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M Satpathy, R Mitra, SP Srinivas; Na+/H+ Exchange in Cultured Bovine Corneal Endothelial Cells: Sensitivity to Protein Kinase C (PKC) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3252.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In corneal endothelial cells, NHE is implicated in control of intracellular pH, volume regulation, and fluid transport. Modulators of NHE include PKC and small GTP binding proteins (Rho) activated by downstream signaling pathways of certain GPCRs (e.g, P2 receptors). This study has examined expression of NHE, RhoA, ROCK (rho kinase; activated downstream of rho), and susceptibility of NHE to modulation by PKC. Methods: BCEC were grown to confluence on glass coverslips. BCECF was used to measure pHi. The extent of activation of the Na+/H+ exchange was measured in terms of recovery rates from an acute acid load induced by the NH4+ pre-pulse technique. Experiments were conducted at 37°C in HCO3- free Ringers. Expression of NHE, RhoA, and ROCK was examined by RT-PCR using total RNA from BCEC. PKC was activated by short-term exposure to its non-specific activator phorbol 12-myristate 13-acetate (PMA). Results: RT-PCR analyses gave positive bands for the expression of NHE-1 and NHE-3 isoforms, RhoA, and ROCK. The expression patterns were confirmed by sequencing the PCR products. NHE-2 was not expressed. Consistent with the expression of NHE-1, the major housekeeping isoform in most cells, recovery rate from an acid load (10 mM NH4+ pre-pulse) was dependent on the presence of Na+, inhibited by HOE 694 (53% at 200 µM) and activated by PMA (500 nM; 10 min exposure; ≷ 240%; n= 12). Prolonged exposure inhibited the recovery rate by 50% (n=6). Conclusion: NHE-1, the housekeeping isoform, is susceptible to PKC. It is susceptible to modulation by cell shrinkage and PKC mediated signaling pathways.
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