Stimulating Camp Production in Human Corneal Epithelial Cells

Home , Mesulergine, Metergoline, Ritanserin, Xylamidine

Pharmacological Characterization of a Serotonin Receptor (5-HT7) Stimulating cAMP Production in Human Corneal Epithelial Cells

Julie Y. Crider,1 Gary W. Williams,1 Colene D. Drace,1 Parvaneh Katoli,1 Michelle Senchyna,2 and Najam A. Sharif 1

PURPOSE. To study the mRNA and pharmacology of a serotonin potency data in the literature for the cloned human 5-HT7 (5-HT) receptor positively coupled to adenylyl cyclase in nor- receptor (r ϭ 0.88). mal, primary (P-CEPI), and immortalized human corneal epi- CONCLUSIONS. These collective data support the presence of a thelial cells (CEPI-17-CL4), by using numerous 5-HT agonists pharmacologically deﬁned, adenylyl cyclase-coupled 5-HT7 re- and antagonists. To determine and compare cloned human ceptor in the CEPI-17-CL4 cells that may have relevance to 5-HT receptor binding afﬁnities of compounds with their 7 physiological and/or pathologic functions of 5-HT7 receptors in functional potency data. the human cornea. (Invest Ophthalmol Vis Sci. 2003;44: METHODS. RT-PCR was used to detect the presence of an mRNA 4837–4844) DOI:10.1167/iovs.02-1292

for the human 5-HT7 receptor in CEPI-17-CL4 cells. Receptor- mediated production of cAMP in cultured cells was measured erotonin (5-HT) is a major neurotransmitter in the mamma- using an enzyme immunoassay. Compound binding afﬁnities Slian central and peripheral nervous system.1–3 The recep- were determined using [3H]-lysergic acid diethylamide ([3H]- tors mediating the many diverse functions associated with 5-HT LSD) binding to cell membranes of human embryonic kidney are currently divided into seven subfamilies (5-HT1–7) which

(HEK-293) cells expressing the cloned human 5-HT7 receptor. have all been cloned and pharmacologically characterized to various degrees.1–3 These membrane proteins, with the excep- RESULTS. RT-PCR revealed the presence of a 5-HT receptor 7 tion of the 5-HT receptor, represent a diverse group of G- mRNA in CEPI-17-CL4 cells. Normal P-CEPI cells generated 3 protein-coupled receptors. The 5-HT receptor subtypes (5- cAMP in response to 5-HT (Ϫlog EC ; pEC ϭ 7.6), 5-carbox- 1 50 50 HT , 5-HT , 5-HT , 5-HT , and 5-HT ) are negatively amidotryptamine (5-CT; pEC ϭ 7.8), 5-methoxy-tryptamine 1A 1B 1D 1E 1F 50 coupled to adenylyl cyclase. The 5-HT (5-HT , 5-HT , and (pEC ϭ 7.0) and 5-methoxy-dimethyl-tryptamine (pEC ϭ 2 2A 2B 50 50 5-HT ) receptor subtypes are coupled to phospholipase C and 5.7). In CEPI-17-CL4 cells, serotonergic agonists also stimulated 2C promote phosphoinositide (PI) turnover and release of intra- cAMP production with different potencies (pEC ): 5-CT 4 50 cellular calcium. The 5-HT receptor is a ligand-gated ion (7.4) Ͼ 5-HT (6.5) Ն 5-methoxy-tryptamine (6.1) Ͼ 5-methoxy- 3 Ն Ͻ ϭ ␣ channel, whereas the 5-HT4, 5-HT6, and 5-HT7 receptors are dimethyl-tryptamine (5.4) 8-OH-DPAT ( 5.0) -methyl- positively coupled to adenylyl cyclase through the G-protein 5-HT (Ͻ5.0). Various 5-HT receptor antagonists inhibited cAMP 5 GS. The 5-HT7 receptor can be distinguished from the 5-HT4 production induced by 5-CT in CEPI-17-CL4 cells with different and 5-HT receptors by its high-affinity and selectivity for Ͼ ϭ 6 potencies (pKi): methiothepin (8.5) mesulergine (8.1) 5-carboximidotryptamine (5-CT).6,7 Ͼ Ն ϭ metergoline (8.0) spiperone (7.4) clozapine (7.2) SB- The 5-HT receptor subtype has been cloned from human Ͼ Ͼ 7 258719 (7.2) mianserin (6.9) ketanserin (6.3). Antagonist and several other species8,9 and is the most recent serotonergic pKi values in P-CEPI cells were methiothepin (8.7), spiperone receptor subtype to be identified and characterized. The 5-HT7 (7.4) and SB-258719 (6.6). The rank order of affinity for dis- receptor exhibits low sequence homology with the other se- 3 placement of [ H]-LSD from the cloned human 5-HT7 receptor rotonin receptors that are positively coupled to adenylyl cy- was: methiothepin Ͼ ritanserin Ͼ mesulergine ϭ clozapine Ն clase. In the transmembrane domain, for instance, the 5-HT7 metergoline ϭ 5-HT Ͼ SB-258719 Ն spiperone Ͼ mianserin Ն receptor expresses approximately 39% homology with the ketanserin. The functional agonist and antagonist potency data 3 5-HT6 receptor and 46% with the 5-HT4 receptor. Three hu- obtained from CEPI-17-CL4 cells correlated well with cloned man 5-HT7 splice variants have been identified: the h5-HT7(a) human 5-HT receptor binding affinity data (r ϭ 0.69), with 7 (long form), h5-HT7(b) (short form), and 5-HT7(d), which differ P-CEPI cell functional data (r ϭ 0.85), and with functional only in their carboxyl terminus regions.10 These splice variants are pharmacologically similar in their coupling to adenylyl

