Acetylcholine prevents intercellular adhesion molecule 1 (CD54)-induced focal adhesion complex assembly in endothelial cells via a nitric oxide-cGMP-dependent pathway

Objective, Nitric oxide (NO) is induced by exposure of endothelial cells (E C) to acetylcholine, where it acts in a paracrine manner to relax smooth mu scle and as a defensive molecule to inhibit the adhesion of leukocytes to E C, The mechanism(s) of the antiadhesive properties of constitutive NO are p oorly understood. In these studies, we found that NO induced by acetylcholi ne exerts autocrine effects, which interfere with normal adhesion mechanism s, Methods. The function of the adhesion molecule intercellular adhesion molec ule 1 (CD54) of EC was measured using latex beads coated with antibody to C D54 as a model for CD54 ligation by the leukocyte beta 2 integrin, Recruitm ent of filamentous actin (F-actin) and of the signaling molecule vasodilato r-stimulated phosphoprotein (VASP) was measured by immunofluorescence micro scopy, Results. Exposure of EC to anti;CD54 beads induced the subplasmalemmal asse mbly of F-actin and VASP, Acetylcholine blocked the anti-CD54 bead-induced translocation of F-actin and VASP; this effect was reversed by inhibition o f NO production. The NO action did not interfere with binding, but complete ly inhibited the assembly of the focal activation complex, which we believe is necessary for firm heterotypic adhesion between leukocyte and EC, Furth er studies indicated that the NO effect was due to its capacity to raise cG MP, Platelet endothelial cell adhesion molecule 1 (CD31, also implicated in leukocyte adhesion) did not mimic CD54 responses. Conclusion. These results indicate that the ligation of endothelial cell CD 54 induces the assembly of subplasmalemmal F-actin and the recruitment of V ASP, NO derived from constitutive nitric oxide synthase acts to disrupt the se CD54-elicited endothelial cell responses. This action may protect vascul ar endothelium from leukocyte-mediated injury.