HUMAN ENDOTHELIAL-CELL STORAGE GRANULES - A NOVEL INTRACELLULAR SITE FOR ISOFORMS OF THE ENDOTHELIN-CONVERTING ENZYME

We have previously shown endothelin (ET)-like immunoreactive staining in Weibel-Palade bodies, storage granules that are an integral compone nt of the regulated secretory pathway in endothelial cells. These stru ctures degranulate after chemical or mechanical stimuli that result in cytosolic calcium influx. We therefore investigated whether the regul ated pathway might be an intracellular site involved in the cleavage o f big ET-1 to the biologically active peptide ET-1 by determining the ultrastructural localization of endothelin-converting enzyme (ECE)-1. A low level of ECE-like immunoreactivity was detected on the cell surf ace of human umbilical vein and coronary artery endothelial cells by s canning electron microscopy. Exogenous big ET-1 was added to permeabil ized and nonpermeabilized cultured human umbilical vein endothelial ce lls, and ECE activity was measured by the detection of ET-like immunor eactivity in the culture supernatant. A marked increase in ECE activit y was observed in permeabilized cells, indicating that ECE may also be expressed in intracellular compartments. Confocal microscopy revealed intense immunofluorescence staining for big ET-1 and the 2 isoforms o f ECE-1 (ECE-1 alpha and ECE-1 beta) in the perinuclear region and in Weibel-Palade bodies of the human umbilical vein endothelial cells. St imulated degranulation of storage granules by the calcium ionophore A2 3187 caused release of ET into the culture supernatants. The findings of this study indicate that big ET-1 is processed to the mature vasoac tive peptide by ECEs located within endothelial storage granules. We h ypothesize that this activity may be important in the regulated mobili zation of ET in human endothelial cells.