cyclase. The 5-HT7(a) and 5-HT7(b) receptor isoforms predomi- 11 1 nate in humans. Studies using the h5-HT7(a) splice variant From the Molecular Pharmacology Unit, Alcon Research, Ltd., 2ϩ 2 produced a stimulation of two Ca /calmodulin-sensitive ad- Fort Worth, Texas; and the School of Optometry, University of Wa- 2ϩ 12 terloo, Waterloo, Ontario, Canada. enylyl cyclase isoforms by increasing intracellular Ca . Hu- Supported by Alcon Research, Ltd. man 5-HT7 receptor splice variants are expressed in numerous Submitted for publication December 17, 2002; revised December peripheral tissues and in the central nervous system, some 3,13 17, 2002; accepted May 5, 2003. differentially. The 5-HT7 receptor is hypothesized to play a Disclosure: J.Y. Crider, Alcon Research, Ltd. (E, F); G.W. Wil- role in several physiological processes including control of liams, Alcon Research, Ltd. (E, F); C.D. Drace, Alcon Research, Ltd. circadian rhythms,14,15 the relaxation of smooth muscle,16 and (E, F); P. Katoli, Alcon Research, Ltd. (E, F); M. Senchyna, None; N.A. the pathophysiology of depression,17 migraine headaches,18 Sharif, Alcon Research, Ltd. (E, F) and schizophrenia.7 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- Despite the wealth of information about the many different ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. roles of 5-HT in the central and peripheral nervous systems Corresponding author: Naj Sharif, Molecular Pharmacology Unit, (see above), the role of 5-HT in the physiology and pathophys- Alcon Research, Ltd. (R2-19), 6201 South Freeway, Fort Worth, TX iology of the eye is less well explored and understood. The 76134-2099; [email protected]. demonstration of serotonergic innervation of the eye19 and the

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presence of 5-HT in the aqueous humor20 has prompted some 3.5) was added for the termination of cAMP synthesis and cell lysis. ␮ ocular serotonergic studies. For instance, 5-HT1A receptors Finally, ice cold 0.1 M sodium acetate (225 L, pH 11.5–12.0) was have been found in the iris-ciliary body (ICB) of the rabbit,21 added to neutralize the samples before analysis by the EIA.27,28 3 and prejunctional 5-HT3 receptors modulating [ H]-norepi- nephrine release have been discovered in bovine and human cAMP Measurements ICBs.22 However, although early studies conducted in rabbit corneal tissues revealed the presence of a 5-HT receptor cou- cAMP production was measured using an EIA kit purchased from pled positively to adenylyl cyclase,23 the pharmacological char- Amersham Pharmacia Biotech (Piscataway, NJ). This assay was con- acterization of this rabbit receptor was not completed because ducted according to the package insert in an automated manner using a robotic workstation (Biomek 2000; Beckman Instruments, Fullerton, of the unavailability of suitable, potent, and selective 5-HT 27,28 receptor agonists and antagonists. We hypothesized that hu- CA). man corneal epithelium may also express such a 5-HT receptor and initiated functional studies on normal human corneal epi- Receptor Binding Studies thelial cells. However, because of the difficulty of obtaining Membranes from human embryonic kidney (HEK-293) cells expressing and propagating these cells, we also explored the use of a the cloned human 5-HT7 receptor (PerkinElmer Life Sciences, Boston, previously immortalized human corneal epithelial cell line MA) were diluted to 16 ␮g protein/mL in 4°C 50 mM Tris buffer (pH (CEPI-17-CL4) that exhibits many of the key genetic, pheno- 7.4). The membranes were resuspended using a tissue disrupter (Poly- typic, morphologic, pharmacological, and physiological fea- tron; Brinkman Instruments, Westbury, NY) equipped with a generator tures of the normal primary corneal epithelial cells and tis- (Ͻ20 seconds; PTA10TS). Drug dilutions were made in 10:10 dimethyl 24,25 sue. In the current communication, we describe the sulfoxide-ethanol (vol/vol), using the robotic workstation (model detailed pharmacological properties of the 5-HT receptor that 2000; Biomek). The diluted compounds (50 ␮L) were then added to a promoted the generation of cAMP in the human CEPI-17-CL4 96-well deep block. A volume of 400 ␮L of receptor preparation was cells, with confirmation of some data in normal human corneal manually added to the 96-well block. The workstation was then used to epithelial cells. In addition, we compared the functional po- add 50 ␮L(2nMfinal concentration) of [3H]-lysergic acid diethylamide tencies of the various serotonergic compounds determined in ([3H]-LSD; Perkin Elmer Life Sciences). Nonspecific binding was de- the CEPI-17-CL4 cells with the receptor binding affinities of fined using 10 ␮M methiothepin. The 96-well blocks were sealed and these compounds at the cloned human 5-HT7 receptor. These incubated in a shaking water bath for 60 minutes at 37°C. The 96-well studies have helped us identify this adenylyl cyclase–coupled blocks were then transferred to a harvester (MachIV; TomTech, Ham- human corneal epithelial 5-HT receptor as the 5-HT7 subtype. den, CT), and the incubations were terminated by rapid vacuum filtration using a glass fiber filter mats (Whatman GF/B; Whatman, MATERIALS AND METHODS Clifton, NJ) previously soaked in 0.3% polyethylenimine. The samples were counted on a beta-counter (Wallac Bigspot; Perkin Elmer) for 3 Tissue Culture minutes at approximately 50% efficiency. Normal, primary human corneal epithelial cells (P-CEPI; passages 2–4; Cascade Biologicals, Portland, OR) isolated from three human donors RT-PCR Analysis of 5-HT7 Receptor mRNA (males; ages 37–55, who had no ocular diseases) were cultured in Oligonucleotide PCR Primers. Sense and anti-sense primers6 medium containing 1% human corneal growth supplement, 100 U/mL for the cloned human 5-HT7 receptor were synthesized at the Central penicillin G, 100 ␮g/mL streptomycin sulfate, and 0.25 ␮g/mL ampho- Facility of the Institute of Molecular Biology and Biotechnology at tericin B (EpiLife; Cascade Biologicals). Culture plates were coated McMaster University (Hamilton, Ontario, Canada). All primers were with 0.1% gelatin to help the cells adhere to the bottom of the plates. designed to span intron–exon boundaries to distinguish between am- These P-CEPI cells were difficult to grow and propagate. plification of mRNA and genomic DNA and were based on published The morphologic, pharmacological, and genetic characterization of cDNA sequences.6 All primers were designed after BLAST retrieval of the simian virus (SV)-40 immortalized human corneal epithelial cells human cDNA sequence of the human 5-HT7 receptor from a GenBank (CEPI-17-CL4 cell) has been previously reported.24,25 These cells (pas- search (http://www.ncbi.nlm.nih.gov/Genbank; provided in the pub- sages 58–158) were cultured in keratinocyte growth medium (KGM) lic domain by the National Center for Biotechnology Information, 29 with 0.15 mM CaCl2. Amphotericin B and gentamicin were replaced by Bethesda, MD), as for other receptors we have worked on in the past. penicillin (100 U/mL) and streptomycin (100 ␮g/mL).24,26 Media and The sense primer sequence was 5Ј-GGA ACA GAT CAA CTA CGG CAG other supplements were purchased from BioWhittaker (Walkersville, AGT-3Ј; the antisense primer sequence was 5Ј-TCT ATT GCT TTA CTG MD). AGC ACT GTC-3Ј. cDNA Synthesis. Total RNA was isolated from CEPI-17-CL4 cells cAMP Production in Cultured Cells using the standard guanidine thiocyanate procedure.29 This RNA was Agonist-dependent cAMP formation was measured by a previously then converted into cDNA in a 10-␮L reverse transcription reaction described method using a sensitive enzyme immunoassay (EIA).27,28 In containing 0.5 ␮g of total RNA; 1ϫ first-strand buffer (75 mM KCl; 50

