Human Ciliary Muscle Cells Research Articles

Human Non-Pigmented Ciliary Epithelium Bradykinin B2-Receptors: Receptor Localization, Pharmacological Characterization of Intracellular Ca2+ Mobilization, and Prostaglandin Secretion

Purpose: To characterize the bradykinin (BK) receptor system in human non-pigmented ciliary epithelium (NPCE) using immunohistochemistry and functional cell-based techniques.Methods: B2-receptor protein expression was studied in sections of human donor eyes and in Cynomolgus monkey eyes using immunohistochemical methods. The pharmacological characteristics of intracellular Ca2+ ([Ca2+]i) mobilization in response to BK and related peptides, and blockade by two antagonists, was studied in primary human (p-h-NPCE) and in immortalized human NPCE (imh-NPCE) cells. Prostaglandins (PGs) release induced by BK was also studied in both cell-types using ELISA assays. Limited studies on primary human ciliary muscle (h-CM) cells and human trabecular meshwork (h-TM) cells and Chinese hamster ovary cells expressing human cloned B2-receptors (CHO-B2) were performed to compare with responses in both the NPCE cell-types.Results: B2-receptor immunoreactivity was observed on human and Cynomolgus monkey NPCE cells on eye sections from both species. BK and related analog peptides differentially activated signaling mechanisms in NPCE cells by mobilizing [Ca2+]i, and the BK-evoked responses were blocked by B2-receptor-selective antagonists, HOE-140 and (S)-WIN-64338. Relative agonist potencies (EC50, nM) in p-h-NPCE cells [and in imh-NPCE cells] were: BK = 3.4 ± 0.4 [6.3 nM]; Hyp3-BK EC50 = 1.7 ± 0.2 [6.0 nM], Lys-BK EC50 = 7.0 ± 0.3 [19.8 nM]; Met-Lys-BK EC50 = 106 ± 57.8 [125 nM]; Des-Arg9-BK EC50 = >10,000 [16 µM]. The antagonist potencies for attenuating BK-induced mobilization of [Ca2+]i in these cells were: HOE-140 (Ki = 7.9 ± 1.8 nM, n = 4) and (S)-WIN-64338 (Ki = 451 ± 44 nM, n = 4). These NPCE cell data correlated well with those obtained for h-CM and h-TM cells, and with B2-receptor binding (r = 0.99, p < 0.0001). However, BK failed to stimulate total PGs production in both NPCE cell-types even though 10% bovine serum increased PG release (by 4.9-fold above baseline), and even though BK stimulated PG release from h-CM, h-TM and in CHO-B2 cells. BK (1 µM) also failed to increase nitric oxide (NO) levels in NPCE cells even though sodium nitropruside increased NO production by 3-fold.Conclusions: Human and monkey NPCE express immunoreactive B2-receptor proteins. These proteins were functionally active, since BK and related peptides potently stimulated mobilization of [Ca2+]i in p-h-NPCE and imNPCE cells that was blocked by two B2-selective antagonists. Down-stream signaling from B2-receptor activation did not appear to involve PG synthesis/release (or NO production) in NPCE cell-types under the present conditions, even though h-CM, h-TM and CHO-B2 cells exhibited robust PG synthesis and release in response to BK.

C6orf176: a novel possible regulator of cAMP-mediated gene expression

cAMP mediates diverse cellular signals including prostaglandin (PG) E(2)-mediated intraocular pressure (IOP)-lowering activity in human ocular ciliary smooth muscle cells (hCSM). We have identified gene regulatory networks and key genes upon activation of the cAMP pathway in hCSM, using novel agonists highly selective for PGE(2) receptor subtypes EP2 or EP4, which are G protein-coupled receptors well known to activate cAMP signaling. Here we describe a novel, EP2/EP4-induced, primate-specific gene of hitherto unknown function, also known as C6orf176 (chromosome 6 open reading frame 176) and recently reclassified as noncoding RNA in NCBI's database. Its expression, as determined by quantitative real-time RT-PCR (qRT-PCR), is dramatically upregulated (>2,000-fold) subsequent to transduction of EP2/EP4/Gs/cAMP signaling not only in hCSM, but also in HEK cells overexpressing the recombinant receptors. Moreover, activation of other IOP lowering, Gs-coupled prostanoid receptors, such as DP1 and IP, as well as a direct activator of adenylyl cyclase, forskolin, also substantially upregulated C6orf176 in hCSM, while FP and TP, which are Gq-coupled prostanoid receptor subtypes, did not. Novel transcript variants carrying open reading frames, derived from an at least 67 kb genomic locus on chromosome 6q27 with putative alternative transcription start sites, were identified. Transcriptional upregulation of transcript variants as well as of two genes expressed in antisense orientation that partially overlap the transcribed C6orf176 region was observed, to varying degrees, subsequent to induction of cAMP signaling using various agonists. Small interfering RNA-mediated C6orf176 gene silencing experiments showed modulation of several cAMP-responsive genes. These transcriptional activities identify C6orf176 as a potential biomarker and/or therapeutic target in context with diseases linked to deregulated cAMP signaling. Also, the cAMP-inducible C6orf176 gene locus could be useful as a model system for studying transcriptional regulation by chromatin and RNA polymerase II.

