Liver Plasma Membranes Research Articles

Insulin-Like Peptides of the Cerebropleural Ganglion of the Mollusc Anodonta cygnea: Isolation, Purification, and Radioligand Analysis

Cerebropleural ganglia from 4000 individuals of the mollusc Anodonta cygnea were submitted to procedures developed for isolation of vertebrate pancreatic insulins: homogenization and extraction, stage-like isoelectrical sedimentation, and ion-exchange chromatography. As a result of purification of the obtained preparation, using high-effective liquid chromatography, there were identified 7 protein peaks differing by time of retention on the reverse-phase sorbent in acetonitryl gradient and designated as insulin-related peptides (IRP), IRP1-IRP7. The material was characterized by the peptide ability to inhibit specific binding of 125I-insulin and of insulin-related factor-1 (125I-IGF-1) by plasma membranes of the rat liver and brain. The IC50 value of peptide concentration (nM) able to replace 50% of the labeled hormone bound with the receptor amounted in the insulin radioreceptor system for IRP1 to 330, for IRP3 to 130, for IRP4 to 17, for IRP5 to130, for IRP6 to 420 nM. Peptide IRP7 at a maximal concentration (104 ng/ml) replaced less than 50% of labeled hormone, whereas in IRP2 no inhibitory ability was detected under these experimental conditions. The IC50 value in the case of 125I-IGF-1 amounted for IRP1, IRP4, and IRP5 to17, for IRP2 to 50, for IRP3 to 83, for IRP6 to 133 nM. IRP7 at a concentration of 104 ng/ml replaced less than 50% of labeled hormone. The same high relative affinity of the peptide IRP4 (12% of activity of standard insulin and IGF-1) to both receptor types is revealed. The results of analysis in two types of hormonal test systems indicate the ability of the insulin-related peptides of the anodonta cerebropleural ganglion to interact with the vertebrate receptor of insulin and IGF-1. This gives grounds to suggest the presence of the metabolic and growth-stimulating properties in these peptides. For the first time, the IGF-1 activity is revealed in insulin-like molecules in invertebrates. Taking into account the chromatographically revealed differences of physicochemical characteristics of individual IRP as well as predominance of their IGF-1-binding properties, there is suggested another organization of the IRP receptor-binding domains in IPR of this mollusc species, as compared with mammalian insulins.

The suppressed expression and functional activity of hepatic P-glycoprotein in rats with protein–calorie malnutrition

The effect of protein-calorie malnutrition (PCM) on the expression and functional activity of P-glycoprotein (P-gp) in the liver of rats was examined. Control protein diets and protein-restricted diets were fed to Sprague-Dawley rats for 4 weeks, and the expression of P-gp in the liver (Western blot), in vitro uptake of a representative substrate of P-gp, daunomycin, into canalicular liver plasma membrane (cLPM) vesicles, and in vivo canalicular excretion of daunomycin were compared between the control and PCM rats. The expression of P-gp in the cLPM vesicles was decreased (22%, p < 0.05) by PCM. Consistent with this result, the in vitro uptake rate (V(max)) was decreased (35%, p < 0.05) by PCM, with constant affinity (K(m)), for the carrier-mediated uptake of daunomycin into cLPM vesicles, resulting in a 33% (p < 0.05) decrease in the intrinsic uptake clearance (CL(int)). The in vivo canalicular excretion clearance (CL(exc)) of daunomycin was also decreased by 79% (p < 0.01) in PCM rats, but the degree was more severe than would be expected from the 22% decrease in the expression of P-gp and the 33% decrease in the uptake of daunomycin (CL(int)). The hepatic level of adenosine 5'-triphosphate, which was decreased by 60% (p < 0.01) in PCM rats, might have contributed to this severe decrease in CL(exc). In summary, the canalicular excretion of P-gp substrates, such as daunomycin, might be reduced in patients with PCM via a mechanism involving the suppression of the expression of P-gp.

Effects of lipopolysaccharide on the biliary excretion of bile acids and organic anions in rats.

