September 2006
Volume 47, Issue 9
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Physiology and Pharmacology  |   September 2006
Human Trabecular Meshwork Cells Express Functional Serotonin-2A (5HT2A) Receptors: Role in IOP Reduction
Author Affiliations
  • Najam A. Sharif
    From Ophthalmology Discovery Research, Alcon Research, Ltd., Fort Worth, Texas.
  • Curtis R. Kelly
    From Ophthalmology Discovery Research, Alcon Research, Ltd., Fort Worth, Texas.
  • Marsha McLaughlin
    From Ophthalmology Discovery Research, Alcon Research, Ltd., Fort Worth, Texas.
Investigative Ophthalmology & Visual Science September 2006, Vol.47, 4001-4010. doi:10.1167/iovs.06-0062
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      Najam A. Sharif, Curtis R. Kelly, Marsha McLaughlin; Human Trabecular Meshwork Cells Express Functional Serotonin-2A (5HT2A) Receptors: Role in IOP Reduction. Invest. Ophthalmol. Vis. Sci. 2006;47(9):4001-4010. doi: 10.1167/iovs.06-0062.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To apply a multidisciplinary approach to the identification and pharmacological characterization of the serotonin (5HT) receptors that mediate functional responses in human trabecular meshwork (h-TM) cells. To correlate in vitro findings with intraocular pressure (IOP) changes in conscious ocular hypertensive cynomolgus monkeys.

methods. Documented methods were used, including reverse transcription–polymerase chain reaction (RT-PCR), phosphoinositide (PI) turnover, and intracellular Ca2+ ([Ca2+]i) mobilization. IOP was measured using standard applanation pneumatonometry.

results. h-TM cells expressed robust mRNA signals for 5HT2A and 5HT2B receptors. 5HT and its analogues stimulated PI turnover and [Ca2+]i mobilization in h-TM cells from multiple donors (20/24 donors’ TM cells responded). The agonist potencies (EC50) of compounds in mobilizing [Ca2+]i were (nM): 5-methoxy tryptamine, 8 ± 4; (R)-DOI, 18 ± 6; α-methyl-5HT, 22 ± 3; 5HT, 40 ± 7; 5-methoxy-dimethyl tryptamine, 64 ± 27; and BW-723C86, 1213 ± 210. These effects were potently blocked by the 5HT2A-receptor-selective antagonist, M-100907 (K i = 1 ± 0.3 nM), but weakly by antagonists of 5HT2B and 5HT2C receptors. Only 5HT2 receptor agonists such as (R)-DOI (300 μg lowered IOP 34.4% from baseline of 38.2 mm Hg; P < 0.001) and α-methyl-5HT (250 μg lowered IOP 30.8% from baseline of 41.8 mm Hg; P < 0.001) lowered IOP in ocular hypertensive cynomolgus monkeys.

conclusions. Strong signals for 5HT2A and 5HT2B receptor mRNAs were detected in h-TM cells. The receptors that coupled to PI hydrolysis and [Ca2+]i mobilization in h-TM cells were the 5HT2A receptor subtype, which also significantly lowered IOP in a primate model. These receptors may mediate the ocular hypotensive actions of 5HT2A agonists.

