Purpose: To develop antibodies that selectively recognize each of the alpha 2-adrenergic receptor (AR) subtypes and to determine the expression and localization of these subtypes in the anterior segment of the human eye.
Methods: Recent studies have shown that there are three subtypes of the alpha 2-ARs, termed alpha 2-C10 (alpha 2A), alpha 2-C2 (alpha 2B), and alpha 2-C4 (alpha 2C). Polymerase chain reaction was used to amplify portions of these receptors fused (in-frame) to a cDNA encoding glutathione-S-transferase (GST). The expressed fusion proteins were used to immunize chickens, and antibodies were generated. Immunofluorescence microscopy was used to localize the alpha 2-AR subtypes in sections of human and rabbit ciliary body. Polymerase chain reaction and dot blot hybridization were used to determine which subtypes were present in RNA from primary cultures of human nonpigmented epithelium (NPE) and rabbit iris-ciliary body (ICB).
Results: Immunofluorescence microscopy of COS cells transfected with the alpha 2-AR subtypes showed that the antibodies raised against the GST-receptor fusion proteins specifically recognized their respective receptor subtypes. In the human ciliary body, alpha 2 B and alpha 2C immunoreactivity were present in the NPE and ciliary muscle. In the rabbit ciliary body, alpha 2A immunoreactivity also was present. Polymerase chain reaction and dot blot hybridization indicated that RNA encoding the alpha 2B and alpha 2C subtypes was present in human NPE and that RNA encoding all three subtypes was present in the rabbit ICB.
Conclusions: Multiple alpha 2-adrenergic subtypes are expressed in the ciliary body. In the human, alpha 2B and alpha 2C predominate, whereas all three are present in the rabbit. This could be important with respect to animal models of glaucoma and to the development of drugs for lowering intraocular pressure.