Expression of multiple Na+,K+‐ATPase genes reveals a gradient of isoforms along the nonpigmented ciliary epithelium: Functional implications in aqueous humor secretion

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Abstract
The Na+,K+‐ATPase α1, α2, and α3 subunit isoforms have been shown to be differentially expressed in the nonpigmented (NPE) and pigmented (PE) cells of the ocular ciliary epithelium (CE)(Martin‐Vasallo et al., J. Cell. Physiol., 141: 243‐252, 1989; Ghosh et al., J. Biol. Chem., 265:2935‐2940, 1990). In this study we analyzed and compared the pattern of expression of the multiple Na+,K+‐ATPase α (α1, α2, α3) subunit genes with the pattern of expression of the Na+,K+‐ATPase β (β1, β2) subunit genes along the bovine CE. We have selected three regions in the CE, referred to as (1) the anterior region of the pars plicata, near the iris; (2) the middle region of the pars plicata; and (3) the posterior region of the pars plana, near the ora serrata. Using isoform‐specific cDNA probes and antibodies for the Na+,K+‐ATPase α1, α2, α3, β1, and β2 subunits on Northern and Western blot analysis, we found that mRNA and polypeptides are expressed in all three CE regions with different abundance. The pattern of expression of α and β isoforms detected along the NPE cell layer suggests a gradient of α1, α2, α3, β1, and β2 mRNAs and polypeptides that correlates with decreasing Na+,K+‐ATPase activity from the most anterior region at the pars plicata towards the posterior region at the ora serrata. We also found marked differences in the pattern of immunolocalization of Na+,K+‐ATPase α1, α2, α3, β1, and β2 subunit isoforms in different regions of the CE. In the anterior region, NPE cells stained intensely at the basal lateral membrane with specific monoclonal and polyclonal antibodies for each of the α (α1, α2, α3) and β (β1, β2) Na,K‐ATPase isoforms. In the middle and posterior regions of the CE, NPE cells showed lower or absent levels of staining with α1, α2, α3, and β1 antibodies, although staining with β2 was abundant. In contrast, PE cells throughout the CE were stained at the basal lateral membrane by antibodies to α1 and β1, while no staining signals were detected with the rest of the antibodies (i.e. α2, α3, and β2). Our results support the conclusion that the three α and two β isoforms of the Na+,K+‐ATPase are differentially expressed in the two cell layers that make up the CE. These results also suggest that the expression of the Na+,K+‐ATPase α and β subunit isoforms may underlie subtle differences along the NPE cell layer in ion transport.