brief, compounds of interest were diluted in ethanol so that the final mM Tris-HCl, [pH 8.3]; 3.0 mM MgCl2); 1.7 mM MgCl2; 1 mM each ␮ ␮ ethanol concentration was 1%, a concentration well tolerated by the dNTP; 10 mM dithiothreitol [dTT]; 2.5 M oligo (dT)18,and5U/ L cells. Cells were seeded at a concentration of 6000 cells per well. On reverse transcriptase [SuperScript II Reverse Transcriptase; Invitrogen- reaching confluence, the cells were rinsed twice with 0.5 mL Dulbec- Gibco, Gaithersburg, MD]). Reactions were incubated at 42°C for 60 co’s modified Eagle’s medium (DMEM)/F-12. The cells were preincu- minutes, heated at 95°C for 5 minutes and then cooled at 4°C for a bated for 20 minutes in the presence or absence of 5-HT receptor minimum of 5 minutes and a maximum of 30 minutes. antagonists in DMEM/F-12 containing 0.8 mM ascorbate and 1.0 mM of Polymerase Chain Reaction. PCR was performed on 5 ␮Lof the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; cDNA preparation, to which was added 44 ␮L of a PCR master mix Sigma-Aldrich, St. Louis, MO) at 23°C. Serotonin receptor agonists containing 1ϫ PCR buffer (55 mM KCl; 13 mM Tris-HCl [pH 8.3]); 1 ␮ were added at the end of this period, and the reaction was allowed to mM MgCl2; 10% dimethylsulfoxide [DMSO]; 1.25 U/50 L DNA poly- proceed for another 15 minutes at 23°C. When the effects of 5-HT merase (AmpliTaq Gold; Applied Biosciences Foster City, CA) with a antagonists were investigated, the agonist, 5-carboxyamidotryptamine thermocycler (1400 GeneAmp; Applied Biosciences) and 0.2 ␮M each (5-CT; 100 nM), was used to stimulate cAMP production. After aspira- sense and antisense primer in a total volume of 50 ␮L. A hot-start PCR tion of the reaction medium, ice cold 0.1 M acetic acid (150 ␮L, pH method was performed in the thermocycler, using the following pa-

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rameters: an initial denaturing step of 10 minutes at 95°C, denaturing at 94°C for 30 seconds, annealing at the optimal temperature (55°C) for 30 seconds, and extending at 72°C for 1 minute. The final poly- merization step was extended an additional 7 minutes. Forty PCR cycles were performed, and suitable precautions were taken to avoid product contamination. PCR setup, amplification, and product pro- cessing were performed using dedicated equipment in separate rooms. In addition, several control reactions were routinely run in parallel during RT-PCR analysis, including RT reactions run in the absence of the reverse transcriptase enzyme, to confirm the absence of genomic DNA and/or cDNA contamination, and RT reactions without RNA to check for reagent contamination. PCR amplification of 1.5 ng of human genomic DNA served as a negative control. Positive control RT-PCR reactions were performed using purchased total human lung or brain RNA (BD Biosciences-Clontech, Palo Alto, CA). PCR amplification reactions were evaluated through electrophoresis of 12 ␮LofPCR product on a 1.5% agarose gels containing 1 ␮g/mL ethidium bromide and visualized by UV transillumination on an imager (GeneGenius Imager; Synoptics Ltd., Cambridge, UK). Initial product identification was made by comparison to the positive control and the molecular weight ladder. Endonuclease digestion was used to confirm product