Prostaglandin E2receptor subtype EP2- and EP4-regulated gene expression profiling in human ciliary smooth muscle cells

Prostanoids are an important class of intraocular pressure (IOP)-lowering antiglaucoma agents that act primarily via increased uveo-scleral aqueous humor outflow through the ciliary body. We have developed two novel PGE(2) analogs that are specific agonists for the PGE(2) receptor subtypes EP2 and EP4, respectively. To identify gene regulatory networks and key players that mediate the physiological effects observed in vivo, we performed genomewide expression studies using human ciliary smooth muscle cells. Quantitative real-time RT-PCR confirmed a largely overlapping gene expression profile subsequent to EP2 and EP4 agonist treatment, with 65 significantly regulated genes identified overall, 5 being specific for the EP2 agonist and 6 specific for the EP4 agonist. We found predicted functional cAMP-response elements in promoter regions of a large fraction of the predominantly upregulated genes, which suggests that the cAMP signaling pathway is the most important intracellular signaling pathway for these agonists in these cells. Several target genes were identified that, as part of complex regulatory networks, are implicated in tissue remodeling processes and osmoregulation (e.g., AREG, LOXL3, BMP2, AQP3) and thus may help elucidate the mechanism of action of these IOP-lowering drugs involving the uveo-scleral outflow path.

Induction of Angiogenic Immediate Early Genes by Activation of FP Prostanoid Receptors in Cultured Human Ciliary Smooth Muscle Cells

Purpose: Agonists of the F prostanoid receptor for prostaglandin F2α exert. exert an ocular hypotensive effect that has been attributed to increased aqueous humor outflow through the uveoscleral pathway. Although tissue remodeling of the ciliary muscle has been described, the signaling mechanisms that link activation of the FP receptor to remodeling of the ciliary muscle are poorly understood. Herein, we describe the identification of novel signaling mechanisms that may contribute to this process.Materials and Methods: Cultures of human ciliary smooth muscle cells were established from fetal eye tissue explants. The cells were validated by their expression of α-smooth muscle-actin and functional FP receptors. Cultures were treated with prostaglandin F2α and examined for the induction of three immediate early genes related to tissue remodeling using Western blot analysis, quantitative real-time polymerase chain reaction, and reporter gene assays.Results: Human ciliary smooth muscle cells express functional FP receptors whose activation up-regulates the expression of early growth response factor-1 and connective tissue growth factor at the mRNA and protein levels. Prostaglandin F2α stimulation also increases the protein expression of hypoxia-inducible factor-1α and activates luciferase reporter plasmids under the control of the hypoxia response element.Conclusions: Early growth response factor-1 and hypoxia-inducible factor-1α are important transcriptional activators of downstream genes involved in tissue remodeling and angiogenesis, whereas connective tissue growth factor is a secreted growth factor that also contributes to these processes. Thus, stimulation of FP receptors in human ciliary smooth muscle cells up-regulates the expression of immediate early genes that may coordinate the remodeling of the ciliary muscle, thereby facilitating aqueous outflow.

Effect of Bimatoprost, Latanoprost, and Unoprostone on Matrix Metalloproteinases and Their Inhibitors in Human Ciliary Body Smooth Muscle Cells

Matrix metalloproteinase (MMP)-mediated turnover of extracellular matrix (ECM) affects outflow resistance in the uveoscleral pathway. The balance of MMPs and tissue inhibitors of metalloproteinases (TIMPs) governs the rate of ECM turnover in many tissues. The hypothesis was that a differential effect on MMPs and TIMPs in ciliary body smooth muscle (CBSM) cells would relate to the relative intraocular pressure-lowering effectiveness of the prostaglandin analogues (PGAs) bimatoprost, latanoprost, and unoprostone. Human CBSM cells isolated from donor corneoscleral rims were incubated for 24 hours with control (0.015% ethanol in DMEM) or the free acid forms of bimatoprost (0.01 or 0.1 microg/mL), latanoprost (0.03 or 0.3 microg/mL), or unoprostone (0.145 or 1.45 microg/mL). Western blot analysis determined the relative protein concentrations of MMP-1, -2, -3. -9, and -24 as well as TIMP-1 through -4. Zymography measured the relative activity levels of MMP-1, -2, -3, and -9. All PGAs increased MMP-1, -3, and -9. Bimatoprost and latanoprost did not change MMP-2. Unoprostone decreased MMP-2 (21% +/- 3%). On zymography, MMP-1 and -2 did not change. Bimatoprost and latanoprost increased MMP-9 activity by 75% +/- 27% and 75% +/- 24%, respectively. MMP-3 activity was not detected on zymography. All PGAs increased TIMP-3, but only unoprostone increased TIMPs1 and -4 by 100% +/- 20% and 61% +/- 11%, respectively. TIMP-2 was unchanged by bimatoprost and latanoprost, but decreased by unoprostone (35% +/- 8%). Decreased MMP-2 with concurrent increases of TIMP-1 and -4 by unoprostone may explain the lower clinical efficacy of unoprostone. The MMP/TIMP balance relates to the observed intraocular pressure-lowering effectiveness in clinical studies with PGAs.

