Isolated Liver Plasma Membranes Research Articles

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.

Modulation by S-Adenosyl-L-Methionine of Hepatic Na+, K+-ATPase, Membrane Fluidity, and Bile Flow in Rats with Ethinyl Estradiol-Induced Cholestasis

Structural and functional changes in the surface membranes of hepatocytes play a pivotal role in the induction and reversion of some forms of drug-induced cholestasis. To elucidate the mechanism by which S-adenosyl-L-methionine (SAMe) leads to a partial reversion of bile flow impairment caused by ethinyl estradiol (EE), female Sprague-Dawley rats were given oral doses of EE (5 mg per kg per day, for 3 days) with and without simultaneous administration of SAMe (25 mg per kg, 3 times per day, for 3 days). Na+,K+-ATPase activity and membrane microviscosity as measured by fluorescent polarization were assayed in isolated liver plasma membranes (LPMs). SAMe administration to normal and EE-treated rats resulted in a marked increase in Na+,K+-ATPase activity and LPM fluidity. EE alone did not cause any change in the physicochemical properties of the LPMs. Hepatic Mg2+-ATPase and gamma-glutamyl transpeptidase activities were not affected by SAMe alone but increased when SAMe was given together with EE. These data indicate that the interaction of in vivo administered SAMe with hepatocyte plasmalemma and its effect on lipid fluidity and enzymes of the LPMs showed a high specificity and an inverse relationship between Na+,K+-ATPase activity and fluorescence polarization values. Furthermore, modulation of hepatic Na+,K+-ATPase was associated with SAMe-induced protection against bile flow impairment due to EE; however, it was not the causative factor for EE-induced cholestasis under the experimental conditions. These findings suggest that changes in surface membrane structure and function might account in part for the reversal by SAMe of EE-induced impairment of bile secretory function.

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.

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.

ATA2-mediated amino acid uptake following partial hepatectomy is regulated by redistribution to the plasma membrane

System A, the Na +-dependent amino acid transport activity, is encoded by the ATA2 gene and up-regulated following partial hepatectomy (PH), and its competitive inhibition interferes with liver regeneration. Rabbit polyclonal antibody was raised against a portion of the ATA2 gene product followed by immunodetection of ATA2 in isolated liver plasma membrane and lysate. The level of ATA2 increased in the plasma membrane following PH, while the relatively high quantity of ATA2 found in liver lysate remained constant. We also have shown that Northern analysis of steady-state ATA2 mRNA revealed no significant change following PH. These data show that ATA2-mediated transport is not regulated by the steady-state level of ATA2 mRNA but is regulated by the amount of ATA2 and redistribution to the plasma membrane. We hypothesize that ATA2 activity is regulated by recruitment of ATA2 protein from an intracellular compartment. In addition, the pattern of expression of System A activity in oocytes, transport kinetics, and sensitivity to chemical modification indicate the presence of a second System A isoform in liver that differs substantially from ATA2.

Sodium dodecyl sulfate-freeze-fracture immunolabeling of gap junctions.

By the mid 1960s, pioneering work using high-resolution electron microscopy, new fixation methods, and negative staining of isolated liver plasma membranes allowed the identification of a geometric subunit pattern likely associated with junctional domains (,). Furthermore, the application of tissue impregnation with electron-dense tracers revealed that the minute “gap” (2 nm wide) between the closely adjoining junctional membranes comprised an hexagonal subunit pattern. This type of membrane-membrane interaction, distinct from tight junctions, adhesion plaques, and desmosomes, was originally called “gap junction” (.

Ligand-induced downregulation of receptor-mediated clearance of hepatocyte growth factor in rats.

The change in tissue uptake clearance of 125I-labeled hepatocyte growth factor (HGF) after an intravenous injection of an excess (120 micrograms/kg) of unlabeled HGF was examined in rats. The heparin-washable component of the hepatic uptake clearance of 125I-HGF was only slightly changed, whereas the heparin-resistant component was significantly reduced 30 min after injection of excess HGF, followed by gradual recovery with a half-life of 3.2 h. Because the former clearance mainly represents 125I-HGF association with heparan sulfate proteoglycan on the cell surface and/or extracellular matrix, whereas the latter includes relatively specific clearance, such as receptor-mediated endocytosis, this result suggests that injection of excess HGF selectively causes downregulation of receptor-mediated HGF clearance in the liver. Downregulation could also be observed for HGF receptor density in isolated liver plasma membrane, assessed by Western blot analysis by means of anti-receptor antibody, 30 min after injection of excess unlabeled HGF, supporting the hypothesis that the overall elimination of HGF from the systemic circulation can be affected by a change in HGF receptor density on the liver cell surface.

