TRANSCYTOSIS IN THE CONTINUOUS ENDOTHELIUM OF THE MYOCARDIAL MICROVASCULATURE IS INHIBITED BY N-ETHYLMALEIMIDE

In a murine heart perfusion system, we were able to ''turn off'' the t ransport of derivatized albumin [dinitrophenylated albumin (DNP-albumi n)] from the perfusate to the tissue, by preperfusing the system with 1 mM N-ethylmaleimide (NEM) for 5 min at 37-degrees, followed by a 5-m in perfusion of DNP-albumin in the presence of NEM. Using a postembedd ing immunocytochemical procedure, we showed that (i) a 30-sec to 1-min treatment of heart vasculature with 1 mM NEM reduces the transendothe lial transport of DNP-albumin and nearly stops it after 5 min, and (ii ) DNP-albumin is detected exclusively in plasmalemmal vesicles (PVs) w hile in transit across endothelial cells. Perfusion with 10 mM dithiot hreitol for 1 min before NEM prevents the inhibition of vesicular tran sport. To quantitate the NEM effect on vesicular transport inhibition, we developed an ELISA and a dot-blot assay for measuring DNP-albumin in supernatants of perfused whole-heart homogenates. The results obtai ned indicate that the treatment of the heart vasculature with 1 mM NEM decreases the vesicular transport of DNP-albumin by 78-80%. Since NEM is known to inhibit the fusion of different types of vesicular carrie rs with their target membranes in other cell types and in in vitro rec onstituted cellular systems, by alkylating a NEM-sensitive factor, we assume that the same mechanism applies in our in situ system. The decr ease of vesicular transport can be explained by NEM preventing the fus ion of recycling vesicles with their targets-i.e., the abluminal and l uminal domains of the plasmalemma. The results open to question previo us interpretations from other laboratories according to which plasmale mmal vesicles are sessile, immobile structures.