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Amritlal Mandal, Mohammad Shahidullah, Nicholas A. Delamere; Acetazolamide Increases cAMP in Cultured Porcine Nonpigmented Ciliary Epithelium and Elicits Subcellular Translocation of H+-ATPase. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5346. doi: https://doi.org/.
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The carbonic anhydrase inhibitor acetazolamide (ACTZ) reduces aqueous humor (AH) secretion and intraocular pressure in species that do or do not concentrate bicarbonate in the AH. Earlier studies showed ACTZ increased the production of cAMP by rat renal cortical slices in vitro (Rodriguez HC et al. J. Clin. Invest. 53:122-130, 1974). Because cAMP signaling in the ciliary body is known to affect AH secretion, studies were carried out to examine the possibility that ACTZ elicits a cAMP response in non pigmented ciliary epithelium (NPE).
Porcine NPE was established in primary culture according to our published method. Using an approach based on centrifugation (Lin PH et al. Biochemistry. 26 :731-736, 1987), a plasma membrane-rich fraction was isolated from the NPE and used for western blot analysis of soluble adenylate cyclase (sAC) and H+-ATPase (V-ATPase) abundance. cAMP was measured by RIA using a commercial kit (Perkin Elmer).
When cultured NPE cells were exposed to ACTZ (500μM) for 10 min the abundance of sAC protein detected in the plasma membrane-rich fraction was doubled, suggesting subcellular sAC translocation. Cells exposed to ACTZ+IBMX for 1 -10 min displayed a rapid increase in cAMP which peaked at 2 min and remained significantly elevated for 10 min. ACTZ treatment for 10 min was found to cause a significant increase in V-ATPase B1 subunit protein in the plasma membrane-rich fraction, pointing to subcellular translocation of H+-ATPase. A similar increase in V-ATPase B1 subunit protein was observed in the plasma membrane-rich fraction obtained from cells exposed for 10 min to 8-Br-cAMP, a cell permeable cAMP analog.
The findings suggest ACTZ increases cAMP in a response that may, in part, involve activation of sAC. Subcellular translocation of H+-ATPase that occurs in cells exposed to ACTZ appears to be a cAMP-dependent response. This raises the possibility that some functional effects of carbonic anhydrase inhibitors on the NPE may be related to cAMP signaling.
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