Expression of the kallikrein/kinin system in human anterior segment

Tissue kallikrein acts on the substrate, low molecular weight kininogen, to liberate bradykinin in a variety of tissues. Bradykinin stimulation of B 2 kinin receptors has been shown to initiate signaling in trabecular meshwork cells and increase conventional outflow facility. The objective of the present study was to determine if the components for kinin generation and response are expressed in tissues of the human anterior segment. Expression of mRNA encoding tissue kallikrein (KK), low molecular weight kininogen, and B 1 and B 2 kinin receptors was examined in human ciliary smooth muscle (CM), trabecular meshwork (TM) and non-pigmented epithelial (NPE) cells using RT-PCR. Expression of component proteins was also investigated by immunohistochemical analyses performed on parasagittal sections of human anterior segment and TM cells, and by immunoblot. KK mRNA was detected in NPE cells and in cultured CM and TM cells from multiple donors. Each cell type also expressed mRNAs encoding both B 1 and B 2 kinin receptors. Immunohistochemical analysis of KK protein in sectioned anterior segment supported the RT-PCR results. Intense KK immunofluorescence was observed in the epithelial lining of the ciliary body and KK protein was also detected in the ciliary muscle. KK protein expression within the TM was demonstrated by analyses of TM tissue and cultured TM cells. The presence of KK along with B 1 and B 2 receptor proteins was confirmed by immunoblots of cell lysates prepared from CM, NPE or TM cells. Finally, both CM and TM cells were found to possess enzymes for bradykinin inactivation. These data demonstrate that key components for kinin generation and regulation are localized within the human anterior segment. Further, multiple cell types express both B 1 and B 2 kinin receptors and are targets for kinin action. The results support the possibility that kinins produced within the eye may contribute to the regulation of aqueous outflow.

Cabergoline: Pharmacology, ocular hypotensive studies in multiple species, and aqueous humor dynamic modulation in the Cynomolgus monkey eyes

The aims of the current studies were to determine the in vitro and in vivo ocular and non-ocular pharmacological properties of cabergoline using well documented receptor binding, cell-based functional assays, and in vivo models. Cabergoline bound to native and/or human cloned serotonin-2A/B/C (5HT2A/B/C), 5HT1A, 5HT7, α2B, and dopamine-2/3 (D2/3) receptor subtypes with nanomolar affinity. Cabergoline was an agonist at human recombinant 5HT2, 5HT1A and D2/3 receptors but an antagonist at 5HT7 and α2 receptors. In primary human ciliary muscle (h-CM) and trabecular meshwork (h-TM) cells, cabergoline stimulated phosphoinositide (PI) hydrolysis (EC50=19±7nM in TM; 76nM in h-CM) and intracellular Ca2+ ([Ca2+]i) mobilization (EC50=570±83nM in h-TM; EC50=900±320nM in h-CM). Cabergoline-induced [Ca2+]i mobilization in h-TM and h-CM cells was potently antagonized by a 5HT2A-selective antagonist (M-100907, Ki=0.29–0.53nM). Cabergoline also stimulated [Ca2+]i mobilization more potently via human cloned 5HT2A (EC50=63.4±10.3nM) than via 5HT2B and 5HT2C receptors. In h-CM cells, cabergoline (1μM) stimulated production of pro-matrix metalloproteinases-1 and -3 and synergized with forskolin to enhance cAMP production. Cabergoline (1μM) perfused through anterior segments of porcine eyes caused a significant (27%) increase in outflow facility. Topically administered cabergoline (300–500μg) in Dutch-belted rabbit eyes yielded 4.5μM and 1.97μM levels in the aqueous humor 30min and 90min post-dose but failed to modulate intraocular pressure (IOP). However, cabergoline was an efficacious IOP-lowering agent in normotensive Brown Norway rats (25% IOP decrease with 6μg at 4h post-dose) and in conscious ocular hypertensive cynomolgus monkeys (peak reduction of 30.6±3.6% with 50μg at 3h post-dose; 30.4±4.5% with 500μg at 7h post-dose). In ketamine-sedated monkeys, IOP was significantly lowered at 2.5h after the second topical ocular dose (300μg) of cabergoline by 23% (p<0.02) and 35% (p<0.004) in normotensive and ocular hypertensive eyes, respectively. In normotensive eyes, cabergoline increased uveoscleral outflow (0.69±0.7μL/min–1.61±0.97μL/min, n=13; p<0.01). However, only seven of the eleven ocular hypertensive monkeys showed significantly increased uveoscleral outflow. These data indicate that cabergoline's most prominent agonist activity involves activation of 5HT2, 5HT1A, and D2/3 receptors. Since 5HT1A agonists, 5HT7 antagonists, and α2 antagonists do not lower IOP in conscious ocular hypertensive monkeys, the 5HT2 and dopaminergic agonist activities of cabergoline probably mediated the IOP reduction observed with this compound in this species.

