Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: melatonin • ciliary processes • receptors: pharmacology/physiology