identity. Briefly, digestion of the 5-HT7 receptor mRNA amplification product was performed using the BclI restriction endonuclease enzyme in a final reaction volume of 25 ␮L. After digestion, products were resolved by 2.5 hours of electrophoresis at 90 V on a 2.0% FIGURE 1. Agonist-induced cAMP production in CEPI-17-CL4 cells. agarose-TBE gel stained with 1 ␮g/mL ethidium bromide. Gels were The cells were incubated with the agonists (six concentrations) for 15 visualized and photographed GeneGenius and GeneSnap software. minutes, the cells lysed and the cAMP generated determined with an Confirmation of appropriate splice products was made by comparison EIA procedure. Representative functional concentration–response to the molecular weight ladder and to the positive control. All RT-PCR curves for several serotonergic receptor agonists in the CEPI-17-CL4 experiments were performed at least three times. cell adenylyl cyclase assay are shown. Data points represent means of two determinations. Summary data from several such experiments are Compounds shown in Table 1. Serotonin receptor agonists and antagonists were either obtained com- mercially from Sigma-Aldrich/ RBI or Tocris (Ballwin, MO) or were slow growth rate and thus the difficulty of propagating these synthesized by the Medicinal Chemistry Group at Alcon Research, Ltd. cells, subsequent studies were conducted with the previously (Ft. Worth, TX). immortalized human corneal epithelial cell line, CEPI-17- CL4.24–26 Data Analysis To define the pharmacological characteristics of the 5-HT receptor stimulating cAMP in the CEPI-17-CL4 cells, we used a Sample optical density (OD) readings were compared with the stan- broad panel of well-known agonist and antagonists that exhibit dard curve and the cAMP content of each sample was evaluated by some level of selectivity for the different 5-HT receptor sub- linear regression analysis using spreadsheet program (Excel; Microsoft, types. Thus, numerous 5-HT receptor agonists stimulated Redmond, WA). Functional potency (EC ,IC ; and K ) was com- 50 50, i cAMP production in CEPI-17-CL4 cells in a concentration-de- puted with a sigmoidal-fit function of the Origin software package Ϯ 29 pendent manner (Fig. 1). Basal levels of cAMP were 1.0 0.1 (Microcal Software, Inc., Northampton, MA), which was also used to pmol/well (n ϭ 44), and these were increased to 9.1 Ϯ 1.2 perform linear regression analysis. Ligand binding and functional an- pmol/well by maximum stimulation by the different agonists tagonist IC values were calculated using ActivityBase (IDBS, Surrey, 50 used in the CEPI-17-CL4 cells. Thus, the various agonists stim- UK) and converted to equilibrium dissociation constants (Ki) as previ- Ϯ ϭ 24,26,29 ulated cAMP by 10.8 1.1-fold (n 44) above basal levels, ously described. All data were subsequently converted to their comparable to that observed in the P-CEPI cells. Table 1 shows –log values (e.g., pK ; pEC ) to permit comparisons with the literature i 50 a summary of the data for the six agonists tested in the CEPI- information and to allow construction of suitable correlation plots. All 17-CL4 cells. The rank order of potency (pEC50) for these functional and binding data are expressed as the mean Ϯ SEM. ϭ Ͼ ϭ Ͼ compounds was: 5-CT (pEC50 7.4) 5-HT (pEC50 6.5) ϭ Ͼ 5-methoxy-tryptamine (pEC50 6.1) 5-methoxy-dimethyl- ϭ Ն Ͻ RESULTS tryptamine (pEC50 5.4) 8-OH-DPAT (pEC50 5.0; Table 1). Whereas 5-CT, 5-HT, and 5-methoxy-tryptamine were full Initial studies of agonist-stimulated cAMP production were agonists, 5-methoxy-dimethyl-tryptamine, 8-OH-DPAT, and ␣ performed in normal, primary human corneal epithelial (P- -methyl-5-HT were partial agonists (Table 1). Similarly, 5-HT4 ϭ Ϯ ϭ Ϯ CEPI) cells. 5-CT (pEC50 7.8 0.2), 5-HT (pEC50 7.6 receptor partial agonists, RS-67333, and RS-67506 were very ϭ Ϯ 0.2), 5-methoxy-tryptamine (pEC50 7.0 0.01), and 5-me- weak or inactive agonists in these cells (Table 1). ϭ Ϯ thoxy-dimethyl-tryptamine (pEC50 5.7 0.4) concentration- Figure 2 shows a representative plot of antagonism of 5-CT dependently induced cAMP accumulation in these P-CEPI cells. (100 nM)–stimulated cAMP production by various 5-HT recep- Basal levels of cAMP in confluent monolayers of P-CEPI cells tor antagonists in CEPI-17-CL4 cells. Table 2 shows a compar- were 1.6 Ϯ 0.4 pmol/well (n ϭ 10) and were elevated to 9.0 Ϯ ison of functional antagonist data obtained for CEPI-17-CL4 ϭ 1.4 pmol/well (n 10) under maximum stimulation by these cells with those obtained for the recombinant human 5-HT7 Ϯ agonists. Thus, these compounds stimulated cAMP by 7.6 receptor and native 5-HT7 receptor in various cells and tissues 1.1-fold above basal levels (n ϭ 10). However, due to the from the literature. The rank order of potency of the 5-HT difficulty of obtaining a steady large supply of normal human receptor antagonists for the CEPI-17-CL4 cell cAMP assay was ϭ Ͼ ϭ ϭ corneal epithelial cells and the problems associated with their methiothepin (pKi 8.5) mesulergine (pKi 8.1) meter-

