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W. M. Dismuke, C. Mbadugha, D. Z. Ellis; NO-Induced Decreases in Cell Volume in Human Primary TM Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2827.
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Nitric Oxide (NO) decreases intraocular pressure (IOP) by increasing outflow facility but the mechanisms of regulation are not known. NO is thought to mediate its actions via activation of soluble guanylate cyclase (sGC) and increases in cGMP. The major route for the outflow of aqueous humor is the trabecular meshwork (TM) and the Schlemm’s canal. Cellular mechanisms thought to regulate outflow facility include cellular contractility and/or cytoskeletal rearrangement and changes in cell volume of TM cells, all of which are not exclusive. Thus decreases in cell volume are associated with increases in outflow facility while increases in cell volume are associated with decreases in outflow facility. This study investigated changes in cell volume in response to NO donors and elevated cGMP levels in cultured primary TM cells.
Human TM cells were isolated after collagenase digestion of TM explants according to the methods of Stamer et al., 1995 and grown on Lab-Tek ll chambered cover glass in DMEM containing serum to 100% confluency and then in serum free DMEM 2-days prior to drug treatment. Cell volume was measured according to the modified protocols of Mitchell et al., 2002 and Bush et al., 2005. Primary human TM cells were loaded with Calcein AM and visualized using a Leica confocal microscope. Calibration of the microscope allowed us to obtain measurements that are expressed as absolute cell volume measured in µm3 using NIH ImageJ software. Images were taken of TM cells that were treated with or without various drugs including NO donors, sGC inhibitors and cGMP analogs.
Exposure of TM cells to the NO-donor, DETA-NO resulted in a time-dependent decrease in cell volume. Interestingly, decreases in cell volume in response to NO donors were not observed in all TM cells. The NO-induced decreases in TM cell volume were abolished by the sGC inhibitor, ODQ. 8-bromo cGMP, a cGMP analog, mimicked the actions of DETA-NO, providing further evidence for the involvement of cGMP in mediating NO-induced decreases in TM cell volume.
DETA-NO resulted in decreases in TM cell volume. ODQ inhibition of the NO-induced decreases in cell volume suggests the involvement of sGC and cGMP in the NO-induced decreases in cell volume. Further evidence for the involvement of cGMP in mediating the NO-induced decreases in TM cell volume was obtained by using 8-Br-cGMP. The ability of DETA-NO to cause decreases in cell volume in some TM cells and not others suggest that the TM cell population is heterogeneous and that different TM cells may be functionally distinct.
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