Role of PKCεin PGF2α-Stimulated MMP-2 Secretion from Human Ciliary Muscle Cells

Studies were designed to examine the roles of individual protein kinase C (PKC) isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-induced matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Studies utilized primary cultures of human ciliary muscle cells. Individual PKC isoforms were detected by Western blotting, using PKC-isoform-specific antibodies. To evaluate MMP-2 secretion, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 4 h and the media analyzed for MMP-2 by Western blotting. To assess ERK1/2 activation, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 5 min and cell lysates analyzed for ERK1/2 phosphorylation by Western blot analysis. To evaluate the roles of individual PKC isoforms, cells were pretreated with PKC inhibitors or siRNAs prior to the addition of PGF(2alpha). In cultured human ciliary muscle cells, the PKC isoforms exhibiting the highest level of expression were PKCalpha, epsilon, iota and lambda. The delta and eta isoforms exhibited moderate levels of expression and beta, gamma, and phi were not detected. The administration of PGF(2alpha) (1 micromol/L) primarily induced the translocation of PKCepsilon from cytosol to the membrane fraction, as well as increased MMP-2 secretion and ERK1/2 phosphorylation. The secretion of MMP-2 was inhibited by pretreatment with the broad-range PKC inhibitor, chelerythrine chloride; however, this response was not blocked by Go-6976, an inhibitor of conventional PKC isoforms. The PGF(2alpha)-induced secretion of MMP-2 was also blocked by pretreatment with the PKCepsilon-selective peptide translocation inhibitor, EAVSLKPT, or the transfection of siRNA-targeting PKCepsilon. The activation of ERK1/2 was inhibited by chelerythrine and the PKCepsilon translocation inhibitor. Human ciliary muscle cells express the alpha, epsilon, iota and lambda PKC isoforms. Stimulation of FP receptors in these cells activates PKCepsilon, resulting in ERK1/2 activation and an eventual increase in MMP-2 secretion. These data support the idea that the activation of FP receptors in vivo modulate uveoscleral outflow through the PKCepsilon-dependent secretion of MMPs.

Cat Iris Sphincter Smooth-Muscle Contraction: Comparison of FP-Class Prostaglandin Analog Agonist Activities

The pharmacologic characteristics of a number of FP-class prostaglandin (PG) analogs were determined by using the cat iris sphincter smooth-muscle-contraction assay. Cumulative concentration-response curves were generated for each compound. The relative agonist potencies (EC(50)) of the compounds were: cloprostenol (0.0012 +/- 0.0004 nM) >> travoprost acid (0.46 +/- 0.13 nM) = bimatoprost acid (0.99 +/- 0.19 nM) > (+/-)-fluprostenol (15.8 +/- 2.6 nM) = PGF(2alpha) (18.6 +/- 1.8 nM) > latanoprost acid (29.9 +/- 1.6 nM) > bimatoprost (140 +/- 45 nM) > S-1033 (588 +/- 39 nM) > unoprostone (UF-021; 1280 +/- 50 nM; n = 4-14). The maximum response induced by travoprost acid (122% +/- 2.3% maximum response relative to PGF(2alpha)) was significantly greater than that induced by all the other PG compounds (P < 0.001 - P < 0.02). Interestingly, the FP-receptor antagonist, AL-8810, behaved as a moderate efficacy partial agonist (EC(50) = 2140 +/- 190 nM; 63 +/- 4.3% maximum response relative to PGF(2alpha)), indicating that the cat iris contains an extremely well-coupled FP-receptor population, and/or the tissue contains an extremely high density of the FP-receptor and/or spare receptors. The cat iris contraction data were well correlated with other FP-receptor-mediated signal-transduction processes, including FP-receptor binding in bovine corpus luteum (r = 0.86), FP-receptor binding in human iris (r = 0.61), phosphoinositide (PI) hydrolysis in human ciliary muscle and trabecular meshwork cells (r = 0.77 - 0.86), PI turnover in rat and mouse cells (r = 0.73 - 0.76) and via cloned human FP-receptor (r = 0.9), and rat uterus contraction (r = 0.84). These data confirm the presence of functional FP-receptors in the cat iris sphincter, which are exquisitely well coupled and which respond to a variety of FP-class PG analogs with differing potencies.

Serotonin-2 (5-HT2) Receptor-Mediated Signal Transduction in Human Ciliary Muscle Cells: Role in Ocular Hypotension