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TABLE 1. Agonist-Stimulated Production of cAMP in CEPI-17-CL4 Cells Compared with Data in the Literature

Agonist Potency (pEC50) Literature Agonist Potencies (pEC50) Reported Receptor in CEPI-17-CL4 Cells for 5-HT7 Receptor-Mediated cAMP Agonist Selectivity (% Max. Response) Production or Tissue Relaxation

Ϯ 30 31 5 22 32 33 5-CT 5-HT7 7.4 0.1 (100%) 7.7 ; 7.8 ; 8.0 ; 8.39 ; 7.24 ; 8.7 5-HT Nonselective 6.5 Ϯ 0.1 (100%) 6.830; 6.6831; 7.15; 6.6922; 6.032; 7.533 5-methoxy-tryptamine Nonselective 6.1 Ϯ 0.1 (123 Ϯ 20%) 6.8631; 6.885; 6.522; 5.0332; 8.1233 5-methoxy-dimethyl-tryptamine Nonselective 5.4 Ϯ 0.1 (53 Ϯ 7%) 5.55 Ͻ Ϯ 30 5 Ͻ 22 33 8-OH-DPAT 5-HT1A 5.0 (17 6%) 6.1 ; 4.2 ; 5 ; 5.25 ␣ Ͻ Ϯ 5 22 Ͻ 32 -methyl-5-HT 5-HT2 5.0 (13 4%) 4.0 ; 5.4 ; 5 Ͻ RS-67333 5-HT4 5.0 — Ͻ RS-67506 5-HT4 5.0 —

Various 5-HT receptor agonists (six concentrations) were tested for their ability to stimulate cAMP production in CEPI-17-CL4 cells. Compounds were incubated with the cells for 15 minutes, the reaction stopped, and the amount of cAMP generated determined with the EIA. Data shown for CEPI-17-CL4 cells are the mean Ϯ SEM from 3 to 17 experiments, each performed in duplicate. The literature data from the indicated ϭ references are shown for comparison purposes. Some key 5-HT analogues yielded the following agonist potencies in P-CEPI cells: 5-CT (pEC50 Ϯ ϭ Ϯ ϭ Ϯ ϭ Ϯ 7.8 0.2), 5-HT (pEC50 7.6 0.2), 5-methoxy-tryptamine (pEC50 7.0 0.01), and 5-methoxy-dimethyl-tryptamine (pEC50 5.7 0.4).

ϭ Ͼ ϭ Ն goline (pKi 8.0) spiperone (pKi 7.4) ritanserin (pKi acteristics of the 5-HT receptor stimulating cAMP in the CEPI- ϭ Ն ϭ ϭ ϭ Ͼ 7.3) clozapine (pKi 7.2) SB-258719 (pKi 7.2) 17-CL4 cells with that of the cloned human 5-HT7 receptor, we ϭ Ͼ ϭ Ͼ mianserin (pKi 6.9) MDL-100907 (pKi 6.6) ketanserin also determined the binding afﬁnities of a broad panel of ϭ (pKi 6.3), and so forth (Table 2). Limited antagonist studies serotonergic agents, mainly antagonists, that exhibit some level in the P-CEPI cells yielded the following antagonist potencies: of selectivity for the different 5-HT receptor subtypes at this ϭ ϭ methiothepin (pKi 8.7), spiperone (pKi 7.4), and SB- human 5-HT7 receptor. Figure 3 shows a representative plot of ϭ 3 258719 (pKi 6.6). All these functional agonist–antagonist [ H]-LSD binding data obtained from cloned human 5-HT7 data from P-CEPI and CEPI-17-CL4 cells compared well with the receptor preparations (expressed in HEK-293 cells) in the cur-

agonist and antagonist potency data reported for the 5-HT7 rent studies. The pKi values for 5-HT, 5-methoxy-dimethyl- receptor in the literature (described later). tryptamine and 8-OH-DPAT were 8.5, 7.9, and 8.5, respectively Because of the difﬁculty of obtaining large preparations of (Table 2). Table 2 also shows a summary of the 5-HT antagonist CEPI-17 cell membranes, all binding studies were conducted binding data compared with functional antagonist data. The 3 with the cloned human 5-HT7 receptor expressed in HEK-293 rank order of potency for displacement of [ H]-LSD from the cell membranes. Thus, to compare the pharmacological char- cloned human 5-HT7 receptor in the current studies was me- ϭ Ͼ ϭ Ͼ thiothepin (pKi 11.1) ritanserin (pKi 10.3) mesul- ϭ ϭ ϭ Ն ergine (pKi 8.8) clozapine (pKi 8.8) metergoline (pKi ϭ Ͼ ϭ Ն ϭ Ͼ 8.6) SB-258719 (pKi 8.0) spiperone (pKi 7.9) ϭ Ն ϭ mianserin (pKi 7.2) ketanserin (pKi 7.1; Table 2). A correlation plot of pEC50;pKi data for the CEPI-17-CL4 cell functional data and pKB or pKi data from the average cAMP data from 5-HT7(a), 5-HT7(b), and 5-HT7(d) receptors expressed in HEK-293 cells10 indicated a high level of correlation (r ϭ 0.88, slope ϭ 1.05, P Ͻ 0.0001) of these two sets of data and thus the pharmacological characteristics of the receptors involved in these systems (Figs. 4A, 4B). The functional cAMP data for numerous agonists and antagonists obtained from CEPI-17-CL4 cells were also well correlated with the [3H]-LSD

binding afﬁnity data obtained from the cloned human 5-HT7 receptor (expressed in HEK-293 cells) from our studies coupled with those reported in the literature (r ϭ 0.69; P Ͻ 0.001; Fig. 4C). Likewise, the CEPI-17-CL4 functional data correlated

well with the 5-HT7 receptor-mediated response data in the literature involving cAMP production through cloned human