Glaucoma is the second major cause of irreversible legal blindness in the human population in the world. 1 It is estimated that 3.3 million people in the United States will have glaucoma by 2020. 1 Because the major risk factor for glaucoma is elevated intraocular pressure (IOP), various pharmaceutical agents have been discovered for treating ocular hypertension, including FP-class prostaglandin analogues, 2 3 β-blockers, α-2 adrenergic agonists, carbonic anhydrase inhibitors and muscarinic agonists (for reviews, see Refs. 4 , 5 ). However, despite the availability of such therapeutic agents, many patients with glaucoma remain either refractory toward several of these drugs or exhibit significant side-effects, such that there continues to be a critical need for novel ocular hypotensive drugs. 
Serotonin (5-hydroxy tryptamine; 5HT), is a major endogenous amine neurotransmitter in the mammalian central nervous system 6 that has also been detected in the aqueous humor 7 8 and iris-ciliary body 9 of human subjects. Furthermore, serotonergic nerves have been shown to innervate various ocular tissues including the iris-ciliary body. 10 Such information has led us to hypothesize that 5HT receptors may play a role in mediating ocular aqueous humor dynamics and thus in modulating IOP. However, even though 5HT1A receptors have been found in the rabbit iris-ciliary body 11 and indeed 5HT1A agonists such as flesinoxan 12 and 5-methyl-urapidil 13 lower IOP in the rabbit, 14 15 the latter agents are ineffective in the monkey. 16 To complicate matters further, another 5HT1A agonist, 5-carboximidotryptamine (5-CT), actually increases IOP in the rabbit. 17 Furthermore, potent α-1 adrenoceptor antagonist activity associated with the 5HT2 antagonists ketanserin 18 19 20 and sarpogrelate (Takenaka H, et al. IOVS 1995;36:ARVO Abstract 3390) in human subjects confound observations of their ocular hypotensive activity. Therefore, there appear to be major species differences in the IOP-lowering activity of the various aforementioned serotonergic agents. However, since initial studies have revealed that certain agonists of the 5HT2 receptor class lower IOP in conscious ocular hypertensive cynomolgus monkeys, 16 we decided to examine the relative abundance of the mRNAs for various 5HT receptor subtypes in human trabecular meshwork (h-TM) cells isolated from several human donor eyes. Then, we studied the functional coupling of the predominant 5HT receptor subtypes present on TM cells using phosphoinositide (PI) turnover and intracellular Ca2+ ([Ca2+]i) mobilization assays involving a broad range of serotonergic agonists and antagonists. Additional biochemical and pharmacological assays were used to extend the data obtained from the aforementioned assays. Furthermore, a variety of serotonergic agonists and antagonists were tested for their ability to lower IOP in the conscious ocular hypertensive cynomolgus monkey and in an effort to correlate the in vitro findings with the in vivo effects of these compounds. 
Materials and Methods
Human Donor Details
h-TM cells were isolated from TM tissue dissected from human donor eyes within 24 to 36 hours after death. Details about the human donors whose TM cells responded to 5HT2 agonists were as follows: age, 48 days to 95 years; gender, 14 males and 10 females; number of 5HT2 agonist-responders, 20 of 24; and responder donors with a history of glaucoma, 6. The donor tissue was obtained and managed in accordance with the guidelines in the Declaration of Helsinki. 
Detection of 5HT Receptor mRNAs
Reverse transcription–polymerase chain reaction (RT-PCR) procedures were used to detect the mRNAs for various 5HT receptors in h-TM cells isolated from eight different human donors’ eyes (ages, 10–75 years) using oligonucleotide primers based on the genomic sequences of the human 5HT receptors obtained from GenBank and as recently described (http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD). 21 PCR amplification products were separated by electrophoresis and visualized by UV light. Positive control tissue samples (whole human brain) were run along with the h-TM cell samples. Endonucleotide digestion was used to confirm the PCR product identities. 
[125I]-DOI and GTP-γ-S35 Binding to Cloned Human 5-HT2 Receptors
Binding of the agonist radioligand [125I]-DOI to cell membranes of Chinese hamster ovary (CHO) cells transfected with full-length clones of human 5HT2A, 5HT2B, and 5HT2C receptors was determined using standard radioligand binding procedures. 16 These studies were conducted at Cerep Inc. (Poitiers, France). 
To determine the functional activity of certain compounds of interest at the cloned human 5HT2A and 5HT2C receptors, a GTP-γ-S35 binding assay was conducted at Cerep Inc., as previously described. 22  
PI Turnover Assays
The relative agonist activity of serotonergic compounds at the 5-HT2 receptor was determined in vitro using the ability of the compounds to stimulate the production of [3H]inositol phosphates in h-TM cells. 23 24 In brief, [3H] myo-inositol-labeled h-TM cells were challenged with the test compounds for 1 hour at 37°C followed by assay termination using ice-cold 0.1 M formic acid. The total [3H]inositol phosphates produced were determined by anion exchange chromatography. 23 24 Concentration-response data were analyzed by a four-parameter nonlinear (sigmoidal) curve fit function (Origin Scientific Graphics software; Microcal Software, Northampton, MA; or Excel-fit; IDBS, Surrey, UK) to determine agonist potency (EC50) and efficacy (E max). 23 24 Serotonin (5-HT) was used as a positive control (standard) agonist compound, and the efficacy of test compounds was compared with that of 5-HT (set at 100%). 
[Ca2+]i Mobilization Assays
The receptor-mediated mobilization of intracellular calcium ([Ca2+]i) was studied with a fluorescence imaging plate reader (FLIPR), as previously described. 25 26 The h-TM cells from numerous donors were grown in a normal medium of DMEM-10% FBS and 10 μg/mL gentamicin. Confluent cell monolayers were trypsinized, centrifuged, and resuspended in normal medium. Cells were seeded in a 50-μL volume at a density of ∼20,000 cells per well in a black walled, 96-well tissue culture plate and grown for 2 days. On the day of the experiment, a vial of FLIPR calcium assay kit dye was resuspended in 50 mL of a FLIPR buffer consisting of Hanks’ balanced salt solution (HBSS), 20 mM HEPES, and 2.5 mM probenecid (pH 7.4). Cells were loaded with the calcium-sensitive dye by addition of an equal volume (50 μL) to each well of the 96-well plate and incubated with dye for 1 hour at 23°C. Compounds were diluted 1:50 in 20% dimethyl sulfoxide (DMSO)-20% ethanol. For dose–response experiments, compounds were diluted 1:50 in FLIPR buffer and serially diluted 1:10 to give a 5- or 8-point concentration–response curve. Other details have been described. 25 26  
Responses were measured as peak fluorescence intensity minus basal and, where appropriate, were expressed as a percentage of a maximum 5-HT-induced response. Data were analyzed as described for the PI turnover experiments. 
Aequorin-Based Intracellular [Ca2+]i Mobilization Assay
Aequorin-based intracellular [Ca2+]i mobilization assays involving CHO-K1 cells transfected with the full-length human 5HT2A-C receptors were used to determine agonist potency of various agents. These studies were performed at Euroscreen (Gosselies, Belgium). In brief, functional response at the 5-HT2 receptor subtypes was determined using CHO-K1 cells stably expressing mitochondrially targeted bioluminescent aequorin, Gα16, and one of either cloned human 5-HT2A, 5-HT2B, or 5-HT2C serotonin receptors. Before testing, cells were loaded in suspension with coelenterazine for 4 to 16 hours and directly injected onto different concentrations of the test compound. Light emitted from the cells was measured 20 to 30 seconds after receptor activation. A luminometer (FDSS-6000; Hamamatsu, Osaka, Japan) was used to record luminescence in response to the test compound. The mean response signal at each of 8 to 11 different concentrations was integrated to provide an estimation of receptor activation, expressed as the EC50. The E max of the response at the 5-HT2A and 5-HT2B receptors was expressed relative to the response of α-methyl-5-HT under the same assay conditions, whereas the efficacy at 5-HT2C was expressed relative to the response of 5-HT. 
