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CELLULAR AND MOLECULAR
Molecular Pharmacology Unit, Alcon Research, Ltd., Fort Worth, Texas
Received January 12, 2006; accepted March 2, 2006.
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Abstract |
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-methyl-5-HT were potent, full agonists (EC50 = 20 and 4.1 nM, respectively), whereas the phenylethylamine, R-(–)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride, was less active (EC50 = 63 nM). Proposed 5-HT2B-selective agonists, BW-723C86 [-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine hydrochloride] and (+)-norfenfluramine, exhibited strong agonist potency and efficacy comparable with 5-HT (EC50 = 18 and 33 nM, respectively) and 
15-fold more potency than (–)-norfenfluramine (EC50 = 500 nM). 5-HT2C receptor agonists m-chlorophenylpiperazine and MK-212 [6-chloro-2-(1-piperaxinyl)pyrazine] were weak agonists in these cells, with potencies of 110 and 880 nM, respectively. A similar rank order of potency was observed in [Ca2+]i mobilization assays (r = 0.9, p < 0.005) in the HUSMC and with contraction of rat stomach fundus strips that contain a 5-HT2B receptor (r = 0.9, p < 0.001). Antagonist studies revealed that a 5-HT2B-selective antagonist, RS-127445 [2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine] (Ki = 0.13 nM), was significantly more effective at inhibiting 5-HT-induced activity than a 5-HT2A antagonist, M-100907 (R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol]) (Ki = 914 nM) and the 5-HT2C antagonists RS-102221 (8-[5-(2,4-dimethoxy-5-(4-trifluoromethylsulfo-amido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione hydrochloride) (Ki = 2.5 µM) and SB-242084 (6-chloro-5-methyl-1-[6-92-methylpyridin-3-yloxy) pyridine-3-ylcarbamoyl] indoline) (Ki = 42.4 nM) in the HUSMC PI turnover assays. Taken together, these studies strongly suggest the presence of a functionally active 5-HT2B receptor subtype in HUSMCs. The physiological role of this receptor in these cells remains to be defined.
, 2002). In particular, the 5-HT2 receptor family comprises three subtypes, 5-HT2A, 5-HT2B, and 5-HT2C, which have been reported to play a major role in a host of central nervous system functions including anxiety, depression, migraine, obesity, and schizophrenia. Due to the involvement of 5-HT2 receptor signaling in these disorders, there has been an increased interest in the therapeutic potential of selective 5-HT2 ligands as antidepressants, anti-obesity drugs, and anxiolytic agents (Hoyer et al., 1994; 2002). Further studies have identified serotonin in human aqueous humor (Veglio et al., 1998) and functionally coupled 5-HT2 receptors in rat retinal pigment epithelial cells (Osborne et al., 1993) and bovine ciliary epithelium (Inoue-Matsuhisa et al., 2003).
These findings suggested a possible role of serotonin and its receptors in aqueous humor dynamics and as a possible target for ocular pathologies. Indeed, it has recently been shown that topical ocular administration of 5-HT2 receptor agonists effectively lowered intraocular pressure in a nonhuman primate model of laser-induced ocular hypertension (May et al., 2003), suggesting a potential utility of 5-HT2 agonists as antiglaucoma therapeutics. However, the current lack of subtype-selective ligands, especially agonists, has made it difficult to ascribe the hypotensive action of these molecules to an individual 5-HT2 receptor subtype.
Recently, the appetite suppressant fenfluramine was withdrawn from the U.S. market because of its association with valvular heart disease (Connolly et al., 1997). Norfenfluramine, the active metabolite of fenfluramine, has been implicated as the causative agent in the observed valvular hyperplasia due to its apparent activity at the 5-HT2B receptor (Fitzgerald et al., 2000), although other biological activities of norfenfluramine have not been ruled out. Because of the potential for unwanted side effects using nonselective agonists, there is great interest in developing 5-HT2 receptor subtype-specific therapeutics that are devoid of 5-HT2B receptor agonist activity.
