Stimulation of G_sand Inhibition of G_iProtein Functions by Minimally Oxidized LDL

Abstract

We have previously shown that treatment of aortic endothelial cells with minimally oxidized LDL (MM-LDL) induces their interaction with monocytes but not neutrophils and that these induced responses are associated with increased cAMP levels. Here we studied the mechanism of by which MM-LDL elevates cAMP levels. Treatment of human aortic endothelial cells with MM-LDL resulted in a saturable dose-dependent increase in cAMP levels. Studies using a combination of pertussis toxin and MM-LDL suggested that part of the cAMP increase was due to the stimulation of Gs complexes. Studies with pertussis toxin-treated membranes in which Gi was completely inhibited were used to directly address the effect of MM-LDL on the Gs pathway. MM-LDL and an oxidized lipid (palmitoyl arachidonyl phosphatidylcholine), the effects of which mimic those of MM-LDL, caused a 40% to 100% increase in cAMP levels in these isolated membranes that was augmented by GTP, thus showing Gs stimulation. These results also show that MM-LDL increases cAMP levels by inhibiting Gi. MM-LDL inhibited ADP ribosylation of Gi by about 30% and completely abolished the ability of serotonin to interact with Gi complexes, whereas direct activation of Gi by mastoparan was not inhibited. This observation suggests that MM-LDL interferes with the interaction of Gi molecules with inhibitory receptors. There was no direct effect of MM-LDL on adenylate cyclase. Overall, these studies show that MM-LDL increases cAMP levels both by stimulating Gi and inhibiting Gi complexes.

Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils

“…Furthermore, exposure of Xenopus oocytes to PGPC did not desensitize the oocyte to the effect of POVPC (data not shown), supporting the hypothesis that the binding sites for the two molecules are different. We previously have reported that MM-LDL increased the levels of cAMP in human aortic endothelial cells by activation of a Gs-coupled receptor and decreasing the endogenous activation of a Gi-coupled receptor (7). The present studies demonstrate that POVPC, but not PGPC, activates a Gs-coupled pathway in Xenopus oocytes (Fig.…”

“…HAEC were incubated with oxidized phospholipids and͞or lipopolysaccharide (LPS) as for cell adhesion experiments, and total RNA was isolated from cells by using the guanidine thiocyanate͞phenol method. Northern blot analysis using E-selectin cDNA was performed as described (7). 36B4 or 28S cDNA were used to control for RNA loading.…”

“…Our group has demonstrated that minimally oxidized low density lipoprotein (MM-LDL) stimulates endothelial cells to bind monocytes, but not neutrophils (4) by a mechanism involving connecting segment 1 (CS-1) fibronectin and ␣4␤1 integrin (5). There are indications that the effects of MM-LDL on endothelial cells are mediated by a receptor, increasing levels of cAMP (6,7).…”

Endothelial Responses to Oxidized Lipoproteins Determine Genetic Susceptibility to Atherosclerosis in Mice

“…40,41 Previous studies have suggested that scavenger receptors are unlikely to be the receptors that mediate MM-LDL-induced inflammatory responses in ECs. 34,42,43 Because they bind, internalize, and degrade oxLDL, scavenger receptors may influence accumulation of oxLDL in arterial walls.…”

Interleukin-10 Blocks Atherosclerotic Events In Vitro and In Vivo

“…Previous work from our laboratory has shown that MM-LDL exerts its effects by initiating an increase in intracellular cAMP. 35 We therefore examined cAMP levels in the presence of IL-10 after stimulation with MM-LDL (Figure 3) or cholera toxin (data not shown) and found them to be elevated to levels equivalent to those in cells identically stimulated but not treated with IL-10. This study suggests that IL-10 acts downstream of the MM-LDL-mediated increase in cAMP.…”