IOP Measurements
IOP was determined in conscious ocular hypertensive cynomolgus monkeys using a pneumatonometer (Alcon Ltd., Fort Worth, TX) after light corneal anesthesia with 0.1% proparacaine. 16 27 Test compounds were formulated in phosphate-buffered saline vehicle containing 0.01% benzalkonium chloride, 0.01% disodium EDTA, 0.05% polysorbate 80, and 0.5% hydroxypropylmethylcellulose and adjusted to pH 7.4. Eyes were rinsed with saline after each measurement. After a baseline IOP measurement, test compound in one 30-μL aliquot was instilled in the test eyes of eight to nine animals per group per study. Vehicle was instilled in the test eyes of five to six additional animals. Subsequent IOP measurements were taken at 1, 3, and 6 hours. A compound was considered efficacious in hypertensive eyes if there was a decrease from baseline IOP of at least 20% after topical administration of test agents. Maximum IOP reduction was calculated as a percentage of the difference from baseline. 
Animal Management
All nonhuman primates were cynomolgus monkeys (Macaca fascicularis) received from the Charles River Primate Corp. (Wilmington, MA) or Hazelton Research Products, Inc. (Denver, PA). Animals were male and female adults that were part of a permanent colony dedicated to glaucoma research. Each animal was permanently identified with a unique number tattooed on the abdomen. Previously, hypertension had been induced in the right eyes of all animals by laser trabeculoplasty. All left eyes were normal and normotensive. Animals had been trained to sit in restraint chairs and conditioned to accept the pressure measurements without chemical restraint. The animals were housed singly in stainless steel squeeze-back suspended wire-bottomed cages, had access to tap water ad libitum, and were fed a standard diet (Certified Laboratory Primate Diet No. 5048; PMI, Brentwood, MO) twice daily and supplemental fresh fruit. No contaminants were known to be present in the diet or drinking water that would interfere with or affect the ocular studies. Lighting in the animal room was controlled to give 14 hours of light and 10 hours of dark each day. Room temperature was maintained at an average 25°C. Humidity was maintained at ≥35%. Animals were transferred from holding cages to restraint chairs using the pole-and-collar method, a procedure to which all animals had been trained. Animals were in the chairs for no longer than 8 hours at a time. Animal studies were conducted in accordance with the resolutions for the use of laboratory animals as adopted by the National Institutes of Health and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Chemicals
Serotonin hydrochloride, R(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide, N,N-dipropyl-5-carboxamidotryptamine maleate, α-methyl-5-hydroxytryptamine maleate, 5-methoxytryptamine hydrochloride, ketanserin, cinanserin, ritanserin, SB-206553, R-(−)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride, N,N-dimethyl-5-methoxytryptamine, and N-methyltryptamine oxylate, were purchased from Sigma/RBI (St. Louis, MO). RS-102221 hydrochloride was obtained from Tocris Cookson (Ellisville, MO). Flesinoxan hydrochloride was obtained from Duphar. α-Methyl-5-methoxytryptamine hydrochloride was obtained from the National Institute of Mental Health’s Chemical Synthesis and Drug Supply Program (SRI International, Menlo Park, CA). Bufotenine was purchased from Biosynth International (Naperville, IL). Oxylate salts were converted to the fumarate salts, which were used for the in vivo studies. M-100907 (R-(+)-α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol) and SB-242084 (6-chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-Indole-1-carboxamide) were synthesized at Alcon. Other common reagents and compounds were purchased from Sigma or Tocris Cookson. 
Results
RT-PCR studies revealed that h-TM cells from multiple donor eyes expressed robust mRNA signals for 5HT2A and 5HT2B receptors but relatively weak signals for 5HT2C, 5HT5, and 5HT7 receptors and no apparent mRNA signals for 5HT3, 5HT4, and 5HT6 receptors (Fig. 1 ; Table 1 ). 
5HT and its analogues stimulated PI turnover (Fig. 2 , top; Table 2 ) and [Ca2+]i mobilization (Figs. 3 4 ; Table 3 ) in h-TM cells isolated from multiple donors’ eyes. In fact, 20 of 24 human donor eye–derived TM cells (including six glaucomatous donors) responded to 5HT2 receptor agonists in these assays. Agonist potency values for several compounds from PI turnover experiments are shown in Table 2 . The PI turnover responses induced by 5HT were potently antagonized by the 5HT2A receptor antagonist M-100907 (K i = 0.13 ± 0.09 nM; n = 5) but less potently by the 5HT2C receptor antagonist RS-102221 (K i = 43 nM; Fig. 2 , bottom). 
The agonist potencies (EC50) of the compounds in [Ca2+]i mobilizing assays in h-TM cells were (nM): 5-methoxy tryptamine, 8; (R)-DOI, 18; α-methyl-5HT, 22; 5HT, 40 ± 7; 5-methoxy-dimethyl tryptamine, 64; and BW-723C86, 1213 (Table 3)
To define the specificity of these agonists for 5HT2 receptor subtypes, we obtained their ligand binding affinities at cloned human 5HT2 receptor subtypes using competition studies involving [125I]-DOI binding to membranes of CHO cells transfected with recombinant 5HT2 receptor subtypes. These data are shown in Table 4 . The rank order of potency of the various agonists in the h-TM cell–based assays and in the cloned human 5HT2A-C receptors was quite similar (Tables 3 5) . In addition, the functional agonist potency of many of the compounds were determined at the recombinant human 5HT2A-C receptors using aequorin-based [Ca2+]i mobilizing assays, and these data are shown in Table 5 . Typically, the potency of compounds were lower in GTP-γ-S35 binding as a functional readout compared with the [Ca2+]i mobilizing assays (Table 5)
The [Ca2+]i mobilizing effects of 5HT2-receptor agonists in h-TM cells were potently blocked by the 5HT2A-receptor–selective antagonist, M-100907 (K i = 1 ± 0.3 nM) but weakly by antagonists of 5HT2B (RS-127445, K i > 10 μM) and 5HT2C (RS-102221, K i = 4.4 ± 1.4 μM) receptors (Fig. 4 , bottom; Table 6 ). Likewise, M-100907 potently antagonized the 5HT-induced PI turnover in h-TM cells (Fig. 2 , bottom). There was good correlation between agonist and antagonist potencies and rank order of activities in the PI turnover and [Ca2+]i mobilizing assays (r = 0.81; P < 0.02; Fig. 5 ). Of note, the only positive correlation between the pharmacology of functional [Ca2+]i mobilizing responses in the h-TM cells and that of the cloned human 5HT2 receptors was with the 5HT2A subtype (r = 0.74, P < 0.05) but not with 5HT2B subtype (r = 0.55, P < 0.55) or 5HT2C subtype (r = 0.22, P < 0.63). 
5HT2 agonists like (R)-DOI, 5HT, α-methyl-5HT, but not 5HT2 antagonists, lowered IOP in conscious ocular hypertensive cynomolgus monkeys (Table 7) . Ligands for other 5HT receptor families including agonists and antagonists for 5HT1A, 5HT1D, 5HT3, 5HT4, 5HT6, and 5HT7 receptors and a 5HT uptake inhibitor were essentially devoid of any ocular hypotensive activity in the lasered monkey eyes (Table 7)
Discussion
The present studies have demonstrated the presence of the mRNAs for 5HT2A and 5HT2B receptors, with a lower signal for 5HT2C receptor mRNA, in h-TM cells isolated from several human donors’ eyes using RT-PCR techniques. Of note, the relative abundance of the other 5HT receptor type mRNA signals (e.g., 5HT3 through 5HT7) was significantly lower than that detected for 5HT2A and 5HT2B (Table 2) . Our results for 5HT2 receptor mRNAs in the h-TM cells were similar to those recently reported for human ciliary body 21 and for human iris and ciliary body tissues (Sharif NA, et al. IOVS 2005;44:ARVO E-Abstract 3688). 