We sought to identify cell systems endogenously expressing human 5-HT2 receptor subtypes to characterize compounds of interest at these therapeutically important receptors. Although compound characterization using cell types with endogenous receptors can be confounded by competing activities at other receptor types, the resulting pharmacology at physiological expression levels and with the native G-protein coupling system may give a more relevant profile than the use of cell lines overexpressing receptors and/or promiscuous G-protein systems. The 5-HT2 receptor subtypes are implicated in serotonin-induced smooth muscle contraction and resulting constriction, presumably through Gq-coupled activation of phospholipase C, PI hydrolysis, and intracellular calcium mobilization. For example, 5-HT2A receptors have been identified in vascular smooth muscle tissue and cells where activation of 2A receptors induces tissue contraction and vasoconstriction. 5-HT2B receptors are highly expressed in rat stomach fundus, and contraction of rat fundic strips is a prototypic assay used to characterize functional agonist activity of compounds at the 5HT2B receptor (Baxter et al., 1994). We tested commercially available cell lines for PI metabolism in response to 5-HT and other prototypic serotonergic compounds. Through our search, we discovered that 5-HT and other agonists induced a reproducible PI turnover response and mobilized intracellular calcium in a human uterine smooth muscle cell (HUSMC). In this study, we describe the pharmacological profile of the 5-HT2 receptor present in HUSMC using prototypic serotonergic agonists and reported subtype-selective 5-HT2 receptor antagonists.
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Materials and Methods |
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-methyl-5-hydroxytryptamine maleate, R-(–)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (R-DOI), and other reagents were purchased from Sigma/RBI (Natick, MA). MK-212, m-chlorophenylpiperazine (mCPP), BW-723C86, and RS-102221 were obtained from Tocris Cookson Inc. (Ellisville, MO). M-100907, SB-242084, RS-127445, R-norfenfluramine, and S-norfenfluramine were synthesized by the Medicinal Chemistry Department of Alcon Research, Ltd. (Fort Worth, TX) or external contract. [3H]Myoinositol (18.5 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). Formic acid, ammonium formate, and LiCl were obtained from Sigma Chemical (St. Louis, MO). AG-1 x 8 anion exchange resin and columns were from Bio-Rad (Hercules, CA). Ecolume scintillation cocktail was obtained from ICN Biomedicals (Costa Mesa, CA). Fluorometric imaging plate reader (FLIPR) and calcium assay kit dyes were from Molecular Devices Corp. (Sunnyvale, CA).
Cell Culture
HUSMCs (catalog no. CC-2562) were obtained from Cambrex (Walkersville, MD). Cell culture media, antibiotics, and trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum was obtained from Hyclone Laboratories (Logan, UT), heat-inactivated at 56°C for 30 min, and stored at –20°C. HUSMCs were cultured in Dulbecco's modified Eagle's medium containing 4.5 g/l glucose, 110 mg/l sodium pyruvate, pyridoxine hydrochloride, and GlutaMax I, supplemented with 10% fetal bovine serum and 10 µg/ml gentamicin sulfate.
In Vitro Functional Assays
Phosphoinositide Hydrolysis Assay. Phosphoinositide hydrolysis (PI turnover) assays of phospholipase C activity were conducted by measurement of the agonist-stimulated production of [3H]inositol phosphates from [3H]myoinositol as described previously (Sharif and Xu, 1996; Griffin et al., 1998). In brief, confluent monolayers of human uterine smooth muscle cells were exposed for 24 to 30 h to 1 to 1.5 µCi of [3H]myoinositol (18.5 Ci/mmol) in 0.5 ml of serum-free media to label cell membrane phospholipids. Cells were rinsed once with Dulbecco's modified Eagle's medium/Ham's F-12 containing 10 mM LiCl before incubation with agonist for 1 h at 37°C. For antagonist experiments, the selected antagonist was preincubated with cells for 15 min before addition of agonist and continued incubation for 1 h at 37°C.
The reaction was quenched by aspiration of the medium and addition of 1 ml of ice-cold 0.1 M formic acid to lyse the cells. Cell sample processing and separation of [3H]inositol phosphates were achieved with anion exchange chromatography. Cell lysates were loaded onto columns containing AG-1 x 8 anion exchange resin. Unincorporated [3H]myoinositol was removed by sequential washing with water and 50 mM ammonium formate. Total [3H]inositol phosphate fraction was eluted from the columns with 1.2 M ammonium formate containing 0.1 M formic acid. The eluate was collected and mixed with 15 ml of scintillation cocktail, and the total [3H]inositol phosphate was determined by liquid scintillation counting on a beta counter at 50% efficiency (LS6000; Beckman Instruments, Carlsbad, CA). Apparent inhibition constants (Ki) values for the functional antagonist experiments using phosphoinositide hydrolysis were calculated from the determined 50% inhibition concentrations (IC50 values) as described elsewhere (Cheng and Prusoff, 1973).
