November 2003
Volume 44, Issue 11
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Physiology and Pharmacology  |   November 2003
Pharmacological Characterization of a Serotonin Receptor (5-HT7) Stimulating cAMP Production in Human Corneal Epithelial Cells
Author Affiliations
  • Julie Y. Crider
    From the Molecular Pharmacology Unit, Alcon Research, Ltd., Fort Worth, Texas; and the
  • Gary W. Williams
    From the Molecular Pharmacology Unit, Alcon Research, Ltd., Fort Worth, Texas; and the
  • Colene D. Drace
    From the Molecular Pharmacology Unit, Alcon Research, Ltd., Fort Worth, Texas; and the
  • Parvaneh Katoli
    From the Molecular Pharmacology Unit, Alcon Research, Ltd., Fort Worth, Texas; and the
  • Michelle Senchyna
    School of Optometry, University of Waterloo, Waterloo, Ontario, Canada.
  • Najam A. Sharif
    From the Molecular Pharmacology Unit, Alcon Research, Ltd., Fort Worth, Texas; and the
Investigative Ophthalmology & Visual Science November 2003, Vol.44, 4837-4844. doi:10.1167/iovs.02-1292
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      Julie Y. Crider, Gary W. Williams, Colene D. Drace, Parvaneh Katoli, Michelle Senchyna, Najam A. Sharif; Pharmacological Characterization of a Serotonin Receptor (5-HT7) Stimulating cAMP Production in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(11):4837-4844. doi: 10.1167/iovs.02-1292.

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      © 2015 Association for Research in Vision and Ophthalmology.

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purpose. To study the mRNA and pharmacology of a serotonin (5-HT) receptor positively coupled to adenylyl cyclase in normal, primary (P-CEPI), and immortalized human corneal epithelial cells (CEPI-17-CL4), by using numerous 5-HT agonists and antagonists. To determine and compare cloned human 5-HT7 receptor binding affinities of compounds with their functional potency data.

methods. RT-PCR was used to detect the presence of an mRNA for the human 5-HT7 receptor in CEPI-17-CL4 cells. Receptor-mediated production of cAMP in cultured cells was measured using an enzyme immunoassay. Compound binding affinities were determined using [3H]-lysergic acid diethylamide ([3H]-LSD) binding to cell membranes of human embryonic kidney (HEK-293) cells expressing the cloned human 5-HT7 receptor.

results. RT-PCR revealed the presence of a 5-HT7 receptor mRNA in CEPI-17-CL4 cells. Normal P-CEPI cells generated cAMP in response to 5-HT (−log EC50; pEC50 = 7.6), 5-carboxamidotryptamine (5-CT; pEC50 = 7.8), 5-methoxy-tryptamine (pEC50 = 7.0) and 5-methoxy-dimethyl-tryptamine (pEC50 = 5.7). In CEPI-17-CL4 cells, serotonergic agonists also stimulated cAMP production with different potencies (pEC50): 5-CT (7.4) > 5-HT (6.5) ≥ 5-methoxy-tryptamine (6.1) > 5-methoxy-dimethyl-tryptamine (5.4) ≥ 8-OH-DPAT (<5.0) = α-methyl-5-HT (<5.0). Various 5-HT receptor antagonists inhibited cAMP production induced by 5-CT in CEPI-17-CL4 cells with different potencies (pKi): methiothepin (8.5) > mesulergine (8.1) = metergoline (8.0) > spiperone (7.4) ≥ clozapine (7.2) = SB-258719 (7.2) > mianserin (6.9) > ketanserin (6.3). Antagonist pKi values in P-CEPI cells were methiothepin (8.7), spiperone (7.4) and SB-258719 (6.6). The rank order of affinity for displacement of [3H]-LSD from the cloned human 5-HT7 receptor was: methiothepin > ritanserin > mesulergine = clozapine ≥ metergoline = 5-HT > SB-258719 ≥ spiperone > mianserin ≥ ketanserin. The functional agonist and antagonist potency data obtained from CEPI-17-CL4 cells correlated well with cloned human 5-HT7 receptor binding affinity data (r = 0.69), with P-CEPI cell functional data (r = 0.85), and with functional potency data in the literature for the cloned human 5-HT7 receptor (r = 0.88).

conclusions. These collective data support the presence of a pharmacologically defined, adenylyl cyclase-coupled 5-HT7 receptor in the CEPI-17-CL4 cells that may have relevance to physiological and/or pathologic functions of 5-HT7 receptors in the human cornea.

