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N.A. Delamere, S. Tamiya, Y. Hou, S.A. Mathews; Studies on Cytoplasmic pH and Src–Family Tyrosine Kinases in Cultured Rabbit Nonpigmented Ciliary Epithelium . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4719.
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© ARVO (1962-2015); The Authors (2016-present)
Past studies from this laboratory pointed to a link between tyrosine kinase function and regulation of active sodium–potassium transport in nonpigmented ciliary epithelium (NPE) (Nakai et al, IOVS 40:2033, 1999). Here, experiments were conducted to test whether dorzolamide, a carbonic anhydrase inhibitor (CAI), causes activation of Src–family tyrosine kinases.
Using a rabbit NPE cell line (a gift from Miguel Coca Prados, Yale) cytoplasmic pH was measured using the fluorescent indicator BCECF. Src activation was probed by western blot analysis using two antibodies, one that recognizes total Src–family protein and one that recognizes only the active kinase that is phosphorylated at Y416.
Phospho–Src western blot band density was increased in cells exposed to dorzolamide (500uM). The maximum response was observed within 10 min. Total Src band density was unchanged. Dorzolamide also lowered cytoplasmic pH. Acetazolamide elicited similar pH and phospho–Src responses. To further study the influence of cytoplasmic pH on Src activation, some cells were exposed to dimethylamiloride (DMA). DMA also lowered cytoplasmic pH and caused an increase in phospho–Src western blot band density.
The results show that CAIs transiently activate at least one member of the Src kinase family. Further studies are required to determine whether the Src response lies downstream of cytoplasmic pH changes that occur on CAI treatment.
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