RECEPTOR-REGULATED TRANSLOCATION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE

The endothelial nitric-oxide synthase (eNOS) is activated by transient increases in intracellular Ca2+ elicited by stimulation of diverse re ceptors, including bradykinin B-2 receptors on endothelial cells. eNOS and B-2 receptors are targeted to specialized signal-transducing doma ins in the plasma membrane termed plasmalemmal caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation, b ut in resting cells, eNOS is tonically inhibited by its interactions w ith caveolin, the scaffolding protein in caveolae, We used a quantitat ive approach exploiting immunofluorescence microscopy to investigate r egulation of the subcellular distribution of eNOS in endothelial cells by bradykinin and Ca2+. In resting cells, most of the eNOS is localiz ed at the cell membrane. However, within 5 min following addition of b radykinin, nearly all the eNOS translocates to structures in the cell cytosol; following more protracted incubations with bradykinin, most o f the cytosolic enzyme subsequently translocates back to the cell memb rane. The bradykinin-induced internalization of eNOS is completely abr ogated by the intracellular Ca2+ chelator BAPTA; conversely, Ca2+-mobi lizing drugs and agonists promote eNOS translocation. These results es tablish that eNOS targeting to the membrane is labile and is subject t o receptor-regulated Ca2+-dependent reversible translocation, providin g another point for regulation of NO-dependent signaling in the vascul ar endothelium.