Serotonin (5-HT) is a major neurotransmitter in the mammalian central and peripheral nervous system. 1 2 3 The receptors mediating the many diverse functions associated with 5-HT are currently divided into seven subfamilies (5-HT1–7) which have all been cloned and pharmacologically characterized to various degrees. 1 2 3 These membrane proteins, with the exception of the 5-HT3 receptor, represent a diverse group of G-protein-coupled receptors. The 5-HT1 receptor subtypes (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F) are negatively coupled to adenylyl cyclase. The 5-HT2 (5-HT2A, 5-HT2B, and 5-HT2C) receptor subtypes are coupled to phospholipase C and promote phosphoinositide (PI) turnover and release of intracellular calcium. 4 The 5-HT3 receptor is a ligand-gated ion channel, whereas the 5-HT4, 5-HT6, and 5-HT7 receptors are positively coupled to adenylyl cyclase through the G-protein GS. 5 The 5-HT7 receptor can be distinguished from the 5-HT4 and 5-HT6 receptors by its high-affinity and selectivity for 5-carboximidotryptamine (5-CT). 6 7  
The 5-HT7 receptor subtype has been cloned from human and several other species 8 9 and is the most recent serotonergic receptor subtype to be identified and characterized. The 5-HT7 receptor exhibits low sequence homology with the other serotonin receptors that are positively coupled to adenylyl cyclase. In the transmembrane domain, for instance, the 5-HT7 receptor expresses approximately 39% homology with the 5-HT6 receptor and 46% with the 5-HT4 receptor. 3 Three human 5-HT7 splice variants have been identified: the h5-HT7(a) (long form), h5-HT7(b) (short form), and 5-HT7(d), which differ 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 predominate in humans. 11 Studies using the h5-HT7(a) splice variant produced a stimulation of two Ca2+/calmodulin-sensitive adenylyl cyclase isoforms by increasing intracellular Ca2+. 12 Human 5-HT7 receptor splice variants are expressed in numerous peripheral tissues and in the central nervous system, some differentially. 3 13 The 5-HT7 receptor is hypothesized to play a role in several physiological processes including control of circadian rhythms, 14 15 the relaxation of smooth muscle, 16 and the pathophysiology of depression, 17 migraine headaches, 18 and schizophrenia. 7  
Despite the wealth of information about the many different roles of 5-HT in the central and peripheral nervous systems (see above), the role of 5-HT in the physiology and pathophysiology of the eye is less well explored and understood. The demonstration of serotonergic innervation of the eye 19 and the presence of 5-HT in the aqueous humor 20 has prompted some ocular serotonergic studies. For instance, 5-HT1A receptors have been found in the iris-ciliary body (ICB) of the rabbit, 21 and prejunctional 5-HT3 receptors modulating [3H]-norepinephrine release have been discovered in bovine and human ICBs. 22 However, although early studies conducted in rabbit corneal tissues revealed the presence of a 5-HT receptor coupled positively to adenylyl cyclase, 23 the pharmacological characterization of this rabbit receptor was not completed because of the unavailability of suitable, potent, and selective 5-HT receptor agonists and antagonists. We hypothesized that human corneal epithelium may also express such a 5-HT receptor and initiated functional studies on normal human corneal epithelial cells. However, because of the difficulty of obtaining and propagating these cells, we also explored the use of a previously immortalized human corneal epithelial cell line (CEPI-17-CL4) that exhibits many of the key genetic, phenotypic, morphologic, pharmacological, and physiological features of the normal primary corneal epithelial cells and tissue. 24 25 In the current communication, we describe the detailed pharmacological properties of the 5-HT receptor that promoted the generation of cAMP in the human CEPI-17-CL4 cells, with confirmation of some data in normal human corneal epithelial cells. In addition, we compared the functional potencies of the various serotonergic compounds determined in the CEPI-17-CL4 cells with the receptor binding affinities of these compounds at the cloned human 5-HT7 receptor. These studies have helped us identify this adenylyl cyclase–coupled human corneal epithelial 5-HT receptor as the 5-HT7 subtype. 
Materials and Methods
Tissue Culture
Normal, primary human corneal epithelial cells (P-CEPI; passages 2–4; Cascade Biologicals, Portland, OR) isolated from three human donors (males; ages 37–55, who had no ocular diseases) were cultured in medium containing 1% human corneal growth supplement, 100 U/mL penicillin G, 100 μg/mL streptomycin sulfate, and 0.25 μg/mL amphotericin B (EpiLife; Cascade Biologicals). Culture plates were coated with 0.1% gelatin to help the cells adhere to the bottom of the plates. These P-CEPI cells were difficult to grow and propagate. 
The morphologic, pharmacological, and genetic characterization of the simian virus (SV)-40 immortalized human corneal epithelial cells (CEPI-17-CL4 cell) has been previously reported. 24 25 These cells (passages 58–158) were cultured in keratinocyte growth medium (KGM) with 0.15 mM CaCl2. Amphotericin B and gentamicin were replaced by penicillin (100 U/mL) and streptomycin (100 μg/mL). 24 26 Media and other supplements were purchased from BioWhittaker (Walkersville, MD). 
cAMP Production in Cultured Cells
Agonist-dependent cAMP formation was measured by a previously described method using a sensitive enzyme immunoassay (EIA). 27 28 In brief, compounds of interest were diluted in ethanol so that the final ethanol concentration was 1%, a concentration well tolerated by the cells. Cells were seeded at a concentration of 6000 cells per well. On reaching confluence, the cells were rinsed twice with 0.5 mL Dulbecco’s modified Eagle’s medium (DMEM)/F-12. The cells were preincubated for 20 minutes in the presence or absence of 5-HT receptor antagonists in DMEM/F-12 containing 0.8 mM ascorbate and 1.0 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich, St. Louis, MO) at 23°C. Serotonin receptor agonists were added at the end of this period, and the reaction was allowed to proceed for another 15 minutes at 23°C. When the effects of 5-HT antagonists were investigated, the agonist, 5-carboxyamidotryptamine (5-CT; 100 nM), was used to stimulate cAMP production. After aspiration of the reaction medium, ice cold 0.1 M acetic acid (150 μL, pH 3.5) was added for the termination of cAMP synthesis and cell lysis. Finally, ice cold 0.1 M sodium acetate (225 μL, pH 11.5–12.0) was added to neutralize the samples before analysis by the EIA. 27 28  
cAMP Measurements
cAMP production was measured using an EIA kit purchased from Amersham Pharmacia Biotech (Piscataway, NJ). This assay was conducted according to the package insert in an automated manner using a robotic workstation (Biomek 2000; Beckman Instruments, Fullerton, CA). 27 28  
Receptor Binding Studies