The fact that the mRNAs for 5HT2 receptors are most probably translated into their respective receptor proteins in the h-TM cells was confirmed by the detection of functional responses to numerous serotonergic agonists known to be activators of 5HT2 receptors including 5HT, α-methyl-5HT, and (R)-DOI (Figs. 2 3 4 ; Tables 2 3 ). 5HT2 agonists stimulated PI turnover and/or mobilized [Ca2+]i in 20 of 24 TM cell strains isolated from up to 24 different human donor eyes, thus indicating a high degree of responsiveness to serotonergic agonists in the current population of donor cells studied. 5HT2A receptor agonists (e.g., α-methyl-5HT, (R)-DOI) or nonselective 5HT2 agonists (e.g., 5-methoxy tryptamine) were the most potent compounds when tested in the PI turnover (Table 2)and [Ca2+]i mobilizing (Table 3)assays in h-TM cells, whereas agonists claimed to be relatively selective for 5HT2B receptors (e.g., BW-723C86) 28 and 5HT2C receptors (MK-212; mCPP) 29 30 31 32 were weak agonists, exhibiting much lower intrinsic activities than other agonists tested (Tables 2 3) . These observations strongly suggest that the predominant receptor being expressed and functionally active in the h-TM cells was the 5HT2A receptor subtype. We tried first to correlate these observations with the relative receptor binding affinities of various compounds at the recombinant human 5HT2A-C receptors but without success. However, a better comparison was found between functional agonist potencies in h-TM and recombinant human 5HT2 receptor subtypes. The functional potencies of various 5HT2 agonists were somewhat different when determined in the aequorin-based [Ca2+]i mobilizing assays using cloned human 5HT2A–C receptors (Table 5)relative to the potency values obtained from h-TM cells (Tables 3 6) . However, in general the relative receptor selectivity indicated by the cloned receptor data helped to confirm the conclusion that the 5HT2A receptor was being predominantly expressed in the h-TM cells. This conclusion was further supported by antagonist data (Figs. 2 4 , bottom; Table 6 ). Thus, the 5HT2A-selective antagonist, M-100907, was the most potent antagonist in the TM cell PI turnover assays and in the [Ca2+]i mobilizing assays, being more than 1000 times more potent that antagonists for the 5HT2B and 5HT2C receptors. 28 29 30 31 32 There was a good correlation (r = 0.81; P < 0.02) between the relative functional potencies of agonists and antagonists mediating PI hydrolysis and mobilizing [Ca2+]i in h-TM cells (Fig. 5) . Likewise, the pharmacology of the [Ca2+]i mobilizing and antagonism of these responses in the h-TM cells correlated well with that of the cloned human 5HT2A receptor (r = 0.74, P < 0.05), but not with cloned human 5HT2B (r = 0.27) or 5HT2C (r = 0.22) receptors. This further confirmed the nature of the predominant functional receptor in h-TM cells. Of note, a 5HT2 receptor also appears to be involved in promoting PI hydrolysis in bovine ciliary epithelium as defined by the pharmacological sensitivity of this system. 33 Similarly, 5HT2 receptors appear to mediate the contraction of bovine ciliary muscle 34 ; hence, the 5HT2 receptor system is present in the target cells and tissues involved in aqueous humor dynamics in different species. Functional receptors of the 5HT3 and 5HT7 receptor family have previously been described in human and bovine iris-ciliary body, 35 36 whereas 5HT1A receptor–binding sites have been detected in rabbit iris-ciliary body tissue homogenates. 11 Therefore, a variety of 5HT receptor families are represented in the ocular tissues of the anterior chamber and are presumably involved in diverse physiological effects in the eye, perhaps including aqueous humor dynamics. 
As a corollary to the in vitro results obtained from isolated human TM cells we wanted to study the effects of various serotonergic agents on their ability to modulate IOP in vivo. Because such studies are difficult to conduct in human subjects, we decided that nonhuman primates would suffice, considering that the cynomolgus monkey model has successfully predicted IOP-lowering efficacy of numerous classes of compounds in human subjects. 3 4 5 27 Accordingly, various 5HT agents were tested for their effects on IOP in conscious ocular hypertensive cynomolgus monkey eyes. It was gratifying to observe that 5HT2 agonists, either 5HT2A-selective or nonselective, were the most efficacious IOP-lowering compounds found among numerous serotonergic agents tested (Table 7) . Thus, the most potent and efficacious agent was (R)-DOI followed by α-methyl-5HT and 5-methoxy-N,N-dimethyl-tryptamine, all with equal or greater efficacy than positive control ocular hypotensive agents, brimonidine, levobetaxolol, pilocarpine, and travoprost (Table 7) . Although one 5HT2C agonist (mCPP) lowered IOP (22.8%, P < 0.05), another 5HT2C agonist, MK-212, was essentially inactive (Table 6) . A 5HT2B-selective agonist, BW-723C86 (18.0%, P > 0.05), also lacked ocular hypotensive activity, further emphasizing that 5HT2A receptors predominantly are involved in lowering IOP in the ocular hypertensive cynomolgus monkey eyes. Whereas 5HT1A agonists such as 8-OH-DPAT, DP-5-CT, and flesinoxan exhibited ocular hypotensive activities in rabbit eyes, 11 12 14 15 17 these compounds, and nonselective 5HT receptor antagonists such as metergoline (16.9%, P > 0.05) and methiothepin (18.3% P < 0.05) had noticeably minimal effects on IOP in cynomolgus monkey eyes (Table 7) . Thus, there are significant species differences in the ocular hypotensive activity of serotonergic compounds, and it is imperative that IOP effects of compounds be determined in humans or at least nonhuman primates to permit some degree of extrapolation to the human situation. It is also clear from data in Table 7that agonists and/or antagonists of non-5HT2 receptor families, such as 5HT3, 5HT4, 5HT6, and 5HT7, were devoid of ocular hypotensive activity, thus further emphasizing the involvement of the 5HT2 receptor in the IOP-lowering activity of certain serotonergic agonist compounds. 
Historically, a few reports have suggested that antagonists of 5HT2 receptors, such as ketanserin and its analogues 18 19 20 37 38 39 and sarpogrelate (Takenaka H, et al. IOVS 1995;36:ARVO Abstract 3390) are ocular hypotensive agents in animals and humans. However, it is clear now that it is the α-1 adrenergic antagonist activity that is responsible for the IOP-lowering actions of these compounds 13 18 40 and not their 5HT2 antagonist activity. Of note, however, numerous 5HT2A-C receptor antagonists, including ketanserin, ritanserin, M-100907, 41 SB-206553, RS-102221, SB-242084, 42 even up to 300 μg topical ocular doses, failed to lower IOP in the conscious ocular hypertensive cynomolgus monkey eyes (Table 7) . Thus, those earlier observations require careful scrutiny. 
Whereas it is not possible to ascribe a precise mechanism of action of 5HT2A agonists for lowering IOP in monkey eyes, one report 43 suggested that an increase in uveoscleral outflow was induced by (R)-DOI in normotensive cynomolgus monkey eyes. Whether uveoscleral outflow, coupled with conventional trabecular meshwork outflow, is activated by 5HT2A agonists in ocular hypertensive monkey eyes remains to be determined. It is worth noting, however, that even though uveoscleral outflow is predominantly stimulated by FP-class prostaglandins, 2 3 4 5 recent observations of FP-receptor-mediated relaxation of endothelin-induced TM contraction 44 supports a conventional outflow component for FP-agonist–mediated IOP reduction. 44 Consequently, we can speculate that a similar conventional outflow component of 5HT2-agonist–mediated ocular hypotension may prevail, because we have demonstrated the presence of 5HT2 receptors in h-TM cells (this study) akin to our previously published work on FP-prostaglandin receptors in h-TM cells. 45 However, much more work is needed to demonstrate the precise mechanism(s) by which 5HT2 agonists lower IOP in ocular hypertensive monkeys. 
In summary, our in vitro studies demonstrated the presence of mRNAs for 5HT2A and 5HT2B receptors in h-TM cells, with a functional expression predominantly of the 5HT2A receptor signal transduction mechanisms, including PI hydrolysis followed by [Ca2+]i mobilization in these cells isolated from several human donor eye TM tissues. These conclusions were derived from extensive pharmacological studies involving numerous agonists and antagonists. As a corollary to these studies, we were able to demonstrate that indeed it is the 5HT2 receptor, again predominantly the 5HT2A subtype, which is involved in lowering IOP in conscious ocular hypertensive cynomolgus monkey eyes. Therefore, 5HT2A receptor agonists represent a novel class of ocular hypotensive agents worthy of further pursuit for glaucoma therapy. 
 
