Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by a decrease in the bioavailability of endothelium-derived nitric oxide. Endothelial nitric-oxide synthase (eNOS) associates with caveolae and is directly regulated by the caveola protein, caveolin. In the present study, we examined the effects of oxidized low density lipoprotein (oxLDL) on the subcellular location of eNOS, on eNOS activation, and on caveola cholesterol in endothelial cells. We found that treatment with 10 g/ml oxLDL for 60 min caused greater than 90% of eNOS and caveolin to leave caveolae. Treatment with oxLDL also inhibited acetylcholine-induced activation of eNOS but not prostacyclin production. oxLDL did not affect total cellular eNOS abundance. Oxidized LDL also did not affect the palmitoylation, myristoylation or phosphorylation of eNOS. Oxidized LDL, but not native LDL, or HDL depleted caveolae of cholesterol by serving as an acceptor for cholesterol. Cyclodextrin also depleted caveolae of cholesterol and caused eNOS and caveolin to translocate from caveolae. Furthermore, removal of oxLDL allowed eNOS and caveolin to return to caveolae. We conclude that oxLDL-induced depletion of caveola cholesterol causes eNOS to leave caveolae and inhibits acetylcholine-induced activation of the enzyme. This process may be an important mechanism in the early pathogenesis of atherosclerosis.Studies in animal models and in humans have shown that hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by an early, selective impairment of endothelium-derived relaxation. This impairment is due to a decrease in bioavailable endothelium-derived nitric oxide (NO) 1 (1). In the initial phase of the disease process, there is impaired responsiveness to receptor-dependent stimuli, such as acetylcholine, whereas responsiveness to receptor-independent stimuli such as the calcium ionophore A23187 is not altered. As such, the early pathogenesis involves attenuated endothelial NO production in response to extracellular stimuli, but the capacity for maximal enzyme activation and the breakdown of NO are not affected. As the disease progresses, there is nonspecific inhibition of NO bioavailability. Although this later inhibition is most likely multifactorial in origin, it is at least partly due to enhanced inactivation of NO by superoxide anions (2-4). These processes result in increased neutrophil adherence to the endothelium, thereby promoting a key step in the pathogenesis of atherosclerosis (5). The chronic inhibition of NO synthesis in rabbit models of hypercholesterolemia accelerates the development of vascular dysfunction and intimal lesions, providing additional evidence that the impairment of NO synthesis promotes atherogenesis (6). In vitro investigations have further shown that oxidized LDL (oxLDL) inhibits NO-mediated responses. Antioxidants reduce both the formation of free radicals and the oxidative modification of LDL that lead to impaired NO-related responses (7). Thus, NO is critically involved ...

Oxidized LDL (oxLDL) depletes caveolae of cholesterol, resulting in the displacement of endothelial nitric-oxide synthase (eNOS) from caveolae and impaired eNOS activation. In the present study, we determined if the class B scavenger receptors, CD36 and SR-BI, are involved in regulating nitric-oxide synthase localization and function. We demonstrate that CD36 and SR-BI are expressed in endothelial cells, co-fractionate with caveolae, and co-immunoprecipitate with caveolin-1. Co-incubation of cells with 10 g/ml high density lipoprotein (HDL) prevented oxLDLinduced translocation of eNOS from caveolae and restored acetylcholine-induced nitric-oxide synthase stimulation. Acetylcholine caused eNOS activation in cells incubated with 10 g/ml oxLDL (10-15 thiobarbituric acid-reactive substances) and blocking antibodies to CD36, whereas cells treated with only oxLDL were unresponsive. Furthermore, CD36-blocking antibodies prevented oxLDL-induced redistribution of eNOS. SR-BI-blocking antibodies were used to demonstrate that the effects of HDL are mediate by SR-BI. HDL binding to SR-BI maintained the concentration of caveola-associated cholesterol by promoting the uptake of cholesterol esters, thereby preventing oxLDL-induced depletion of caveola cholesterol. We conclude that CD36 mediates the effects of oxLDL on caveola composition and eNOS activation. Furthermore, HDL prevents oxLDL from decreasing the capacity for eNOS activation by preserving the cholesterol concentration in caveolae and, thereby maintaining the subcellular location of eNOS.Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by an early, selective decrease in the bioavailability of endothelium-derived nitric oxide (NO) (1). Responsiveness to receptor-dependent stimuli, such as acetylcholine, is decreased in the initial phase of the disease process, whereas responsiveness to receptor-independent stimuli such as the calcium ionophore A23187 is not altered. Therefore, the early pathogenesis is characterized by attenuated endothelial NO production in response to extracellular stimuli, even though the capacity for maximal enzyme activation and the breakdown of NO are not affected. As the disease progresses, nonspecific inhibition of NO bioavailability occurs, which is at least partly due to enhanced inactivation of NO by superoxide anions (2-4). The chronic inhibition of NO synthesis in rabbit models of hypercholesterolemia accelerates the development of vascular dysfunction and intimal lesions, providing additional evidence that the impairment of NO synthesis promotes atherogenesis (5). In vitro investigations have further shown that oxLDL 1 inhibits NO-mediated responses (6). Numerous studies have demonstrated that the endothelial isoform of NO synthase (eNOS) is localized in plasmalemmal caveolae and that caveolin is a negative regulator of eNOS enzymatic activity (7). Caveolae are lipid domains that typically represent about 1-4% of the total plasma membrane surface area (8). The structure and function of caveolae is dep...

