AGONIST-INDUCED RISE IN INTRACELLULAR CALCIUM OF HENS EPITHELIAL-CELLS - EFFECTS ON THE ACTIN CYTOSKELETON

Primary cultures of rabbit and skate lens epithelia were used to inves tigate the effect of calcium release from intracellular stores upon th e actin cytoskeleton. Primary cultures were loaded with fura-2 AM and intracellular calcium, i.e. (Ca2+)i, quantitated using a Hamamatsu Pho tonics digital imaging system. Agonists used were bombesin, inositol-1 ,4,5-trisphosphate (IP3), thapsigargin (Tg), neuropeptide Y (NPY) and calcium chloride. Recordings were typically made on seven cells in eac h case. We found that IP3 caused a 6-8-fold immediate release of (Ca2)i in rabbit cells, but skate cells showed no response unless permeabi lized with saponin, whereupon an increase of about 50% occurred. Tg in duced release from internal stores in rabbit cells, but had no effect on skate cells. Bombesin caused a large increase in (Ca2+)i release in both, while NPY had no effect in either. Skate cells incubated in cal cium-free EGTA-Ringer's solution responded rapidly to addition of 5 mM CaCl2, whereas only three of 35 rabbit cells responded, and in gradua l fashion. After calcium imaging, the cells were fixed and stained wit h rhodamine phalloidin or with an antibody against IP3 receptor (IP3R) conjugated to FITC. Fluorescence microscopy revealed that the actin c ytoskeleton had reorganized from the normal stress fiber pattern into polygonal networks. Tg caused the same structures to form in rabbit ce lls, but bombesin had no effect. IP3 receptor was located intracellula rly, presumably on endoplasmic reticulum, and was not associated with plasma membranes. The rapid response of rabbit cells may have been cau sed by the DMSO in which fura-2 was dissolved. We have found an intere sting difference in agonist-induced calcium release between rabbit and skate cells. The latter may utilize either a Ca-Na exchanger or capac itative calcium entry, which could reflect a difference in lens accomm odative mechanisms. This seems relevant in view of the fact that the r abbit lens accommodates through change in shape, whereas the skate len s does so through translation of position.