Abstract
Purpose::
The purpose of this study is to demonstrate early activation of the protein kinase C gamma pathway in the streptozotocin-induced diabetic rat lens.
Methods::
Twelve-week old male and female Spraque-Dawley rats were injected with 80 mg/kg (body weight) of streptozotocin (N-[methylnitrosocarbamoyl]-D-glucosamine) intraperitoneally. The streptozotocin was dissolved in 0.9 % saline (pH 4.5) at a concentration of 50 mg/mL. Diabetes was defined as a non-fasting blood glucose level of at least 450 mg/dL. All experiments were done in 1-3 day streptozotocin diabetic rats. PKC gamma activation was measured by enzyme activity assay and by Western blotting to show autophosphorylation on Thr514. PKCgamma-phosphorylation of Cx43 on Ser368 was determined using anti-phospho-S368 Cx43 antibody. Activation of PLCgamma1 was also determined by Western blotting using anti-phosphoThr783 antisera. Lens gap junction activity was determined by microinjection-lucifer yellow dye transfer assay. Electron microscopy was applied to affirm fiber cell damage in the streptozotocin diabetic lenses.
Results::
In the lenses of 3-day streptozotocin-induced diabetic rats we observed that PKC gamma enzyme was activated. PKC gamma could be further activated by 400 nM Phorbol ester (TPA), but the protein levels remained constant. Western blots showed that activation of PKC gamma may be due to activation of PLC gamma 1 and subsequent elevation of endogenous diacylglycerol, a native PKC activator. Activated PKC gamma phosphorylated Cx43 on Ser368 and this subsequently inhibited lens gap junction dye transfer activity in the diabetic lenses. Furthermore, early damage occurred in the inner cortex in elongated fiber cells in the lens of streptozotocin-induced diabetic rats, but not in the normal control lenses.
Conclusions::
Early activation of PLCgamma1 was observed at 3 days of diabetes. This resulted in activation of PKCgamma, phosphorylation of Cx43 on Ser368 and in early inhibition of dye transfer. Defective signaling from PKCgamma to Cx43 was observed after 3 days of diabetes in the rat lens.
Keywords: cell-cell communication • cataract • diabetes