The aim of this study was to characterize the serotonin (5-hydroxytryptamine; 5-HT) receptors that mediate phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) mobilization in isolated cells of human ciliary muscle (h-CM) from multiple donors using a variety of agonists and antagonists. An additional aim was to visualize the mRNAs and receptor binding sites for 5-HT2 receptors in human ciliary body (h-CB), CM, and other tissues by reverse transcriptase polymerase chain reaction and quantitative autoradiography techniques, respectively, and to correlate with ocular hypotensive activity of such compounds. CBs isolated from several donor eyes revealed the presence of 5-HT(2A2C) receptor mRNAs. [3H]-5-HT and [3H]-ketanserin autoradiography on sections of human eyes revealed a high density of 5-HT2 receptor binding sites in the iris, ciliary epithelium, and longitudinal CM. In isolated h-CM cells, the agonists alpha-methyl-5-HT (EC50=63+/-17 nM), 5-HT (EC50=85+/-16 nM), (R)-DOI (EC50=165+/-47 nM), and 5-methoxy alpha-methyl tryptamine (EC50=1200+/-270 nM) differentially stimulated PI turnover. These agonists also mobilized [Ca2+]i in h-CM cells with the following potencies (EC50s): 5-methoxy-tryptamine=42+/-11 nM; alpha-methyl-5-HT=36+/-11 nM; (R)-DOI=120 nM; 5-HT=130+/-36 nM; MK-212=470 nM; mCPP>1 microM; BW723C86=1766 nM. The agonist-induced [Ca2+]i mobilization in h-CM cells was potently blocked by the 5-HT2A-selective antagonist M-100907 (IC50=1.2+/-0.4 nM) but less potently by the antagonists for 5-HT2B (RS-127445, IC50>10 microM) and 5-HT2C (RS-102221, IC50=5.8+/-2.3 microM) receptors. In conclusion, h-CB, h-CM, and CM cells express mRNAs and proteins for 5-HT2 receptor subtypes, of which the predominant functionally active subtype is the 5-HT2A receptor, as defined by agonist and antagonist activities. These receptors may be responsible for mediating the intraocular pressure reduction observed in recent literature with a number of 5-HT2 agonists, such as (R)-DOI, alpha-methyl-5HT, and AL-34662.

Preclinical Pharmacology of AL-12182, a New Ocular Hypotensive 11-Oxa Prostaglandin Analog

The aim of this study was to determine selected in vivo ocular properties of AL-12182 (5,6-dihydro-4,5-didehydro-11-deoxy-11-oxa-16-(3-chlorophenoxy)-omega-tetranor-PGF(2alpha) isopropyl ester) and the in vitro profile of its free acid, AL-12180. Previously documented radioligand binding and functional assays involving human ciliary muscle cells (h-CM), human trabecular meshwork (h-TM) and other cells, and porcine ocular arteries were utilized. For in vivo procedures, we utilized rabbits, cats, and nonhuman primates to measure hyperemia, pupil diameter, and intraocular pressure (IOP), respectively. AL-12180 exhibited the highest affinity for the FP-receptor (K(i) = 143 +/- 36 nM) and much lower affinity for DP-, EP(3)-, IP-, and TP-receptors, and for several nonprostanoid receptors, enzymes, neurotransmitter uptake sites, ion channels, and other regulatory sites. AL-12180 activated phospholipase C-mediated phosphoinositide hydrolysis (potency, EC(50) = 13.7-42.7 nM) through the FP-receptor in a variety of cells, such as h-CM, h-TM cells, human embryonic kidney cells expressing the cloned human ciliary body FP-receptor (HEK-FP), mouse 3T3 cells, and rat vascular smooth muscle cells. AL-8810, an FP-antagonist, blocked the effects of AL-12180 in h-CM cells (IC(50) = 8.7 microM). AL-12180 also stimulated the mobilization of intracellular Ca(2+) ([Ca(2+)](i)) in h-TM cells (EC(50) = 111 +/- 36 nM), h-CM cells (EC(50) = 11 nM), and in host cells expressing the cloned human ciliary body FP-receptor (EC(50) = 5.9 +/- 3.1 nM). AL-12180 lacked significant agonist activity at DP-, EP(2)-, EP(4)-, IP-, and TP-receptors in cell-based assays. However, AL-12180 contracted porcine central retinal and short posterior ciliary arteries in vitro with micromolar potencies that appeared to involve TP-receptor activation. in vivo, AL-12182 elicited dose-related hyperemia in the rabbit eye, miosis in the cat eye, and ocular hypotension in the nonhuman primate eye. AL-12180 is a relatively potent and selective FP-receptor agonist whose isopropyl ester prodrug (AL-12182) lowers IOP by as much as 40% following topical ocular dosing in a laser-induced nonhuman primate model of ocular hypertension.

Acute Effects of PGF2αon MMP-2 Secretion from Human Ciliary Muscle Cells: A PKC- and ERK-Dependent Process

Studies were designed to evaluate the cellular mechanisms associated with prostaglandin (PG)F(2alpha)-induced matrix metalloproteinase (MMP)-2 secretion from human ciliary muscle (HCM) cells. The secretion and activity of MMP-2 was determined by Western blot analysis and zymography, using conditioned medium and HCM cells. ERK1/2 activity was measured by in-gel kinase assay and Western blot analysis with anti-phospho-ERK1/2 antibodies. PGF(2alpha) increased the secretion of MMP-2 in a dose-dependent manner with an EC(50) of 2.7 x 10(-8) M. The addition of 1 muM PGF(2alpha) also increased MMP-2 secretion in a time-dependent manner with maximum secretion occurring at 4 hours after administration. At 4 hours, the maximum increase in MMP-2 secretion and activity were 112% +/- 32% and 88% +/- 18%, respectively. The secretory action of PGF(2alpha) was inhibited by pretreatment with a protein kinase C (PKC) inhibitor, chelerythrine chloride; the FP receptor antagonist, AL-8810; and the MEK inhibitor, PD-98059. The addition of PGF(2alpha) and latanoprost acid increased ERK1/2 activity by 117% +/- 12% and 75% +/- 9%, respectively. The PGF(2alpha)- and latanoprost-acid-induced ERK1/2 activation was blocked by the presence of PKC inhibitors and downregulation of PKC by prolonged incubation with a phorbol ester. These data provide evidence that FP receptor activation leads to an increase in the secretion and activation of MMP-2 through PKC- and ERK1/2-dependent pathways. FP-agonist-induced activation of ERK1/2 was blocked by PKC inhibitors, indicating that PKC activation is required for ERK1/2 activation and MMP-2 secretion from HCM cells. In the ciliary muscle, the functional responses to ERK1/2 activation include secretion of MMP-2, supporting the hypothesis that increases in uveoscleral outflow facility induced by PG administration involves the secretion and activation of MMP-2.

