Abstract
Purpose: :
To determine whether endothelin-1 (ET-1) alters the intracellular redox state of retinal microvessels.
Methods: :
Freshly isolated retinal microvessels of rats were exposed to ET-1 (100 nM), and semi-quantitative measurements of the changes in the level of intracellular superoxide. The changes in the ethidium fluorescence of retinal pericytes were recorded by time-lapse photography. The roles played by BQ-123 and BQ-788, receptor antagonists for ETA and ETB receptors respectively, on the changes induced by ET-1was determined. In addition, the effects of apocynin (10 µM), myr-PKC (1.0 µM), allopurinol (100 µM), rotenone (10 µM) and L-NAME (100 µM) on the changes induced by ET-1 were evaluated. The effect of phorbol 12-myristate 13-acetate (PMA, 10 nM), a protein kinase C (PKC) activator, on the microvessels was also determined.
Results: :
The ethidium fluorescence-positive regions of the blood vessels coincided with the location of retinal pericytes. The intracellular superoxide levels were significantly increased after the addition of ET-1 to the medium, and this elevation was suppressed by apocynin and myr-PKC. Other enzyme inhibitors did not change the level significantly. The ET-1-induced increase of superoxide was significantly suppressed when BQ-123 (1.0 µM) and BQ-788 (1.0 µM) were simultaneously added. PMA (10 nM) mimicked the effect of ET-1.
Conclusions: :
ET-1 increases the production of superoxide in the retinal microvessel cells by activating NADPH oxidase, and an activation of PKC may be involved in the mechanism. Thus, ET-1 probably augments its vasoconstrictive effects through the formation of superoxides and the decrease in the bioavailability of nitric oxide.
Keywords: drug toxicity/drug effects • oxidation/oxidative or free radical damage • blood supply