Membranes from human embryonic kidney (HEK-293) cells expressing the cloned human 5-HT7 receptor (PerkinElmer Life Sciences, Boston, MA) were diluted to 16 μg protein/mL in 4°C 50 mM Tris buffer (pH 7.4). The membranes were resuspended using a tissue disrupter (Polytron; Brinkman Instruments, Westbury, NY) equipped with a generator (<20 seconds; PTA10TS). Drug dilutions were made in 10:10 dimethyl sulfoxide-ethanol (vol/vol), using the robotic workstation (model 2000; Biomek). The diluted compounds (50 μL) were then added to a 96-well deep block. A volume of 400 μL of receptor preparation was manually added to the 96-well block. The workstation was then used to add 50 μL (2 nM final concentration) of [3H]-lysergic acid diethylamide ([3H]-LSD; Perkin Elmer Life Sciences). Nonspecific binding was defined using 10 μM methiothepin. The 96-well blocks were sealed and incubated in a shaking water bath for 60 minutes at 37°C. The 96-well blocks were then transferred to a harvester (MachIV; TomTech, Hamden, CT), and the incubations were terminated by rapid vacuum filtration using a glass fiber filter mats (Whatman GF/B; Whatman, Clifton, NJ) previously soaked in 0.3% polyethylenimine. The samples were counted on a beta-counter (Wallac Bigspot; Perkin Elmer) for 3 minutes at approximately 50% efficiency. 
RT-PCR Analysis of 5-HT7 Receptor mRNA
Oligonucleotide PCR Primers.
Sense and anti-sense primers 6 for the cloned human 5-HT7 receptor were synthesized at the Central Facility of the Institute of Molecular Biology and Biotechnology at McMaster University (Hamilton, Ontario, Canada). All primers were designed to span intron–exon boundaries to distinguish between amplification of mRNA and genomic DNA and were based on published cDNA sequences. 6 All primers were designed after BLAST retrieval of human cDNA sequence of the human 5-HT7 receptor from a GenBank search (http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), as for other receptors we have worked on in the past. 29 The sense primer sequence was 5′-GGA ACA GAT CAA CTA CGG CAG AGT-3′; the antisense primer sequence was 5′-TCT ATT GCT TTA CTG AGC ACT GTC-3′. 
cDNA Synthesis.
Total RNA was isolated from CEPI-17-CL4 cells using the standard guanidine thiocyanate procedure. 29 This RNA was then converted into cDNA in a 10-μL reverse transcription reaction containing 0.5 μg of total RNA; 1× first-strand buffer (75 mM KCl; 50 mM Tris-HCl, [pH 8.3]; 3.0 mM MgCl2); 1.7 mM MgCl2; 1 mM each dNTP; 10 mM dithiothreitol [dTT]; 2.5 μM oligo (dT)18, and 5 U/μL reverse transcriptase [SuperScript II Reverse Transcriptase; Invitrogen-Gibco, Gaithersburg, MD]). Reactions were incubated at 42°C for 60 minutes, heated at 95°C for 5 minutes and then cooled at 4°C for a minimum of 5 minutes and a maximum of 30 minutes. 
Polymerase Chain Reaction.
PCR was performed on 5 μL of cDNA preparation, to which was added 44 μL of a PCR master mix containing 1× PCR buffer (55 mM KCl; 13 mM Tris-HCl [pH 8.3]); 1 mM MgCl2; 10% dimethylsulfoxide [DMSO]; 1.25 U/50 μL DNA polymerase (AmpliTaq Gold; Applied Biosciences Foster City, CA) with a thermocycler (1400 GeneAmp; Applied Biosciences) and 0.2 μM each sense and antisense primer in a total volume of 50 μL. A hot-start PCR method was performed in the thermocycler, using the following parameters: 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 polymerization 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 processing 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 μL of PCR product on a 1.5% agarose gels containing 1 μg/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 μL. After digestion, products were resolved by 2.5 hours of electrophoresis at 90 V on a 2.0% agarose-TBE gel stained with 1 μg/mL ethidium bromide. Gels were visualized and photographed GeneGenius and GeneSnap software. Confirmation of appropriate splice products was made by comparison to the molecular weight ladder and to the positive control. All RT-PCR experiments were performed at least three times. 
Compounds
Serotonin receptor agonists and antagonists were either obtained commercially from Sigma-Aldrich/ RBI or Tocris (Ballwin, MO) or were synthesized by the Medicinal Chemistry Group at Alcon Research, Ltd. (Ft. Worth, TX). 
Data Analysis
Sample optical density (OD) readings were compared with the standard curve and the cAMP content of each sample was evaluated by linear regression analysis using spreadsheet program (Excel; Microsoft, Redmond, WA). Functional potency (EC50, IC50,; and K i) was computed with a sigmoidal-fit function of the Origin software package (Microcal Software, Inc., Northampton, MA), 29 which was also used to perform linear regression analysis. Ligand binding and functional antagonist IC50 values were calculated using ActivityBase (IDBS, Surrey, UK) and converted to equilibrium dissociation constants (K i) as previously described. 24 26 29 All data were subsequently converted to their –log values (e.g., pKi; pEC50) to permit comparisons with the literature information and to allow construction of suitable correlation plots. All functional and binding data are expressed as the mean ± SEM. 
Results
Initial studies of agonist-stimulated cAMP production were performed in normal, primary human corneal epithelial (P-CEPI) cells. 5-CT (pEC50 = 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) concentration-dependently induced cAMP accumulation in these P-CEPI cells. Basal levels of cAMP in confluent monolayers of P-CEPI cells were 1.6 ± 0.4 pmol/well (n = 10) and were elevated to 9.0 ± 1.4 pmol/well (n = 10) under maximum stimulation by these agonists. Thus, these compounds stimulated cAMP by 7.6 ± 1.1-fold above basal levels (n = 10). However, due to the difficulty of obtaining a steady large supply of normal human corneal epithelial cells and the problems associated with their slow growth rate and thus the difficulty of propagating these cells, subsequent studies were conducted with the previously immortalized human corneal epithelial cell line, CEPI-17-CL4. 24 25 26  
To define the pharmacological characteristics of the 5-HT receptor stimulating cAMP in the CEPI-17-CL4 cells, we used a broad panel of well-known agonist and antagonists that exhibit some level of selectivity for the different 5-HT receptor subtypes. Thus, numerous 5-HT receptor agonists stimulated cAMP production in CEPI-17-CL4 cells in a concentration-dependent manner (Fig. 1) . Basal levels of cAMP were 1.0 ± 0.1 pmol/well (n = 44), and these were increased to 9.1 ± 1.2 pmol/well by maximum stimulation by the different agonists used in the CEPI-17-CL4 cells. Thus, the various agonists stimulated cAMP by 10.8 ± 1.1-fold (n = 44) above basal levels, comparable to that observed in the P-CEPI cells. Table 1 shows a summary of the data for the six agonists tested in the CEPI-17-CL4 cells. The rank order of potency (pEC50) for these compounds was: 5-CT (pEC50 = 7.4) > 5-HT (pEC50 = 6.5) > 5-methoxy-tryptamine (pEC50 = 6.1) > 5-methoxy-dimethyl-tryptamine (pEC50 = 5.4) ≥ 8-OH-DPAT (pEC50 < 5.0; Table 1 ). Whereas 5-CT, 5-HT, and 5-methoxy-tryptamine were full agonists, 5-methoxy-dimethyl-tryptamine, 8-OH-DPAT, and α-methyl-5-HT were partial agonists (Table 1) . Similarly, 5-HT4 receptor partial agonists, RS-67333, and RS-67506 were very weak or inactive agonists in these cells (Table 1)
