Prostaglandin E2 receptor 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.

The prostanoid EP2 receptor agonist butaprost increases uveoscleral outflow in the cynomolgus monkey.

PURPOSE: To investigate the ocular hypotensive effect of the prostanoid EP2 receptor agonist butaprost and to establish its mechanism of action. METHODS: All experiments were performed in cynomolgus monkeys after topical application of butaprost (0.1%). The effects of butaprost on aqueous humor flow were determined by fluorophotometry. Total outflow facility was measured by the two-level, constant-pressure perfusion method, and uveoscleral outflow was determined by perfusion of FITC-labeled dextran through the anterior chamber. Effects on ocular morphology were studied after tissue fixation with transcardial perfusion by paraformaldehyde and immersion fixation of the globe, in animals subjected to long-term treatment with butaprost. Conscious ocular normotensive monkeys and monkeys with unilateral ocular hypertension were used for intraocular pressure (IOP) studies. RESULTS: Butaprost had no significant effect on aqueous humor flow or total outflow facility in ocular normotensive monkeys. Uveoscleral outflow was significantly higher in the butaprost treated eyes than in vehicle treated eyes, 1.03 +/- 0.20 vs. 0.53 +/- 0.18 microL.min(-1). After a 1-year treatment with butaprost, the morphology of the ciliary muscle was changed, showing increased spaces between ciliary muscle bundles and the apparent formation of new outflow channels. In many instances, changes were observed in the trabecular meshwork as well. Butaprost, in a single 0.1% dose, decreased IOP significantly in ocular normotensive monkeys and reduced IOP in laser-induced glaucomatous monkey eyes to the same level as that in the ocular normotensive contralateral eyes. CONCLUSIONS: The prostanoid EP2 receptor agonist butaprost appears to lower IOP by increasing uveoscleral outflow, according to both physiological and morphologic findings. Although the prostanoid EP2 receptor is structurally and functionally distinct from the FP receptor, the effects of EP2 and FP receptor stimulation on aqueous humor outflow are similar.

Prostanoid EP4 receptor stimulation produces ocular hypotension by a mechanism that does not appear to involve uveoscleral outflow.

PURPOSE: As part of a systematic elucidation of the pharmacology of prostaglandin's (PG) effects on intraocular pressure in the monkey, the prototypical selective prostanoid EP(4) receptor agonist (3,7-dithia PGE(1)) was examined. It was found to be highly efficacious in nonhuman primates, and its mechanism of ocular hypotensive activity was investigated. METHODS: Intraocular pressure (IOP) was measured by pneumatonometry in conscious monkeys restrained in custom-designed chairs. All other animal experiments were performed in animals sedated with ketamine or anesthetized with ketamine/diazepam and given drug or vehicle for various lengths of time. Aqueous flow was determined by fluorophotometry. Total outflow facility was measured by the two-level, constant-pressure method and by 2-minute tonography in both normotensive and hypertensive monkey eyes. Uveoscleral outflow was measured by perfusing the anterior chamber with FITC-labeled dextran for 30 minutes at a fixed IOP of approximately 15 mm Hg. Isometric responses to drugs were measured in longitudinal and radial preparations of monkey and human isolated ciliary smooth muscle specimens. RESULTS: The selective EP(4) receptor agonist 3,7-dithia PGE(1) and an isopropyl ester prodrug thereof reduced IOP in monkeys. A single dose of 3,7-dithia PGE(1) isopropyl ester, at a 0.01% or 0.1% dose, decreased IOP in the glaucomatous monkey in the range of 40% to 50%. Studies on total outflow facility by the two-level, constant-pressure perfusion method and tonography indicated that EP(4) receptor stimulation facilitated aqueous humor outflow facility. No effect on aqueous flow was apparent. In contrast to all PGs and prostamides studied to date, 3,7-dithia PGE(1) exerted no effect on uveoscleral outflow measured directly. Moreover, it did not relax longitudinal or radial preparations of isolated human or monkey ciliary muscles. CONCLUSIONS: The EP(4) receptor agonist 3,7-dithia PGE(1) is a highly efficacious IOP-lowering drug in monkeys. It has no effect on uveoscleral outflow but does increase total outflow facility, which accounts for a substantial proportion of the ocular hypotensive activity.

The prevention of colitis by E Prostanoid receptor 4 agonist through enhancement of epithelium survival and regeneration.

Inflammatory bowel disease (IBD) is often triggered and/or exacerbated by nonsteroidal anti-inflammatory drugs (NSAIDs). Among various prostanoids affected by NSAIDs, prostaglandin E2 (PGE2), in particular, seems to play critical roles in IBD via the EP4 receptor, one of the four PGE2 receptor subtypes (EP1-4). An EP4 agonist, [[3-[[(1R,2S,3R)-3-hydroxy-2-[(1E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]-1-butenyl]-5-oxocyclopentyl]thio]propyl]thio]-acetic acid, C22H30O6S2 (ONO-AE1-329), for example, when topically applied, has been reported to ameliorate typical colitis symptoms by suppressing the production of cytotoxic cytokines in the dextran sodium sulfate (DSS)-induced colitis model. EP4 agonists are also known, however, for their ability to protect epithelial cells from apoptosis in vitro, which may contribute to the protection of mucosal barrier functions. To investigate this potential application, we have tested another EP4-selective agonist in the DSS-indomethacin mouse colitis model. 7-[2-(3-Hydroxy-4-phenyl-but-1-enyl)-6-oxo-piperidin-1-yl]-heptanoic acid methyl ester, C23H33NO4 (AGN205203), an analog from the 8-azapiperidinone series of EP4 agonists, is metabolically and chemically more stable than the ONO agonist, because of its lack of oxidizable sulfur atoms in the alpha-chain and of 11-OH group, a potential source of beta-elimination reaction. Treatment of mice subcutaneously with AGN205203 at 3 mg/kg/day minimized colitis symptoms, such as weight loss, diarrhea, and colonic bleeding. Further histological examination of colons revealed healthy surface columnar epithelial cells free of erosion and ulceration compared with those without the drug treatment. At cellular level, the drug treatment decreased colon epithelial apoptosis, prevented goblet cell depletion, and promoted epithelial regeneration. AGN205203 may be unique among known EP4 agonists for its metabolic and chemical stability, and it is amenable to systemic applications for the prevention and recovery of IBD.

