Abstract
Purpose:
Homeostasis of intracellular calcium is crucial for lens architecture and transparency. The identity of specific channel proteins regulating calcium influx in lens, however, is not completely clear. Here we examined the expression and distribution profile of L-type calcium channels (LTCC) and explored their role in morphological integrity and transparency of the mouse lens.
Methods:
cDNA microarray, RT-PCR, immunoblotting and immunofluorescence analyses were performed to characterize the expression and distribution profile of LTCCs in mouse lens. Mouse lens LTCC activity was targeted using specific pharmacological inhibitors including felodipine and nifedipine and their effects on transparency were tested in cultured lenses. The time-dependent effects of felodipine on lens architecture, protein profile, aquaporin-0 phosphorylation and myosin light chain (MLC) phosphorylation were evaluated using mass spectrometry and immunoblotting analysis.
Results:
Expression and distribution of Ca (V) 1.2 and 1.3 channel proteins are associated with both epithelium and cortical fiber cells in the mouse lens. Inhibition of LTCCs with felodipine or nifedipine resulted in subcapsular and cortical cataract formation in association with decreased lens weight. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling changes reported for altered aquaporin-0 activity. Protein changes in both soluble and membrane enriched fractions of felodipine treated lenses revealed decreases in beta-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in felodipine-treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and in levels of connexin-50, and decreases in MLC phosphorylation and in the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein.
Conclusions:
Collectively, these observations reveal a crucial role for LTCC activity in regulation of expression, activity and stability of aquaporin-0, connexins, cytoskeletal proteins, and the contractile activity of lens, all of which have a vital role in maintaining lens function and architecture.
Keywords: 569 ion channels •
445 cataract •
715 signal transduction: pharmacology/physiology