5-HT7 receptors and rat astrocyte 5-HT7 receptors, and canine cerebral artery relaxation data, coupled with [3H]-5-HT release from bovine ICB data (r ϭ 0.71, P Ͻ 0.0001; Fig. 4B). Finally, as expected, the CEPI-17-CL4 functional data correlated highly with agonist and antagonist data obtained from P-CEPI cells (r ϭ 0.85, slope ϭ 0.81, P Ͻ 0.02; Fig. 4D). Molecular biological studies using RT-PCR techniques re- FIGURE 2. Inhibition of agonist-induced cAMP production in CEPI-17- vealed the presence of the mRNA for the human 5-HT7 recep- CL4 cells. Various serotonergic antagonists were preincubated with tor in CEPI-17-CL4 cells (Fig. 5), thus conﬁrming and supple- the cells for 20 minutes before the addition of 5-CT (100 nM). The menting the pharmacological data presented herein for these assays were continued for another 15 minutes, the cells lysed, and the cAMP generated determined with an EIA procedure. Representative cells. In addition, the mRNA for the human 5-HT7 receptor was functional inhibition curves for a range of serotonergic receptor antag- also detected in biopsy specimens of human conjunctival and onists in the CEPI-17-CL4 cells are shown. Data points represent means corneal tissues (Senchyna M, et al., unpublished observations, of two determinations. Summary data from several such experiments 2000), using RT-PCR assays. Limited RT-PCR studies to study are shown in Table 2. mRNAs for other 5-HT receptors revealed the absence of

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3 TABLE 2. Antagonism of 5-CT–Mediated cAMP Production in CEPI-17-CL4 Cells and Competition for [ H]-LSD Binding to Cloned Human 5-HT7 Receptors: Comparison with 5-HT7 Receptor Functional Data in the Literature

Inhibition of cAMP Inhibition of cAMP Production 3 Production in or Tissue Relaxation Via 5-HT7 [ H]-LSD Binding to Cloned Reported 5-HT CEPI-17-CL4 Cells Receptors (pKi;pKb) from the Human 5-HT7 Receptor Antagonist Receptor Selectivity (pKi) Literature (pKi)

Methiothepin Nonselective 8.5 Ϯ 0.1 8.130; 9.2910; 8.4533; 7.9831 11.1 Ϯ 0.4 Ϯ 30 10 31 32 Ϯ Mesulergine 5-HT2C 8.1 0.2 7.5 ; 8.18 ; 7.58 ; 7.3 8.8 0.4 Ϯ 10 33 Ϯ Metergoline 5-HT1, 5-HT2 8.0 0.3 8.60 ; 7.1 8.6 0.1 Ϯ 10 33 32 32 Ϯ Spiperone 5-HT1A, 5-HT2A 7.4 0.1 7.95 ; 6.03 ; 6.6 ; 6.9 7.9 0.2 Ϯ 10 31 Ϯ Ritanserin 5-HT1, 5-HT2 7.3 0.1 7.74 ; 7.2 10.3 0.2 Ϯ 30 33 31 32 Ϯ Clozapine 5-HT7, 5-HT6 7.2 0.03 7.2 ; 6.65 ; 7.03 ; 7.1 8.8 0.1 Ϯ 30 10 Ϯ SB-258719 5-HT7 7.2 0.3 7.0 ; 7.72 8.0 0.1 Mianserin Nonselective 6.9 Ϯ 0.1 6.0533; 6.431 7.2 Ϯ 0.0 Ϯ MDL-100907 5-HT2A 6.6 0.2 —— Ϯ 30 10 33 Ϯ Ketanserin 5-HT2 6.3 0.1 6.3 ; 6.95 ; 5.3 7.1 0.1 Ϯ BW501C67 5-HT2A 6.1 0.2 —— Ϯ WB-4101 5-HT1A 5.5 0.2 —— Ϯ 31 Way 100635 5-HT1A 5.5 0.2 5 — Ϯ Xylamidine 5-HT2A 5.4 0.03 —— Ϯ RS-127445 5-HT2B 5.4 0.2 —— ␣ Ϯ BMY-7378 5-HT1A; 1d 5.3 0.1 —— Ͻ RS-102221 5-HT2C 5.00 —— Ͻ SB-203186 5-HT4 5.00 —— Ͻ SB-242084 5-HT2C 5.00 —— Ͻ Y-25130 5-HT3 5.00 —— Ͻ RS-23597-190 5-HT4 5.00 —— Ͻ RS-39604 5-HT4 5.00 —— Ͻ Ro-04-6790 5-HT6 5.00 ——

CEPI-17-CL4 cells were incubated with the antagonist under study at various concentrations for 20 minutes before addition of 100 nM 5-CT to stimulate cAMP production. The assays were continued for another 15 minutes, the cells lysed, and the cAMP produced measured with the EIA. The functional assay data are mean Ϯ SEM from three to eight experiments performed in duplicate. Limited antagonist studies in the P-CEPI cells ϭ ϭ ϭ yielded the following antagonist potencies: methiothepin (pKi 8.7), spiperone (pKi 7.4) and SB-258719 (pKi 6.6). Competition binding 3 assays were performed using [ H]-LSD and HEK-293 cell membranes expressing the recombinant human 5-HT7 receptor. The pKi values for the Ϯ Ϯ Ϯ agonists 5-HT, 5-methoxy-dimethyl-tryptamine and 8-OH-DPAT were 8.5 0.1, 7.9 0.2, and 8.5 0.3, respectively. The pKi values for other 9 ϭ ϭ ϭ 3 Ϯ agonists from the literature were 5-CT, pKi 9.5; 5-methoxy-trptamine, pKi 8.8; and 8-OH-DPAT, pKi 7.5. [ H]-LSD binding data are mean SEM from 3 to 16 experiments.