Figure 2 shows a representative plot of antagonism of 5-CT (100 nM)–stimulated cAMP production by various 5-HT receptor antagonists in CEPI-17-CL4 cells. Table 2 shows a comparison of functional antagonist data obtained for CEPI-17-CL4 cells with those obtained for the recombinant human 5-HT7 receptor and native 5-HT7 receptor in various cells and tissues from the literature. The rank order of potency of the 5-HT receptor antagonists for the CEPI-17-CL4 cell cAMP assay was methiothepin (pKi = 8.5) > mesulergine (pKi = 8.1) = metergoline (pKi = 8.0) > spiperone (pKi = 7.4) ≥ ritanserin (pKi = 7.3) ≥ clozapine (pKi = 7.2) = SB-258719 (pKi = 7.2) > mianserin (pKi = 6.9) > MDL-100907 (pKi = 6.6) > ketanserin (pKi = 6.3), and so forth (Table 2) . Limited antagonist studies in the P-CEPI cells yielded the following antagonist potencies: methiothepin (pKi = 8.7), spiperone (pKi = 7.4), and SB-258719 (pKi = 6.6). All these functional agonist–antagonist data from P-CEPI and CEPI-17-CL4 cells compared well with the agonist and antagonist potency data reported for the 5-HT7 receptor in the literature (described later). 
Because of the difficulty of obtaining large preparations of CEPI-17 cell membranes, all binding studies were conducted with the cloned human 5-HT7 receptor expressed in HEK-293 cell membranes. Thus, to compare the pharmacological characteristics of the 5-HT receptor stimulating cAMP in the CEPI-17-CL4 cells with that of the cloned human 5-HT7 receptor, we also determined the binding affinities of a broad panel of serotonergic agents, mainly antagonists, that exhibit some level of selectivity for the different 5-HT receptor subtypes at this human 5-HT7 receptor. Figure 3 shows a representative plot of [3H]-LSD binding data obtained from cloned human 5-HT7 receptor preparations (expressed in HEK-293 cells) in the current studies. The pKi values for 5-HT, 5-methoxy-dimethyl-tryptamine and 8-OH-DPAT were 8.5, 7.9, and 8.5, respectively (Table 2) . Table 2 also shows a summary of the 5-HT antagonist binding data compared with functional antagonist data. The rank order of potency for displacement of [3H]-LSD from the cloned human 5-HT7 receptor in the current studies was methiothepin (pKi = 11.1) > ritanserin (pKi = 10.3) > mesulergine (pKi = 8.8) = clozapine (pKi = 8.8) ≥ metergoline (pKi = 8.6) > SB-258719 (pKi = 8.0) ≥ spiperone (pKi = 7.9) > 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 cells 10 indicated a high level of correlation (r = 0.88, slope = 1.05, P < 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 = 0.69; P < 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 = 0.71, P < 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 = 0.85, slope = 0.81, P < 0.02; Fig. 4D ). 
Molecular biological studies using RT-PCR techniques revealed the presence of the mRNA for the human 5-HT7 receptor in CEPI-17-CL4 cells (Fig. 5) , thus confirming and supplementing the pharmacological data presented herein for these cells. In addition, the mRNA for the human 5-HT7 receptor was also detected in biopsy specimens of human conjunctival and corneal tissues (Senchyna M, et al., unpublished observations, 2000), using RT-PCR assays. Limited RT-PCR studies to study mRNAs for other 5-HT receptors revealed the absence of 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 Cl 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 2 3 4 Accordingly, the current studies have provided functional pharmacological 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 observed a high potency for 5-CT (pEC50 = 7.4–7.8) in P-CEPI and CEPI-17-CL4 cells, a serotonergic-agonist known to exhibit a relatively high potency and selectivity for the 5-HT7 receptor. 1 2 3 4 5 6 7 8 9 10 11 The rank order of potency in the current CEPI-17-CL4 cells studies of 5-CT > 5-HT > 8-OH-DPAT for the stimulation of adenylyl cyclase was highly corroborated by similar cAMP data from both the human 5-HT7(a) 30 and 5-HT7(b) splice variants 33 expressed in HEK-293 cells (Table 1) . Similarly, Adham et al. 5 reported an identical rank order of potency (5-CT > 5-HT > 5-methoxy-tryptamine > 5-methoxy-dimethyl-tryptamine > 8-OH-DPAT > α-methyl-5-HT) for stimulation of cAMP production by the recombinant human 5-HT7(a) receptor expressed in murine fibroblasts. The profile of receptor antagonist potency data for the inhibition of 5-CT–stimulated cAMP production also supported the presence of functional 5-HT7 receptors in the CEPI-17-CL4 cells. We observed a rank order of antagonist potency of methiothepin > spiperone = metergoline = clozapine = SB-258719 > ketanserin in the CEPI-7-CL4 cells, and similar findings were noted for the inhibition of 5-HT–stimulated cAMP production in murine fibroblasts expressing the recombinant human 5-HT(7a) receptor isoform. 5 In addition, our agonist and antagonist potency data from the CEPI-17-CL4 cells compared well with data obtained for the 5-HT7 receptor mediating relaxation of the canine cerebral arteries (Tables 1 2)
Further evidence supporting the notion that a functionally active 5-HT7 receptor mediates production of cAMP in the CEPI-17-CL4 cells included the following: The 5-HT4 receptor-selective antagonists, 2 RS-23597, RS-39604, and SB-203186, were weak or inactive at inhibiting 5-CT–stimulated cAMP production in CEPI-17-CL4 cells (pKi < 5), thus ruling out the presence of 5-HT4 receptors in these cells; likewise, the relatively weak antagonist potency of the 5-HT6 receptor antagonist, Ro-04-6790, 22 38 in the CEPI-17-CL4 cells ruled out the presence of a 5-HT6 receptor in these cells. However, SB-258719, 22 30 39 a 5-HT7 receptor antagonist exhibiting a 100-fold selectivity for the 5-HT7 receptor, 22 30 39 was a potent inhibitor of 5-CT–stimulated cAMP production (pKi = 7.2; Table 2 ). This finding, along with other high antagonist potency data (e.g., for methiothepin, metergoline, and spiperone) typical of 5-HT7 receptors in other systems (Table 2) , supports the identification of a 5-HT7 receptor in the CEPI-17-CL4 (and P-CEPI) cells. This conclusion was further supported by the detection of an mRNA of the cloned human 5-HT7 receptor in CEPI-17-CL4 cells (Fig. 5) . In addition, mRNA corresponding to the cloned human 5-HT7 receptor was observed in human 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 [3H]-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 > mesulergine > clozapine > spiperone > ritanserin > 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. 
The expression of 5-HT7 receptors in human corneal epithelial cells suggests a role for this receptor is maintaining the normal physiology of the ocular surface. Serotonin has been implicated in the regulation of aqueous humor dynamics in the eye 19 20 21 22 and prejunctional 5-HT7 receptors have been hypothesized to play a role in intraocular pressure regulation. 22 Serotonin, which is present in human tears, 37 increases cAMP levels in corneas, which results in increased Cl transport. 36 All these findings suggest that the presence of functional 5-HT7 receptors in corneal epithelial cells may play a role in fluid/mucin secretion on the ocular surface. However, further work is necessary to expand on these findings. 
Taken together, our pharmacological and molecular biological data have demonstrated that P-CEPI and CEPI-17-CL4 cells express a functional 5-HT7 receptor positively coupled to adenylyl 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 pharmacological aspects of the human ocular 5-HT7 receptor. 
Figure 1.
 