Finding of endocannabinoids in human eye tissues: implications for glaucoma.

Cannabinoid CB(1) receptors are involved in ocular physiology and may regulate intraocular pressure (IOP). However, endocannabinoid levels in human ocular tissues of cornea, iris, ciliary body, retina, and choroid from normal and glaucomatous donors have not been investigated. Anandamide (N-arachidonoylethanolamine; AEA), 2-arachidonoylglycerol (2-AG), and the anandamide congener, palmitoylethanolamide (PEA), were detected in all the human tissues examined. In eyes from patients with glaucoma, significantly decreased 2-AG and PEA levels were detected in the ciliary body, an important tissue in the regulation of IOP. The findings suggest that these endogenous compounds may have a role in this disease, particularly with respect to regulation of IOP.

Bimatoprost and prostaglandin F(2 alpha) selectively stimulate intracellular calcium signaling in different cat iris sphincter cells.

Bimatoprost is a synthetic analog of prostaglandin F(2 alpha) ethanolamide (prostamide F(2 alpha)), and shares a pharmacological profile consistent with that of the prostamides. Like prostaglandin F(2 alpha) carboxylic acid, bimatoprost potently lowers intraocular pressure in dogs, primates and humans. In order to distinguish its mechanism of action from prostaglandin F(2 alpha), fluorescence confocal microscopy was used to examine the effects of bimatoprost, prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) on calcium signaling in resident cells of digested cat iris sphincter, a tissue which exhibits contractile responses to both agonists. Constant superfusion conditions obviated effective conversion of bimatoprost. Serial challenge with 100 nM bimatoprost and prostaglandin F(2 alpha) consistently evoked responses in different cells within the same tissue preparation, whereas prostaglandin F(2 alpha) and 17-phenyl prostaglandin F(2 alpha) elicited signaling responses in the same cells. Bimatoprost-sensitive cells were consistently re-stimulated with bimatoprost only, and prostaglandin F(2 alpha) sensitive cells could only be re-stimulated with prostaglandin F(2 alpha). The selective stimulation of different cells in the same cat iris sphincter preparation by bimatoprost and prostaglandin F(2 alpha), along with the complete absence of observed instances in which the same cells respond to both agonists, strongly suggests the involvement of distinct receptors for prostaglandin F(2 alpha) and bimatoprost. Further, prostaglandin F(2 alpha) but not bimatoprost potently stimulated calcium signaling in isolated human embryonic kidney cells stably transfected with the feline- and human-prostaglandin F(2 alpha) FP-receptor and in human dermal fibroblast cells, and only prostaglandin F(2 alpha) competed with radioligand binding in HEK-feFP cells. These studies provide further evidence for the existence of a bimatoprost-sensitive receptor that is distinct from any of the known prostaglandin receptor types.

Vascular activities of prostaglandins and selective prostanoid receptor agonists in human retinal microvessels.

Prostanoid analogs have recently been introduced into clinical use for the management of increased intraocular pressure (IOP). This class of compounds is known to exert effects on vascular components and some endogenous parent prostaglandins have been shown to alter regional ocular blood flow and exhibit significant vasoactive properties in isolated ocular blood vessels, so the possibility exists that prostanoids could affect the ocular microcirculation either by absorption into the systemic circulation or by direct localized activity on the retinal microvasculature. Thus, the aim of this study was to examine systematically the effects of a broad variety of agonists that exhibit preferential activity at EP(1)-, EP(2)-, EP(3)-, FP-, DP-, IP-, and TP-prostanoid receptor sites on microvessel caliber in the microvasculature associated with human retinal tissues grafted into the hamster cheek pouch membrane. The selective DP-receptor agonist, BW245C and the selective TP-receptor agonist, U-46619, were the only compounds tested that exhibited significant vasoactive effects relative to baseline resting diameters in retinal microvessels. A dose-dependent increase in arteriolar caliber was elicited by BW245C over a concentration range of 10(-8)-10(-4)M at the tested 5- and 10-min timepoints. U-46619 evoked a sharp decrease in microvessel diameter within a 10(-7)-10(-4)M gamut, with the dose-response profiles at 5- and 10-min timepoints remaining essentially parallel over the tested range of concentrations. In contrast to the vasoconstriction induced by U-46619, retinal microvessel calibers were not markedly affected by AGN 192093, a thromboxane-like agonist with additional unique properties. No significant changes in human retinal arteriolar diameters relative to baseline were observed in response to a broad panel of parent and derived compounds known to be selective for EP-, FP- and IP-prostanoid receptors.