Isolation and microsequencing of a novel cotinine receptor.

1. Nicotine and its main derivative. cotinine, are reported to have distinct central activities in mammals. In this study, the cotinine receptor was separated by biochemical procedures including radio receptor, affinity-chromatography, SDS-PAGE, and N-terminal sequencing assays. 2. Consistently, the results showed that distinctive cotinine receptors exist in different tissues of mammals. In rat brain, the affinity chromatography and [125I]cotinine receptor essays were used to isolate a 40-kDa protein (p40) with higher affinity for cotinine than alpha-bungarotoxin and nicotine. The N-terminus amino acid sequences of the p40 and its internal tryptic peptides showed no identity to recently described protein sequences, with the exception of homology to the human p205 synovial fluid protein. 3. These results, in agreement with other behavioral studies, are the first molecular evidence for distinctive nicotine and cotinine receptors in mammals.

Evidence that nicotine acetylcholine receptors are not the main targets of cotinine toxicity.

The toxicity of nicotine, cotinine and their mixtures was studied in Mus musculus mice as well their effects on growth after repetitive administration to young mice. The affinity constants of the two alkaloids for the nicotinic acetylcholine receptors (nAChRs) of Torpedo and rat brain membranes were determined. The administration of these alkaloids produced distinct symptoms of intoxication. Nicotine was 100-fold more toxic than cotinine and 10-fold more rapid than cotinine at producing respiratory arrest. The affinity of nicotine for both subtypes of nAChRs was > 100-fold higher than that of cotinine. Repetitive administrations of nicotine caused weight loss, whereas that of cotinine caused weight gain (P < 0.01). The administration of the two alkaloids as mixtures to mice caused significantly (P < 0.01) higher mortality than theoretically expected. Furthermore, hexamethonium pretreatment reduced by 2-fold (P < 0.01) the toxicity of nicotine but enhanced by 1.6-fold (P < 0.01) that of cotinine and was without effects on toxicity of mixtures. We suggest that nAChRs are not the main targets of cotinine toxicity.

Effect of nicotine and cotinine on the susceptibility to in vitro oxidation of LDL in healthy non smokers and smokers.

Cigarette smoke of which the major component is nicotine plays an important role in the development of cardiovascular diseases. To study the effect of in vitro incubation of LDL with nicotine and its metabolite, cotinine on a copper-induced peroxidation, we monitored the formation of conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances production. The LDL studied were taken from six non-smokers (aged 41.5 years) and six smokers who consumed at least ten cigarettes per day (40.7 years). LDL oxidation with CuSO4 showed that cigarette smoking promotes LDL susceptibility to peroxidative modification. During the peroxidation of LDL with nicotine (O to 5 mmol/1) and CuSO4 (5 micromol/l), the formation of hydroperoxides decreased when nicotine concentrations increased and the production of TBARS increased in a concomitant manner. The results showed that the presence of nicotine destabilized the production of hydroperoxides in LDL and increased the formation of secondary oxidation products. On the other hand, cotinine had no effect on LDL oxidative susceptibility in smokers and non-smokers.

Physico-chemical properties of copper-oxidized very low density lipoproteins.

The aim of our study was to investigate the effect of Cu2+ catalyzed oxidation on VLDL physico-chemical properties and secondary structure of apo B-100. Incubation of very low density lipoproteins with copper ions resulted in a decrease in tryptophan and lysine residues parallel to lipid peroxidation products, conjugated dienes and TBARS. Fluorescence polarization showed an increase in the molecular order at the lipoprotein surface of VLDL, as demonstrated by the increase in Pf values of DPH. The secondary structure of apo B-100 was investigated by infrared spectroscopy. Increased order and structural changes, as observed after oxidative stress on VLDL, could be of relevance in the abnormal interactions between lipoproteins and cell membranes.

Nicotine is more efficient than cotinine at passing the blood-brain barrier in rats.

1. Nicotine and its main metabolite, cotinine, were reported to have distinct behavioral activities in mammals. 2. In this study, cotinine was synthesized without detectable nicotine contamination to compare the ability of nicotine and cotinine to pass the blood-brain barrier (BBB) in rats. 3. The alkaloids were extracted from plasma and brain tissues by methanol, identified by thin-layer chromatography, and quantified by high-pressure liquid chromatography and radioimmunoassays. 4. Consistently, the three methods showed that the passage of cotinine was time, route of administration, and dose dependent and that nicotine was more efficient than cotinine to pass the BBB. 5. The results suggest that these alkaloids may have central activities that probably result from their actions at distinct molecular levels.

Effect of desialylation on low density lipoproteins: comparative study before and after oxidative stress.

