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A. Mandal, M. Shahidullah, C. Beimgraben, N. A. Delamere; Endothelin-1 Inhibits Na,K-ATPase Activity Through a Src Family Kinase Mediated Mechanism in Porcine Lens Epithelium. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4608.
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The Na,K-ATPase is vital for the maintenance of lens transparency. Studies in intact porcine lens suggested the involvement of tyrosine kinases in short-term regulation of Na,K-ATPase (Bozulic, et al. 2004). In vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs) resulted in modification of Na,K-ATPase activity (Bozulic, et al. 2005). Here, we examined the effect of ET-1, an endothelin receptor agonist, on Na,K-ATPase function and SFK activation in the intact porcine lens.
Porcine lenses were incubated in bicarbonate buffered Krebs’ solution at 37°C equilibrated with 5% CO2 and 95% air for a period of 0 to 30 min. Epithelia were carefully peeled off, homogenized in RIPA buffer and examined by western blot. Na,K-ATPase function in the intact lens was measured using ouabain-sensitive potassium (86Rb) uptake and by measurement of ouabain-sensitive ATP hydrolysis.
ET-1 caused a significant reduction in Na,K-ATPase activity as determined by direct measurement of ATP hydrolysis by intact lenses. ET-1 also caused a significant reduction in ouabain-sensitive potassium (86Rb) uptake. Both responses were abolished when lenses were pretreated with 10 µM PP2, a selective inhibitor of Src family kinases. Significant SFK phosphorylation in ET-1 treated lenses was determined as a ~ 61 kDa band recognized by the active loop Tyr416-specific antibody. Furthermore, the phosphotyrosine band density was significantly reduced when an antibody against the C-terminal inhibitory Tyr527-specific antibody was used. ET-1 caused a significant ERK1/2 phosphorylation, which also was abolished by PP2.
The data point to an ET-1 mediated SFK activation which is associated with an inhibition of Na,K-ATPase pump activity. SFK activation appears to precede ERK1/2 activation in porcine lens. It remains to be determined whether SFK mediated ERK1/2 activation plays a role in modulating Na,K-ATPase activity.ReferencesBozulic, L. D., et al. (2004). Am. J. Physiol. Cell Physiol. 286(1): C90-96.Bozulic, L. D. et al .(2005). Invest. Ophthalmol. Vis. Sci. 46(2): 618-622.
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