P.3.136 Asenapine has a unique human receptor binding signature

The aims of the current studies were to determine the in vitro and in vivo ocular and non-ocular pharmacological properties of cabergoline using well documented receptor binding, cell-based functional assays, and in vivo models. Cabergoline bound to native and/or human cloned serotonin-2A/B/C (5HT2A/B/C), 5HT1A, 5HT7, α2B, and dopamine-2/3 (D2/3) receptor subtypes with nanomolar affinity. Cabergoline was an agonist at human recombinant 5HT2, 5HT1A and D2/3 receptors but an antagonist at 5HT7 and α2 receptors. In primary human ciliary muscle (h-CM) and trabecular meshwork (h-TM) cells, cabergoline stimulated phosphoinositide (PI) hydrolysis (EC50 = 19 ± 7 nM in TM; 76 nM in h-CM) and intracellular Ca2+ ([Ca2+]i) mobilization (EC50 = 570 ± 83 nM in h-TM; EC50 = 900 ± 320 nM in h-CM). Cabergoline-induced [Ca2+]i mobilization in h-TM and h-CM cells was potently antagonized by a 5HT2A-selective antagonist (M-100907, Ki = 0.29–0.53 nM). Cabergoline also stimulated [Ca2+]i mobilization more potently via human cloned 5HT2A (EC50 = 63.4 ± 10.3 nM) than via 5HT2B and 5HT2C receptors. In h-CM cells, cabergoline (1 μM) stimulated production of pro-matrix metalloproteinases-1 and -3 and synergized with forskolin to enhance cAMP production. Cabergoline (1 μM) perfused through anterior segments of porcine eyes caused a significant (27%) increase in outflow facility. Topically administered cabergoline (300–500 μg) in Dutch-belted rabbit eyes yielded 4.5 μM and 1.97 μM levels in the aqueous humor 30 min and 90 min post-dose but failed to modulate intraocular pressure (IOP). However, cabergoline was an efficacious IOP-lowering agent in normotensive Brown Norway rats (25% IOP decrease with 6 μg at 4 h post-dose) and in conscious ocular hypertensive cynomolgus monkeys (peak reduction of 30.6 ± 3.6% with 50 μg at 3 h post-dose; 30.4 ± 4.5% with 500 μg at 7 h post-dose). In ketamine-sedated monkeys, IOP was significantly lowered at 2.5 h after the second topical ocular dose (300 μg) of cabergoline by 23% (p < 0.02) and 35% (p < 0.004) in normotensive and ocular hypertensive eyes, respectively. In normotensive eyes, cabergoline increased uveoscleral outflow (0.69 ± 0.7 μL/min–1.61 ± 0.97 μL/min, n = 13; p < 0.01). However, only seven of the eleven ocular hypertensive monkeys showed significantly increased uveoscleral outflow. These data indicate that cabergoline's most prominent agonist activity involves activation of 5HT2, 5HT1A, and D2/3 receptors. Since 5HT1A agonists, 5HT7 antagonists, and α2 antagonists do not lower IOP in conscious ocular hypertensive monkeys, the 5HT2 and dopaminergic agonist activities of cabergoline probably mediated the IOP reduction observed with this compound in this species.

Human ciliary muscle cell responses to FP-class prostaglandin analogs: phosphoinositide hydrolysis, intracellular Ca2+ mobilization and MAP kinase activation.

