Abstract
Abstract: :
Purpose: Gap junction activity is controlled by PKC γ catalyzed phosphorylation. The purpose of this project is to determine if PKC γ also controls retinal Cx 36 gap junctions. Methods: PKC γ enzyme activity assay was performed on the R28 neuronal cell line using a peptide assay kit. Dose curves for enzyme activity were performed using different levels of phorbol–12 myristate 13 acetate (TPA), hydrogen peroxide, lens epithelium–derived growth factor (LEDGF), pigment epithelium derived factor (PEDGF), or brain derived neurotrophic factor (BDNF). Eyes were disected from six week old mice (b6129pf2/j100903 control and B6;129p–Prkcctm1St1 KOγ) and retinas were removed within an hour at 4 °C. Retina tissue was lysed in 0.5% Triton lysis buffer with 1% 3–[(3–Cholamidopropyl0dimethylammonio]–1–propanesulfonate (CHAPS). Whole retina extracts were treated with 200 nM of TPA or 100 µM of hydrogen peroxide then were immuno–precipitated with anti–rabbit Cx36 (Zymed) and blotted it with anti–rabbit phosphoserine (Chemicon). Results: Dose curve results for PKC γ enzyme activity assay that was performed on R28 cells showed 120% activation at 200 nM TPA and 90% increase at 100 µM hydrogen peroxide. R28 cells treated with LEDGF, BDNF and PEDF did not show any significant increase in activity. Whole retina extract from control or KOγ mice were treated with 200 nM of TPA or 100 µM hydrogen peroxide. Cx36 from whole retina extract showed no detectable phosphorylation of Cx36 in all KOγ retina’s from either treated or untreated fractions. Phosphorylation of Cx 36 detected in control retina that were treated with TPA or hydrogen peroxide. No phosphorylation of Cx 36 was observed in untreated control retina extracts. Conclusions: Both TPA and hydrogen peroxide cause PKCγ activation and Cx36 phosphorylation in control mouse retina. This is not observed in the PKC γ knockout mouse retina.
Keywords: gap junctions/coupling • retina • retinal degenerations: cell biology