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KY Allen, M Satpathy, R Mitra, BY J T Yue, SP Srinivas; Na+/H+ Exchange in Cultured Bovine Trabecular Meshwork (TM) Cells: Response to Hyperosmotic Stress and Activation of PKC . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3247.
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Purpose: In non-epithelial cells, NHE is implicated in control of intracellular pH, volume regulation, cellular proliferation, and mechanotransduction upon stretching. Expression, activity, and turnover of NHE isoforms are regulated by growth factors, GPCRs, and corticosteroids through protein kinases (e.g., PKC and MAP kinases) and small GTP binding proteins. In this study, we have characterized NHE expression and activity in TM cells because of their relevance to pathophysiology of POAG, glucocorticoid induced increase in IOP, and pharmacology of outflow pathway. Methods: Bovine TM cells (BTMC) were grown to confluence on glass coverslips. BCECF was used to measure pHi. 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 using HCO3- Ringers. Hyperosmotic solutions were obtained by addition of sucrose. Expression of NHE isoforms was examined by RT-PCR and Western Blot techniques. PKC was activated by short-term exposure to its non-specific activator phorbol 12-myristate 13-acetate. Results: RT-PCR analyses using total RNA from BTMC gave positive bands for the expression of NHE-1 and NHE-3 isoforms. The expression patterns were further confirmed by sequencing the PCR products. NHE-2 was not expressed. Recovery rate from an acid load (10 mM NH4+ pre-pulse) was dependent on the presence of Na+ and activated by PKC (10 min exposure; 500 nM; ∼70%; n = 4). Recovery rate was also higher by 130% under hyperosmotic conditions at pH values close to 7.4. Conclusion: BTMC cells express NHE-1, the housekeeping isoform, found ubiquitously in many epithelial and non-epithelial cell types. It is susceptible to modulation by cell shrinkage and PKC mediated signaling pathways.
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