Phospholipase C induced phosphoinositide (PI) turnover, intracellular Ca(2+) ([Ca(2+)](i)) mobilization and mitogen-activated protein (MAP) kinase activation by FP-class prostaglandin analogs was studied in normal human ciliary muscle (h-CM) cells. Agonist potencies obtained in the PI turnover assays were: travoprost acid ((+)-fluprostenol; EC(50) = 2.6 +/- 0.8 nM) > bimatoprost acid (EC(50) = 3.6 +/- 1.2 nM) > (+/-)-fluprostenol (EC(50) = 4.3 +/- 1.3 nM) >> prostaglandin F(2 alpha) (PGF(2 alpha)) (EC(50) = 134 +/- 17 nM) > latanoprost acid (EC(50) = 198 +/- 83 nM) > S-1033 (EC(50) = 2930 +/- 1420 nM) > unoprostone (EC(50) = 5590 +/- 1490 nM) > bimatoprost (EC(50) = 9600 +/- 1100 nM). Agonist potencies in h-CM cells correlated well with those previously obtained for the cloned human ciliary body-derived FP receptor (r = 0.96, p< 0.001) and that present on h-TM cells (r = 0.94, p< 0.0001). Travoprost acid, PGF(2 alpha) and unoprostone also stimulated [Ca(2+)](i) mobilization in h-CM cells with travoprost acid being the most potent agonist. MAP kinase activity was stimulated in the h-CM cells with the following rank order of activity (at 100 nM): travoprost acid > PGF(2 alpha) > latanoprost acid > PGD(2) > bimatoprost > latanoprost = bimatoprost acid = fluprostenol > PGE(2) = S-1033 > unoprostone > PGI(2). The PI turnover, [Ca(2+)](i) mobilization and MAP kinase activation induced by several of these agonists was blocked by the FP receptor antagonist, AL-8810 (11 beta-fluoro-15-epiindanyl PGF(2 alpha)) (e.g. K(i) = 5.7 microM versus PI turnover). These studies have characterized the biochemical and pharmacological properties of the native FP prostaglandin receptor present on h-CM cells using three signal transduction mechanism assays and a broad panel of FP-class agonist analogs (including free acids of bimatoprost, travoprost and latanoprost) and the FP receptor antagonist, AL-8810.

Comparison of Prostaglandin F2α, Bimatoprost (Prostamide), and Butaprost (EP2 Agonist) on Cyr61 and Connective Tissue Growth Factor Gene Expression

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.

Effects of prostaglandin analogues on human ciliary muscle and trabecular meshwork cells.

To determine the effects of prostaglandin F(2alpha) analogues on gene expression of human ciliary muscle (HCM) and trabecular meshwork (HTM) cells. Cultures of HCM and HTM cells were established from five different donors treated for 9 days with 10 microg/mL of either latanoprost (free acid) or prostaglandin F(2alpha) ethanolamide and compared with control cells. The mRNA from the cells of the five individual donors was pooled and analyzed by using gene microarrays. Gene expression changes were confirmed by either real-time PCR or relative quantitative PCR. Approximately 12 genes showed a twofold or greater change in expression under experimental conditions. Four of these may alter outflow. Aquaporin-1 and versican were downregulated in the HCM, whereas IGF1 and fibroleukin were upregulated in HTM. Expression levels of TNFSF10 and promelanosome-concentrating hormone also increased in the treated HTM cells. The mRNA levels for the prostaglandin FP receptor were downregulated in the ciliary muscle cells. Optineurin and alphaB-crystallin levels remained unchanged, but myocilin in the HTM cells was decreased in some samples. Both analogues changed gene expression similarly in either HCM or HTM cells, but the changes appeared to be cell specific, perhaps indicating that other transcription factors are influential. Outflow of aqueous humor may be increased by the prostaglandin analogues by alterations in the extracellular matrix. Other changes may influence cellular metabolism, such as the increases in IGF1, tumor necrosis factor superfamily-10 and promelanosome-concentrating hormone.

Studies on receptor binding and signal transduction pathways of unoprostone isopropyl.

We examined the binding characteristics of unoprostone isopropyl and its metabolite, M1 (M1), in bovine corpus luteum membranes, mobilization of intracellular calcium in human ciliary muscle cells and cyclic AMP generation in rabbit iris-ciliary body. The ligand binding assay of 3H-unoprostone isopropyl and M1 did not demonstrate any specific binding of these compounds in the bovine corpus luteum membranes. However, there was a high specific binding of prostaglandin F2alpha. Competitive ligand binding studies showed that neither the docosanoid, unoprostone isopropyl, nor M1 binds to prostaglandin receptor sites. In human ciliary muscle cells that express EP1, EP2 and FP receptors, unoprostone isopropyl did not increase the mobilization of intracellular calcium nor was it able to generate cyclic AMP at low concentrations in rabbit iris-ciliary body. Similar observations were made with M1 on the above signal transduction pathways. From these results, it is concluded that unoprostone isopropyl and M1 do not bind to prostaglandin (PG) receptor sites in the bovine corpus luteum membranes and do not have affinity for PG receptors linked to intracellular calcium and cyclic AMP second messenger systems.

Herpes Simplex Virus Mediated Gene Transfer to Primate Ocular Tissues

We evaluated the feasibility of delivering a gene into monkey eyes using a replication-competent herpes simplex virus (HSV) type 1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene. To determine the efficiency of in vitro HSV-mediated gene transfer, cultured human trabecular meshwork (HTM) and human ciliary muscle (HCM) cells were infected with hrR3 and β-galactosidase activity was measured histochemically. Six cynomolgus monkey eyes received viral injections into the anterior chamber (2×107pfu) and/or the vitreous (5×107pfu), and the distribution of cells expressing lacZ was evaluated. In vitro, both cultured HTM and HCM cells displayed multiplicity-dependent β-galactosidase activity. In vivo, intracameral and/or intravitreal injection resulted in transgene expression in TM cells and in non-pigmented ciliary epithelial cells (NPE), but not in CM cells. Transgene expression was also detected in retinal pigmented epithelial (RPE) cells and sporadic retinal ganglion cells (RGC) in eyes receiving virus intracamerally and intravitreally respectively. We observed significant inflammation in the anterior chamber, TM and CM in virus-injected eyes, along with mild vitritis and retinitis. This study demonstrates successful gene transfer using hrR3 as a vector in human ocular cells and in ocular tissues in living monkeys. Further investigation of the etiology of the inflammatory response, possible cytotoxicity, and limited duration of transgene expression is necessary in order to make this technique clinically applicable.

