TISSUE KALLIKREIN ACTIVITY AND KININ RELEASE IN HUMAN ENDOTHELIAL-CELLS

The kininogenase, tissue kallikrein (EC 3.4.21.8), has been identified in different blood vessels. The enzyme was mainly found in vascular s mooth muscle cells. It is not known whether it is present and function ally active in vascular endothelial cells. The following study investi gates the presence of tissue kallikrein in endothelial cells from huma n umbilical veins and pulmonary arteries. Tissue kallikrein was demons trated in three ways: 1) by immunostaining in endothelial cells; 2) by measurement of tissue kallikrein activity using a colorimetric assay; 3) by the measurement of kinin release in intact and homogenised endo thelial cells with a radioimmunoassay. Immunostaining demonstrated the presence of tissue kallikrein in endothelial cells from human umbilic al veins and endothelial cells from human pulmonary arteries. Tissue k allikrein-like activity, measured by the degradation of D-Val-cyclohex yl-Ala-Arg-4-nitraniline, was 3.57 +/- 0.5 mU/10(6) endothelial cells from human umbilical veins and 7.52 +/- 0.84 mU/10(6) endothelial cell s from human pulmonary arteries. Intracellular kinin concentrations we re 424 +/- 83 pg/10(6) cells in endothelial cells from human umbilical veins and 576 +/- 146 pg/10(6) cells in endothelial cells from human pulmonary arteries, and they increased in a time-dependent manner afte r homogenisation. The increase was abolished by aprotinin (1000 kIU), an inhibitor of tissue kallikrein in both cell types. Addition of exog enous kallikrein (5 mU) to homogenised cells led to a five fold increa se of kinin concentrations after five minutes, indicating a sufficient resource of cellular kininogen. Removal of extracellularly bound kini nogen by washing with dextran sulphate (100 mg/l) resulted in an appro ximately 75% reduction of the cellular kinin release. This suggests th at extracellular kininogen is the main source of endothelial kinin pro duction. The present data demonstrate the presence of tissue kallikrei n in human endothelial cells and provide evidence for the ability of h uman endothelial cells to release kinins.