Liver Plasma Membrane Fractions Research Articles

Human Hepatic Transporter Signature Peptides for Quantitative Targeted Absolute Proteomics: Selection, Digestion Efficiency, and Peptide Stability.

Quantitative targeted absolute proteomics (QTAP) quantifies proteins by measuring the signature peptides produced from target proteins by trypsin digestion. The selection of signature peptides is critical for reliable peptide quantification. The purpose of this study was to comprehensively assess the digestion efficiency and stability of tryptic peptides and to identify optimal signature peptides for human hepatic transporters and membrane marker proteins. The plasma membrane fraction of the human liver was digested at different time points and the peptides were comprehensively quantified using quantitative proteomics. Transporters and membrane markers were quantified using the signature peptides by QTAP. Tryptic peptides were classified into clusters with low digestion efficiency, low stability, and high digestion efficiency and stability. Using the cluster information, we found that a proline residue next to the digestion site or the peptide position in or close to the transmembrane domains lowers digestion efficiency. A peptide containing cysteine at the N-terminus or arginine-glycine lowers peptide stability. Based on this information and the time course of peptide quantification, optimal signature peptides were identified for human hepatic transporters and membrane markers. The quantification of transporters with multiple signature peptides yielded consistent absolute values with less than 30% of coefficient variants in human liver microsomes and homogenates. The signature peptides selected in the present study enabled the reliable quantification of human hepatic transporters. The QTAP protocol using these optimal signature peptides provides quantitative data on hepatic transporters usable for integrated pharmacokinetic studies.

Glucose transporter expression and regulation following a fast in the ruby-throated hummingbird, Archilochus colubris.

Hummingbirds, subsisting almost exclusively on nectar sugar, face extreme challenges to blood sugar regulation. The capacity for transmembrane sugar transport is mediated by the activity of facilitative glucose transporters (GLUTs) and their localisation to the plasma membrane (PM). In this study, we determined the relative protein abundance of GLUT1, GLUT2, GLUT3 and GLUT5 via immunoblot using custom-designed antibodies in whole-tissue homogenates and PM fractions of flight muscle, heart and liver of ruby-throated hummingbirds (Archilochus colubris). The GLUTs examined were detected in nearly all tissues tested. Hepatic GLUT1 was minimally present in whole-tissue homogenates and absent win PM fractions. GLUT5 was expressed in flight muscles at levels comparable to those of the liver, consistent with the hypothesised uniquely high fructose uptake and oxidation capacity of hummingbird flight muscles. To assess GLUT regulation, we fed ruby-throated hummingbirds 1 mol l-1 sucrose ad libitum for 24 h followed by either 1 h of fasting or continued feeding until sampling. We measured relative GLUT abundance and concentration of circulating sugars. Blood fructose concentration in fasted hummingbirds declined (∼5 mmol l-1 to ∼0.18 mmol l-1), while fructose-transporting GLUT2 and GLUT5 abundance did not change in PM fractions. Blood glucose concentrations remained elevated in fed and fasted hummingbirds (∼30 mmol l-1), while glucose-transporting GLUT1 and GLUT3 in flight muscle and liver PM fractions, respectively, declined in fasted birds. Our results suggest that glucose uptake capacity is dynamically reduced in response to fasting, allowing for maintenance of elevated blood glucose levels, while fructose uptake capacity remains constitutively elevated promoting depletion of blood total fructose within the first hour of a fast.

A high fat diet induces sex-specific differences in hepatic lipid metabolism and nitrite/nitrate in rats

