| Lens fiber cells differentiated from the epithelial cells at the equator of the lens gradually lose their organelles during their migration to the nucleus, making the lens a unique organ in that there appears to be no protein turnover in the center throughout one's lifespan. The center of lens changes its shape to enable focusing by the human eye as well as is the main area where post-translational modifications take place. These two features leave the nucleus of the lens prone to age-related changes.Studies have shown that there is a marked increase in the stiffness of the human lens with age This is most pronounced in the nucleus, which speeds up the development of presbyopia and cataract. Since there is no measurable difference in the water contents of the nuclear regions of the lenses, this marked increase in stiffness is not due to compaction of the lens nucleus. Other studies have shown that 50% of the soluble proteins in the lens become insoluble in middle age, increasing the stiffness of the lens. In erythrocytes and bile canalicular cells, it has also been reported that aging is accompanied by a decrease in cell membrane fluidity.What happens to the human lens cell membrane during aging? Will it become more condensed or more fluid? Will the alterations contribute to the hardening of the lens tissue? This study investigated the cell membrane fluidity of human lenses of different ages, and the factors that influence it, aiming to answer these questions. Purpose To examine human lens cell membrane fluidity as a function of age.Methods Human lenses, aged 22-83 years, were assessed for membrane fluidity using Laurdan with two-photon confocal microscopy. Laurdan intensity values were measured across equatorial sections and GP values were calculated. The contralateral lenses of those used for Laurdan staining were dissected into 4 regions. Cholesterol contents in these regions were determined by mass spectrometry.Results In the young lenses (20-40 years) GP values across the sections remained constant as a function of distance from the lens center. As a function of age, in the central part of the lens, there was a progressive decrease in the GP values. No clear pattern of cholesterol was observed either across each individual lens, or in the central lens regions, as a function of age.Conclusions Membrane fluidity in the central part of the lens increases progressively as a function of age. However, the major membrane constituent, cholesterol, is not responsible for these changes.Purpose To investigate whether human lens cell membrane fluidity can be affected by soluble crystallins changes.Methods lens pairs, aged 30,41, and 63 years, were employed with one lens of the pair incubated for 20 h at 50℃while the other was stored at-80℃. Subsequent sectioning, Laurdan labeling, and imaging was performed as described above for each lens. Nuclear samples were obtained from the remaining tissue and soluble protein contents were determined using gel filtration HPLC. A 38 year old lens pair was used. One lens was sliced and sections were incubated at 50℃for 0,4,8,16, or 24 h before Laurdan staining. The nucleus of other lens was divided into 5 parts and incubated as for the slides. Soluble protein contents were determined.Results GP value in the nucleus of the heated lens was significantly lower than that of the unheated control. In the intact lenses, heating led to a decrease in the content of soluble proteins. The 38 year lens pair experiment showed that GP values changed in parallel with the concentrations of soluble proteins.Conclusions Proteins such as alpha crystallin, and possibly other soluble crystallins, could be involved in modulating lens cell membrane fluidity.Purpose To confirm those membrane fluidity changes were due to a direct effect of crystallins on membranes.Methods One hour prior to imaging, Laurdan was added to the 0.1mg/ml DHSM vesicles. Purified alpha, beta, and gamma crystallins were dissolved in the buffer to a concentration of 12.5,5,2.5, or 1.25mg/ml, 10μL of which was then mixed with 240μL of DHSM vesicles and incubated at 25℃for 30 min. GP values of the samples were determined by a fluorometer.Results Both alpha and beta crystallins markedly affected fluidity in a concentration-dependent manner. In contrast gamma crystallin had no effect.Conclusions Alpha and beta, but not gamma, crystallins had a direct effect on membrane fluidity... |
PDF Full Text Request