5-HT1A, 5-HT2A, 5-HT2B, and 5-HT2C mRNAs in CEPI-17-CL4 cells (data not shown).

DISCUSSION There is a relative paucity of information on the effects of serotonergic agonists on ocular cells such as the corneal epithelium. The initial work of Neufeld et al.23 in the rabbit cornea suggested that this tissue contained a serotonergic receptor but the investigators were unable to identify the subtype of this receptor pharmacologically because of the unavailability of potent and 5-HT receptor-selective compounds. 5-HT was shown to increase cAMP levels in incubated rabbit corneas, which subsequently resulted in ClϪ secretion.36 A physiological or pathologic role for 5-HT on the ocular surface was further suggested by the detection of 5-HT in human tears.37 We hypothesized that of the family of 5-HT receptors, the receptor type mediating the aforementioned effects in the

cornea involved either the 5-HT4, 5-HT6, or 5-HT7 receptor because these are the only 5-HT receptors known to stimulate cAMP production by activating adenylyl cyclase.1–4 Accord- ingly, the current studies have provided functional pharmaco-

logical evidence for the presence of a 5-HT7 receptor that is positively coupled to adenylyl cyclase in the CEPI-17-CL4 cells

and also in normal primary human corneal epithelial cells. We 3 ϭ FIGURE 3. [ H]-LSD binding to cloned human 5-HT7 receptors ex- observed a high potency for 5-CT (pEC50 7.4–7.8) in P-CEPI pressed in HEK-293 cell membranes. Competition assays were con- and CEPI-17-CL4 cells, a serotonergic-agonist known to exhibit ducted with a range of serotonergic agents (eight concentrations) 3 a relatively high potency and selectivity for the 5-HT7 recep- using [ H]-LSD (2 nM ﬁnal). Data points represent means of two 1–11 tor. The rank order of potency in the current CEPI-17-CL4 determinations in duplicate. Summary afﬁnity data from several such cells studies of 5-CT Ͼ 5-HT Ͼ 8-OH-DPAT for the stimulation experiments are shown in Table 2.

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FIGURE 4. (A) Correlation of serotonergic agonist and antagonist potency data from CEPI-17-CL4 cells and functional literature data for the cloned

human 5-HT7 receptors. The correlation plot depicts functional potency data (pEC50;pKi) (cAMP production) from the CEPI-17-CL4 cells and similar pEC50;pKB;pKi data from the adenylyl cyclase (cAMP production) assays conducted on the cloned human 5-HT7(a), 5-HT7(b), and 5-HT7(d) receptors expressed in HEK-293 cells.5,10,30,33 (B) Correlation of serotonergic agonist and antagonist potency data from CEPI-17-CL4 cells and

functional data in the literature for the cloned human 5-HT7 receptor and the 5-HT7 receptor in various cells and tissues of different species. The correlation plot depicts functional potency data (pEC50;pKi) from the CEPI-17-CL4 cells and similar pEC50;pKB;pKi data from the adenylyl cyclase 5,10,30,33 assays conducted on the cloned human 5-HT7(a), 5-HT7(b), and 5-HT7(c) receptors expressed in HEK-293 cells, and functional assays 31 22 32 performed on native 5-HT7 receptor in brain astrocytes, bovine ICB, and canine cerebral arteries. (C) Correlation of the functional agonist and antagonist data from CEPI-17-CL4 cells and that of binding data from the current studies ([3H]-LSD) and from the literature ([3H]-LSD and [3H]-5-HT) 6,9 3 using the cloned human 5-HT7 receptor. Assays to measure cAMP accumulation in CEPI-17-CL4 cells and [ H]-LSD binding to the cloned human 5-HT7 receptor were conducted. The correlation details were changed to the following when the two outlying data points (for ritanserin and methiothepin) were omitted from the linear regression analysis: r ϭ 0.82, slope ϭ 0.96, P Ͻ 0.001. (D) Correlation of the functional agonist–antagonist data from P-CEPI and CEPI-17-CL4 cells from the current studies. Assays to measure cAMP accumulation in CEPI-17-CL4 cells and P-CEPI cells were conducted. r ϭ 0.85, slope ϭ 0.81, P Ͻ 0.02.

of adenylyl cyclase was highly corroborated by similar cAMP 5-HT Ͼ 5-methoxy-tryptamine Ͼ 5-methoxy-dimethyl-trypta- 30 Ͼ Ͼ ␣ data from both the human 5-HT7(a) and 5-HT7(b) splice vari- mine 8-OH-DPAT -methyl-5-HT) for stimulation of cAMP 33 ants expressed in HEK-293 cells (Table 1). Similarly, Adham production by the recombinant human 5-HT7(a) receptor ex- et al.5 reported an identical rank order of potency (5-CT Ͼ pressed in murine ﬁbroblasts. The proﬁle of receptor antago-