Men and women differ substantially with regard to the severity of insulin resistance (IR) but the underlying mechanism(s) of how this occurs is poorly characterized. We investigated whether a high fat (HF) diet resulted in sex-specific differences in nitrite/nitrate production and lipid metabolism and whether these variances may contribute to altered obesity-induced IR.Male and female Wistar rats were fed a standard laboratory diet or a HF diet for 10 weeks. The level of plasma nitrite/nitrate, as well as free fatty acid (FFA), in both plasma and liver lysates were assessed. The levels of inducible nitric oxide (NO) synthase (iNOS), p65 subunit of NFκB, total and phosphorylated forms of Akt, mTOR and PDK-1 in lysates, and the levels of glucose transporter 2 (Glut-2) and fatty acid translocase/cluster of differentiation 36 (FAT/CD36) in plasma membrane fractions of liver were assessed.HF-fed male rats exhibited a significant increase in plasma nitrite/nitrate, and hepatic FFA and FAT/CD36 levels compared with controls. They also displayed a relative decrease in iNOS and Glut-2 levels in the liver. Phosphorylation of Akt (at Ser473 and Thr308), mTOR and PDK-1 was also reduced. HF-fed female rats exhibited increased levels of NFκB-p65 in liver compared with controls, while levels of Glut-2, FAT/CD36 and Akt phosphorylation at Thr308 and PDK-1 were decreased.Our results reveal that altered lipid and glucose metabolism in obesity, lead to altered iNOS expression and nitrite/nitrate production. It is likely that this mechanism contributes to sex-specific differences in the development of IR.

RAPID NONGENOMIC EFFECTS OF CORTISOL ON MEMBRANE FLUIDITY AND PHOSPHORYLATION IN RAINBOW TROUT LIVER.

Event Abstract Back to Event RAPID NONGENOMIC EFFECTS OF CORTISOL ON MEMBRANE FLUIDITY AND PHOSPHORYLATION IN RAINBOW TROUT LIVER. Laura Dindia1 and Mathilakath Vijayan1* 1 University of Waterloo, Canada While rapid steroid signaling is thought to play a key role in cellular function, little is known about the nongenomic role of cortisol in hepatic tissue. Therefore, we studied the nongenomic effects of cortisol on the biophysical properties of the liver plasma membrane and investigated the effects on the phosphorylation profile of protein kinase A (PKA), protein kinase C (PKC) and AKT kinase substrate proteins. The effect of cortisol on membrane fluidity was examined by measuring fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in isolated liver plasma membrane fractions. In vitro, physiological (100 ng/ml) and pharmacological (500 and 1000 ng/ml) cortisol concentrations were shown to rapidly increase hepatic membrane fluidity. Moreover, cortisol rapidly induced phosphorylation of several PKC substrate proteins. These results indicate for the first time that stress-induced plasma cortisol levels modulates liver membrane fluidity, which may lead to rapid intracellular signaling in rainbow trout. Acknowledgements This research was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to MMV, and a NSERC Post-graduate Scholarship (PGS D) to LD.

Biochemical separation of Na+,K+-ATPase from a "purified" light density, "canalicular"-enriched plasma membrane fraction from rat liver.

Cytochemical studies suggest that Na+,K+-ATPase is localized to sinusoidal and lateral portions of the hepatocyte plasma membrane whereas Mg++-ATPase and alkaline phosphatase are luminal or canalicular membrane markers. To validate further these cytochemical findings, we have isolated from the nuclear pellet of rat liver homogenates a liver plasma membrane (LPM) fraction enriched in all three enzyme markers, as previously described (Biochimica et Biophysica Acta 1975; 401:59-52). Following tight Dounce homogenization, the vesiculated membrane preparation was further separated on a multiple-step discontinuous sucrose density gradient (d 1.12 to 1.22). Na+,K+-ATPase activity "dissociated" from Mg++-ATPase activity, sedimenting at densities of 1.14 and greater. Further studies were carried out in two-step discontinuous sucrose gradients (1.13 and d greater than 1.13), and a light density fraction (d 1.13) was further characterized in calcium-free media (since addition of calcium increased contamination with intracellular membranes). Electron microscopy demonstrated a homogeneous vesicular membrane population in contrast to the heavy density fraction (d greater than 1.13) which contained membrane sheets and junction complexes as well as vesicles. The light density fraction was highly enriched n Mg++-ATPase (42.1 x homogenate specific activity) and alkaline phosphatase (64.6 x homogenate), 3 to 4 times their activities in the original LPM. In contrast, Na+,K+-ATPase activity in the light density fraction, diminished 16-fold from values in the original unfractionated LPM. All but 15% of total Na+,K+-ATPase activity in the original LPM could be accounted for in unwashed preparations. Neither cholesterol/phospholipid ratios nor an analysis of peptides on sodium dodecyl sulfate gel electrophoresis demonstrated differences in the composition of the light vs. heavy density subfractions, although there were relative increases in several peptide bands in the light density subfraction. These studies provide further supporting biochemical evidence for the concept that Na+,K+-ATPase resides on different membrane domains than does Mg++-ATPase and alkaline phosphatase and further characterizes a vesiculated membrane preparation highly enriched in putative "canalicular" enzyme markers.