Agonist-induced cAMP production in CEPI-17-CL4 cells. The cells were incubated with the agonists (six concentrations) for 15 minutes, the cells lysed and the cAMP generated determined with an EIA procedure. Representative functional concentration–response curves for several serotonergic receptor agonists in the CEPI-17-CL4 cell adenylyl cyclase assay are shown. Data points represent means of two determinations. Summary data from several such experiments are shown in Table 1 .
Figure 1.
 
Agonist-induced cAMP production in CEPI-17-CL4 cells. The cells were incubated with the agonists (six concentrations) for 15 minutes, the cells lysed and the cAMP generated determined with an EIA procedure. Representative functional concentration–response curves for several serotonergic receptor agonists in the CEPI-17-CL4 cell adenylyl cyclase assay are shown. Data points represent means of two determinations. Summary data from several such experiments are shown in Table 1 .
Table 1.
 
Agonist-Stimulated Production of cAMP in CEPI-17-CL4 Cells Compared with Data in the Literature
Table 1.
 
Agonist-Stimulated Production of cAMP in CEPI-17-CL4 Cells Compared with Data in the Literature
Agonist Reported Receptor Selectivity Agonist Potency (pEC50) in CEPI-17-CL4 Cells (% Max. Response) Literature Agonist Potencies (pEC50) for 5-HT7 Receptor-Mediated cAMP Production or Tissue Relaxation
5-CT 5-HT7 7.4 ± 0.1 (100%) 7.730 ; 7.831 ; 8.05 ; 8.3922 ; 7.2432 ; 8.733
5-HT Nonselective 6.5 ± 0.1 (100%) 6.830 ; 6.6831 ; 7.15 ; 6.6922 ; 6.032 ; 7.533
5-methoxy-tryptamine Nonselective 6.1 ± 0.1 (123 ± 20%) 6.8631 ; 6.885 ; 6.522 ; 5.0332 ; 8.1233
5-methoxy-dimethyl-tryptamine Nonselective 5.4 ± 0.1 (53 ± 7%) 5.55
8-OH-DPAT 5-HT1A <5.0 (17 ± 6%) 6.130 ; 4.25 ; <522 ; 5.2533
α-methyl-5-HT 5-HT2 <5.0 (13 ± 4%) 4.05 ; 5.422 ; <532
RS-67333 5-HT4 <5.0
RS-67506 5-HT4 <5.0
Figure 2.
 