Aim of our study was to investigate the effect of the desialylation induced by neuraminidase treatment on low density lipoprotein susceptibility to peroxidative stress induced by incubation with copper ions. Our results show that peroxidative stress induces the formation of aggregates that was not observed in desialylated low density lipoproteins. An increase in thiobarbituric reactive substances and a decrease in polyunsaturated fatty acids content have been shown in oxidized LDL. These modifications were less pronounced in oxidized low density lipoproteins previously treated by neuraminidase. The present data suggest a lower susceptibility to peroxidative stress in previously desialylated low density lipoproteins.

Desialylated low density lipoproteins and atherosclerosis.

Oxidative modification of LDL is accompanied by a number of compositional and structural changes, now well known. In addition, other atherogenic modifications of LDL exist, such as desialylation. The present article summarizes the recent data related to desialylated LDL and to the presence of these LDL in blood plasma of patients with coronary atherosclerosis. In addition, this review examines the sensitivity of these LDL to peroxidative stress.

Effect of hydrogen fluoride inhalation on lipid metabolism in guinea pigs.

The action of fluoride in vivo (exposure 96 hrs to 7 mg/m3) on the metabolism of cyclic AMP and relationship between cAMP and lipid metabolism was investigated. The mean values for cAMP, non esterified fatty acids and cholesterol were significantly increased after hydrogen fluoride exposure. cAMP is directly responsible for the increased lipolysis. In animals exposed to HF, theophylline injection causes increases of non esterified fatty acids and not produces modification of cholesterol level.

Effects of inhaled HF on lipid metabolism in guinea pigs.

Guinea pigs were exposed to hydrogen fluoride (10 mg/m3) for 84 hr. Plasma triglyceride and cholesterol levels were significantly increased in the treated animals with respect to the controls. An increase in triglyceride levels was also observed in the liver. The metabolism of plasma and hepatic lipids was studied using incorporation of [1-14C]acetate. Compared to the control group, triglyceride synthesis was accelerated in the liver of the fluoride group. Extrahepatic lipoprotein lipase activity however was decreased in the fluoride group; this could be related to the decrease in apoprotein C.

Influence of dicarboxylic phosphatidylcholines on phosphatidylcholine liposomes as revealed by gel chromatography and electron microscopy.

The effect of dicarboxylic phosphatidylcholines (glutarylphosphatidylcholine) on the structural changes of phosphatidylcholine liposomes is examined by using multilamellar liposomes prepared with egg phosphatidylcholine or dipalmitoylphosphatidylcholine and by varying the surface charge by addition of dicetyl phosphate. Investigations are performed by gel chromatography and electron microscopy. Glutarylphosphatidylcholine is in micellar form (rod-like micelles or globular micelles). The structures obtained depend on the fatty acid saturation of liposomes and on the charge of liposome (addition or not of dicetyl phosphate). With egg phosphatidylcholine/glutarylphosphatidylcholine dispersions, an aspect more similar to myelinic figures than liposomes is observed, while in the presence of dicetyl phosphate, liposomes similar to control egg phosphatidylcholine liposomes are obtained. Gel chromatography on Sepharose 4B and turbidity measurements prove that dicetyl phosphate increases the stability of egg phosphatidylcholine/glutarylphosphatidylcholine mixtures. On the other hand, in dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, incorporation of dicetyl phosphate destabilizes bilayer structure and the formation of mixed micelles occurs. Viscosity measurement shows, in the presence of dicetyl phosphate, an increased fluidity for dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, in agreement with the micellar organization. These data confirm that the disorganization of liposomal membranes by dicarboxylic phosphatidylcholine depends on the fatty acid composition of phosphatidylcholine and on the presence of dicetyl phosphate.

Affinity chromatography of arterial lysosomal cholesterol ester hydrolase.

The glycoproteic nature of rabbit aortic lysosomal cholesterol ester hydrolase has been demonstrated by affinity chromatography on Concanavalin A-Sepharose. After chromatography, the enzyme lacks synthesizing activity. This activity is restored by addition of deactivated lysosomes containing more endogenous cholesterol. On the other hand, a hypothesis for the activation role of bis(monoacylglyceryl) phosphate is suggested.

Influence of dicarboxylic phosphatidylcholines on the stability and phase transition of phosphatidylcholine liposomes.