Figure 1.
 
5HT2 receptor subtype mRNA signals in TM cells. h-TM cells isolated from four to five donor eye TM tissues were processed for RT-PCR studies to determine the presence of the mRNAs for the different 5HT2 receptor subtypes and the housekeeping enzyme glycerol-3-phosphate dehydrogenase (G3PDH). Human brain tissue isolates were used as positive controls for each receptor subtype. Note the high level of expression of 5HT2A and 5HT2B receptor mRNAs.
Figure 1.
 
5HT2 receptor subtype mRNA signals in TM cells. h-TM cells isolated from four to five donor eye TM tissues were processed for RT-PCR studies to determine the presence of the mRNAs for the different 5HT2 receptor subtypes and the housekeeping enzyme glycerol-3-phosphate dehydrogenase (G3PDH). Human brain tissue isolates were used as positive controls for each receptor subtype. Note the high level of expression of 5HT2A and 5HT2B receptor mRNAs.
Table 1.
 
5HT Receptor mRNAs Found in Human Ocular (h-TM) Cells and Other Anterior Uveal Tissues
Table 1.
 
5HT Receptor mRNAs Found in Human Ocular (h-TM) Cells and Other Anterior Uveal Tissues
5HT2A 5HT2B 5HT2C 5HT3 5HT4 5HT5 5HT6 5HT7
h-TM cells +++ +++ + + +
h-Ciliary body +++ ++ ++ ND ND ND ND +++
Figure 2.
 
Production of [3H]-IPs in h-TM cells in response to 5HT agonists and antagonists. Top: generation of [3H]-IPs in h-TM cells in response to two agonists, serotonin, and α-methyl serotonin, after the cells had been preincubated with [3H]-myo-inositol for 24 hours at 37°C. Bottom: antagonism of the 5HT-induced PI hydrolysis by a 5HT2A-selective antagonist (M-100907) and by a 5HT2C-selective antagonist (RS-102221). Data are the mean ± SEM from more than three experiments, except for RS-102221 which is from one experiment, for illustrative purposes.
Figure 2.
 
Production of [3H]-IPs in h-TM cells in response to 5HT agonists and antagonists. Top: generation of [3H]-IPs in h-TM cells in response to two agonists, serotonin, and α-methyl serotonin, after the cells had been preincubated with [3H]-myo-inositol for 24 hours at 37°C. Bottom: antagonism of the 5HT-induced PI hydrolysis by a 5HT2A-selective antagonist (M-100907) and by a 5HT2C-selective antagonist (RS-102221). Data are the mean ± SEM from more than three experiments, except for RS-102221 which is from one experiment, for illustrative purposes.
Table 2.
 
Serotonergic Agonist Potencies and Intrinsic Activities in h-TM Cell PI Turnover Assays
Table 2.
 
Serotonergic Agonist Potencies and Intrinsic Activities in h-TM Cell PI Turnover Assays
Compound Reported or Purported Receptor Selectivity Agonist Potency in PI Turnover Assays (EC50, nM) Intrinsic Activity (E max, % Relative to 5HT)
(R)-DOI 5HT2A agonist 13 ± 5 78 ± 10
α-methyl-5HT 5HT2A agonist 140 ± 60 128 ± 16
5-methoxy-α-methyl tryptamine Nonselective agonist 147 ± 35 110 ± 8
5-methoxy tryptamine Nonselective agonist 181 ± 72 95 ± 20
5-methoxy-dimethyl tryptamine Nonselective agonist 247 ± 107 62 ± 12
5HT Nonselective agonist 364 ± 75 107 ± 4
BW-723C86 5HT2B agonist >1000 58
Figure 3.
 
Mobilization of [Ca2+]i in h-TM cells in response to different concentrations of serotonin (top) and α-methyl serotonin (bottom). The data shown represent the actual traces for [Ca2+]i mobilization over seconds on exposure to the various concentrations of these agonists. The peak response amplitudes from such assays were used to construct the concentration–response curves in Figure 4(top).
Figure 3.
 
Mobilization of [Ca2+]i in h-TM cells in response to different concentrations of serotonin (top) and α-methyl serotonin (bottom). The data shown represent the actual traces for [Ca2+]i mobilization over seconds on exposure to the various concentrations of these agonists. The peak response amplitudes from such assays were used to construct the concentration–response curves in Figure 4(top).
Figure 4.
 
Mobilization of [Ca2+]i in h-TM cells. Top: concentration–response curves to serotonin and α-methyl serotonin for [Ca2+]i mobilization; bottom: antagonism of 5HT-induced response by various 5HT2 receptor subtype-selective antagonists. Data are the mean ± SEM from more than three experiments for the agonist and antagonist studies.
Figure 4.
 
Mobilization of [Ca2+]i in h-TM cells. Top: concentration–response curves to serotonin and α-methyl serotonin for [Ca2+]i mobilization; bottom: antagonism of 5HT-induced response by various 5HT2 receptor subtype-selective antagonists. Data are the mean ± SEM from more than three experiments for the agonist and antagonist studies.
Table 3.
 
Serotonergic Agonist Potency in the h-TM Cell [Ca2+]i Mobilization Assays
Table 3.
 
Serotonergic Agonist Potency in the h-TM Cell [Ca2+]i Mobilization Assays
Compound Reported or Purported Receptor Selectivity Agonist Potency; [Ca2+]i Mobilization (EC50, nM) Intrinsic Activity; [Ca2+]i Mobilization (Emax, % relative to 5HT)
(R)-DOI 5HT2A agonist 18 ± 6
α-Methyl-5HT 5HT2A agonist 22 ± 3 112 ± 5
5-Methoxy tryptamine Nonselective agonist 8 ± 4 118 ± 14
5-Methoxy-dimethyl tryptamine Nonselective agonist 64 ± 27 68 ± 5
5HT Nonselective agonist 40 ± 5 107 ± 5
BW-723C86 5HT2B agonist 1213 ± 210 54 ± 6
mCPP 5HT2C agonist >1000 15 ± 4
MK-212 5HT2C agonist >1000 25
Table 4.
 
Serotonergic Agonist Affinities at Cloned Human 5HT2 Receptor Subtypes
Table 4.
 
