Ward M. Peterson

Localization of ocular P2Y2 receptor gene expression by in situ hybridization.

The objective of this study was to determine the cellular localization of P2Y(2) receptor gene expression in rabbit and primate ocular tissues using the technique of non-isotopic in situ hybridization. Fresh frozen whole eye from a New Zealand White rabbit and whole eye and eyelid from a rhesus macaque were cut into 5 microm thick sections and mounted onto glass slides. In situ hybridization was performed on ocular cryosections using digoxigenin-labeled P2Y(2) receptor riboprobes. Alkaline phosphatase-conjugated anti-digoxigenin antibody was used to localize riboprobe hybridization, which was subsequently visualized by staining with a precipitating alkaline phosphatase substrate. Cytoplasmic staining indicative of antisense riboprobe hybridization to P2Y(2) receptor mRNA was observed in the palpebral and bulbar conjunctival epithelium, including goblet cells, the corneal epithelium, and in meibomian gland sebaceous and ductal cells. Staining was also observed in both layers of the ciliary body epithelium, subcapsular epithelium of the lens, and corneal endothelium. In the posterior eye, staining was observed in various layers of the retina, including ganglion cell, inner nuclear, inner segment and retinal pigment epithelium layers, in the optic nerve head, and in a variety of structures within the choroid. No specific staining of sense riboprobe was seen on any of the ocular structures. These results demonstrate that the P2Y(2) receptor gene is expressed in a variety of ocular cells types and suggest that P2Y(2) receptors are associated with diverse physiological functions throughout the eye.

Effect of 5-mca-nat, a Putative Melatonin Mt3 Receptor Agonist, on Intraocular Pressure in Glaucomatous Monkey Eyes

Purpose5-MCA-NAT, a putative melatonin MT3 receptor agonist, reduced intraocular pressure (IOP) in ocular normotensive rabbit eyes. This study evaluates the effect of topical application of 5-MCA-NAT on IOP in monkey eyes with laser-induced unilateral glaucoma. MethodsA multiple-dose study was performed in 8 glaucomatous monkey eyes. One 25-μL drop of 5-MCA-NAT (2%) was applied topically to the glaucomatous eye at 9:30 am and 3:30 pm for 5 consecutive days. IOP was measured hourly for 6 hours beginning at 9:30 am for one baseline day, one vehicle-treated day, and treatment days 1, 3, and 5 with 5-MCA-NAT. ResultsCompared with vehicle treatment, twice daily administration of 5-MCA-NAT for 5 days reduced (P < 0.05) IOP from 1 hour to 5 hours after the first dose, and the IOP-lowering effects were shown to last at least 18 hours following administration, based on IOP measurements made after the fourth and eighth doses. The ocular hypotensive effect of 5-MCA-NAT was enhanced with repeated dosing. The maximum reduction (P < 0.001) of IOP occurred at 3 hours after each morning dose, and was 4.0 ± 0.5 (mean ± SEM) mm Hg (10%) on day 1, 5.6 ± 0.8 mm Hg (15%) on day 3, and 7.0 ± 1.1 mm Hg (19%) on day 5. Adverse ocular or systemic side effects were not observed during the 5 days of treatment. Conclusions5-MCA-NAT, a putative melatonin MT3 receptor agonist, reduces IOP in glaucomatous monkey eyes. Melatonin agonists with activity on the putative MT3 receptor may have clinical potential for treating elevated IOP.

Expression Profiling after Retinal Detachment and Reattachment : A Possible Role for Aquaporin-0

PURPOSE Retinal detachment (RD) is associated with acute visual loss caused by anatomic displacement of the photoreceptors and with chronic visual loss/disturbance caused by retinal remodeling and photoreceptor cell death, which may occur even after successful reattachment. The P2Y(2) receptor agonist INS37217 improves the rate of retinal reattachment in animal models of induced RD, and has been shown to also significantly enhance the rate of ERG recovery in a mouse model of RD. The identification of genes modulated by INS37217 may allow further drug discovery for treating RD and edema. METHODS To identify genes involved in RD and subsequent reattachment, a retinal microarray screen was performed using a mouse model of RD in the presence or absence of INS37217. RESULTS Ninety-two genes were identified as differentially expressed across three time points, most of which were upregulated in the presence of this agonist. Furthermore, it was shown that RD alters the expression of aquaporin-0 (AQP-0), and this modulation is prevented by treatment with INS37217. The presence of AQP-0 in retinal bipolar cells was also demonstrated, whereas it was previously thought to be specific to the lens. Mice lacking functional alleles of AQP-0 had a phototransduction deficit as assessed by electroretinography; however, their photoreceptor structure was normal, indicative of a problem with signal transmission between neurons. CONCLUSIONS This study establishes the genes involved in RD and reattachment, and also demonstrates for the first time a physiologically significant role for AQP-0 in retinal function.

Potency and Duration of Action of Synthetic P2Y2 Receptor Agonists on Schirmer Scores in Rabbits

P2Y2 receptors belong to the family of G-protein coupled receptors and are activated by nucleotides such as ATP and UTP.1,2 Activation of P2Y2 receptors (P2Y2-Rs) on various epithelial cell types enhances chloride, fluid and mucin secretion and thereby stimulates the body’s natural mucosal lubrication and clearance mechanisms.3,4 On the ocular surface, P2Y2-R mRNA is localized in various epithelial cells in the eyelid, cornea and conjunctiva. Previous work using conventional non-isotopic in situ hybridization techniques demonstrated localization of P2Y2-R mRNA in the palpebral conjunctiva of the eyelid, the epithelium of the lid margin and the meibomian gland; the corneal epithelium and adjacent bulbar conjunctiva, particularly the conjunctival goblet cells.5