LOCALIZATION OF ALPHA(2)-ADRENERGIC RECEPTOR SUBTYPES IN THE ANTERIORSEGMENT OF THE HUMAN EYE WITH SELECTIVE ANTIBODIES

Purpose, To develop antibodies that selectively recognize each of the alpha(2)-adrenergic receptor (AR) subtypes and to determine the expres sion and localization of these subtypes in the anterior segment of the human eye. Methods, Recent studies have shown that there are three su btypes of the alpha(2)-ARs, termed alpha(2)-C10 (alpha(2)A), alpha(2)- C2 (alpha(2)B), and alpha(2)-C4 (alpha(2)C). 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 p roteins were used to immunize chickens, and antibodies were generated. Immunofluorescence microscopy was used to localize the alpha(2)-AR su btypes in sections of human and rabbit ciliary body. Polymerase chain reaction and dot blot hybridization were used to determine which subty pes were present in RNA from primary cultures of human nonpigmented ep ithelium (NPE) and rabbit iris-ciliary body (ICE). Results, Immunofluo rescence microscopy of COS cells transfected with the alpha(2)-AR subt ypes 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(2)C immunoreactivity we re present in the NPE and ciliary muscle. In the rabbit ciliary body, alpha(2)A immunoreactivity also was present. Polymerase chain reaction and dot blot hybridization indicated that RNA encoding the alpha(2)B and alpha(2)C subtypes was present in human NPE and that RNA encoding all three subtypes was present in the rabbit ICE. Conclusions, Multipl e alpha(2)-adrenergic subtypes are expressed in the ciliary body. In t he human, alpha(2)B and alpha(2)C predominate, whereas all three are p resent in the rabbit. This could be important with respect to animal m odels of glaucoma and to the development of drugs for lowering intraoc ular pressure.