein-mediated effects.

Taken together, these results show that the h5-HT_4a and h5-HT_4b-receptor variants do activate partially different intracellular processes (Fig. 8, A and B). The characteristic sum of these differences may result in pharmacological differences of compounds as described in different tissues (Dumuis et al., 1988; Bockaert et al., 1997). Although possible differences in the localization of 5-HT_4a and 5-HT_4b receptors in brain tissue are still controversial (Gerald et al., 1995; Bender et al., 2000), saturation binding experiments performed on membranes from different human brain regions have revealed a higher fraction of agonist binding sites in the substantia nigra (0.54) than in other brain regions such as the frontal cortex (0.27), the striatum (0.25), or the hippocampus (0.16) (Bonaventure et al., 2000). These values parallel very closely the findings in the present cellular study. Although further studies on cell- and tissue-specific 5-HT₄ receptor expression patterns are required; the above findings together with the reported tissue-specific differences in agonist efficacy provide a physiological context of our study. We have evaluated the influence of receptor levels on the observed responses, however, for the Ca²⁺-response, promiscuous coupling cannot be completely ruled out. However, we have shown that it is not cAMP-dependent in HEK cells and has different thresholds and pharmacology on both receptor isoforms. It is important to state that a direct comparison of tissue expression levels (guinea pig striatum ~200 fmol/mg; Van den Wyngaert et al., 1997) to data derived on recombinant cells is problematic, because the physiological concentration of neurotransmitter receptors in the synaptic cleft is certainly higher than the tissue average observed in binding studies.

View larger version (45K): [in this window] [in a new window]   Fig. 8.   Model of the signal transduction pathways of h5-HT4a and h-5HT4b receptor splice variants. A summarizes the AC activation pathway. h5-HT4b receptor-induced cAMP formation results from a balance between stimulatory (Gs) and inhibitory (Gi/o) G-protein activation, whereas h5-HT4a receptors induce AC activation only via Gs-proteins. For both splice variants, AC activation is only slightly dependent on the chemical class of agonists. In contrast, calcium influx (B) is largely specific for the h5-HT4a receptor, and its efficacy is strongly dependent on the compound structure, preferring benzamide-like structures to indoleamines. Pointed arrows represent putative pathways; dashed arrows represent inhibitory effects.

On the receptor level the differences in the functional profile of the investigated reference compounds in cAMP and Ca²⁺ measurements favor different forms of R* for each receptor isoform and signaling pathway, which can be stabilized to a different degree by the compounds investigated. In general, further understanding of the fine-tuning of G-protein-coupled receptor isoform-associated signaling and its compound specificity could, in the future, lead to G-protein-coupled receptor isoform-specific (i.e., "super") drugs with potentially broader safety margins.