In an earlier study it was reported that thrombin significantly reduces the rate of Na,K-adenosine triphosphatase (ATPase)-mediated ion transport by porcine lens. Because thrombin stimulates the release of endogenous endothelin (ET)-1 stores from some tissues, and because ET-1 can cause Na,K-ATPase inhibition, this study was designed to determine whether thrombin causes release of ET-1 from the lens.
Rabbit lenses exposed to 0.5 mM hydrogen peroxide for 1 h and then incubated for an additional 19 h demonstrated small yet significant changes in electrolyte balance. Active cation pumping by the lens, as determined by 86Rb uptake studies, was partially impaired following 1 h hydrogen peroxide exposure; the cation pump further deteriorated during the subsequent 19-hour incubation. Passive membrane permeability of the lens, measured by 86Rb efflux, was elevated following 1 h hydrogen peroxide but no further increase in leakiness was observed during the subsequent 19-hour incubation. These findings suggest that even relatively brief exposure to oxidative damage initiates a chain of events from which the lens is unable to recover.
The anion transport inhibitor DIDS is known to reduce aqueous humor secretion but questions remain about anion dependence of the effect. In some tissues, DIDS is reported to cause Na-K-ATPase inhibition. Here, we report on the ability of DIDS to inhibit Na-K-ATPase activity in nonpigmented ciliary epithelium (NPE) and investigate the underlying mechanism. Porcine NPE cells were cultured to confluence on permeable supports, treated with drugs added to both sides of the membrane, and then used for (86)Rb uptake measurements or homogenized to measure Na-K-ATPase activity or to detect protein phosphorylation. DIDS inhibited ouabain-sensitive (86)Rb uptake, activated Src family kinase (SFK), and caused a reduction of Na-K-ATPase activity. PP2, an SFK inhibitor, prevented the DIDS responses. In BCECF-loaded NPE, DIDS was found to reduce cytoplasmic pH (pHi). PP2-sensitive Na-K-ATPase activity inhibition, (86)Rb uptake suppression, and SFK activation were observed when a similar reduction of pHi was imposed by low-pH medium or an ammonium chloride withdrawal maneuver. PP2 and the ERK inhibitor U0126 prevented robust ERK1/2 activation in cells exposed to DIDS or subjected to pHi reduction, but U0126 did not prevent SFK activation or the Na-K-ATPase activity response. The evidence points to an inhibitory influence of DIDS on NPE Na-K-ATPase activity by a mechanism that hinges on SFK activation associated with a reduction of cytoplasmic pH.
Potential difference, resistance, cation content, and 86Rb efflux were measured in frog lenses maintained in normal or calcium-free EGTA Ringer's solution. Exposure of the lens to calcium-free solution resulted in a rapid fall in potential and resistance, together with a twofold increase in 86Rb efflux rate. These rapid changes were not due to an alteration in cation distribution between the lens and its environment. However, the alteration in 86Rb efflux rate could be explained on the basis of the fall in potential. These findings suggested that removal of calcium from the bathing medium caused a rapid increase in sodium permeability alone. This suggestion was substantiated by the results of experiments where the response of the lens to low calcium solution was determined in a medium in which 90% of the sodium had been replaced by sucrose.