Lipopolysaccharide is known to be a cause of cholestasis associated with sepsis. It has also recently been reported to down-regulate the basolateral and canalicular transporters. The aim of the present study was to examine simultaneously the effect of lipopolysaccharide on the biliary excretion of typical substrates of bile salt export pump and multidrug resistance protein 2 in vivo, and the effect of lipopolysaccharide on the amount of these transporters. After intravenous administration of O127:B8-derived lipopolysaccharide (2.5 mg/kg), the biliary excretion of taurocholate and various organic anions was studied, and the protein levels of bile salt export pump and multidrug resistance protein 2 in the crude liver membrane was determined by western blot analysis. Lipopolysaccharide decreased the biliary excretion of tracer amounts of taurocholate, leukotriene C4, taurolithocholate-3-sulfate and temocapril without affecting bile flow. The biliary excretion of high doses of taurocholate and sulfobromophthalein was markedly inhibited by lipopolysaccharide. Lipopolysaccharide decreased bile salt export pump levels in the liver plasma membrane fraction to 48% of control rats, and markedly decreased multidrug resistance protein 2 levels to 17% of control rats. These findings support the hypothesis that down-regulation of the canalicular transporters by lipopolysaccharide causes the impairment of the biliary excretion of bile acids and organic anions in cholestasis of sepsis prior to the decrease of bile flow.

Effect of Kaiyu Qingwei granule on insulin receptor in liver and skeletal muscular cell membrane in diabetes mellitus rats

Objective: To investigate the effect of Kaiyu Qingwei granule (KYQWG) on the insulin binding capacity of liver and skeletal muscular cell membrane and serum insulin-like growth factor-1 (IGF-1) in streptozotocin-induced diabetic rats.Methods: Rats in four experimental groups were investigated: the control group, the model group, the KYQWG group and the Metformin group. The insulin binding rate (IBR) of liver and skeletal muscular cell membrane was detected by receptor-ligand radiometric method and changes of serum levels of glucose, insulin and IGF-1 were observed before and after 4 weeks of medication.Results: The KYQWG group had a lower blood glucose level and IBR of liver and muscular cell membrane, as compared with those in the model group (P<0.01 or P<0.05), and a higher level of IGF-1 than that in the model group(P<0.01), but had no obvious changes in the serum level of insulin.Conclusion: KYQWG may increase the serum level of IGF-1 in diabetic rats, thus to decrease the insulin resistance at ante-receptor sites and improve the sugar metabolic disturbance in rats with diabetes mellitus.

Hepatic cell membrane damage during cold preservation sensitizes liver grafts to rewarming injury.

Increasing levels of glycerol in extracellular fluid indicate cell membrane disintegration, and can be continuously monitored with microdialysis. The aim of this study was to monitor liver cell membrane damage during liver transplantation in a pig model. Thirteen donor and recipient pairs were divided into two groups; group I, with a liver ischemia time of 5 h (n = 6), and group II, with 15 h of ischemia (n = 7). Microdialysis samples from the liver graft were collected at 20-min intervals during donor operation, cold preservation, implantation, and reperfusion in the recipient. Glycerol concentrations were analyzed. During cold preservation, a continuous increase in glycerol levels was observed. This increase correlated with the time of cold ischemia (r2 = 0.88). During implantation, an accelerated increase of glycerol was observed, with a higher acceleration in group II (P < 0.005). During the first hour after reperfusion, a higher release of glycerol was observed in group II (P = 0.0005). Our data show a constant glycerol release during cold storage, indicating a continuous injury to the cell membranes over time. Improvement in cell membrane protection during cold preservation could lead to improvement in liver transplantation outcome and acceptance of an extended period of cold ischemia.