Inhibition of agonist-induced cAMP production in CEPI-17-CL4 cells. Various serotonergic antagonists were preincubated with the cells for 20 minutes before the addition of 5-CT (100 nM). The assays were continued for another 15 minutes, the cells lysed, and the cAMP generated determined with an EIA procedure. Representative functional inhibition curves for a range of serotonergic receptor antagonists in the CEPI-17-CL4 cells are shown. Data points represent means of two determinations. Summary data from several such experiments are shown in Table 2 .
Figure 2.
 
Inhibition of agonist-induced cAMP production in CEPI-17-CL4 cells. Various serotonergic antagonists were preincubated with the cells for 20 minutes before the addition of 5-CT (100 nM). The assays were continued for another 15 minutes, the cells lysed, and the cAMP generated determined with an EIA procedure. Representative functional inhibition curves for a range of serotonergic receptor antagonists in the CEPI-17-CL4 cells are shown. Data points represent means of two determinations. Summary data from several such experiments are shown in Table 2 .
Table 2.
 
Antagonism of 5-CT–Mediated cAMP Production in CEPI-17-CL4 Cells and Competition for [3H]-LSD Binding to Cloned Human 5-HT7 Receptors: Comparison with 5-HT7 Receptor Functional Data in the Literature
Table 2.
 
Antagonism of 5-CT–Mediated cAMP Production in CEPI-17-CL4 Cells and Competition for [3H]-LSD Binding to Cloned Human 5-HT7 Receptors: Comparison with 5-HT7 Receptor Functional Data in the Literature
Antagonist Reported 5-HT Receptor Selectivity Inhibition of cAMP Production in CEPI-17-CL4 Cells (pKi) Inhibition of cAMP Production or Tissue Relaxation Via 5-HT7 Receptors (pKi; pKb) from the Literature [3H]-LSD Binding to Cloned Human 5-HT7 Receptor (pKi)
Methiothepin Nonselective 8.5 ± 0.1 8.130 ; 9.2910 ; 8.4533 ; 7.9831 11.1 ± 0.4
Mesulergine 5-HT2C 8.1 ± 0.2 7.530 ; 8.1810 ; 7.5831 ; 7.332 8.8 ± 0.4
Metergoline 5-HT1, 5-HT2 8.0 ± 0.3 8.6010 ; 7.133 8.6 ± 0.1
Spiperone 5-HT1A, 5-HT2A 7.4 ± 0.1 7.9510 ; 6.0333 ; 6.632 ; 6.932 7.9 ± 0.2
Ritanserin 5-HT1, 5-HT2 7.3 ± 0.1 7.7410 ; 7.231 10.3 ± 0.2
Clozapine 5-HT7, 5-HT6 7.2 ± 0.03 7.230 ; 6.6533 ; 7.0331 ; 7.132 8.8 ± 0.1
SB-258719 5-HT7 7.2 ± 0.3 7.030 ; 7.7210 8.0 ± 0.1
Mianserin Nonselective 6.9 ± 0.1 6.0533 ; 6.431 7.2 ± 0.0
MDL-100907 5-HT2A 6.6 ± 0.2
Ketanserin 5-HT2 6.3 ± 0.1 6.330 ; 6.9510 ; 5.333 7.1 ± 0.1
BW501C67 5-HT2A 6.1 ± 0.2
WB-4101 5-HT1A 5.5 ± 0.2
Way 100635 5-HT1A 5.5 ± 0.2 531
Xylamidine 5-HT2A 5.4 ± 0.03
RS-127445 5-HT2B 5.4 ± 0.2
BMY-7378 5-HT1A; α1d 5.3 ± 0.1
RS-102221 5-HT2C <5.00
SB-203186 5-HT4 <5.00
SB-242084 5-HT2C <5.00
Y-25130 5-HT3 <5.00
RS-23597-190 5-HT4 <5.00
RS-39604 5-HT4 <5.00
Ro-04-6790 5-HT6 <5.00
Figure 3.
 
[3H]-LSD binding to cloned human 5-HT7 receptors expressed in HEK-293 cell membranes. Competition assays were conducted with a range of serotonergic agents (eight concentrations) using [3H]-LSD (2 nM final). Data points represent means of two determinations in duplicate. Summary affinity data from several such experiments are shown in Table 2 .
Figure 3.
 
[3H]-LSD binding to cloned human 5-HT7 receptors expressed in HEK-293 cell membranes. Competition assays were conducted with a range of serotonergic agents (eight concentrations) using [3H]-LSD (2 nM final). Data points represent means of two determinations in duplicate. Summary affinity data from several such experiments are shown in Table 2 .
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.5103033 (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 assays conducted on the cloned human 5-HT7(a), 5-HT7(b), and 5-HT7(c) receptors expressed in HEK-293 cells,5103033 and functional assays performed on native 5-HT7 receptor in brain astrocytes,31 bovine ICB,22 and canine cerebral arteries.32 (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) using the cloned human 5-HT7 receptor.69 Assays to measure cAMP accumulation in CEPI-17-CL4 cells and [3H]-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 = 0.82, slope = 0.96, P < 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 = 0.85, slope = 0.81, P < 0.02.
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.5103033 (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 assays conducted on the cloned human 5-HT7(a), 5-HT7(b), and 5-HT7(c) receptors expressed in HEK-293 cells,5103033 and functional assays performed on native 5-HT7 receptor in brain astrocytes,31 bovine ICB,22 and canine cerebral arteries.32 (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) using the cloned human 5-HT7 receptor.69 Assays to measure cAMP accumulation in CEPI-17-CL4 cells and [3H]-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 = 0.82, slope = 0.96, P < 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 = 0.85, slope = 0.81, P < 0.02.
Figure 5.
 