Intracellular Calcium Mobilization Assay. Intracellular calcium mobilization [Ca2+]i induced by 5-HT and other serotonergic compounds in HUSMCs was studied using a FLIPR instrument (Molecular Devices) (Schroeder and Neagle, 1996). Evaluation of the functional agonist activity of test compounds was performed using a protocol described previously (Kelly et al., 2003; May et al., 2003). In brief, confluent cell monolayers of human uterine smooth muscle cells were trypsinized, pelleted, and seeded at a density of 20,000 cells per well in black-walled, 96-well tissue culture plates and grown to confluence. The fluorescence response of HUSMCs was enhanced by growing the cells in medium containing 10% dialyzed fetal bovine serum for 2 to 3 days followed by incubation in serum-free medium overnight before the experiment. For the experiment, cells were loaded with a calcium-sensitive fluorescent dye provided in a calcium assay kit (Molecular Devices). The dye was reconstituted in FLIPR buffer (Hanks' balanced salt solution buffered with 20 mM HEPES, pH 7.4, 2.5 mM probenecid) and incubated with cells at 23°C for 1 h. Test compounds were diluted in 25% dimethyl sulfoxide/25% ethanol, and further dilutions were prepared in FLIPR buffer and evaluated in concentration-response formats. Agonist-stimulated intracellular calcium mobilization was measured on a FLIPR I system monitoring real-time changes in cellular fluorescence (
ex = 488 nm, em = 540 nm) upon agonist additions. Calibration of the instrument was performed using manufacturer's standard procedures.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-methyl-5-HT exhibited approximately 5-fold stronger potency than 5-HT (EC50 = 4.1 ± 2.1 nM, Emax = 93 ± 12%) in the assay. The tryptamine analog BW-723C86, described as a 5-HT2B-selective agonist, exhibited strong agonist potency and efficacy comparable with 5-HT (EC50 = 18 ± 2.5 nM, Emax = 104 ± 4%). The phenylethylamine ligand R-DOI, considered to be a relatively selective and potent 5-HT2A receptor agonist, was a full agonist with a modest potency weaker than 5-HT, -methyl-5-HT, and BW-723C86. The piperazines mCPP and MK-212 exhibited functional potencies of 110 ± 32 and 880 ± 320 nM, respectively, with mCPP consistently displaying partial agonist efficacy (Emax = 37 ± 5%). MK-212 had the weakest potency of the compounds tested. The (+)- and (–)-isomers of norfenfluramine, the active metabolite of fenfluramine, were tested for agonist activity in PI turnover dose response. (+)-Norfenfluramine displayed very good potency comparable with 5-HT and 15-fold better than (–)-norfenfluramine (Fig. 1B; Table 1).
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Intracellular Calcium Mobilization. The human uterine smooth muscle cells were tested for mobilization of intra-cellular calcium in response to 5-HT and selected serotonergic agonists. As expected, 5-HT at 10 µM elicited a rapid increase in relative fluorescence compared with basal fluorescence in HUSMCs indicative of increased intracellular calcium ([Ca2+]i) as monitored in real time using the FLIPR instrument. Surprisingly, the relative fluorescence change measuring calcium mobilization in response to 5-HT and other serotonergic compounds was lower than anticipated based on the robust, consistent phosphoinositide metabolism response in these cells. It was also significantly lower than the 5-HT-induced fluorescence change observed with endogenous 5-HT2 receptors of rat aortic smooth muscle (A7r5) cells (Doyle et al., 1986; May et al., 2003). The HUSMCs exhibited good viability and adequate loading of the calcium-sensitive dye based on a very robust fluorescence increase in response to 10 µM histamine observed in these cells. The consistency and magnitude of the 5-HT-induced [Ca2+]i mobilization were improved by growing the cells in media with 10% dialyzed serum for 2 to 3 days and then overnight in serum-free medium before the experiment. All serotonergics tested induced increases in relative fluorescence in HUSMC in a concentration-dependent manner (Figs. 2 and 3, A and B), and potency and efficacy values were determined from the generated curve fits (Table 2). The rank order potencies of the tested compounds were similar to those observed using PI hydrolysis as the functional readout. 5-HT and -methyl 5-HT were the most potent agonists in the calcium assay. The potencies of BW-723C86 and (+)-norfenfluramine were weaker than observed in the PI experiments but comparable with the activity of R-DOI. MK-212, mCPP, and (–)-norfenfluramine were weak agonists for calcium mobilization as they were for PI hydrolysis (EC50 > 1 µM). mCPP showed partial agonist efficacy in the assay (Emax = 54%).
