Agonist-induced rise in intracellular calcium of lens epithelial cells: effects on the actin cytoskeleton.
Primary cultures of rabbit and skate lens epithelia were used to investigate the effect of calcium release from intracellular stores upon the actin cytoskeleton. Primary cultures were loaded with fura-2 AM and intracellular calcium, i.e. (Ca2+)i, quantitated using a Hamamatsu Photonics 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 each 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 permeabilized with saponin, whereupon an increase of about 50% occurred. Tg induced 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 calcium-free EGTA-Ringer's solution responded rapidly to addition of 5 mM CaCl2, whereas only three of 35 rabbit cells responded, and in gradual fashion. After calcium imaging, the cells were fixed and stained with rhodamine phalloidin or with an antibody against IP3 receptor (IP3R) conjugated to FITC. Fluorescence microscopy revealed that the actin cytoskeleton had reorganized from the normal stress fiber pattern into polygonal networks. Tg caused the same structures to form in rabbit cells, but bombesin had no effect. IP3 receptor was located intracellularly, presumably on endoplasmic reticulum, and was not associated with plasma membranes. The rapid response of rabbit cells may have been caused by the DMSO in which fura-2 was dissolved. We have found an interesting difference in agonist-induced calcium release between rabbit and skate cells. The latter may utilize either a Ca-Na exchanger or capacitative calcium entry, which could reflect a difference in lens accommodative mechanisms. This seems relevant in view of the fact that the rabbit lens accommodates through change in shape, whereas the skate lens does so through translation of position.
Rafferty
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Northwestern University, Chicago, IL.
Rafferty
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KA
Ito
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D000065:Accommodation, Ocular; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001839:Bombesin / Q000494:pharmacology; D002118:Calcium / Q000819:agonists* / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002478:Cells, Cultured; D007294:Inositol / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007904:Lens Cortex, Crystalline / Q000378:metabolism*; D009478:Neuropeptide Y / Q000494:pharmacology; D011817:Rabbits; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D015398:Signal Transduction; D012860:Skates, Fish; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D000199:Actins; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009478:Neuropeptide Y; D018160:Receptors, Cytoplasmic and Nuclear; D013729:Terpenes; D007294:Inositol; D019284:Thapsigargin; D002122:Calcium Chloride; D001839:Bombesin; D002118:Calcium