To further examine calcium-dependence of the response, lenses were exposed to either HC067047 or BAPTA. The strategy was as follows: HC067047 inhibits the initiating step in the response, TRPV4-mediated calcium entry. BAPTA is a cytoplasmic calcium chelator that suppresses an increase of cytoplasmic calcium even though calcium entry occurs. Importantly, SFK activation was inhibited by BAPTA, HC067047, and a PKA inhibitor H89. Taken together, the findings point to cAMP as a link between TRPV4 channel-mediated calcium entry, activation of calcium-dependent adenylyl cyclases, a rise of cAMP and SFK activation. In accordance with this proposed role of calcium-dependent adenylyl cyclases, and the dependence of Na,K-ATPase activity on SFK activation,
3 the doubling of Na,K-ATPase activity in the epithelium of lenses exposed to hyposmotic solution was prevented both by H89 and by BAPTA. In an earlier report, phosphorylation of Src has been linked to PKA in the bovine sperm.
44 As a direct test of cAMP-dependent SFK activation, intact lenses were exposed to 8-pCPT-cAMP, a cell permeable stable analog of cAMP, and SFK phosphorylation was confirmed to occur. In summary, our results point to the following sequence of events in lenses exposed to hyposmotic solution: TRPV4 activation and calcium entry followed by calcium-mediated production of cAMP, PKA-dependent SFK activation, and a subsequent increase of Na,K-ATPase activity (
Fig. 9). Regulation of Na,K-ATPase activity in the lens epithelium has significance because the lens is made up almost entirely of differentiated fiber cells that have little or no Na,K-ATPase activity. The fiber cells, however, are well coupled, and as a consequence active transport by Na,K-ATPase in relatively few cells in the epithelium is able to regulate cytoplasmic sodium and potassium levels in the entire structure. If sodium and potassium levels are not properly controlled, osmotic forces cause the cells to swell and this impairs lens transparency. The findings here are consistent with our earlier proposal that TRPV4 enables the epithelium to sense swelling and adjust Na,K-ATPase activity accordingly.
3 The discovery of SFK-mediated regulation of Na,K-ATPase through the integration of TRPV4-mediated calcium entry and cAMP signaling illustrates a complex regulatory mechanism. The complexity may be greater still because different SFKs may have different effects on Na,K-ATPase activity.
45 Earlier we showed that at least four SFK members (Src, Fyn, Yes, Hck) are expressed in the porcine lens epithelium and that endothelin-1 (ET-1) activated Fyn but inhibited Src and Hck, and had no effect on Yes.
45 In regard to pathology, maintenance of Na,K-ATPase activity is significant in relation to cataract, the most common cause of age-related vision loss. In the human lens, a progressive increase of sodium and water content and a decrease potassium is associated with cortical cataract formation.
46 In other words, cataract is associated with failure of Na,K-ATPase-mediated active transport to keep pace with ion leakage.