The phosphatidylethanolamine N-methyltransferase pathway is quantitatively not essential for biliary phosphatidylcholine secretion

The phosphatidylethanolamine N-methyltransferase (PEMT) pathway of phosphatidylcholine (PC) biosynthesis is not essential for the highly specific acyl chain composition of biliary PC. We evaluated whether the PEMT pathway is quantitatively important for biliary PC secretion in mice under various experimental conditions. Biliary bile salt and PC secretion were determined in mice in which the gene encoding PEMT was inactivated (Pemt(-/-)) and in wild-type mice under basal conditions, during acute metabolic stress (intravenous infusion of the bile salt tauroursodeoxycholate), and during chronic metabolic stress (feeding a taurocholate-containing diet for 1 week). The activity of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of PC biosynthesis via the CDP-choline pathway, and the abundance of multi-drug-resistant protein 2 (Mdr2; encoded by the Abcb4 gene), the canalicular membrane flippase essential for biliary PC secretion, were determined. Under basal conditions, Pemt(-/-) and wild-type mice exhibited similar biliary secretion rates of bile salt and PC ( approximately 145 and approximately 28 nmol/min/100 g body weight, respectively). During acute or chronic bile salt administration, the biliary PC secretion rates increased similarly in Pemt(-/-) and control mice. Mdr2 mRNA and protein abundance did not differ between Pemt(-/-) and wild-type mice. The cytidylyltransferase activity in hepatic lysates was increased by 20% in Pemt(-/-) mice fed the basal (bile salt-free) diet (P < 0.05). We conclude that the biosynthesis of PC via the PEMT pathway is not quantitatively essential for biliary PC secretion under acute or chronic bile salt administration.

The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8(+/+), Abcg8(+/-), and Abcg8(-/-) mice with hydrophilic and hydrophobic bile salts. In Abcg8(-/-) mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8(-/-) mice at a much lower infusion rate compared with Abc8(-/-) and Abcg8(+/-) mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45% in cholesterol content under these conditions in Abcg8(-/-) mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.

Proteomic analysis of mouse liver plasma membrane: Use of differential extraction to enrich hydrophobic membrane proteins

To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.

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.

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.

Familial intrahepatic cholestasis 1: studies of localization and function

Mutations in the FIC1 gene constitute the molecular defect in familial intrahepatic cholestasis I (Fic1 [Byler's disease]) and benign recurrent intrahepatic cholestasis. This report describes the localization of Fic1 in rat liver and intestine, as well as biochemical and transfection studies that support its function as an energy-dependent aminophospholipid translocase. Immunocytochemistry of rat liver and immunoblotting of membrane fractions localized Fic1 to the canalicular, but not basolateral, plasma membrane domain. In the small intestine, Fic1 was localized to the apical membrane of epithelial cells. The distribution of Fic1 in liver plasma membrane fractions from control and taurocholate-treated rats correlated positively with adenosine triphosphate (ATP)-dependent aminophospholipid (phosphatidyl-serine) translocase activity. In canalicular membrane vesicles, translocase activity had an initial velocity of 3.3 nmol phosphatidylserine (PS) translocated per milligram of protein per minute and a K(m) (ATP) = 1.2 mmol/L; was inhibited by vanadate, N-ethylmaleimide, sodium azide, and calcium; and was unidirectional (i.e., from the outer to the inner canalicular plasma membrane leaflet). Transient transfection of CHOK1 cells with FIC1 cDNA resulted in appearance of FIC1 in membrane preparations and energy-dependent PS translocation in cells. These studies indicate that FIC1 is a canalicular P-type ATPase that participates in maintaining the distribution of aminophospholipids between the inner and outer leaflets of the plasma membrane. How this process produces cholestasis is under study.

Biochemical analysis of a caveolae-enriched plasma membrane fraction from rat liver.