Taurolithocholic Acid Exerts Cholestatic Effects via Phosphatidylinositol 3-Kinase-dependent Mechanisms in Perfused Rat Livers and Rat Hepatocyte Couplets

Taurolithocholic acid (TLCA) is a potent cholestatic agent. Our recent work suggested that TLCA impairs hepatobiliary exocytosis, insertion of transport proteins into apical hepatocyte membranes, and bile flow by protein kinase Cepsilon (PKCepsilon)-dependent mechanisms. Products of phosphatidylinositol 3-kinases (PI3K) stimulate PKCepsilon. We studied the role of PI3K for TLCA-induced cholestasis in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHC). In IPRL, TLCA (10 micromol/liter) impaired bile flow by 51%, biliary secretion of horseradish peroxidase, a marker of vesicular exocytosis, by 46%, and the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione, by 95% and stimulated PI3K-dependent protein kinase B, a marker of PI3K activity, by 154% and PKCepsilon membrane binding by 23%. In IRHC, TLCA (2.5 micromol/liter) impaired canalicular secretion of the fluorescent bile acid, cholylglycylamido fluorescein, by 50%. The selective PI3K inhibitor, wortmannin (100 nmol/liter), and the anticholestatic bile acid tauroursodeoxycholic acid (TUDCA, 25 micromol/liter) independently and additively reversed the effects of TLCA on bile flow, exocytosis, organic anion secretion, PI3K-dependent protein kinase B activity, and PKCepsilon membrane binding in IPRL. Wortmannin also reversed impaired bile acid secretion in IRHC. These data strongly suggest that TLCA exerts cholestatic effects by PI3K- and PKCepsilon-dependent mechanisms that are reversed by tauroursodeoxycholic acid in a PI3K-independent way.

Forthcoming papers and review topics

We have developed a new rapid cell-free assay for endocytic clathrin-coated vesicle formation using highly purified rat liver plasma membrane sheets. After incubation in the presence of cytosol and nucleotides, released vesicles were collected by high-speed centrifugation and incorporated cargo receptors were detected by Western blotting. Three different cargo receptors were internalized into vesicles while a receptor, known to be excluded from coated pits, was not. The recruitment of cargo receptors into the vesicle fraction was cytosol, ATP and temperature-dependent and was enhanced by addition of GTP. Vesicle formation in this assay was confirmed by subcellular fractionation and EM analysis. Plasma membranes stripped of their endogenous coat proteins with 0.5 m Tris retained vesicle formation activity, which was highly dependent on clathrin and dynamin. Coat proteins and dynamin were not sufficient for clathrin-coated vesicle formation, and other peripheral membrane proteins recruited from the cytosol are required. The nonhydrolyzable ATP analogue, AMPPNP did not support clathrin-coated vesicle formation; however, surprisingly, GTP gamma S was as effective as GTP. This assay will provide a powerful tool to dissect the minimum machinery and to probe the hierarchy of events involved in cargo selection and endocytic clathrin-coated vesicle formation.

N-glycosylation controls functional activity of Oatp1, an organic anion transporter.

Rat Oatp1 (Slc21a1) is an organic anion-transporting polypeptide believed to be an anion exchanger. To characterize its mechanism of transport, Oatp1 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. Protein was present at high levels in isolated S. cerevisiae secretory vesicles but had minimal posttranslational modifications and failed to exhibit taurocholate transport activity. Apparent molecular mass (M) of Oatp1 in yeast was similar to that of unmodified protein, approximately 62 kDa, whereas in liver plasma membranes Oatp1 has an M of approximately 85 kDa. To assess whether underglycosylation of Oatp1 in yeast suppressed functional activity, Oatp1 was expressed in Xenopus laevis oocytes with and without tunicamycin, a glycosylation inhibitor. With tunicamycin, M of Oatp1 decreased from approximately 72 to approximately 62 kDa and transport activity was nearly abolished. Mutations to four predicted N-glycosylation sites on Oatp1 (Asn to Asp at positions 62, 124, 135, and 492) revealed a cumulative effect on function of Oatp1, leading to total loss of taurocholate transport activity when all glycosylation sites were removed. M of the quadruple mutant was approximately 62 kDa, confirming that these asparagine residues are sites of glycosylation in Oatp1. Relatively little of the quadruple mutant was able to reach the plasma membrane, and most remained in unidentified intracellular compartments. In contrast, two of the triple mutants tested (N62/124/135D and N124/135/492D) were present in the plasma membrane fraction yet exhibited minimal transport activity. These results demonstrate that both membrane targeting and functional activity of Oatp1 are controlled by the extent of N-glycosylation.