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Phosphoinositide Hydrolysis Antagonist Studies. To better characterize the 5-HT2 receptor subtype responsible for PI hydrolysis in human uterine smooth muscle cells, we tested subtype-selective antagonists for the ability to inhibit the 5-HT-mediated PI metabolism. The agonist activity of a submaximal concentration of 5-HT (30 nM) was challenged using increasing concentrations of the selective 5-HT2A antagonist, M-100907, the 5-HT2C-selective antagonists, SB-242084 and RS-102221, and the 5-HT2B-selective antagonist, RS-127445. All 5-HT2 receptor antagonists tested inhibited the 5-HT-induced PI hydrolysis in HUSMCs in a concentration-dependent fashion (Fig. 4). However, there were greater than 2 orders of magnitude separation in potency between RS-127445 and the other antagonists tested. The 5-HT2B antagonist was by far the most potent inhibitor of the 5-HT agonist response, exhibiting a subnanomolar IC50 (Fig. 4). The IC50 values derived from the antagonist concentration-response studies were converted into Ki values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973) (Table 3). The 5-HT2C receptor-selective antagonists SB-242084 and RS-102221 were much less effective inhibitors in these cells, producing apparent Ki values approximately 400- and >10,000-fold weaker than the potency of RS-127445, respectively (Table 3). The potent 5-HT2A receptor antagonist, M-100907, was also a very weak inhibitor of the 5-HT response in this system (Ki = 914 nM), which was nearly 10,000-fold weaker than the potency of the 5-HT2B antagonist.
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As mentioned above, the rank order potencies of serotonergic agonists were comparable using either PI hydrolysis or mobilization of intracellular calcium as the functional readout. A correlation plot of pEC50 data for PI turnover (Table 1) against pEC50 data for functional Ca2+ mobilization (Table 2) shows a high level of agreement (r = 0.9, p < 0.005) between these two sets of data (Fig. 5A). The [Ca2+]i mobilization pEC50 data from the current study in HUSMCs also correlated with previous FLIPR functional data using cloned human 5-HT2B receptors expressed in CHO-K1 cells (r = 0.84, p < 0.01) (Table 2) (Porter et al., 1999). The functional PI data (pEC50; pKi), obtained with selected agonists in HUSMCs in the present study, correlated most strongly with the reported functional data (pEC50; pA2) for these same compounds inducing the 5-HT2B-mediated contraction of rat stomach fundus tissues obtained by Baxter et al. (1994) and Baxter (1996) (r = 0.91, p < 0.001) (Table 1; Fig. 5B).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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A panel of prototypic serotonergic agonists with reported selectivity at the various 5-HT2 receptor subtypes was profiled using PI turnover and [Ca2+]i mobilization assays in HUSMC. There is a paucity of available 5-HT2 subtype-selective agonists, and the compounds reported in the literature to exhibit selectivity give conflicting results in diverse assay systems. Selectivity is further complicated by observed species-dependent differences in agonist activity (Wainscott et al., 1996). However, the strong potency of reported 2B-selective agonists and the rank order profile of agonists from our results suggested that a 5-HT2B receptor was present in human uterine smooth muscle cells.
5-HT and -methyl-5-HT were potent, full agonists as measured by PI turnover and [Ca2+]i mobilization in the uterine cells, whereas R-DOI, a prototypic 2A agonist, was less active. Although both 5-HT and -methyl-5-HT are considered nonselective 5-HT2 agonists, previous studies comparing cloned human 5-HT2 receptor subtypes have shown -methyl-5-HT and, to a lesser extent, 5-HT to have a propensity to selectively activate 5-HT2B receptors (Porter et al., 1999; Jerman et al., 2001). R-DOI exhibited less activity than might be expected if the receptor was a 5-HT2A receptor subtype.
