We first used two Ca
2+-indicator dyes (Fluo3-AM and Calcium Green2-AM; Molecular Probes, Eugene, OR) to determine whether an increase in external Ca
2+ induces an increase in internal Ca
2+ in fiber cell membrane vesicles. Although the Ca
2+ sensitivity of fluorescence was not calibrated, after external Ca
2+ was increased, there was a time-dependent increase in fluorescence that indicated internal Ca
2+ was significantly increasing (data not shown). These results were consistent with the Ca
2+ effects being either intracellular or extracellular, and if the effect was internal, then it might involve calmodulin. Two calmodulin inhibitors, TFP (75 μM) and CDZ (8 μM), were used to determine whether calmodulin inhibition would eliminate the effect of Ca
2+ on AQP0 in rabbit lens fiber cell membrane vesicles
(Fig. 6) . As can be seen, either inhibitor greatly reduced the effect of elevated Ca
2+ on AQP0 water permeability (compare these data with those in
Fig. 3 ), but neither altered the response to a decrease in external pH. These data suggest that calmodulin is indeed involved in the increase in water permeability that occurs when Ca
2+ is elevated, that the Ca
2+ effect is intracellular, and that the sites of Ca
2+ and pH regulation are physically different. Given the previous finding that Ca
2+ and pH can saturate the same factor to increase water permeability, this factor appears to be regulated by two different binding sites.