Effect of in vivo administered hexachlorobenzene on epidermal growth factor receptor levels, protein tyrosine kinase activity, and phosphotyrosine content in rat liver

In the present study, the effects of hexachlorobenzene (HCB) on epidermal growth factor receptor (EGFR) content of liver microsomes and plasma membrane, and on EGFR-tyrosine kinase activity in the microsomal fraction were investigated. In addition, we studied the parameters of the tyrosine kinase signalling pathway such as protein tyrosine kinase (PTK) activity and phosphotyrosine content in microsomal and cytosolic protein. To determine whether the observed alterations were correlated with a manifestation of overt toxicity, a single very low dose of HCB (1 mg/kg body wt) and two much higher doses (100 and 1000 mg/kg body wt), the highest being toxicologically significant in that it reduced serum thyroxine (T 4) and inhibited uroporphyrinogen decarboxylase (URO-D) (EC 4.1.1.37) activity, were tested. Our results demonstrated that liver microsomes of rats treated with HCB had higher levels of EGFR than untreated rats; treated rats also had less EGFR present in hepatocyte plasma membrane fractions than did untreated rats. HCB altered the phosphotyrosine content and protein phosphorylation of some microsomal and cytosolic proteins in a biphasic dose–response relationship. At the low dose, phosphorylation and phosphotyrosine content of several microsomal proteins were increased; however, these effects were diminished or reversed at the higher doses. Our results suggest that chronic HCB treatment produces a down-regulation of the EGFR and a dose-dependent increase in EGFR-tyrosine kinase activity in the microsomal fraction. This effect may contribute to the alteration of membrane and cytosolic protein tyrosine phosphorylation. The level of sensitivity encountered in our studies is extraordinary, occurring at 1/10 to 1/1000 the doses of HCB known to cause other toxicological lesions.

A comparison of hepatoprotective activities of aminoguanidine and N-acetylcysteine in rat against the toxic damage induced by azathioprine

Azathioprine (AZA) is an important drug used in the therapy of autoimmune system disorders. It induces hepatotoxicity that restricts its use. The rationale behind this study was the proven efficacy of N-acetylcysteine (NAC; a replenisher of sulfhydryls) and reports on the antioxidant potential of aminoguanidine (AG; an iNOS inhibitor), that might be useful to protect against the toxic implications of AZA. AG (100 mg/kg; i.p.) or NAC (100 mg/kg; i.p.) were administered to the Wistar male rats for 7 days and after that AZA (15 mg/kg, i.p.) was given as a single dose. This caused an increase in the activity of hepatic aminotransferases (AST and ALT) in the serum 24 h after AZA treatment. AZA (7.5 or 15 mg/kg, i.p.) also caused an increase in rat liver lipid peroxides and a lowering of reduced glutathione (GSH) contents. In the other part of experiment, protective effects of AG and NAC were observed on AZA induced hepatotoxicity. NAC significantly protected against the toxic effects produced by AZA. Pretreatment with NAC prevented any change in the activities of both the aminotransferases after AZA. This pretreatment also resulted in a significant decline in the contents of lipid peroxides and a significant elevation in GSH level was evident after AZA treatment. In the group with AG pretreatment the activities of AST and ALT did not increase significantly after AZA when compared to control. However, the lipid peroxides and GSH levels did not have any significant difference when compared to AZA group. These observations also indicate that the improvement in the GSH levels by NAC is the most significant protective mechanism rather than any other mechanistic profile. The protective effect of AG against the enzyme leakage seems to be through the liver cell membrane permeability restoration and is independent of any effects on liver GSH contents.