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conjunctival/corneal biopsy tissues (Senchyna M, et al., unpublished observations, 2000). Results of the current [3H]-LSD–binding studies performed

using the cloned human 5-HT7 receptor were similar to the binding proﬁles reported in the literature. Displacement of 3 [ H]-HT for the three human 5-HT7(a), 5-HT7(b) and 5-HT7(d) splice variants produced pKi values of between 8.8 and 8.9 for serotonin and 7.4 and 7.6 for 8-OH-DPAT,10 compared with

pKi values of 8.3 and 7.0 for the same compounds, respectively, in our studies. Krobert et al.10 observed a rank order of afﬁnity for 5-HT receptor antagonists of methiothepin Ͼ mesulergine Ͼ clozapine Ͼ spiperone Ͼ ritanserin Ͼ ketanserin

for all three human 5-HT7(a), 5-HT7(b), and 5-HT7(d) splice variants. These results compared favorably with our current studies, with the exception of a higher potency in our studies for ritanserin. However, this disparity may be attributed to differences in assay conditions between the two studies. FIGURE 5. Visualization of the mRNA for human 5-HT7 receptor in The expression of 5-HT7 receptors in human corneal epi- CEPI-17-CL4 cells and other cells and tissues. Total RNA isolated from thelial cells suggests a role for this receptor is maintaining the CEPI-17-CL4 cells was reverse transcribed into cDNA. Similar cDNAs were generated for other control tissues (e.g., rat brain, hamster normal physiology of the ocular surface. Serotonin has been uterus). PCR was performed on the cDNAs, using the sense and implicated in the regulation of aqueous humor dynamics in the eye19–22 and prejunctional 5-HT receptors have been hypoth- antisense primers for the human 5-HT7 receptor and the PCR products 7 size fractionated on agarose gels, visualized under UV light, and then esized to play a role in intraocular pressure regulation.22 Sero- 37 photographed. The PCR product size for the human 5-HT7 receptor tonin, which is present in human tears, increases cAMP levels Ϫ 36 was 772 bp. Sense and antisense primers for the human 5-HT7 receptor in corneas, which results in increased Cl transport. All these were constructed as previously described6,35 (see the Methods section findings suggest that the presence of functional 5-HT7 recep- for sequences) and the PCR procedure conducted as for other receptor 29,35 tors in corneal epithelial cells may play a role in fluid/mucin systems. Lanes 1, 2: nonpigmented ciliary epithelial cells; lanes 3, secretion on the ocular surface. However, further work is 4: CEPI-17-CL4 cells; lanes 5, 6: hamster uterus smooth muscle; lane 7: molecular weight ladder; lane 8: no RNA added (negative control 1); necessary to expand on these findings. lane 9: no reverse transcriptase added (negative control 2); lane 10: Taken together, our pharmacological and molecular biolog- human brain; lane 11, 12: G3PDH house-keeping enzyme. Long ar- ical data have demonstrated that P-CEPI and CEPI-17-CL4 cells

rows: position of the 5-HT7 mRNA; short arrows: position of the express a functional 5-HT7 receptor positively coupled to ad- house-keeping enzyme (G3PDH). enylyl cyclase. Furthermore, these observations suggest that CEPI-17-CL4 cells represent a suitable and predictive in vitro cellular system for further study of the biochemical and phar-

nist potency data for the inhibition of 5-CT–stimulated cAMP macological aspects of the human ocular 5-HT7 receptor. production also supported the presence of functional 5-HT7 receptors in the CEPI-17-CL4 cells. We observed a rank order of Acknowledgments antagonist potency of methiothepin Ͼ spiperone ϭ metergoline ϭ clozapine ϭ SB-258719 Ͼ ketanserin in the CEPI-7-CL4 The authors thank Jesse May for a critical review of the manuscript and cells, and similar findings were noted for the inhibition of Tom Dean for his generous support which enabled us to conduct these 5-HT–stimulated cAMP production in murine fibroblasts ex- studies. 5 pressing the recombinant human 5-HT(7a) receptor isoform. In addition, our agonist and antagonist potency data from the References CEPI-17-CL4 cells compared well with data obtained for the 1. Hoyer D, Clarke DE, Fozard JR, et al. VII. International union of 5-HT7 receptor mediating relaxation of the canine cerebral arteries (Tables 1, 2). pharmacology classification of receptors for 5-hydroxytryptamine Further evidence supporting the notion that a functionally (serotonin). Pharm Rev. 1994;46:157–203. active 5-HT receptor mediates production of cAMP in the 2. Barnes NM, Sharp T. A review of central 5-HT receptors and their 7 function. Neuropharmacology. 1999;38:1083–1152. CEPI-17-CL4 cells included the following: The 5-HT4 receptor- 2 3. Vanhoenacker P, Haegeman G, Leysen JE. 5-HT7 receptors: Cur- selective antagonists, RS-23597, RS-39604, and SB-203186, rent knowledge and future prospects. Trends in Pharmacol Sci. were weak or inactive at inhibiting 5-CT–stimulated cAMP 2000;21:70–77. Ͻ production in CEPI-17-CL4 cells (pKi 5), thus ruling out the 4. Gerhardt CC, van Heerikuizen, H. Functional characteristic of presence of 5-HT4 receptors in these cells; likewise, the rela- heterologously expressed 5-HT receptors. Eur J Pharmacol. 1997; tively weak antagonist potency of the 5-HT6 receptor antago- 334:1–23. nist, Ro-04-6790,22,38 in the CEPI-17-CL4 cells ruled out the 5. Adham N, Zgoombick JM, Bard J, Branchek TA. Functional char-

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