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

Abstract

After endothelial damage in vivo, there is an induction of nitric oxide synthase (NOS) in the underlying smooth muscle cells. We hypothesized that intrinsic factors could induce NOS independently of blood elements. This was tested using an in vitro organ culture technique. Rat aortas with endothelium removed before 24-h organ culture (ERB) failed to constrict to phenylephrine after culture, whereas with endothelium removal after culture there was a normal constrictor response. Constrictor activity in ERB aortas was restored by the concomitant treatment with either the protein synthesis inhibitor cycloheximide (1 μM) or the NOS inhibitorl- N 5-(1-iminoethyl)ornithine hydrochloride (l-NIO, 100 μM). 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 application ofl-NIO subsequent to the 24-h organ culture, which suggests that other effects during culture contributed to the diminished tissue response. When ERB aortas were treated with reduced glutathione (GSH, 3 mM for 24 h), acute application of l-NIO then fully restored the constrictor effect. This suggests that peroxynitrite produced during culture may in part be responsible for loss of constrictor effects, and this was substantiated by the presence of nitrated tyrosine residues in aortic proteins and also widespread DNA damage, which was prevented by bothl-NIO and GSH. Thus some of the immediate (24-h) effects of endothelium removal involve intrinsic mechanisms resulting in iNOS synthesis, which leads to both nitric oxide and peroxynitrite generation, with resultant tissue damage and loss of contractile function.

Cardiac performance in Salmo salar with infectious salmon anaemia (ISA): putative role of nitric oxide

“…This appears similar to therapeutic strategies adopted in animal models and in humans with septic shock. In such models the induction of iNOS, following endothelial damage (Binko et al 1999), in vivo endotoxin injection (Kelly et al 1996), or activation of pro-inflammatory cytokines (Finkel et al 1992) with consequent generation of large amounts of NO, is responsible for a reversible defect in myocardial function (Vallance et al 2000).…”

Incubation of rat aortic rings produces a specific reduction in agonist-evoked contraction: effect of age of donor

“…This difficulty can be overcome by incubation of intact segments. Incubation of aortic segments produces a decrease in phenylephrine-evoked contraction that is restored by NOS inhibition or glutathione (Binko et al, 1999) and stimulates nitrate production that is blocked by N G -nitro-L-arginine methyl ester (L-NAME) (Bishop-Bailey et al, 1997).…”

“…Oxidative stress was indirectly evaluated by measuring anti-oxidant capacity: glutathione-related (glutathione Stransferases (GSTs, EC 2.5.1.18), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione reductase (GSSR, EC 1.6.4.2) and catalase (EC 1.11.1.6) activity. The impact of oxidative stress on vasomotion was assessed by co-incubation with the anti-oxidants reduced glutathione (GSH) (Binko et al, 1999) or pyrrolidine dithiocarbamate (PDTC) (Moellering et al, 1999). The role of iNOS was appraised by measurement of iNOS mRNA expression (compared to cyclo-oxygenase (COX-2) expression) and the impact of L-NAME on contraction.…”

Effects of elevated perfusion pressure and pulsatile flow on human saphenous veins isolated from diabetic and non-diabetic patients

“…14,15 Oxidative stress Similar to other published studies, no immunohistochemical expression of nitrotyrosine in the venous wall was observed. 11,12 NO can react with the superoxide anion (O 2 − ) to produce peroxynitrite (ONOO − ), 20 which can cause nitration of tyrosine residues in proteins. Therefore, the absence of nitrotyrosine staining provides indirect evidence that oxidative stress does not have a crucial role.…”