Dietary Eritadenine and Ethanolamine Depress Fatty Acid Desaturase Activities by Increasing Liver Microsomal Phosphatidylethanolamine in Rats

The effects of eritadenine, a constituent of the Lentinus edodes mushroom, and ethanolamine, the base constituent of phosphatidylethanolamine (PE), on fatty acid desaturase activities and lipid profiles were investigated comparatively in rats. Rats were fed a control diet or a diet supplemented with either eritadenine (0.05 g/kg) or ethanolamine (8 g/kg) for 14 d. Eritadenine and ethanolamine had marked hypocholesterolemic effects. The concentration of liver microsomal PE was significantly increased and the ratio of phosphatidylcholine (PC) to PE was significantly decreased by both eritadenine and ethanolamine. These changes in phospholipid profile were also observed in the mitochondria and plasma membranes in the liver. The activities of the Delta5-, Delta6- and Delta9-desaturases in liver microsomes were significantly decreased by eritadenine and ethanolamine; there was a significant correlation between the activity of Delta5- or Delta6-desaturase and the proportion of PE in the total phospholipids or the PC/PE ratio. Reflecting decreased Delta5- and Delta6-desaturase activities, the 20:4(n-6)/18:2(n-6) ratio was significantly decreased by eritadenine and ethanolamine in PC of the liver microsomes, mitochondria and plasma membranes. Although the 20:4(n-6)/18:2(n-6) ratio of liver microsomal PE was also significantly decreased by eritadenine and ethanolamine, the fatty acid composition of phosphatidylinositol and phosphatidylserine was less affected by these compounds. Eritadenine and ethanolamine increased the proportion of 16:0-18:2 and decreased the proportion of 18:0-20:4 in liver PC. The results suggest that dietary eritadenine and ethanolamine might lead to decreases in desaturase activities and changes in fatty acid and molecular species composition of PC through an increase in liver microsomal PE.

Lymphosarcoma-induced alterations in hepatic adrenergic receptors: implications to the hypoglycemia of cancer cachexia.

A highly malignant transplantable rat lymphosarcoma was studied to determine the involvement of hepatic adrenergic receptors in the development of the hypoglycemia of cancer cachexia. Following inoculation of Fischer 344 rats with lymphosarcoma cells, rats were examined at 2 and 4 weeks, at the pre-cachexic stage; 6 weeks, at the transitional stage; and 7 weeks, at the cachexic hypoglycemic stage of lymphosarcoma progression. Death occurred by the 8th week. Blood glucose levels in lymphosarcoma-bearing rats relative to control rats were: unaffected at week 2; significantly reduced 8% at weeks 4 and 6; and reduced 24% at week 7. Alpha1 adrenergic receptor binding to plasma membranes isolated from the livers of lymphosarcoma-bearing rats was: 114, 89, 67 and 30% of control at weeks 2, 4, 6, and 7, respectively. Kinetic analysis indicated that the lymphosarcoma-induced decrease at week 7 was due to a decrease in numbers of receptors with no change in affinity: B(max)(control): 1411.1 fmol/mg: Kd(control): 0.44 nm; B(max)(lympho): 345.5 fmol/mg; Kd(lympho): 0.50 nm. Alpha2 adrenergic receptor binding to plasma membranes isolated from the livers of lymphosarcoma-bearing rats was: 130, 137, 243 and 212% of control at weeks 2,4, 6, and 7, respectively. The pattern of changes in hepatic alpha1, alpha2 and beta adrenergic receptors at week 6 was comparable to that of 17 day fetal liver: a decrease in alpha1 and beta and an increase in alpha2. Hepatic adrenergic receptor changes occurred in the absence of liver damage and were not due to contamination of the liver plasma membrane fractions with lymphosarcoma cells. Plasma insulin levels displayed modest (10-15%), but not statistically significant, increases post-inoculation after week 4. Plasma glucagon levels fluctuated post-inoculation until week 7 where they were significantly increased: 202% of control. Plasma T3 and T4 levels displayed an early and steady decline after lymphosarcoma inoculation: T3: unchanged at week 2 and significantly decreased 14, 44 and 50% at weeks 4, 6 and 7, respectively. T4 increased 20% at week 1; decreased 9% at week 4 and significantly decreased thereafter: 55 and 49% at weeks 6 and 7, respectively. We propose that the development of the hypoglycemia of cancer cachexia in this lymphosarcoma model is due primarily to an early and progressive thyroid hormone dependent decrease in the number of hepatic alpha1 adrenergic receptors, compounded by an increase and decrease, respectively, in the hepatic beta and alpha2 adrenergic receptors.