We isolated and characterized a subcellular fraction derived from the blood-sinusoidal plasma membrane of hepatocytes enriched in caveolin and containing several of the molecular components described to be present in caveolae isolated from other cell types. A morphological study by electron microscopy revealed that it was composed of caveolae-attached membrane profiles. Immunoelectron microscopy of isolated fraction showed the specific labeling of internal caveolae membranes with anti-caveolin antibody. Finally, one- and two-dimensional electrophoresis and Western blotting were used for the biochemical analysis of this new rat liver plasma membrane fraction. From the biochemical and the morphological characterization, we conclude that the caveolae-enriched plasma membrane fraction is a plasma membrane fraction, which originates from specialized regions of the sinusoidal plasma membrane, enriched in caveolae.

Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles. III. Specificity and stereospecificityDedicated as a memorial to Prof. Govind Sethu Rao, University of Bonn, who suddenly passed away on 31 Aug. 1997.

In previous papers we provided evidence for a glucocorticoid (GC) responsive site in a highly purified rat liver plasma membrane (PM) fraction, which has proved to be osmotically active, `right side-out' vesicles, free of CBG, glucocorticoid receptors (GR) and ATP ( J. Steroid Biochem. Molec. Biol. 42 (1992) 737–756 and 757–771). This site, now called `GC importer', mediates active transmembrane transport of corticosterone (B). Pronounced specificity, including stereo- and enantiomeric specificity, of ligand-GC importer interaction was demonstrated by competition assays using 54 different steroidal hormones and molecules. Important structural prerequisites for ligands with high specificity for the GC importer are plane C 21-steroid hormones with 1-ene and/or 4-ene or 5 α-reduced configuration, and/or OH-group(s) at C11 β>C17 α>C21. Unexpectedly, other preferred ligands are C17 α-ethynyl steroids like estrogens with an OH- or OCH 3-group at C3 (EE 2, mestranol) as well as progestins with C3–OH and 4-ene configuration (ethynodiol). C 21-steroids with 11 α-OH, 11-keto, 16 α-CH 3, 16 β-CH 3, 16 α-OH or 5 β-reduced configuration are low specificity ligands. The importer even displays different specificity for enantiomers (levonorgestrel> l-norgestrel). Altogether, the GC importer preferentially recognizes active GC and natural progestins which act as GC-antagonist ( e.g. prednisolone>11 β-cortisol=B≥progestins). Synthetic GC-agonists ( e.g. dexamethasone, betamethasone, triamcinolone), most synthetic progestins, biologically inactive GC ( e.g. 11 α-cortisol, prednisone, cortisone, 11-dehydro-B), mineralocorticoids (aldosterone), natural estrogens ( e.g. E 1, E 2, E 3), DES and vitamin D 3 derivatives do not interact with the GC importer. Osmotic shrinkage experiments revealed that interaction of high as well as low specificity ligands with the GC importer comprises reversible binding and transport through the PM. The ligand specificity profile of the GC importer and the GR exhibit pronounced differences, suggesting that both GC recognizing sites are different proteins. Performing immunoblotting, using specific mono- and polyclonal antibodies directed against the intracellular rat GR, of the PM pretreated with the membrane protein solubilizing detergent CHAPSO, we found that specific steroid binding to the PM site is not due to contamination with GR. Colchicine, daunorubicine, quinine, reserpine, verapamil and vinblastine, representatives of lipophilic xenobiotics which are known to be transported out of cells by the glycoprotein P170, did not compete with B for uptake into PM-vesicles, indicating that the GC importer is not a member of the ABC/mdr superfamily. The GC importer seems to be an additional link in the chain of steroid signal transduction and may be functionally involved in the action of natural GC-agonists and GC-antagonists.

Identification of autophosphorylation sites in c-Yes purified from rat liver plasma membranes.