Visualization of the mRNA for human 5-HT7 receptor in CEPI-17-CL4 cells and other cells and tissues. Total RNA isolated from CEPI-17-CL4 cells was reverse transcribed into cDNA. Similar cDNAs were generated for other control tissues (e.g., rat brain, hamster uterus). PCR was performed on the cDNAs, using the sense and antisense primers for the human 5-HT7 receptor and the PCR products size fractionated on agarose gels, visualized under UV light, and then photographed. The PCR product size for the human 5-HT7 receptor was 772 bp. Sense and antisense primers for the human 5-HT7 receptor were constructed as previously described635 (see the Methods section for sequences) and the PCR procedure conducted as for other receptor systems.2935Lanes 1, 2: nonpigmented ciliary epithelial cells; lanes 3, 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); lane 9: no reverse transcriptase added (negative control 2); lane 10: human brain; lane 11, 12: G3PDH house-keeping enzyme. Long arrows: position of the 5-HT7 mRNA; short arrows: position of the house-keeping enzyme (G3PDH).
Figure 5.
 
Visualization of the mRNA for human 5-HT7 receptor in CEPI-17-CL4 cells and other cells and tissues. Total RNA isolated from CEPI-17-CL4 cells was reverse transcribed into cDNA. Similar cDNAs were generated for other control tissues (e.g., rat brain, hamster uterus). PCR was performed on the cDNAs, using the sense and antisense primers for the human 5-HT7 receptor and the PCR products size fractionated on agarose gels, visualized under UV light, and then photographed. The PCR product size for the human 5-HT7 receptor was 772 bp. Sense and antisense primers for the human 5-HT7 receptor were constructed as previously described635 (see the Methods section for sequences) and the PCR procedure conducted as for other receptor systems.2935Lanes 1, 2: nonpigmented ciliary epithelial cells; lanes 3, 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); lane 9: no reverse transcriptase added (negative control 2); lane 10: human brain; lane 11, 12: G3PDH house-keeping enzyme. Long arrows: position of the 5-HT7 mRNA; short arrows: position of the house-keeping enzyme (G3PDH).
 
