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F. Huete Toral, A. Crooke, J. Pintor; Melatonin and 5-MCA-NAT Reduce Chloride Efflux in Rabbit Cilliary Non-Pigmented Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):162.
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To investigate if the hypotensive effect of melatonin and 5-MCA-NAT is due to a reduction in the efflux of chloride in cilliary non-pigmented epithelial cells.
Immortalized NPE cells, kindly provided by Dr. Coca-Prados, were seeded in high glucose DMEM until they were confluent. Chloride measurements were performed with MQAE incubated at 1 mM during 20 h before the experiments performed. Measurements were carried out by incubating at high Cl- (130 mM, 10 min), then washed and changed by a non-chloride buffer (Cl- is changed by nitrate). Stern-Volmer constant for chloride efflux in NPE cells was calculated by means of the double ionophore technique. After that cells were challenged with single doses (100 uM) of melatonin, 5-MCA-NAT and IIK7. Dose-response analysis was performed by assaying the agonists at concentrations ranging from 0.1 uM to 150 uM.
NPE cells presented changes in the intracellular chloride concentrations when they were treated with fixed concentrations of melatonin agonists at 100 uM. Melatonin reduced the Cl- efflux 75±5 % and 5-MCA-NAT (MT3 agonist) 70±7 % of control (n=8). IIK7 (MT2 agonist), presented a bi-phasic effect first increasing a chloride influx followed by a chloride efflux. Concentration-response curves for melatonin and 5-MCA-NAT presented pD2 values of 5.8 ± 0.5 and 5.5 ± 0.8, respectively (n=8). Stern-Volmer constant showed a value of 1.86 M-1, which helped to calculate an intracellular chloride concentration of 129.43 mM.
The application of melatonins reduced IOP as we have previously described. Here we have demonstrated that at least in part, the reduction of IOP can be explained as a reduction in the chloride efflux from the NPE cells, since this ion is one of the most important in governing the net flow of water in the ciliary processes.
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