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Uttio Roy Chowdhury, Cindy K. Bahler, Cheryl R. Hann, Minhwang Chang, Michael F. Romero, Michael P. Fautsch; Additive Effect of Latanoprost-Free Acid and Diazoxide, an ATP-Sensitive Potassium (KATP) Channel Opener, on Outflow Facility in Human Anterior Segment Culture. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4642.
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Previous studies from our laboratory have shown that the KATP channel opener diazoxide increases outflow facility in cultured human anterior segments. The current study was performed to investigate whether diazoxide and latanoprost-free acid (LFA) use similar mechanisms to increase outflow facility.
Cultured human anterior segments (range 44-88 years; n=6) were perfused with diazoxide (20 µM) or LFA (100 nM) for 24 hours. At 24 hours, the same anterior segments received both diazoxide and LFA for an additional 24 hours. Fellow anterior segments were perfused with vehicle. Median outflow facility was calculated at 24 and 48 hours post-treatment and significance determined by Wilcoxon signed-rank test. Patch clamping (inside-out) was used to assess the current responses (KATP activity) to LFA (100 nM) and diazoxide (100 µM) on primary normal human trabecular meshwork (NTM) cells. Diazoxide and vehicle-treated cell lysates isolated from serum starved NTM cells were screened for changes in protein phosphorylation using Phosphor antibody arrays (Full Moon Systems, CA, USA). Western blotting of select proteins was used to confirm results of Phosphor antibody array.
Diazoxide or LFA significantly increased outflow facility (µl/min/mmHg) from 0.14 (quartiles 0.12, 0.15) to 0.26 (0.23, 0.31; p=0.03) in cultured human anterior segments. Addition of the second drug to these eyes further increased outflow facility to 0.46 (0.25, 0.50; p=0.06) indicating a different mode of action for each drug. No change in vehicle treated fellow eyes was noted (p>0.5). Inside-out patch experiments on diazoxide-treated NTM cells showed increased KATP channel opening compared to vehicle-treated. Conversely, inside-out patch experiments with LFA treated NTM cells showed no KATP channel opening suggesting a different mechanism of action between diazoxide and LFA. Diazoxide-treated NTM cells showed increased expression of proteins involved in cell survival pathways. Diazoxide decreased serum-deprived activation of AMPK-α1, a key sensor of cellular energy homeostasis, indicating a role for cellular protection from stress.
Diazoxide increases outflow facility in human anterior segment culture by a mechanism distinct from that of latanoprost. Activation of cell survival pathways and decreased AMPK-α1 expression by diazoxide may indicate a protective effect of diazoxide treatment.
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