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 (EC) to acetylcholine, where it acts in a paracrine manner to relax smooth muscle and as a defensive molecule to inhibit the adhesion of leukocytes to EC. The mechanism(s) of the antiadhesive properties of constitutive NO are poorly understood. In these studies, we found that NO induced by acetylcholine exerts autocrine effects, which interfere with normal adhesion mechanisms.


METHODS
The function of the adhesion molecule intercellular adhesion molecule 1 (CD54) of EC was measured using latex beads coated with antibody to CD54 as a model for CD54 ligation by the leukocyte beta2 integrin. Recruitment of filamentous actin (F-actin) and of the signaling molecule vasodilator-stimulated phosphoprotein (VASP) was measured by immunofluorescence microscopy.


RESULTS
Exposure of EC to anti-CD54 beads induced the subplasmalemmal assembly of F-actin and VASP. Acetylcholine blocked the anti-CD54 bead-induced translocation of F-actin and VASP; this effect was reversed by inhibition of NO production. The NO action did not interfere with binding, but completely inhibited the assembly of the focal activation complex, which we believe is necessary for firm heterotypic adhesion between leukocyte and EC. Further studies indicated that the NO effect was due to its capacity to raise cGMP. Platelet endothelial cell adhesion molecule 1 (CD31, also implicated in leukocyte adhesion) did not mimic CD54 responses.


CONCLUSIONS
These results indicate that the ligation of endothelial cell CD54 induces the assembly of subplasmalemmal F-actin and the recruitment of VASP. NO derived from constitutive nitric oxide synthase acts to disrupt these CD54-elicited endothelial cell responses. This action may protect vascular endothelium from leukocyte-mediated injury.
Clancy
R M
RM
Hospital for Joint Diseases and Department of Medicine, New York University School of Medicine, New York 10003, USA.
Abramson
S B
SB
D000109:Acetylcholine / Q000494:pharmacology*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology* / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000648:ultrastructure; D022001:Focal Adhesions / Q000187:drug effects*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000502:physiology*; D008840:Microfilament Proteins / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014471:Umbilical Veins; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D018799:Intercellular Adhesion Molecule-1; D009569:Nitric Oxide; D006152:Cyclic GMP; D000109:Acetylcholine