The lens generates an internal circulation of solute, which is followed by fluid to create a micro circulatory system for this avascular organ. The circulation depends on fiber cell gap junctions to conduct intracellular fluxes of ions and perhaps fluid, which flow from the center to surface of the lens. Extracellular fluxes move from the surface to center of the lens along the spaces between fiber cells. The intracellular and extracellular fluxes are linked by fiber cell membranes, which express nonselective cation channels, chloride channels and the water channel AQP0. The transmembrane flux of solute creates small transmembrane osmotic gradients that generate the circulation of fluid. The circulation is critical for homeostasis in central fiber cells, which rely on the inwardly directed extracellular fluid flow to carry essential nutrients, amino acids and antioxidants into the lens. We have spent many years characterizing the molecular basis of the lens circulation and have made enormous progress (reviewed in Mathias et al., 2008), but there are still questions, as outlined in aims 1 and 2 of this proposal. In the last grant period, we began the process of putting together what we have learned to understand generation of the age-onset central cataract. Based on our findings, the overall hypothesis for this proposal is: The age-onset nuclear cataract is a consequence of compromise of the lens circulation due to cumulative oxidative damage to transport proteins.
Aim 3 of this proposal will directly address this hypothesis. Moreover, we need to understand how a normally functioning circulation depends on lens transport parameters, and how these parameters can be altered by cumulative oxidative damage.
Aim 1 is to further characterize gap junction coupling in the normal lens. It involves testing 2 hypotheses. 1a) Intracellular water flow in the lens is mediated by gap junctions. 1b) The equator to poles variation in gap junction coupling conductance is due to regulation of Cx50 channels through the MAP kinase pathway.
Aim 2 is to further characterize lens water channels. It involves testing 2 hypotheses. 2a) AQP0 has a specialized role other than providing a water channel. 2b) AQP0 is physiologically regulated by pH and [Ca2+]i.
Aim 3 is to characterize the effects of cumulative oxidative damage on lens transport and intracellular homeostasis. It involves testing 2 hypotheses. 3a) Oxidative stress in the lens causes reductions in gap junction coupling conductance, leading to compromise of the circulation and eventual cataracts. 3b) Oxidation-mediated reductions in lens gap junction coupling are initiated through activation of PKC3. To achieve these aims we will use standard biochemical techniques and several specialized techniques that were developed by us. The distribution of functional gap junction channels will be evaluated using whole lens impedance techniques. Osmotic studies of isolated fiber cell membrane vesicles will determine water permeability and its regulation by pH and calcium. Microelectrode based techniques will be used to measure the spatial distribution of intracellular hydrostatic pressure, and intracellular sodium and calcium in intact lenses. Most studies involve lenses from genetically altered mice that express modified transport proteins or other enzymes.
The work described in this proposal is designed to further our understanding of the lens circulation, and how cumulative oxidative damage to transport proteins can compromise the circulation and lead to age-onset central cataracts. Our hypothesis is oxidative damage to gap junction coupling in the lens is the first step that initiates a cascade of events leading to central cataracts. If so, there are pharmacological interventions that can up regulate gap junction coupling, such as inhibitors of PKC3, and these could potentially prevent development of the cataract.
|Scheiblin, David A; Gao, Junyuan; Caplan, Jeffrey L et al. (2014) Beta-1 integrin is important for the structural maintenance and homeostasis of differentiating fiber cells. Int J Biochem Cell Biol 50:132-45|
|Hall, James E; Mathias, Richard T (2014) The aquaporin zero puzzle. Biophys J 107:10-5|
|Gao, Junyuan; Wang, Huan; Sun, Xiurong et al. (2013) The effects of age on lens transport. Invest Ophthalmol Vis Sci 54:7174-87|
|Gao, Junyuan; Sun, Xiurong; Moore, Leon C et al. (2013) The effect of size and species on lens intracellular hydrostatic pressure. Invest Ophthalmol Vis Sci 54:183-92|
|Gao, Junyuan; Sun, Xiurong; Moore, Leon C et al. (2011) Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling. J Gen Physiol 137:507-20|
|Kumari, S Sindhu; Eswaramoorthy, Subramaniam; Mathias, Richard T et al. (2011) Unique and analogous functions of aquaporin 0 for fiber cell architecture and ocular lens transparency. Biochim Biophys Acta 1812:1089-97|
|Das, Satyabrata; Wang, Huan; Molina, Samuel A et al. (2011) PKC?, role in lens differentiation and gap junction coupling. Curr Eye Res 36:620-31|
|Fudge, Douglas S; McCuaig, John V; Van Stralen, Shannon et al. (2011) Intermediate filaments regulate tissue size and stiffness in the murine lens. Invest Ophthalmol Vis Sci 52:3860-7|
|Shi, Yanrong; De Maria, Alicia B; Wang, Huan et al. (2011) Further analysis of the lens phenotype in Lim2-deficient mice. Invest Ophthalmol Vis Sci 52:7332-9|
|Varadaraj, K; Kumari, S S; Mathias, R T (2010) Transgenic expression of AQP1 in the fiber cells of AQP0 knockout mouse: effects on lens transparency. Exp Eye Res 91:393-404|
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