CELL-SPECIFIC EXPRESSION OF THE HUMAN NA-ATPASE BETA-2 SUBUNIT ISOFORM IN THE NONPIGMENTED CILIARY EPITHELIUM(,K+)

Purpose. To evaluate the patterns of expression of beta subunit isofor ms of the Na+,K+-ATPase and H+,K+-ATPase in the human eye and to deter mine the cell-specific distribution of the beta 2 subunit in the human ciliary epithelium. Methods, Total RNA extracted from human ocular ti ssues was screened by Northern blot analysis with cDNA probes for the human Na+,K+-ATPase subunit isoforms (beta 1 and beta 2) or the H+,K+- ATPase (alpha and beta) subunits. Antibodies were raised to the amino and carboxyl terminal regions of the human beta 2 isoform. Polymerase chain reaction was used to verify the expression of beta 2 subunit in nonpigmented ciliary epithelial cells (NPE). Results. Transcripts for the Na+,K+-ATPase beta 1 and beta 2 subunit isoforms were present at d ifferent levels in all the ocular tissues except the lens, which expre ssed only beta 1. No transcripts for the alpha or beta subunits of the H+,K+-ATPase were detected in the eye. Isoform beta 2 specific anti-p eptide antibodies V15E (N-terminus) and A18R (C-terminus) recognized a 55- to 60-kDa protein in the ciliary epithelium and the core protein of 32 kDa after N-glycanase treatment. Immunocytochemical localization within the ciliary epithelium indicates that the Na+,K+-ATPase beta 2 isoform is expressed preferentially in the NPE cells. The expression of Na+,K+-ATPase beta 2 isoform in the human NPE cell line, ODM-2, was confirmed by polymerase chain reaction amplification and Southern blo t analysis. Conclusions. The Na+,K+-ATPase beta 2 subunit isoform, but not H+,K+-ATPase, was expressed widely in ocular tissues of the human eye. The restricted cellular distribution of beta 2 isoform within th e NPE cells represents an important differential gene marker associate d with the multiple alpha subunit isoforms of Na+,K+-ATPase.