Evidence using immunoelectron microscopy for regulated and constitutive pathways in the transport and release of endothelin.

We investigated the distribution of endothelin (ET)-like immunoreactivity in the human coronary artery and examined sites linked to the storage and release of intracellular proteins. Intense ET-like immunoreactivity was observed at the light-microscope level in luminal coronary artery endothelial cells. A low level of staining also was detected in the outer medial smooth-muscle layer and diffusely within the adventitia. Immunoelectron microscopy was used to determine the ultrastructural localisation of ET in the endothelium. Positive ET-like immunoreactive staining was detected in secretory vesicles at the ultrastructural level. Quantitative immunoelectron microscopy revealed that ET-like immunoreactivity was predominantly localised to the cytoplasmic matrix (including secretory vesicles) and Weibel-Palade bodies (endothelial cell-specific storage granules). Labelling was detected in 44% of Weibel-Palade body profiles positively identified by using antisera to von Willebrand factor and in cytoplasm surrounding these structures. A low level of immunoreactive staining was associated with mitochondria, whereas the cell nucleus and Golgi complex showed little or no positive staining. These findings indicate that ET is released from human coronary artery endothelial cells via two distinct secretory pathways. We propose that ET is continuously transported in and released from secretory vesicles by the constitutive secretory pathway. ET may also be stored in Weibel-Palade bodies and released after an appropriate stimulus by the regulated pathway. Positive immunoreactivity was also observed in plasmalemmal vesicles (50- to 60-nm diameter), indicating a role for these structures in endocytosis.