Endothelium removal induces iNOS in rat aorta in organ culture, leading totissue damage

After endothelial damage in vivo, there is an induction of nitric oxide syn thase (NOS) in the underlying smooth muscle cells. We hypothesized that int rinsic factors could induce NOS independently of blood elements. This was t ested using an in vitro organ culture technique. Rat aortas with endotheliu m removed before 24-h organ culture (ERB) failed to constrict to phenylephr ine after culture, whereas with endothelium removal after culture there was a normal constrictor response. Constrictor activity in ERB aortas was rest ored by the concomitant treatment with either the protein synthesis inhibit or cycloheximide (1 mu M) or the NOS inhibitor L-N-5-(1-iminoethyl)ornithin e hydrochloride (L-NIO, 100 mu M). The ERB aortas also had an elevated NOS activity and induced NOS (iNOS) immunoreactivity. The constrictor response to phenylephrine in ERB aortas was only partially restored by acute applica tion of L-NIO subsequent to the 24-h organ culture, which suggests that oth er effects during culture contributed to the diminished tissue response. Wh en ERB aortas were treated with reduced glutathione (GSH, 3 mM for 24 h), a cute application of L-NIO then fully restored the constrictor effect. This suggests that peroxynitrite produced during culture may in part be responsi ble for loss of constrictor effects, and this was substantiated by the pres ence of nitrated tyrosine residues in aortic proteins and also widespread D NA damage, which was prevented by both L-NEO and GSH. Thus some of the imme diate (24-h) effects of endothelium removal involve intrinsic mechanisms re sulting in iNOS synthesis, which leads to both nitric oxide and peroxynitri te generation, with resultant tissue damage and loss of contractile functio n.