The authors thank Jesse May for a critical review of the manuscript and Tom Dean for his generous support which enabled us to conduct these studies. 
Hoyer, D, Clarke, DE, Fozard, JR, et al (1994) VII. International union of pharmacology classification of receptors for 5-hydroxytryptamine (serotonin) Pharm Rev 46,157-203 [PubMed]
Barnes, NM, Sharp, T. (1999) A review of central 5-HT receptors and their function Neuropharmacology 38,1083-1152 [CrossRef] [PubMed]
Vanhoenacker, P, Haegeman, G, Leysen, JE. (2000) 5-HT7 receptors: Current knowledge and future prospects Trends in Pharmacol Sci 21,70-77 [CrossRef]
Gerhardt, CC, van Heerikuizen, H. (1997) Functional characteristic of heterologously expressed 5-HT receptors Eur J Pharmacol 334,1-23 [CrossRef] [PubMed]
Adham, N, Zgoombick, JM, Bard, J, Branchek, TA. (1998) Functional characterization of the recombinant human 5-hydroxytryptamine7(a) receptor isoform coupled to adenylate cyclase stimulation J Pharm Exp Ther 287,508-514
Bard, JA, Zgombick, J, Adham, N, Vaysse, P, Branchek, TA, Weinshank, RL. (1993) Cloning of a novel human serotonin receptor (5-HT7) positively linked to adenylate cyclase J Biol Chem 268,23422-23246 [PubMed]
Roth, BL, Craigo, SC, Choudhary, MS, et al (1994) Binding of typical and atypical antipsychotic agents to 5-hydroxytrptamine-6 and 5-hydroxytryptamine-7 receptors J Pharmacol Exp Ther 268,1403-1410 [PubMed]
Ruat, M, Traiffort, E, Leurs, R, et al (1993) Molecular cloning, characterization, and localization of a high-affinity serotonin receptor (5-HT7) activating cAMP formation Proc Natl Acad Sci USA 90,8547-8551 [CrossRef] [PubMed]
Shen, Y, Monsma, FJ, Metcalf, MA, Jose, PA, Hamblin, MW, Sibley, DR. (1993) Molecular cloning and expression of a 5-hydroxytryptamine-7 serotonin receptor subtype J Biol Chem 268,18200-18204 [PubMed]
Krobert, KA, Bach, T, Syversveen, T, Kvingedal, AM, Levy, FO. (2001) The cloned human 5-HT7 receptor splice variants: a comparative characterization of their pharmacology, function and distribution Naunyn-Schmiedebergs Arch Pharm 363,620-632 [CrossRef]
Heiddmann, DEA, Metchalf, MA, Kohen, R, Hamblin, MW. (1997) Four 5-hydroxy-tryyptamine7 (5-HT7) receptor isoforms in human and rat produced by alternative splicing: species differences due to altered intron-exon organization J Neurochem 68,1372-1378 [PubMed]
Baker, LP, Nielsen, MD, Impey, S, et al (1998) Stimulation of type 1 and type 8 Ca2+/calmodulin-sensitive adenylyl cyclases by the Gs-coupled 5-hydroxytryptamine subtype 5-HT7A receptor J Biol Chem 273,17469-17476 [CrossRef] [PubMed]
Heidmann, DEA, Szot, P, Kohen, R, Hamblin, MW. (1998) Function and distribution of three rat 5-hydroxytryptamine7 (5-HT7) receptor isoforms produced by alternative splicing Neuropharmacology 37,1621-1632 [CrossRef] [PubMed]
Lovenberg, TW, Baron, B, De Lecea, L, et al (1993) A novel adenylyl cyclase-activating serotonin receptor (5-HT7) implicated in the regulation of mammalian circadian rhythms Neuron 11,449-458 [CrossRef] [PubMed]
Ying, S-W, Rusak, B. (1997) 5-HT7 receptors mediate serotonergic effects on light-sensitive suprachiasmatic nucleus neurons Brain Res 755,246-254 [CrossRef] [PubMed]
Schoeffter, P, Ullmer, C, Bobirnac, I, Gabbiana, G, Lobbert, H. (1996) Functional, endogenously expressed 5-hydroxytrptamine 5-ht7 receptors in human vascular smooth muscle cells Br J Pharmacol 117,993-994 [CrossRef] [PubMed]
Sleight, AJ, Carolo, C, Petit, N, Zwingelstein, C, Bourson, A. (1995) Identification of 5-HT7 receptor binding sites in rat hypothalamus Mol Pharmacol 47,99-103 [PubMed]
Fozard, JR. (1995) the 5-hydroxytryptamine-nitric oxide connection: the key in the initiation of migraine? Arch Int Pharmacdyn 329,111-119
Tobin, AB, Unger, W, Osborne, NN. (1988) Evidence for presence of serotonergic nerves and receptors in the iris-ciliary body complex of the rabbit J Neurosci 8,3713-3721 [PubMed]
Martin, XD, Brennan, MC, Lichter, PR. (1988) Serotonin in human aqueous humor Ophthalmology 95,1221-1226 [CrossRef] [PubMed]
Chidlow, G, DeSantis, LM, Sharif, NA, Osborne, NN. (1995) Characteristics of [3H]-5-hydroxytryptamine binding to iris-ciliary body tissue of the rabbit Invest Ophthalmol Vis Sci 36,2238-2245 [PubMed]
Harris, LC, Awe, SO, Opere, CA, Leday, AM, Ohia, SE, Sharif, NA. (2001) [3H]Serotonin release from bovine iris-ciliary body: pharmacology of pre-junctional serotonin (5HT7) autoreceptors Exp Eye Res 73,59-67 [CrossRef] [PubMed]
Neufeld, AH, Ledgard, SE, Jumblatt, MM, Klyce, SD. (1982) Serotonin-stimulated cyclic AMP synthesis in the rabbit corneal epithelium Invest Ophthalmol Vis Sci 23,193-198 [PubMed]
Sharif, NA, Wiernas, TK, Howe, WE, Griffin, BW, Offord, EA, Pfeifer, AMA. (1998) Human corneal epithelial cell functional responses to inflammatory agents and their antagonists Invest Ophthalmol Vis Sci 39,2562-2571 [PubMed]
Offord, E, Sharif, NA, Mace, K, et al (1999) Immortalized human corneal epithelial cells for ocular toxicity and inflammation studies Invest Ophthalmol Vis Sci 40,1091-1101 [PubMed]
Wiernas, TK, Griffin, BW, Sharif, NA. (1997) The expression of functionally-coupled B2-bradykinin receptors in human corneal epithelial cells and their pharmacological characterization with agonists and antagonists Br J Pharmacol 121,649-656 [CrossRef] [PubMed]
Crider, JY, Griffin, BW, Sharif, NA. (1999) Prostaglandin DP receptors positively coupled to adenylyl cyclase in embryonic bovine tracheal (EBTr) cells: pharmacological characterization using agonists and antagonists Br J Pharmacol 127,204-210 [CrossRef] [PubMed]
Crider, JY, Griffin, BW, Sharif, NA. (1998) Prostaglandin-stimulated adenylyl cyclase activity via a pharmacologically defined EP2 receptor in human nonpigmented ciliary epithelial cells J Ocular Pharmacol 14,293-304 [CrossRef]
Sharif, NA, Senchyna, M, Xu, SX. (2002) Pharmacological and molecular biological (RT-PCR) characterization of functional TP prostanoid receptors in immortalized human non-pigmented ciliary epithelial cells J Ocul Pharmacol Ther 18,141-162 [CrossRef] [PubMed]
Thomas, DR, Gittins, SA, Collin, LL, et al (1998) Functional characterisation of the human cloned 5-HT7 receptor (long form): antagonist profile of SB-258719 Br J Pharmacol 124,1300-1306 [CrossRef] [PubMed]
Hirst, WD, Price, GW, Rattray, M, Wilkin, GP. (1997) Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes Br J Pharmacol 120,509-515 [CrossRef] [PubMed]
Terron, JA, Falcon-Neri, A. (1999) Pharmacological evidence for the 5-HT7 receptor mediating smooth muscle relaxation in canine cerebral arteries Br J Pharmacol 127,609-616 [CrossRef] [PubMed]
Jasper, JR, Kosaka, A, To, ZP, Chang, DJ, Eglen, RM. (1997) Cloning, expression and pharmacology of a truncated splice variant of the human 5-HT7 receptor (ht-HT7(b) ) Br J Pharmacol 122,126-132 [CrossRef] [PubMed]
Ullmer, C, Schmuck, K, Kalkman, HO, Lubbert, H. (1995) Expression of serotonin receptor mRNA in blood vessels FEBS Lett 370,215-221 [CrossRef] [PubMed]
Kyveris, A, Maruscak, E, Senchyna, M. (2002) Optimization of RNA isolation from human ocular tissues and analysis of prostanoid receptor mRNA expression using RT-PCR Mol Vis 8,51-58 [PubMed]
Klyce, SD, Palkama, KA, Harkonen, M, et al (1982) Neural serotonin stimulates chloride transport in the rabbit corneal epithelium Invest Ophthalmol Vis Sci 23,181-192 [PubMed]
Martin, XD, Brennan, MC. (1994) Serotonin in human tears Eur J Ophthalmol 4,159-165 [PubMed]
Bentley, JC, Bourson, A, Boess, FG, et al (1999) Investigation of stretching behavior induced by selective 5-HT6 receptor antagonist, Ro-04-6790, in rats Br J Pharmacol 126,1537-1542 [CrossRef] [PubMed]
Forbes, IT, Dabbs, S, Duckworth, DM, et al (1998) (R)-3, N-dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)-propyl]-benzene sulfonamide: The first selective 5-HT7 receptor antagonist J Med Chem 41,655-657 [CrossRef] [PubMed]
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