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 component of the regulated secretory pathway in endothelial cells. These structures degranulate after chemical or mechanical stimuli that result in cytosolic calcium influx. We therefore investigated whether the regulated pathway might be an intracellular site involved in the cleavage of 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 surface of human umbilical vein and coronary artery endothelial cells by scanning electron microscopy. Exogenous big ET-1 was added to permeabilized and nonpermeabilized cultured human umbilical vein endothelial cells, and ECE activity was measured by the detection of ET-like immunoreactivity in the culture supernatant. A marked increase in ECE activity 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 of ECE-1 (ECE-1alpha and ECE-1beta) in the perinuclear region and in Weibel-Palade bodies of the human umbilical vein endothelial cells. Stimulated degranulation of storage granules by the calcium ionophore A23187 caused release of ET into the culture supernatants. The findings of this study indicate that big ET-1 is processed to the mature vasoactive peptide by ECEs located within endothelial storage granules. We hypothesize that this activity may be important in the regulated mobilization of ET in human endothelial cells.