The reportedly 5-HT2C-selective piperazines, mCPP and MK-212, exhibited a weak potency in the agonist assays, and mCPP consistently produced partial efficacy. Our findings are in good agreement with previous studies showing mCPP to be a weak partial agonist at the 2B receptor (Baxter et al., 1994; Porter et al., 1999; Rothman et al., 2000; Vickers et al., 2001). Others have observed that mCPP displays no intrinsic agonist activity and acts as an antagonist at the cloned human 5-HT2B receptor (Thomas et al., 1996; Wood et al., 1997). As a weak partial agonist at the 2B receptor, mCPP may behave as a functional antagonist in some assay systems.
The tryptamine analog BW-723C86 has been shown to be a selective agonist for rat (Baxter, 1996; Vickers et al., 2001) and recombinant human (Porter et al., 1999; Jerman et al., 2001) 5-HT2B receptors, although others have found BW-723C86 to be nonselective in receptor binding (Knight et al., 2004) and functional (Cussac et al., 2002) assays. We found that BW-723C86 was a potent agonist for PI hydrolysis and calcium mobilization in HUSMCs. This result was in contrast to the very weak 5-HT2 agonist activity of BW-723C86 that we have observed using the same functional assays in A7r5 rat vascular smooth muscle cells and primary human ocular trabecular meshwork cells (unpublished data). Likewise, (+)- and (–)-norfenfluramine gave functional profiles consistent with the observed 5HT2B-selective potency reported by Porter et al. (1999) at the cloned human 5-HT2B receptor and similar to the activities for the norfenfluramine isomers observed by others in a variety of systems (Fitzgerald et al., 2000; Rothman et al., 2000; Setola et al., 2003). The high-agonist potencies of the BW-723C86 compound and (+)-nor-fenfluramine exhibited here strongly suggested the presence of a 5-HT2B receptor in the HUSMCs.
A prior study using ketanserin as antagonist suggested that 5-HT2A receptors mediate a serotonin-dependent collagenase induction in rat uterine smooth muscle (Rydelek-Fitzgerald et al., 1993), whereas we found evidence of a 2B receptor in human uterine smooth muscle. It would be interesting to test currently available subtype-selective antagonists for the ability to inhibit 5-HT induction of collagenase in rat uterine cells. It is unclear if rat and human uterine smooth muscle express different 5-HT2 receptor subtypes or possess both 2A and 2B with distinct functions. Rydelek-Fitzgerald et al. (1993) do note that 5-HT induction of collagenase was unique to the uterine smooth muscle because no such response was observed in rat aortic smooth muscle. In human uterine tissue, 5-HT2B receptor mRNA was expressed at high levels, and the receptor was cloned from uterine cDNA libraries (Kursar et al., 1994) as well as from the human neuroblastoma cell line SH-SY5Y (Schmuck et al., 1994). The presence of high levels of 5-HT2B receptor mRNA in human uterine tissue supports our contention that the 5-HT2 receptor signal transduction we observe in HUSMCs can be attributed to the 5-HT2B receptor subtype.
5-HT and -methyl-5-HT showed stronger potencies for calcium mobilization than PI hydrolysis, whereas BW-723C86 and the other agonists tested were stronger in PI hydrolysis. The reasons for these discrepancies is unclear, although it is tempting to speculate that the increased potency of the nonselective agonists may involve activity at other 5-HT receptors or a greater role of these agonists for the influx of extracellular calcium. Despite these discrepancies, the rank order potencies of agonists derived from our two functional assays are in close agreement (r = 0.9, p < 0.005; Fig. 5A). Agonist functional data from our studies were also compared with existing data in the literature obtained using rat fundus contraction (Table 1) (Baxter et al., 1994) and cloned human 5-HT2B receptor (Table 2) (Porter et al., 1999). For a majority of compounds, the potency values were increased in the rat tissue contraction assay versus PI hydrolysis (Table 1). This is perhaps not surprising considering the inherent differences in assay conditions, including readout, tissue versus cell culture, and potential species differences. In fact, we observed differences in absolute potency within calcium mobilization between cloned and endogenous human 2B receptors (Table 2), possibly due to differences in receptor density or coupling efficiency. However, our data here are in good agreement with those obtained from cloned human 5-HT2B receptors (r = 0.84, p < 0.01) and from rat stomach fundus contraction studies (r = 0.91, p < 0.001; Fig. 5B), where endogenous 5HT2B receptors are present.
