Evidence for the formation of endothelin by lysed red blood cells from endogenous precursor

The release of endothelin from various blood cell fractions was investigated. Human as well as rat blood cell fractions homogenized by sonification were incubated in buffer for up to 60 min. Neither in platelet nor leukocyte homogenates from either species could immunoreactive endothelin be detected. In contrast, homogenates of red blood cells from both species showed a rapid and time-dependent rise of immunoreactive endothelin levels, reaching a peak at 15 min and decreasing thereafter. However, at time point 0 no immunoreactive endothelin could be detected. Reverse phase high performance liquid chromatography showed immunoreactive endothelin to consist of endothelin-1 as well as big endothelin-1. The release of immunoreactive endothelin in human and rat homogenates was concentration-dependently inhibited by the protease inhibitors, leupeptin, phosphoramidon, chymostatin and pepstatin A in order of increasing potency. Intact red blood cells did not incorporate [125I]endothelin-1 nor did they transform exogenous big endothelin-1 to endothelin-1. However, haemolysis of red blood cells with hypotonic saline (0.2%) or incubation with pore-forming staphylococcal alpha-toxin induced the release of immunoreactive endothelin into the buffer samples. Thus, apart from the indirect vasoconstrictor, haemoglobin, red blood cells can also liberate the direct vasoconstrictor, endothelin, a finding expected to be of considerable pathophysiological significance.