Effects of endothelin on cultured bovine retinal microvascular pericytes

The endothelins, a recently discovered family of peptides, include endothelin-1, the most potent vasoconstrictor known. Retinal microvascular pericytes are thought to be contractile cells analogous to the smooth muscle cells of larger vessels, but the physiologic stimulus for their contraction is unknown. We hypothesized that the endothelins might serve as such stimuli.

The intracellular free/bound Ca++ ratio increases rapidly immediately before muscle cell contraction. We evaluated changes in this ratio in cultured bovine retinal microvascular pericytes and, for comparison, three other types of ocular cells. We loaded the cells with the calcium-sensitive, fluorescent dye Indo-1. Using a laser cytometer, we monitored the time course of changes in fluorescence of individual cells in response to several putative vasoactive agents, including, in particular, endothelin-1, 2, and 3.

Endothelin-1 (ET-1) produced a rapid rise in the free/bound Ca++ ratio, followed by a slow decline. The response occurred at ET-1 concentrations as low as 1 x 10(-12) mol/l, and was graded in amplitude and concentration dependent. After an initial application of ET-1, repeat applications yielded no response. Endothelin-2 was less effective than ET-1, and ET-3 had no effect at all, but both agents blocked the response to ET-1. Several other agents also raised the free/bound Ca++ ratio, but were substantially less effective than ET-1. When any of these agents, except for histamine, was added after even a submaximal concentration of ET-1, no response was observed, but ET-1 applied after these agents produced a large response. Histamine could elicit a rise in the free/bound Ca++ ratio after application of ET-1 to cultured pericytes. The ET-1 response occurred in Ca(++)-free medium and in medium containing 10(-4) mol/l verapamil or nifedipine, indicating that the results we observed are due primarily to the release of free Ca++ from bound intracellular stores. Endothelin-1 produced a similar change in the free/bound Ca++ ratio in cultured bovine RPE cells, but not in retinal microvascular endothelial cells or lens epithelial cells.

ET-1 is at least three orders of magnitude more effective in producing the release of free intracellular Ca++ than other agents tested. It appears to act through specific cell surface receptors, which can be blocked by prior application of other endothelin isopeptides, but not by structurally dissimilar molecules. However, with the exception of histamine, all of these agents appear to act through a common intracellular pathway, because application of ET-1 blocks the subsequent effect of the other agents tested, except histamine. Alternatively, ET-1 may be capable of desensitizing the receptors for these agents without occupying the receptor sites. Because cultured retinal pericytes are extremely sensitive to ET-1 in a response closely linked to muscle cell contraction, ET-1 must be considered a highly plausible agonist for pericyte contraction in vivo.