Serotonergic Agonist Affinities at Cloned Human 5HT2 Receptor Subtypes
Compound Inhibition Constant at 5HT2A Receptor (K i; nM) Inhibition Constant at 5HT2B Receptor (K i; nM) Inhibition Constant at 5HT2C Receptor (K i; nM)
(R)-DOI 1 ± 0.1 18 ± 3 4 ± 1
5HT 8 ± 2 13 ± 5 8 ± 3
α-Methyl-5HT 12 ± 2 13 ± 3 7 ± 1
5-Methoxy-dimethyl tryptamine 15 ± 2 52 ± 2 42 ± 8
Table 5.
 
Serotonergic Agonist Potencies for Mobilizing [Ca2+]i and/or GTP-γ-S35 Binding to Human Cloned 5HT2 Receptors
Table 5.
 
Serotonergic Agonist Potencies for Mobilizing [Ca2+]i and/or GTP-γ-S35 Binding to Human Cloned 5HT2 Receptors
Compound Reported or Purported Selectivity Potency at Cloned 5HT2A Receptor (EC50; nM) Potency at Cloned 5HT2B Receptor (EC50; nM) Potency at Cloned 5HT2C Receptor (EC50; nM)
(R)-DOI 5HT2A 1 ± 0.2 4 ± 1 1 ± 0.1
0.3 ± 0.1* 17 ± 2*
5HT Nonselective 6 ± 4 7 ± 4 0.2 ± 0.1
63 ± 11* 44 ± 11
α-Methyl-5HT 5HT2A 5 ± 2 7 ± 0.2 3 ± 1
146 ± 29* 39 ± 6
5-Methoxy-dimethyl tryptamine Nonselective 152 ± 60* ND 226 ± 39*
BW723C86 5HT2B 63 ± 22 3 ± 1 9 ± 3
mCPP 5HT2C 32 ± 8 26 ± 2 2 ± 1
MK-212 5HT2C 529 ± 198 230 ± 77 8 ± 3
Figure 5.
 
Correlation plot for the relative potencies of agonists and antagonists determined in h-TM cells in the PI turnover and [Ca2+]i mobilization assays. pEC50 = −Log EC50; pIC50 = −Log IC50.
Figure 5.
 
Correlation plot for the relative potencies of agonists and antagonists determined in h-TM cells in the PI turnover and [Ca2+]i mobilization assays. pEC50 = −Log EC50; pIC50 = −Log IC50.
Table 6.
 
5HT2 Receptor Subtype Selective Antagonist Potencies against h-TM Cell Functional Responses
Table 6.
 
5HT2 Receptor Subtype Selective Antagonist Potencies against h-TM Cell Functional Responses
5HT2 Antagonist Reported Receptor Selectivity Antagonist Potency in [Ca2+]i Mobilization Assays (K i, nM)
M-100907 5HT2A 1 ± 0.3
RS-127445 5HT2B >10,000
RS-102221 5HT2C 4,400 ± 1,400
SDZ-SER082 5HT2B/5HT2C 1,000 ± 100
Table 7.
 
IOP-Lowering Activities of Selected Compounds in Conscious Ocular Hypertension Monkeys
Table 7.
 
IOP-Lowering Activities of Selected Compounds in Conscious Ocular Hypertension Monkeys
Reported/Purported Receptor Selectivity Topical Ocular Dose (μg/eye) Peak % IOP Reduction (SEM)
Compound
 (R)-8-OH-DPAT 5HT1A agonist 300 6.3 (4.5)*
 DP-5-CT 5HT1A agonist; 5HT7 agonist 500 11.9 (1.7)*
 Flesinoxan 5HT1A agonist; α1 agonist 250 11.6 (1.9)*
 GR-43175C 5HT1D agonist 250 2.8 (2.0)
500 6.4 (3.3)
 MDL-73005EF 5HT1A antagonist 500 8.3 (5.0)
 (R)-DOI 5HT2A agonist 50 21.3 (3.3), †
100 34.4 (5.0), †
300 27.7 (5.8), †
300 34.4 (6.7), †
 α-methyl-5HT 5HT2A agonist 150 30.9 (3.8), †
250 30.8 (7.7), †
 BW723C86 5HT2B agonist 150 16.6 (5.3)
300 18.0 (5.0)
 mCPP 5HT2C agonist 300 22.8 (6.0), †
300 19.1 (3.7)
 MK-212 5HT2C agonist 150 13.0 (3.0)
300 12.1 (3.2)
 5HT Nonselective 5HT agonist 250 18.0 (5.1)
 5-methoxy-N,N-dimethyl tryptamine Nonselective 5HT2 agonist 25 17.2 (4.2), †
100 27.2 (7.3), †
300 30.2 (4.4), †
300 30.5 (5.9), †
 Clozapine Mixed 5HT agonist; muscarinic agonist 150 14.3 (4.6)
 Ketanserin 5HT2 antagonist; α1 antagonist 300 3.4 (6.4)*
 Cinanserin 5HT2 antagonist 100 9.2 (2.3)
300 6.8 (2.2)
 Ritanserin 5HT2 antagonist; α1 antagonist 300 10.2 (3.6)*
 M-100907 5HT2A antagonist 300 15.8 (3.8)*
 SB-206553 5HT2B/C antagonist 150 15.1 (4.5)
300 13.4 (4.4)
 RS-102221 5HT2C antagonist 300 7.9 (2.6)
 SB-242084 5HT2C antagonist 300 12.2 (1.7), †
 Bufotenine 5HT3 agonist 30 13.8 (3.0)
100 8.9 (5.0)
300 26.1 (5.1), †
 Quipazine 5HT3 antagonist 300 12.9 (5.1)
 R/S-Zacopride 5HT3 antagonist; 5HT4 agonist 500 8.6 (1.8)
 SDZ-205557 5HT4 antagonist 300 14.7 (3.4)
 SB-258510A 5HT6 antagonist 300 20.1 (5.0), †
 (+)-SB-258719 5HT7 antagonist 300 12.8 (3.3)
 Methiothepin Nonselective 5HT antagonist 30 18.3 (4.0), †
100 10.0 (4.1)
 Metergoline Nonselective 5HT antagonist 250 16.9 (3.6)
 Fluoxetine (caused intense discomfort) 5HT uptake inhibitor 100 22.3 (6.3), †
Positive controls
 Brimonidine α2-agonist 150 31.2 (6.3), †
 Levobetaxolol β-antagonist 150 25.9 (3.2), †
 Pilocarpine Muscarinic agonist 150 27.9 (6.0), †
150 29.7 (4.6), †
300 34.0 (3.8), †
300 35.4 (5.0), †
600 28.6 (3.7), †
600 32.1 (4.1), †
 Travoprost Prostaglandin FP agonist 0.1 22.7 (5.8), †
0.3 25.7 (5.1), †
1 28.7 (3.5), †
 Latanoprost Prostaglandin FP agonist 1 32.3 (3.6), †
3 25.7 (5.1), †
10 28.8 (5.8), †
The authors thank Debbie Lane and Robin Chambers for providing h-TM cells; Allan Shepard for isolating total RNA from some of the cell samples for RT-PCR studies conducted at University of Waterloo (Canada) under contract with Michelle Senchyna and Chris May; and Daniel Scott, Tony Wallace, and Terri Cage for their expert technical assistance with the monkey IOP studies. 
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Figure 1.
 