Action of ion flux regulators on membrane of liver cells in primary culture

The present paper deals with the interaction of some ion flux regulators and metabolic inhibitors upon the generation of the membrane potential in isolated liver cell culture. This study is the basis of an accurate selection of inhibitors of specific cellular metabolic pathways, which control the ion transport of membrane. This selection will allow to improve both the fluorescence microscopy study methods of membrane transport and the evaluation techniques concerning the action of some metabolic modulators such as the hormones. [...]

Loss of early insulin secretion leads to postprandial hyperglycaemia.

A loss of early-phase insulin response is common in Type II diabetic patients and in people with impaired glucose metabolism. It is hypothesized that this alteration is not simply a marker for the risk of developing diabetes, rather it is a an important pathogenetic mechanism causing excessive postprandial hyperglycaemia. Relevant literature on the epidemiology, physiopathology, and therapeutic intervention related to loss of early insulin secretion and postprandial hyperglycaemia has been analysed. In response to intravenous glucose, insulin secretion is biphasic. This behaviour translates as a rapid release of insulin into the blood stream in response to the ingestion of carbohydrates or a mixed meal. The rapid increase in portal insulin concentration and the avid binding of the hormone to its receptor on liver cell membranes, account for a prompt suppression of endogenous glucose production and reduction of the rate of increase in plasma glucose concentrations. This has been supported by experimental studies carried out in both animals and humans: the selective abolition of early insulin secretion in healthy subjects results in impaired glucose tolerance, excessive glucose excursions, and possible hampering of the thermic effects of ingested carbohydrates. In non-diabetic subjects, the loss of early insulin secretion is a determinant for developing diabetes. The critical role of the early-phase insulin response in determining postprandial hyperglycaemia, is supported by an amelioration of glucose tolerance by restoring the acute rise in plasma insulin concentrations after the ingestion of both glucose and a mixed meal. This amelioration in plasma glucose profile can prevent late hyperglycaemia and hyperinsulinaemia. Therapeutic approaches aimed at restoring a physiological pattern of insulin secretion could prove effective in reducing postprandial glucose excursions particularly in the early stage of Type II diabetes.

Evaluation of the toxicological effects of perfluorooctane sulfonic acid in the common carp ( Cyprinus carpio)

In the present study we evaluated the toxicological effects of a scarcely documented environmental pollutant, perfluorooctane sulfonic acid (PFOS), on selected biochemical endpoints in the common carp, Cyprinus carpio. Juvenile organisms were exposed to PFOS through a single intraperitoneal injection (liver concentrations ranging from 16 to 864 ng/g after 5 days of exposure) and after 1 and 5 days effects were assessed in liver and serum of the exposed organisms. The investigation of the hepatotoxicity of PFOS included the determination of the peroxisome proliferating potential (peroxisomal palmitoyl CoA oxidase and catalase activity) and the compounds influence on the average DNA basepair length (ABPL) by agarose gel electrophoresis. Total antioxidant activity (TAA), cholesterol and triglyceride levels were monitored in the serum. After 1 day of exposure the ABPL was significantly increased in the 270 and 864 ng/g treatment groups. After 5 days of exposure significant increases relative to the control were observed for the 16, 270 and 864 ng/g treatment groups. Enzyme leakage from the liver was investigated by measurement of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the serum. At 561, 670 and 864 ng/g PFOS a significant increase in serum ALT activity became apparent after 5 days of exposure with values ranging from 159 to 407% relative to the control. For serum AST activity a significant increase for the 864 ng/g treatment group was observed with a value of 112% relative to the control. Determination of the polymorphonuclear leukocyte migration into liver tissue as assessed through myeloperoxidase (MPO) activity in liver, was used as an indicator for inflammation. It appeared that inflammation was not involved in the observed membranous enzyme leakage for the 561, 670 and 864 ng/g PFOS treatment groups. The results of this study suggest that PFOS induces inflammation-independent enzyme leakage through liver cell membranes that might be related to cell necrosis. Furthermore, results show that PFOS does not significantly affects serum antioxidant levels nor does it clearly induce peroxisome proliferation in carp. This study also points out that PFOS might interfere with homeostasis of the DNA metabolism. The results of these biochemical analyses were used to perform an initial hazard assessment study indicating that PFOS levels observed in tissues of wildlife populations could induce a clear rise in serum transaminase levels indicative for disruption of hepatocyte membrane integrity.