Subtype-selective antagonists were used to more clearly define the 5-HT2 receptor subtype responsible for the observed 5-HT response in HUSMCs. In contrast to the lack of selective 5-HT2 agonists, potent subtype-specific antagonists are available and routinely used to assign pharmacological actions of potential therapeutics to individual receptor subtypes. M-100907, a potent and selective antagonist for 5-HT2A receptors (Kehne et al., 1996), was ineffective as an inhibitor of 5-HT-induced phosphoinositide hydrolysis in HUSMCs (pKi = 6.0). This is 1000-fold weaker than binding affinities reported for M-100907 using cloned human 5-HT2A receptor, rat cortex tissues, or other 5-HT2A systems (Kehne et al., 1996).
Likewise, the 5-HT2C-selective antagonists RS-102221 (Bonhaus et al., 1997) and SB-242084 (Kennett et al., 1997) weakly inhibited the 5-HT response, exhibiting potencies substantially less than would be expected from a 5-HT2C-mediated response. Previous studies have identified RS-102221 as a 5-HT2C-selective compound with nanomolar affinity for human 5-HT2C receptor (pKi = 8.4) as well as nanomolar antagonist potency in a cell-based microphysiometry assay (pA2 = 8.1) (Bonhaus et al., 1997). We found RS-102221 to be a very weak inhibitor of 5-HT action in HUSMCs, with a pKi = 5.6, approximately 300-fold weaker than observed in 5-HT2C systems. A second 5-HT2C-specific antagonist, SB-242084, was a more effective inhibitor (pKi = 7.4), yet this potency was 50- to 100-fold weaker than the affinity and potency reported in 5-HT2C systems and correlated very well with binding affinities reported for this compound at cloned human 5-HT2B receptors (pKi = 7.0) (Kennett et al., 1997).
In contrast, the 5-HT2B receptor antagonist RS-127445 was an extremely potent inhibitor of 5-HT-induced PI turnover in HUSMCs (pKi = 9.9), and these results compared favorably with previous studies where RS-127445 displayed subnanomolar affinity at the 5-HT2B receptor (pKi = 9.5) and potently inhibited both 5-HT-evoked inositol phosphate formation (pKB = 9.5) as well as increases in intracellular calcium (pIC50 = 10.4) in cells expressing human recombinant 5-HT2B receptors (Bonhaus et al., 1999). RS-127445 has demonstrated 1000-fold selectivity for the 5-HT2B subtype over other 5-HT2 subtypes (Bonhaus et al., 1999) and has recently been identified as the most selective 2B receptor antagonist currently available (Knight et al., 2004).
Taken together, the pharmacological profile of the serotonergic agonists and subtype-selective antagonists presented here, as well as the strong correlation with existing functional data for these compounds in a variety of assay systems, provide compelling evidence for the presence of a functional 5-HT2B receptor in these cultured human uterine smooth muscle cells. The human uterine smooth muscle cells provide a convenient cell-based system for studying the biochemical and pharmacological properties of a human endogenous 5-HT2B receptor and allow for profiling the bioactivity of compounds of interest.
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Footnotes |
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ABBREVIATIONS: 5-HT, 5-hydroxytryptamine, serotonin; PI, phosphoinositide; HUSMC, human uterine smooth muscle cell; R-DOI, R-(–)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride; MK-212, 6-chloro-2-(1-piperaxinyl)pyrazine; mCPP, m-chlorophenylpiperazine; BW-723C86, -methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine hydrochloride; RS-102221, 8-[5-(2,4-dimethoxy-5-(4-trifluoromethylsulfo-amido)-phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione hydrochloride; M-100907, R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol]; SB-242084, 6-chloro-5-methyl-1-[6-92-methylpyridin-3-yloxy) pyridine-3-ylcarbamoyl] indoline; RS-127445, 2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine; FLIPR, fluorometric imaging plate reader.
Address correspondence to: Dr. Curtis R. Kelly, Molecular Pharmacology (R2-43), Alcon Research, Ltd., 6201 South Freeway, Fort Worth, TX 76134. E-mail: curtis.kelly{at}alconlabs.com
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, 5-HT; 
, 
, BW-723C86; 
, R-DOI. B, 
, (+)-norfenfluramine; 
, (–)-norfenfluramine; 
, MK-212; 
, mCPP. Responses induced by the test compounds were represented as a percentage of the maximal response generated by 10 µM 5-HT. Data are mean ± S.E.M. from n 
3 independent experiments.