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are oxidized. The oxidation of 1,2-propanediol is competitive with that of ethanol. The enzyme also oxidizes monohalo, but not dior trihalo derivatives of ethanol, and reduces monochloro, dichloro, and trihalo derivatives of acetaldehyde. Both the polyhalo derivatives of ethanol and of acetaldehyde inhibit ethanol oxidation. These enzymatic findings are pertinent to the metabolism of these alcohols and aldehydes in man.

Aldehyde dehydrogenase was partially purified from human liver. During purification, activity was resolved into one major and one minor species by DEAE-cellulose column chromatography; the properties of the predominant form were investigated.Aldehydes are oxidized when NAD+, but not NADP+, is the electron acceptor, maximal activity occurring between pH 9 and 10. Several aliphatic aldehydes and hydroxyaldehydes served as substrates for the enzyme. Benzaldehyde also was oxidized, but at a comparatively low rate. Aliphatic aldehydes carrying negatively charged groups are not oxidized. The enzyme is sensitive to low concentrations of two sulfhydryl reagents, p-chloromercuribenzoate and mercuric ions; this inhibition was reversed with sulfhydryl compounds. Like other aldehyde dehydrogenases, the human liver enzyme is inhibited by arsenite and the inhibition is potentiated by mercaptoethanol. Only 35% inhibition was produced by disulfiram at 40 μM; and diethyldithiocarbamate, its metabolic reduction product, had no effect on activity below 10 mM.

Human liver aldehyde dehydrogenase was inhibited by aromatic chelating agents. However, structurally related compounds with much lower metal-complexing ability displayed affinities for enzyme essentially equal to those of their respective chelating analogues. Inhibition was competitive with respect to the coenzyme. It is suggested that hydrophobic interactions between the inhibitors and the coenzyme-binding site of the enzyme are responsible for the observed effects on activity.

In order to increase the retention of drug activity, regiospecific coupling has been used to synthesize conjugates of methotrexate (MTX, 1) with normal rabbit IgG (NRG) and a mouse anti-human renal cancer monoclonal IgG (Dal K-20). MTX gamma-methyl ester (4) was produced either by selective esterification of MTX or by coupling of 4-amino-4-deoxy-N10-methylpteroic acid (2) with suitable glutamic acid derivatives. The MTX gamma-methyl ester (4) was then converted to the corresponding hydrazide 6. An amide-linked conjugate was formed when the MTX gamma-hydrazide (6) was converted to reactive acylating species 7 by using tert-butyl nitrite or trifluoroacetaldehyde, which were reacted with nucleophilic centers, presumably epsilon-amino groups, in native IgG. A hydrazone-linked conjugate was formed when MTX gamma-hydrazide (6) was reacted directly with IgG that had first been oxidized with periodate to form polyaldehyde IgG. The regiospecifically synthesized conjugates were somewhat more effective inhibitors in vitro of dihydrofolate reductase and of colony formation by human renal cancer (Caki-1) cells than were control nonregiospecific conjugates.

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