c-Yes was purified 322-fold from a rat liver plasma membrane fraction to a single 60-kDa band on SDS-PAGE. The purified protein contained essentially no phosphotyrosine residues and was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues with a concomitant increase in the protein-tyrosine kinase activity. The autophosphorylated c-Yes was extensively digested by trypsin and the resultant two major phosphopeptides, peptides I and II, were purified by HPLC on a reversed-phase C-18 column. The amino acid sequence of peptide I was determined to be LIEDNEYTAR, which is identical with the sequence from Leu-418 through Arg-427 of mouse c-Yes, indicating that one of the autophosphorylation sites corresponds to Tyr-424 of the mouse c-Yes. After partial determination of the N-terminal sequence of 10 amino acid residues of peptide II, the 230 bp sequence of rat cDNA that encodes the N-terminal 76 amino acid residues of c-Yes covering peptide II, was determined. From the predicted amino acid sequence, the sequence of peptide II was assumed to be from Tyr-16 through Lys-46, YTPENPTEPVNTSAGHYGVEHATAATTSSTK. The purified c-Yes phosphorylated the tyrosine residue of synthetic peptides covering Tyr-32 and its surrounding sequence but did not phosphorylate peptides covering Tyr-16 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.

Characterization of the binding of diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) to rat liver cell membranes

Diadenosine polyphosphates present in the extracellular environment can, through interaction with appropriate purinoceptors, influence a range of cellular activities. Here we have investigated the nature of the ligand:receptor interactions involved in diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A)-mediated stimulation of glycogen breakdown in isolated rat liver cells. [2-3H]Ap4A showed specific binding to both intact isolated liver cells and plasma membrane fractions prepared from isolated liver cells. HPLC analysis confirmed that binding was mediated by intact Ap4A and not by potential breakdown products (e.g. ATP, adenosine, etc.). Binding of [2-3H]Ap4A, to isolated liver cell plasma membrane preparations, was successfully displaced by a range of both naturally occurring and synthetic diadenosine polyphospates with the rank order potency Ap4A > or = Ap5A > Ap6A > Ap3A > Ap2A. [2-3H]Ap4A binding was not displaced by P1 effectors but was successfully displaced by a range of P2 effectors with the rank order potency 2-methylthio-ATP > ATP > ATP > or = adenosine 5'-[alpha beta-methylene]triphosphate > adenosine 5'-[beta gamma-methylene]triphospate. These findings are consistent with the interaction of Ap4A with a P2y-like subclass of purinoceptor and are discussed in relation to (1) the known purinoceptor populations in liver cell plasma membranes and (2) observations concerning the binding of diadenosine polyphosphates to purinoceptors in other tissues.

Covalent Modification of Rat Liver Dipeptidyl Peptidase IV (CD26) by the Nonsteroidal Anti-Inflammatory Drug Diclofenac

Diclofenac is a nonsteroidal anti-inflammatory drug that has been implicated in several cases of severe hepatotoxicity. Our previous study showed that diclofenac metabolites bound covalently and selectively to rat liver plasma membrane proteins with estimated monomeric masses of 110, 140, and 200 kDa. We report here that we have identified the 110 kDa diclofenac-labeled protein in rat liver as dipeptidyl peptidase IV, also known as CD26. In addition, we found that the activity of dipeptidyl peptidase IV in liver plasma membrane fractions was lowered after diclofenac treatment of rats. These results suggest that the hepatotoxicity associated with diclofenac might be due, in part, to the covalent modification of dipeptidyl peptidase IV.

Chronic ethanol administration increases the amounts of α and β subunits of G-proteins in liver plasma membrane fractions: [formula omitted] Liver Study Unit, VA Medical Center and the University of Nebraska Medical Center, Omaha NE 68105

Chronic ethanol administration increases the amounts of α and β subunits of G-proteins in liver plasma membrane fractions: [formula omitted] Liver Study Unit, VA Medical Center and the University of Nebraska Medical Center, Omaha NE 68105

Insulin-Induced Proteolysis of the Insulin Receptor alpha-Subunit from Rat Liver does not Occur in vivo but is Prevented in vitro by Blood Serum Proteinase Inhibitors