Effect of continuous administration of a cholinergic agonist on [3H]4-DAMP binding and m3 mRNA expression in cultured human ciliary muscle cells.

It is well known that chronic topical administration of cholinergic agonists results in a subsensitization in ciliary muscle-mediated increases in outflow facility and accommodation in monkey eyes in vivo. These physiologic changes are apparently mediated by the M3 subtype receptor. However, the nature of this subsensitization remains unclear. This study investigated the effect of the continuous presence of carbachol, a muscarinic agonist, on the expression of the muscarinic receptor subtype m3 and the binding of [3H]4-DAMP in cultured human ciliary muscle cells (H7CM). The H7CM cell line, derived from the ciliary muscle of a one-day-old human infant, was used in this study. Confluent monolayers were treated individually with 1 mM carbachol for 2, 6, 24 and 48 hours. The level of mRNA encoding muscarinic receptor subtype m3 was measured by RNase protection. For confirmation, a receptor binding assay was done using [3H]4-DAMP, a radioligand selective for M3 subtype receptors. At each timepoint, results were compared with untreated controls. Treatment with carbachol resulted in a down regulation ranging from 23.4% to 34.8% of m3 mRNA expression at all time points. All [3H]4-DAMP binding assay results also decreased, ranging from 24.5% to 31.0%.

The acute and long-term effect of olanzapine compared with placebo and haloperidol on serum prolactin concentration

The aims of the current studies were to determine the in vitro and in vivo ocular and non-ocular pharmacological properties of cabergoline using well documented receptor binding, cell-based functional assays, and in vivo models. Cabergoline bound to native and/or human cloned serotonin-2A/B/C (5HT2A/B/C), 5HT1A, 5HT7, α2B, and dopamine-2/3 (D2/3) receptor subtypes with nanomolar affinity. Cabergoline was an agonist at human recombinant 5HT2, 5HT1A and D2/3 receptors but an antagonist at 5HT7 and α2 receptors. In primary human ciliary muscle (h-CM) and trabecular meshwork (h-TM) cells, cabergoline stimulated phosphoinositide (PI) hydrolysis (EC50 = 19 ± 7 nM in TM; 76 nM in h-CM) and intracellular Ca2+ ([Ca2+]i) mobilization (EC50 = 570 ± 83 nM in h-TM; EC50 = 900 ± 320 nM in h-CM). Cabergoline-induced [Ca2+]i mobilization in h-TM and h-CM cells was potently antagonized by a 5HT2A-selective antagonist (M-100907, Ki = 0.29–0.53 nM). Cabergoline also stimulated [Ca2+]i mobilization more potently via human cloned 5HT2A (EC50 = 63.4 ± 10.3 nM) than via 5HT2B and 5HT2C receptors. In h-CM cells, cabergoline (1 μM) stimulated production of pro-matrix metalloproteinases-1 and -3 and synergized with forskolin to enhance cAMP production. Cabergoline (1 μM) perfused through anterior segments of porcine eyes caused a significant (27%) increase in outflow facility. Topically administered cabergoline (300–500 μg) in Dutch-belted rabbit eyes yielded 4.5 μM and 1.97 μM levels in the aqueous humor 30 min and 90 min post-dose but failed to modulate intraocular pressure (IOP). However, cabergoline was an efficacious IOP-lowering agent in normotensive Brown Norway rats (25% IOP decrease with 6 μg at 4 h post-dose) and in conscious ocular hypertensive cynomolgus monkeys (peak reduction of 30.6 ± 3.6% with 50 μg at 3 h post-dose; 30.4 ± 4.5% with 500 μg at 7 h post-dose). In ketamine-sedated monkeys, IOP was significantly lowered at 2.5 h after the second topical ocular dose (300 μg) of cabergoline by 23% (p < 0.02) and 35% (p < 0.004) in normotensive and ocular hypertensive eyes, respectively. In normotensive eyes, cabergoline increased uveoscleral outflow (0.69 ± 0.7 μL/min–1.61 ± 0.97 μL/min, n = 13; p < 0.01). However, only seven of the eleven ocular hypertensive monkeys showed significantly increased uveoscleral outflow. These data indicate that cabergoline's most prominent agonist activity involves activation of 5HT2, 5HT1A, and D2/3 receptors. Since 5HT1A agonists, 5HT7 antagonists, and α2 antagonists do not lower IOP in conscious ocular hypertensive monkeys, the 5HT2 and dopaminergic agonist activities of cabergoline probably mediated the IOP reduction observed with this compound in this species.