RECEPTOR-BINDING AND INTERNALIZATION OF A UNIQUE BIOLOGICALLY-ACTIVE ANGIOTENSIN-II COLLOIDAL GOLD CONJUGATE - MORPHOLOGICAL ANALYSIS OF ANGIOTENSIN-II PROCESSING IN ISOLATED VASCULAR STRIPS

It is well recognized that following binding of ligand, many receptors undergo a process of agonist-induced receptor-mediated endocytosis (RME) or internalization. We recently characterized the intracellular pathway and kinetics of angiotensin II (AII)-RME in cultured explant-derived rat aortic vascular smooth muscle cells (VSMCs). To definitively study vascular internalization of AII, however, it is critical to examine AII binding and uptake in intact, differentiated VSM. For the present study, we used a unique, biologically active AII-colloidal gold conjugate to qualitatively examine, by transmission electron microscopy, the ultrastructural details of AII binding and internalization in intact VSM. Strips of isolated, cleaned, and denuded rat aortic smooth muscle were incubated with AII-gold probe for 2 h at 4 degrees C to allow binding of the complex without simultaneous internalization. After rinsing to remove unbound AII-gold, the strips were incubated at 37 degrees C (5-90 min) to initiate internalization. These studies show that AII initially binds over the entire surface of medial VSMCs. Following binding, the AII is internalized via small receptosomes which likely represent clathrin-coated vesicles. By 20 min after internalization, gold particles are evident within large lysosome-like vesicles deep within the VSM. There was no evidence of association of gold particles with the Golgi network at any of the time periods examined. Gold particles were occasionally observed in perinuclear regions after 90 min at 37 degrees C, although this did not appear to represent a typical pattern. AII-gold binding and internalization were selective: control probe (no AII) did not internalize; Losartan potassium (selective AT(1) receptor antagonist) effectively competed for AII-gold binding and internalization.