Integration of solid-phase extraction membranes for sample multiplexing: application to rapid protein identification from gel-isolated protein extracts.

The present report describes the design and application of a dual sprayer system for high-throughput proteome analysis. This system comprises parallel solid-phase extraction cartridges used for preconcentration and desalting of proteolytic digests prior to nanoelectrospray mass spectrometry analyses. Tryptic peptides from in-gel digest of protein bands/spots are first adsorbed on styrene divinyl benzene membrane and subsequently eluted with a short plug of organic buffer prior to infusion to the mass spectrometer at a flow rate of typically 500 nL/min. Tryptic peptide eluting from the membrane are analyzed by the mass spectrometer by moving in turn each sprayer in front of the sampling orifice. Sequential injection, preconcentration and analyses of tryptic digests are typically achieved with a throughput of up to 3.5 min/sample and a detection limit of approximately 8-80 fmol per injection. Replicate injections of peptide mixtures indicated that reproducibility of peak areas ranged from relative standard deviations (RSD) of 1.1% to 4.5%. The application of this device is demonstrated for digests of gel-isolated proteins obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of rat liver plasma membrane and from two-dimensional gel electrophoresis of total cell lysate extracts from human prostatic cancer cell.

Sucrose feeding effects inhibition of γ-glutamyltranspeptidase in the liver of the rat: possible mediation by thyroid hormone

The effect of sucrose on Fischer 344 rat liver γ-glutamyltranspeptidase (γGT) was studied: in adults fed sucrose for 3 weeks; and rats exposed to sucrose from the 18th day of gestation to the 40th day after birth. Rats fed regular rodent chow served as controls. Sucrose caused mild lipemia; and in the liver an increase in size and fat build-up without damage. In adult sucrose-fed rats, compared to controls, plasma glucose levels were increased: 1.12-, 1.40- and 1.13-fold after 24, 48 h and 3 week consumption of sucrose, respectively. Insulin levels were unaltered for the first week of sucrose consumption but increased from control levels: 16% at 1 week, and 2.0-fold at 3 weeks. The T 3 levels were comparable to control levels 24 h after the sucrose was started and were increased: 1.22-, 1.13- and 1.12-fold at 48 h, 1 and 3 weeks, respectively. The T 4 levels were comparable at all time points between sucrose-fed and control rats. Liver γGT activity exhibited a steady decrease from control levels: after 24, 48 h, 1 and 3 weeks of sucrose feeding the decrease was 5, 8, 21 and 37%, respectively in homogenates; and 10, 17, 24 and 41%, respectively in plasma membranes. Perinatal sucrose exposure effected in 40-day-old rats, compared controls: a 1.09-fold increase in plasma glucose; no change in plasma insulin; an increase of 1.15- and 1.39-fold in plasma levels of total and free T 3, respectively; a decrease of 20 and 14% in plasma levels of total and free T 4, respectively. γGT activity was decreased in liver plasma membranes isolated from sucrose-exposed rats relative to those of control: 80% in the male; 82% in the female. Relative specific activities of γGT were the same in both males: 15.4 and 16.1 in control and sucrose-exposed male rats, respectively; and females: 14.1 and 15.4 in control and sucrose-exposed female rats, respectively. γGT was 2-fold higher in the livers of female relative to male rats in sucrose-exposed and control groups. Kidney γGT activities were the same in control and sucrose-exposed rats. The involvement of T 3 in the sucrose-induced decrease in liver γGT is discussed.