5HT2 receptor subtype mRNA signals in TM cells. h-TM cells isolated from four to five donor eye TM tissues were processed for RT-PCR studies to determine the presence of the mRNAs for the different 5HT2 receptor subtypes and the housekeeping enzyme glycerol-3-phosphate dehydrogenase (G3PDH). Human brain tissue isolates were used as positive controls for each receptor subtype. Note the high level of expression of 5HT2A and 5HT2B receptor mRNAs.
Figure 1.
 
5HT2 receptor subtype mRNA signals in TM cells. h-TM cells isolated from four to five donor eye TM tissues were processed for RT-PCR studies to determine the presence of the mRNAs for the different 5HT2 receptor subtypes and the housekeeping enzyme glycerol-3-phosphate dehydrogenase (G3PDH). Human brain tissue isolates were used as positive controls for each receptor subtype. Note the high level of expression of 5HT2A and 5HT2B receptor mRNAs.
Figure 2.
 
Production of [3H]-IPs in h-TM cells in response to 5HT agonists and antagonists. Top: generation of [3H]-IPs in h-TM cells in response to two agonists, serotonin, and α-methyl serotonin, after the cells had been preincubated with [3H]-myo-inositol for 24 hours at 37°C. Bottom: antagonism of the 5HT-induced PI hydrolysis by a 5HT2A-selective antagonist (M-100907) and by a 5HT2C-selective antagonist (RS-102221). Data are the mean ± SEM from more than three experiments, except for RS-102221 which is from one experiment, for illustrative purposes.
Figure 2.
 
Production of [3H]-IPs in h-TM cells in response to 5HT agonists and antagonists. Top: generation of [3H]-IPs in h-TM cells in response to two agonists, serotonin, and α-methyl serotonin, after the cells had been preincubated with [3H]-myo-inositol for 24 hours at 37°C. Bottom: antagonism of the 5HT-induced PI hydrolysis by a 5HT2A-selective antagonist (M-100907) and by a 5HT2C-selective antagonist (RS-102221). Data are the mean ± SEM from more than three experiments, except for RS-102221 which is from one experiment, for illustrative purposes.
Figure 3.
 
Mobilization of [Ca2+]i in h-TM cells in response to different concentrations of serotonin (top) and α-methyl serotonin (bottom). The data shown represent the actual traces for [Ca2+]i mobilization over seconds on exposure to the various concentrations of these agonists. The peak response amplitudes from such assays were used to construct the concentration–response curves in Figure 4(top).
Figure 3.
 
Mobilization of [Ca2+]i in h-TM cells in response to different concentrations of serotonin (top) and α-methyl serotonin (bottom). The data shown represent the actual traces for [Ca2+]i mobilization over seconds on exposure to the various concentrations of these agonists. The peak response amplitudes from such assays were used to construct the concentration–response curves in Figure 4(top).
Figure 4.
 
Mobilization of [Ca2+]i in h-TM cells. Top: concentration–response curves to serotonin and α-methyl serotonin for [Ca2+]i mobilization; bottom: antagonism of 5HT-induced response by various 5HT2 receptor subtype-selective antagonists. Data are the mean ± SEM from more than three experiments for the agonist and antagonist studies.
Figure 4.
 
Mobilization of [Ca2+]i in h-TM cells. Top: concentration–response curves to serotonin and α-methyl serotonin for [Ca2+]i mobilization; bottom: antagonism of 5HT-induced response by various 5HT2 receptor subtype-selective antagonists. Data are the mean ± SEM from more than three experiments for the agonist and antagonist studies.
Figure 5.
 
Correlation plot for the relative potencies of agonists and antagonists determined in h-TM cells in the PI turnover and [Ca2+]i mobilization assays. pEC50 = −Log EC50; pIC50 = −Log IC50.
Figure 5.
 
Correlation plot for the relative potencies of agonists and antagonists determined in h-TM cells in the PI turnover and [Ca2+]i mobilization assays. pEC50 = −Log EC50; pIC50 = −Log IC50.
Table 1.
 
5HT Receptor mRNAs Found in Human Ocular (h-TM) Cells and Other Anterior Uveal Tissues
Table 1.
 
5HT Receptor mRNAs Found in Human Ocular (h-TM) Cells and Other Anterior Uveal Tissues
5HT2A 5HT2B 5HT2C 5HT3 5HT4 5HT5 5HT6 5HT7
h-TM cells +++ +++ + + +
h-Ciliary body +++ ++ ++ ND ND ND ND +++
Table 2.
 
Serotonergic Agonist Potencies and Intrinsic Activities in h-TM Cell PI Turnover Assays
Table 2.
 
Serotonergic Agonist Potencies and Intrinsic Activities in h-TM Cell PI Turnover Assays
Compound Reported or Purported Receptor Selectivity Agonist Potency in PI Turnover Assays (EC50, nM) Intrinsic Activity (E max, % Relative to 5HT)
(R)-DOI 5HT2A agonist 13 ± 5 78 ± 10
α-methyl-5HT 5HT2A agonist 140 ± 60 128 ± 16
5-methoxy-α-methyl tryptamine Nonselective agonist 147 ± 35 110 ± 8
5-methoxy tryptamine Nonselective agonist 181 ± 72 95 ± 20
5-methoxy-dimethyl tryptamine Nonselective agonist 247 ± 107 62 ± 12
5HT Nonselective agonist 364 ± 75 107 ± 4
BW-723C86 5HT2B agonist >1000 58
Table 3.
 
Serotonergic Agonist Potency in the h-TM Cell [Ca2+]i Mobilization Assays
Table 3.
 
Serotonergic Agonist Potency in the h-TM Cell [Ca2+]i Mobilization Assays
Compound Reported or Purported Receptor Selectivity Agonist Potency; [Ca2+]i Mobilization (EC50, nM) Intrinsic Activity; [Ca2+]i Mobilization (Emax, % relative to 5HT)
(R)-DOI 5HT2A agonist 18 ± 6
α-Methyl-5HT 5HT2A agonist 22 ± 3 112 ± 5
5-Methoxy tryptamine Nonselective agonist 8 ± 4 118 ± 14
5-Methoxy-dimethyl tryptamine Nonselective agonist 64 ± 27 68 ± 5
5HT Nonselective agonist 40 ± 5 107 ± 5
BW-723C86 5HT2B agonist 1213 ± 210 54 ± 6
mCPP 5HT2C agonist >1000 15 ± 4
MK-212 5HT2C agonist >1000 25
Table 4.
 
