Multiple forms of 22 kDa caveolin-1 alpha present in bovine lens cells could reflect variable palmitoylation.

Two-dimensional immunoblots of immunoprecipitated caveolin-1 from cultured bovine lens epithelial cells revealed four to five-22 kDa forms of caveolin-1 alpha with isoelectric points of between pH values 5.5 and 6.6. Fibre cell membrane recovered from fresh bovine lenses displayed an even greater number of multiforms, some with isoelectric point pH values as low as about 4. Caveolin-1 can be both phosphorylated and palmitoylated. None of the caveolin-1 alpha multiforms were labelled following culture of the lens epithelial cells with 32P-orthophosphate nor were they recognized by either caveolin-specific phosphotyrosine antibody or protein anti-phosphoserine antibody and treatment of lens fibre cell membrane with phosphatase did not alter the two-dimensional profile of immunoreactive caveolins. However, short-term incubation of BLEC with 3H-palmitate labelled some of the immunoprecipitated caveolin-1 multiforms. We suggest that the observed spectrum of caveolin multiforms could reflect variable palmitoylation of its three cysteine residues and result in populations of caveolin-1 alpha molecules with separate physical and functional properties.

Concentration and distribution of ubiquinone (coenzyme Q), the endogenous lipid antioxidant, in the rat lens: effect of treatment with simvastatin.

PURPOSE: Ubiquinone (Ub) is the only known endogenously synthesized lipid soluble antioxidant. It is synthesized from intermediates in the cholesterol metabolic pathway. Our goal was to identify the Ubs and determine the concentration and distribution of Ubs in the rat lens and the effect of treatment with simvastatin, a cholesterol synthesis inhibitor, on lens levels. METHODS: Intact lenses and separated lens fractions from young rats were homogenized in organic solvents, the Ubs recovered, and identified by HPLC analysis. Rats were fed Ub-10 to determine effects of supplementation on tissue levels. Sprague-Dawley (SD) and Chbb:Thom (CT) rats were treated with simvastatin, an inducer of cataracts in CT rats, to determine its effects on lens Ubs. RESULTS: Ubiquinone-9 (9 isoprenes in its hydrocarbon tail) was the main Ub in the rat lens. The intact lens contained about 3.0 microg Ub/g lens wet weight of which 80-90% was Ub-9 and the remainder Ub-10. No reduced Ubs were detected. Although the epithelial fraction contained the highest Ub concentration (about 8 microg/g), the cortex and nucleus combined accounted for about 90% of the lens' total content. Dietary supplementation with Ub-10 markedly increased the Ub-10 concentration in liver but not lens. Treatment with simvastatin decreased lens Ubs of both SD and CT rats by about 20%. CONCLUSIONS: The abundance of mitochondria in lens epithelium likely accounted for its high level of Ubs; but, finding most of the lens' total Ub in the cortex plus nucleus also suggests roles in maintaining the fiber cell membrane. The decrease in lens Ubs caused by simvastatin is interpreted to reflect a response to drug induced cellular stress rather than to inhibition of the cholesterol synthesis pathway.

Direct perturbation of lens membrane structure may contribute to cataracts caused by U18666A, an oxidosqualene cyclase inhibitor.

Induction of cataracts in experimental animals is a common toxic feature of oxidosqualene cyclase (OSC) inhibitors. U18666A has been shown to produce irreversible lens damage within a few weeks of treatment. Drug actions, besides reducing the availability of cholesterol, could contribute to cataract formation. Cholesterol added to cultures of lens epithelial cells could only partially overcome the growth-inhibiting effects of U18666A. In view of this finding and the fact that U18666A and other OSC inhibitors are highly lipophilic cationic tertiary amines, we tested the hypothesis that the cataractogenic effect of U18666A is related to direct perturbation of lens membrane structure and function. Based on changes in the anisotropy of fluorescent probes, U18666A incorporated into bovine lens lipid model membranes increased membrane structural order and, using small-angle x-ray diffraction, U18666A was shown to intercalate into the lens lipid model membranes and produce a broad condensing effect on membrane structure. Also, exposure of cultured lens epithelial cells and intact rat lenses to U18666A induced apoptosis. Induction of apoptosis may begin by intercalation of U18666A into cell membranes. By increasing membrane structural order, U18666A may also increase light scatter, thus directly contributing to lens opacification.

Distribution of caveolin-1 in bovine lens and redistribution in cultured bovine lens epithelial cells upon confluence.

The distribution of caveolin-1 in the lens and lens epithelial cells was determined to assess possible roles in cholesterol trafficking, cell to cell communication and signal transduction. Bovine lenses and cultured bovine lens epithelial cells (BLEC) were divided into subcellular fractions and the distribution of proteins recognized by three different caveolin-1 antibodies determined. The immunolocalization of caveolin-1 in the lens epithelium and in subconfluent and confluent cultured BLEC was probed by fluorescence microscopy and laser scanning confocal microscopy. EGF induced phosphorylation of caveolin-1 was detected by Western blotting with an anti-phosphotyrosine antibody to immunoprecipitated caveolin-1 from BLEC and human cancer cells. Monomeric caveolin-1 of about 26 kDa was detected in the epithelial cell membrane of cultured BLEC and fresh epithelia and in the plasma membrane fraction of lens cortical fiber cells. Caveolin-1 of cultured BLEC redistributed from the cytoplasm to plasma membrane as the cells proceeded from subconfluent to confluent states. The apparent abundance of caveolin-1 in cortical fiber cell plasma membrane is consistent with possible roles in distribution of lens membrane cholesterol and membrane structure. The presence of caveolin-1 in the plasma membrane of epithelial cells at - but not before - confluency is consistent with a role of caveolin-1 in cell to cell communications. EGF stimulated phosphorylation of caveolin-1 in human A431 cells but not lens cells.