ABSTRACT
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.
Subject(s)
Epithelial Cells/chemistry , Eye/metabolism , RNA, Messenger/analysis , Receptors, Purinergic P2/genetics , Animals , Ciliary Body/chemistry , Conjunctiva/chemistry , Cornea/chemistry , Endothelium, Corneal/chemistry , Epithelium, Corneal/chemistry , Gene Expression , Goblet Cells/chemistry , In Situ Hybridization/methods , Lens, Crystalline/chemistry , Macaca mulatta , Meibomian Glands/chemistry , Rabbits , Receptors, Purinergic P2Y2ABSTRACT
OBJECTIVE: To determine the expression of P2Y(2) receptors in vaginal and cervical tissues and the effects of P2Y(2) receptor agonists INS45973 and INS365 on vaginal moisture. DESIGN: Pilot in vivo and histological study using animal subjects. SETTING: Experimental laboratory research. ANIMAL(S): Female New Zealand White rabbits were used for in vivo studies and female cynomolgus monkey (Macaca fascicularis) was used for in situ hybridization. INTERVENTION(S): Rabbits were kept intact or ovariectomized. Two weeks after ovariectomy, animals received daily vaginal instillation of vehicle or drugs for 16 days. MAIN OUTCOME MEASURE(S): Vaginal moisture was assessed in rabbits on 4 separate days during the treatment period. The P2Y(2) receptor mRNA distribution was assessed by in situ hybridization of monkey vagina and cervix. RESULT(S): Compared to control, vaginal moisture was significantly diminished in ovariectomized animals treated with vehicle. INS365 (8.1%) and INS45973 (0.9%) increased vaginal moisture in ovariectomized animals to levels that were comparable to or significantly higher than control animals, respectively. In situ hybridization studies indicated that P2Y(2) receptor mRNA was localized to endocervical and cervical gland, epithelium, and stratified squamous epithelium of the vagina. CONCLUSION(S): INS45973 and INS365 may interact with P2Y(2) receptors in the cervix and vagina to stimulate vaginal moisture in the estrogen (E)-deprived state. The P2Y(2) receptor agonists provide a potential nonhormonal alternative for treating vaginal dryness in postmenopausal women.
