Mechanism of calcium-induced disintegrative globulization of rat lens fiber cells

Purpose. To study the role of calcium and calcium-dependent processes in the disintegrative globulization of isolated single rat lens cortical fibers. Methods. The authors isolated viable and morphologically intact single fiber cells from rat lens cortex and studied the effect of 1 mM [Ca ²⁺](o) on the globulization of fiber cells from the outer and inner cortex. They investigated the effects of the calcium-channel blocker, verapamil, an inhibitor of calcium transport, lanthanum; various protease inhibitors Na ⁺- free and K ⁺-free media; calcium ionophore, A23187; and calcium chelator, BAPTA, on the globulization of fiber cells exposed to 1 mM [Ca ²⁺](o). Results. Perfusion with Ringer's containing 1 mM [Ca ²⁺](o), caused disintegration and globulization of the isolated fibers in 32.3 ± 1 minute, and the addition of 10 μM A23187 to the superfusing solution reduced the time to complete globulization (t(K)) to 19.4 ± 0.3 minutes. However, the addition of protease inhibitors, leupeptin, calpain inhibitor I, E-64, pepstatin (0.5 mM each) to the superfusing solution, increased I(K) to 105 ± 3.5, 84.2 ± 7.8, 60.7 ± 3.5, and 48.3 ± 3.1 minutes, respectively. The t(K) also increased (96.4 ± 3.5 minutes) when the fibers were preincubated with BAPTA-AM or when they were exposed to 1 mM [Ca ²⁺](o) in Na ⁺-or K ⁺- free Ringer's solution (t(K) = 66.7 ± 5.3 and 58.9 ± 3.9 minutes, respectively or in Ringer's solution containing 1 mM [Ca ²⁺](o) + 50 μM verapamil (t(K) = 49.3 ± 3.8 minutes). Single fibers isolated from the outer cortex of the lens were less susceptible to extracellular calcium than those isolated from the inner cortex. Conclusions. Increased calcium influx and the attendant elevation of [Ca ²⁺](i) are necessary for disintegrative globulization of lens fiber cells. Calcium influx appear to be mediated partially by the L-type calcium channels and the background calcium leak. Protection by protease inhibitors suggests that membrane fragmentation, caused by elevated [Ca ²⁺](o) results from proteolytic damage to the fiber cytoskeleton. Besides underscoring the central role of calcium homeostasis in preserving the morphologic integrity of the cortical fibers, this study suggests a possible cellular mechanism for the formation of supranuclear cataract.