Ubiquinol inhibition of neutral sphingomyelinase in liver plasma membrane: specific inhibition of the Mg 2+-dependent enzyme and role of isoprenoid chain

In this work, the specificity of ubiquinol as inhibitor of the neutral sphingomyelinases present at the plasma membrane (Mg 2+-dependent and -independent) and structural requirements for such inhibition have been studied. Our results have shown that ubiquinol specifically inhibits Mg 2+-dependent neutral sphingomyelinase activity in isolated liver plasma membranes, but no significant participation of the Mg 2+-independent enzyme was observed. Both the reduction state of the (hydro)quinone ring and the length of the hydrophobic side chain were important determinants in neutral sphingomyelinase inhibition. Ubiquinols inhibited the nSMase more efficiently than ubiquinones, and hydrophobic homologs with six or nine isoprene units were the most effective inhibitors. Inhibition of nSMase by ubiquinols displayed similarities with inhibition by manumycin and the hydroquinones F11334's, suggesting that these compounds could act as structural analogs of ubiquinol. Beyond its participation in mitochondrial energy metabolism, and as antioxidant, this novel role for ubiquinol as a neutral sphingomyelinase inhibitor should be considered an important factor to regulate lipid signaling at the plasma membrane that could be related to its beneficial effects on cells, tissues, and organisms.

The interaction of anthocyanins with bilitranslocase

Bilitranslocase (TC 2.A.65.1.1) is an organic anion membrane carrier expressed at the sinusoidal domain of the liver plasma membrane and in epithelial cells of the gastric mucosa. Its substrates are sulfobromophthalein, bilirubin, and nicotinic acid. This work reports on the identification of a new class of bilitranslocase substrates, i.e., anthocyanins. Seventeen out thes 20 compounds tested behaved as competitive inhibitors of bilitranslocase transport activity ( K I =1.4–22 μM ). Their structure–activity relationship reveals that mono- and di-glucosyl anthocyanins, the anthocyanin species occurring in food, are better ligands than the corresponding aglycones. Moreover, the first interaction of anthocyanins with the carrier occurs through hydrophilic moieties, such as the 3-glucosyl moiety and the B ring for the monoglucosides, through the 5-glucosyl moiety and the A ring for the diglucosides, and through either the B or the A ring for the aglycones. These findings suggest that bilitranslocase could play a role in the bioavailability of anthocyanins.

Purification and Characterization of a Doxorubicin-inhibited NADH-quinone (NADH-ferricyanide) Reductase from Rat Liver Plasma Membranes

Plasma membrane-associated redox systems play important roles in regulation of cell growth, internal pH, signal transduction, apoptosis, and defense against pathogens. Stimulation of cell growth and stimulation of the redox system of plasma membranes are correlated. When cell growth is inhibited by antitumor agents such as doxorubicin, capsaicin, and antitumor sulfonylureas, redox activities of the plasma membrane also are inhibited. A doxorubicin-inhibited NADH-quinone reductase was characterized and purified from plasma membranes of rat liver. First, an NADH-cytochrome b(5) reductase, which was doxorubicin-insensitive, was removed from the plasma membranes by the lysosomal protease, cathepsin D. After removal of the NADH-cytochrome b(5) reductase, the plasma membranes retained a doxorubicin-inhibited NADH-quinone reductase activity. The enzyme, with an apparent molecular mass of 57 kDa, was purified 200-fold over the cathepsin D-treated plasma membranes. The purified enzyme had also an NADH-coenzyme Q(0) reductase (NADH: external acceptor (quinone) reductase; EC 1.6.5.) activity. Partial amino acid sequence of the enzyme showed that it was unique with no sequence homology to any known protein. Antibody against the enzyme (peptide sequence) was produced and affinity-purified. The purified antibody immunoprecipitated both the NADH-ferricyanide reductase activity and NADH-coenzyme Q(0) reductase activity of plasma membranes and cross-reacted with human chronic myelogenous leukemia K562 cells and doxorubicin-resistant human chronic myelogenous leukemia K562R cells. Localization by fluorescence microscopy showed that the reaction was with the external surface of the plasma membranes. The doxorubicin-inhibited NADH-quinone reductase may provide a target for the anthracycline antitumor agents and a candidate ferricyanide reductase for plasma membrane electron transport.