The effect of dicarboxylic phosphatidylcholines (glutaryl phosphatidylcholine) on the stability and phase transition of phosphatidylcholine liposomes is examined by using liposomes prepared with egg phosphatidylcholine or dipalmitoyl phosphatidylcholine and by varying the surface charge by addition of dicetyl phosphate. Light-scattering and osmotic behaviour studies showed that the stability of liposomes containing dicarboxylic phosphatidylcholine is influenced by the charge and the fatty acid saturation of the liposomes. Increasing the glutaryl phosphatidylcholine-to-phosphatidylcholine molar ratio in liposomes caused the formation of mixed glutaryl phosphatidylcholine/phosphatidylcholine micelles. The sensitivity of the lipid bilayers towards glutaryl phosphatidylcholine action increases with the fatty acid saturation of liposomes. Dipalmitoyl phosphatidylcholine liposomes are most sensitive to the dicarboxylic phosphatidylcholine effect. Dicetyl phosphate addition enhances the solubilization of liposomes prepared from saturated phospholipids. The effect of increasing concentrations of glutaryl phosphatidylcholine on the gel-to-liquid crystal thermal transition of dipalmitoyl phosphatidylcholine was observed. Glutaryl phosphatidylcholine modifies the thermal phase transition of the constituents of the liposome. The presence of dicetyl phosphate in liposomes affects the phase transition temperature of these liposomes. It is suggested that the formation of the mixed micelles is responsible for the phase transition modifications. These data show that the solubilization of liposomes by dicarboxylic phosphatidylcholines depends on the fatty acid composition of phosphatidylcholine and on the presence of dicetyl phosphate.

[Biochemistry of atherogenesis: effects of prostaglandins (author's transl)]. / Rôle des lipides, des thromboxanes et des prostacyclines dans l'athérosclérose.

Endothelial damage results in increased permeability to serum lipoproteins, adhesion of platelets which release a growth - stimulating protein for smooth muscle cells and a number of vasoactive substances, including thromboxanes. Smooth muscle cells migrate from media, proliferate and accumulate lipid. "Atherogenic" lipoproteins enhance arterial accumulation of cholesterol. After infiltration of serum lipoproteins, smooth muscle cells become into lipid - loaded foam cells which die. Cell necrosis produce the amorphous lipid deposits which form the nucleus of the atherosclerosis plaque. The accompanying proliferative and inflammatory response in the developing plaque is associated with increased prostaglandin synthesis. Calcification and ulceration appear. Thromboxane production leads to platelet aggregation and thrombus formation. Antiplatelet and antiinflammatory drugs have beneficial effects by blocking aggregation, inhibiting the vasospasm associated with microthrombus formation and thromboxane release and modifying the inflammatory response by blocking prostaglandin and thromboxane synthesis in the developing plaque.

[Changing relative proportions of apolipoproteins of VLDL in chronic male alcoholics (author's transl)]. / Etude des apolipoprotéines CII et CIII des VLDL lors d'une consommation alcoolique chronique excessive.

Apoproteins of very low density lipoproteins of plasma from male chronic alcoholics and healthy men serving as controls were investigated. VLDL were delipidated using diethyl ether-ethanol and were separated by polyacrylamide gel electrophoresis. The average relative concentration of apo Cll, Clll1 and Clll2 are investigated. The proportion of apo Cll is reduced, and that of apo Clll increased (particularly Clll1) in male chronic alcoholics compared to the normal and the ratio of apo Clll1 to apo cll was significantly increased. The Cll/Clll1 + Clll2 ratio is highly significantly reduced in patients. Changes in apo Clll1/Cll ratio in relation to hypertriglyceridaemia are envisaged. Furthermore apo Cll is believed to activate and apo Clll to inhibit lipoprotein lipase activity "in vitro".

Fatty acid composition of aortic lipids in male and female rabbits.

Aortic tissue of adult New-Zealand male and female white rabbits was studied. After thin-layer chromatography of cholesterol esters, triglycerides, free fatty acids, phosphatidylethanolamines and phosphatidylcholines, their fatty acid compositions are determined. Phosphatidylcholines and phosphatidylethanolamines contain important quantities of arachidonic acid. The level of linoleic acid in these phospholipids is higher in male animals than in female. Conversely the level of arachidonic acid is higher in females than in males.

Inhibition of lecithin:cholesterol acyltransferase in human plasma by dicarboxylic lecithins.

The effect of dicarboxylic lecithins on the lecithin:cholesterol acyltransferase [LCAT] activity was studied with a partially purified preparation [approximately 24-fold]. The presence of dicarboxylic lecithins is followed by the inhibition of LCAT. The inhibition is proportional to the concentration of glutaryl-lecithin and is about 50% with 0.16 mM glutaryl-lecithin. For a higher concentration, the inhibition is total. The inhibition of LCAT activity was of a mixed type. It can be explained by a combination of competitive inhibition and interaction of glutaryl-lecithin with phospholipid, which changes the physical properties of lipid, making it less available for conversion into cholesteryl ester.