Blocking chloride channels in the rat lens: Localized changes in tissue hydration support the existence of a circulating chloride flux

PURPOSE. To investigate the effects of inhibitors of chloride channels on l ens volume and tissue architecture under isotonic conditions. METHODS. Rat lenses were maintained in organ culture under isotonic conditi ons in the presence of various putative chloride channel inhibitors. The ef fect of an inhibitor on lens wet mass and tissue morphology was determined by weighing and histologic examination, respectively. RESULTS. Exposure to 100 mu M of either 5-nitro-2- (3-phenylpropylamino) be nzoic acid (NPPB) or 4,4 '-diisothiocyanatostilbene-2,2'-disulfonic acid (D IDS) caused an increase in wet mass and severe tissue disruption in the len s equatorial region. Two distinctly different zones of tissue damage were e vident: a peripheral zone of fiber cell swelling and an inner zone of exten sive tissue breakdown. Extracellular space dilations caused the extensive t issue damage in the inner zone and preceded the peripheral fiber cell swell ings. That the observed effects were a consequence of the inhibition of chl oride channels was supported by (1) the effectiveness of NPPB at the lower dose of 10 mu M, (2) the absence of any NPPB effect in chloride-free medium , and (3) an identical effect after exposure to tamoxifen, an inhibitor of the chloride channel regulator p-glycoprotein. CONCLUSIONS. Study results indicate that chloride channels are active in th e lens under isotonic conditions. The spatial and temporal pattern of morph ologic changes that was observed is consistent with a steady state efflux o f chloride ions and water from peripheral fiber cells and a corresponding i nflux into fiber cells deeper in the lens. These observations may therefore represent the first visualization of the chloride flux postulated by other s to be a component of the lens internal circulation system.