Serotonergic Agonist Affinities at Cloned Human 5HT2 Receptor Subtypes
Table 4.
 
Serotonergic Agonist Affinities at Cloned Human 5HT2 Receptor Subtypes
Compound Inhibition Constant at 5HT2A Receptor (K i; nM) Inhibition Constant at 5HT2B Receptor (K i; nM) Inhibition Constant at 5HT2C Receptor (K i; nM)
(R)-DOI 1 ± 0.1 18 ± 3 4 ± 1
5HT 8 ± 2 13 ± 5 8 ± 3
α-Methyl-5HT 12 ± 2 13 ± 3 7 ± 1
5-Methoxy-dimethyl tryptamine 15 ± 2 52 ± 2 42 ± 8
Table 5.
 
Serotonergic Agonist Potencies for Mobilizing [Ca2+]i and/or GTP-γ-S35 Binding to Human Cloned 5HT2 Receptors
Table 5.
 
Serotonergic Agonist Potencies for Mobilizing [Ca2+]i and/or GTP-γ-S35 Binding to Human Cloned 5HT2 Receptors
Compound Reported or Purported Selectivity Potency at Cloned 5HT2A Receptor (EC50; nM) Potency at Cloned 5HT2B Receptor (EC50; nM) Potency at Cloned 5HT2C Receptor (EC50; nM)
(R)-DOI 5HT2A 1 ± 0.2 4 ± 1 1 ± 0.1
0.3 ± 0.1* 17 ± 2*
5HT Nonselective 6 ± 4 7 ± 4 0.2 ± 0.1
63 ± 11* 44 ± 11
α-Methyl-5HT 5HT2A 5 ± 2 7 ± 0.2 3 ± 1
146 ± 29* 39 ± 6
5-Methoxy-dimethyl tryptamine Nonselective 152 ± 60* ND 226 ± 39*
BW723C86 5HT2B 63 ± 22 3 ± 1 9 ± 3
mCPP 5HT2C 32 ± 8 26 ± 2 2 ± 1
MK-212 5HT2C 529 ± 198 230 ± 77 8 ± 3
Table 6.
 
5HT2 Receptor Subtype Selective Antagonist Potencies against h-TM Cell Functional Responses
Table 6.
 
5HT2 Receptor Subtype Selective Antagonist Potencies against h-TM Cell Functional Responses
5HT2 Antagonist Reported Receptor Selectivity Antagonist Potency in [Ca2+]i Mobilization Assays (K i, nM)
M-100907 5HT2A 1 ± 0.3
RS-127445 5HT2B >10,000
RS-102221 5HT2C 4,400 ± 1,400
SDZ-SER082 5HT2B/5HT2C 1,000 ± 100
Table 7.
 
IOP-Lowering Activities of Selected Compounds in Conscious Ocular Hypertension Monkeys
Table 7.
 
IOP-Lowering Activities of Selected Compounds in Conscious Ocular Hypertension Monkeys
Reported/Purported Receptor Selectivity Topical Ocular Dose (μg/eye) Peak % IOP Reduction (SEM)
Compound
 (R)-8-OH-DPAT 5HT1A agonist 300 6.3 (4.5)*
 DP-5-CT 5HT1A agonist; 5HT7 agonist 500 11.9 (1.7)*
 Flesinoxan 5HT1A agonist; α1 agonist 250 11.6 (1.9)*
 GR-43175C 5HT1D agonist 250 2.8 (2.0)
500 6.4 (3.3)
 MDL-73005EF 5HT1A antagonist 500 8.3 (5.0)
 (R)-DOI 5HT2A agonist 50 21.3 (3.3), †
100 34.4 (5.0), †
300 27.7 (5.8), †
300 34.4 (6.7), †
 α-methyl-5HT 5HT2A agonist 150 30.9 (3.8), †
250 30.8 (7.7), †
 BW723C86 5HT2B agonist 150 16.6 (5.3)
300 18.0 (5.0)
 mCPP 5HT2C agonist 300 22.8 (6.0), †
300 19.1 (3.7)
 MK-212 5HT2C agonist 150 13.0 (3.0)
300 12.1 (3.2)
 5HT Nonselective 5HT agonist 250 18.0 (5.1)
 5-methoxy-N,N-dimethyl tryptamine Nonselective 5HT2 agonist 25 17.2 (4.2), †
100 27.2 (7.3), †
300 30.2 (4.4), †
300 30.5 (5.9), †
 Clozapine Mixed 5HT agonist; muscarinic agonist 150 14.3 (4.6)
 Ketanserin 5HT2 antagonist; α1 antagonist 300 3.4 (6.4)*
 Cinanserin 5HT2 antagonist 100 9.2 (2.3)
300 6.8 (2.2)
 Ritanserin 5HT2 antagonist; α1 antagonist 300 10.2 (3.6)*
 M-100907 5HT2A antagonist 300 15.8 (3.8)*
 SB-206553 5HT2B/C antagonist 150 15.1 (4.5)
300 13.4 (4.4)
 RS-102221 5HT2C antagonist 300 7.9 (2.6)
 SB-242084 5HT2C antagonist 300 12.2 (1.7), †
 Bufotenine 5HT3 agonist 30 13.8 (3.0)
100 8.9 (5.0)
300 26.1 (5.1), †
 Quipazine 5HT3 antagonist 300 12.9 (5.1)
 R/S-Zacopride 5HT3 antagonist; 5HT4 agonist 500 8.6 (1.8)
 SDZ-205557 5HT4 antagonist 300 14.7 (3.4)
 SB-258510A 5HT6 antagonist 300 20.1 (5.0), †
 (+)-SB-258719 5HT7 antagonist 300 12.8 (3.3)
 Methiothepin Nonselective 5HT antagonist 30 18.3 (4.0), †
100 10.0 (4.1)
 Metergoline Nonselective 5HT antagonist 250 16.9 (3.6)
 Fluoxetine (caused intense discomfort) 5HT uptake inhibitor 100 22.3 (6.3), †
Positive controls
 Brimonidine α2-agonist 150 31.2 (6.3), †
 Levobetaxolol β-antagonist 150 25.9 (3.2), †
 Pilocarpine Muscarinic agonist 150 27.9 (6.0), †
150 29.7 (4.6), †
300 34.0 (3.8), †
300 35.4 (5.0), †
600 28.6 (3.7), †
600 32.1 (4.1), †
 Travoprost Prostaglandin FP agonist 0.1 22.7 (5.8), †
0.3 25.7 (5.1), †
1 28.7 (3.5), †
 Latanoprost Prostaglandin FP agonist 1 32.3 (3.6), †
3 25.7 (5.1), †
10 28.8 (5.8), †
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