Insulin binding to rat liver plasma membranes promotes the action of a plasma membrane proteinase that degrades the 135-kDa alpha-subunit of the insulin receptor to a 120kDa product. It has been proposed that this proteolysis may be the initial step leading to down-regulation of the hepatic insulin receptor [Lipson, K. E., Kolhatkar, A. A. & Donner, D. B. (1988) J. Biol. Chem. 263, 10495-10501]. Our results confirm that liver plasma membrane fractions from intact or perfused rat liver and from isolated rat hepatocytes do have a proteinase that degrades the alpha-subunit of the insulin receptor, whose activity increases significantly in the presence of insulin. In contrast to these in vitro results, plasma membranes and Golgi fractions isolated from liver at different times after a single intravenous injection of insulin to rats only contained intact alpha-subunits of the insulin receptor. Insulin administration was associated with a rapid and reversible translocation of insulin receptors from the plasma membranes to Golgi fractions but did not affect their total numbers or the receptor half-life. Incubation of rat liver plasma membranes with human, bovine or rat blood sera totally blocked alpha-subunit proteolysis both in the absence or presence of insulin. The three major serum proteinase inhibitors, alpha 1-antitrypsin, alpha 2-macroglobulin and antithrombin III, blocked alpha-subunit proteolysis of the insulin receptor to a varying extent. alpha 1-antitrypsin exhibited the highest potency, decreasing the amount of 120-kDa product by 70%. When both alpha 1-antitrypsin and antithrombin III were present, inhibition increased to 85%. Thus, the absence of proteolysis of the hepatic insulin receptor in vivo could be mostly accounted for by the antiproteolytic activity of blood serum. These findings suggest that insulin receptor alpha-subunit proteolysis does not represent a mechanism involved in the down-regulation of the insulin receptor in liver.

Detection and characterization of ceramide-1-phosphate phosphatase activity in rat liver plasma membrane.

A calcium-dependent ceramide (Cer) kinase was recently detected in human leukemia (HL-60) cells (Kolesnick, R.N., and Hemer, M.R. (1990) J. Biol. Chem. 265, 18803-18808) where it may function in terminating the regulatory effects of Cer, and in synaptic vesicles (Bajjalieh, S. M., Martin, T. F. J., and Floor, E. (1989) J. Biol. Chem. 264, 14354-14360). We now demonstrate that the addition of both Cer-1-phosphate (Cer-1-P) and a short-acyl chain analog of Cer-1-P,N-hexanoylsphingosine-1-phosphate (C6-Cer-1-P) to cultured cells and a variety of subcellular fractions results in rapid degradation to Cer and C6-Cer, respectively. The Cer-1-P phosphatase activity is enriched in a rat liver plasma membrane fraction and appears to be distinct from the phosphatase that hydrolyzes phosphatidic acid (PA), PA phosphohydrolase, as shown by the difference in sensitivity of Cer-1-P and PA hydrolysis to propranolol, detergent, and heat treatment. Moreover, the Km of Cer-1-P hydrolysis is 10-fold lower than the Km of PA hydrolysis in plasma membrane. PA is a noncompetitive inhibitor of Cer-1-P hydrolysis, with an inhibition constant 1-1.5-fold higher than the Km of Cer-1-P hydrolysis. In contrast, Cer-1-P does not inhibit PA hydrolysis. Finally, we describe the synthesis of a novel analog of Cer-1-P which is not hydrolyzed in vitro and in vivo and is internalized in cultured cells by endocytosis. These results are discussed in relation to the possible roles of Cer-1-P in regulating intracellular levels of Cer.

Liver Membrane Composition after Short-Term Parenteral Nutrition With and Without Taurine in Guinea Pigs: The Effect to Taurine

Having recently demonstrated that taurine supplementation prevents total parenteral nutrition (TPN)-induced cholestasis, we chose to use this model to examine plasma membrane composition in relation to bile formation. Male guinea pigs received daily a mixture of glucose and of the amino acid solution Travasol with or without added taurine (1.2 mM). After 3 days, bile was collected and liver plasma membrane fractions enriched in sinusoidal lateral membrane and bile canalicular membrane domains were isolated. In animals receiving TPN alone, bile flow and biliary secretory rate of bile acid and bicarbonate decreased significantly compared with controls. Although membrane ATPases (Na+K+ and Mg+) were unchanged, TPN induced an increase in the lipid to protein ratio and a decrease of polyunsaturated fatty acids, in conjunction with a higher content of diene conjugates in sinusoidal lateral membrane fractions. Taurine corrected these changes and, in addition, reduced significantly the cholesterol to phospholipid ratio in both membrane fractions. The data show that changes in liver cell membranes occur in TPN-induced cholestasis and suggest that free radical injury may play a role. As taurine prevented cholestasis as well as membrane changes, it is suggested that taurine should be added to amino acid solutions used for parenteral nutrition.