319 Decreases of 5-HT 7 mRNA expression and cyclic amp formation by differentiation in rat cortical astrocytes

It has been proposed that an abnormality in the regulation of cytosolic-free Ca2+ may be the cause of some forms of insulin resistance. In support of this proposition, it was reported that phospholipase C-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by liver plasma membranes from obese patients with non-insulin-dependent diabetes mellitus (NIDDM) was abnormally augmented (Thakker et al., J. Biol. Chem. 264, 7169–7175). The objective of this investigation was to determine if a novel antidiabetic agent, pioglitazone, ameliorated hepatic insulin resistance in KKAy mice and to identify any alterations in PIP2-phospholipase C activity of liver plasma membranes that may accompany changes in insulin sensitivity. Treatment of KKAy mice for 4 days with pioglitazone (20 mg/kg per day) decreased blood glucose and insulin and improved a variety of indices of hepatic insulin resistance, but did not alter the rate of PIP2 hydrolysis by liver plasma membranes. Acute treatment of isolated liver plasma membranes with pioglitazone (1–100 μM) also failed to alter PIP2-phospholipase C activity. Furthermore, the specific activity, Ca2+-requirement, pH-dependence and sensitivity to guanosine 5′-thiotriphosphate of the PIP2-phospholipase C in KKAy liver membranes were indistinguishable from those of C57BL/6J (normal) mice. Among C57BL/6J and KKAy mice fed either a control or pioglitazone-supplemented diet, there was no correlation between PIP2-phospholipase C activity in isolated liver membranes and either glucose or insulin concentrations in the circulation. These data indicate that an alteration in PIP2-phospholipase C activity of liver plasma membranes is neither a cause nor an obligatory consequence of insulin resistance in KKAy mice or its amelioration by pioglitazone. Alterations of liver membrane phospholipase C activity in NIDDM, therefor, may reflect diabetic pathology other than the insulin resistance associated with this disease.

Distribution of chlordecone to liver plasma membranes and recovery from hepatobiliary dysfunction in rats

Chlordecone (CD) impairs biliary excretion of organic anions (including phenolphthalein glucuronide (PG), imipramine polar metabolites, and taurocholate) without evidence of hepatocellular necrosis in rats. In this study we investigated the hypothesis that CD-induced hepatobiliary dysfunction is dependent on CD concentration in liver plasma membranes where it inhibits active transport in vitro. Rats were treated by gavage (0 or 60 mg CD/kg in corn oil) 24 or 72 h prior to bile duct cannulation. Biliary excretion of PG, a marker of hepatic organic anion transport, and [ 14C]mannitol, a marker of passive transcellular permeability, was determined. Biliary excretion of PG decreased approximately 25% in rats 24 h after CD treatment, however rats recovered control PG excretion rates 72 h after CD treatment. Recovery of PG excretion occured despite higher liver homogenate [ 14C]CD concentrations at 72 h than at 24 h after [ 14C]CD treatment. Biliary clearance of [ 14C]mannitol decreased both 24 h and 72 h after treatment. Even though the amount of [ 14C]CD retained in the liver was greater at 72 h than at 24 h after treatment, the concentration of [ 14C]CD in isolated liver plasma membranes (LPM) was the same (3.5–3.9 nmol/mg protein) at both times. There was a significant reduction in 5′-nucleotidase activity of LPM at 24 h but not at 72 h after CD. This study demonstrated no correlation between recovery from CD-induced hepatobiliary dysfunction and whole liver accumulation. Altered subcellular [ 14C]CD distribution (reduced LPM-to-homogenate concentration ratio) was coincident with recovery.

Hepatic insulin resistance in KKA y mice and its amelioration by pioglitazone do not involve alterations in phospholipase C activity

It has been proposed that an abnormality in the regulation of cytosolic-free Ca 2+ may be the cause of some forms of insulin resistance. In support of this proposition, it was reported that phospholipase C-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2) by liver plasma membranes from obese patients with non-insulin-dependent diabetes mellitus (NIDDM) was abnormally augmented (Thakker et al., J. Biol. Chem. 264, 7169–7175). The objective of this investigation was to determine if a novel antidiabetic agent, pioglitazone, ameliorated hepatic insulin resistance in KKA y mice and to identify any alterations in PIP 2-phospholipase C activity of liver plasma membranes that may accompany changes in insulin sensitivity. Treatment of KKA y mice for 4 days with pioglitazone (20 mg/kg per day) decreased blood glucose and insulin and improved a variety of indices of hepatic insulin resistance, but did not alter the rate of PIP 2 hydrolysis by liver plasma membranes. Acute treatment of isolated liver plasma membranes with pioglitazone (1–100 μM) also failed to alter PIP 2-phospholipase C activity. Furthermore, the specific activity, Ca 2+-requirement, pH-dependence and sensitivity to guanosine 5′-thiotriphosphate of the PIP 2-phospholipase C in KKA y liver membranes were indistinguishable from those of C57BL/6J (normal) mice. Among C57BL/6J and KKA y mice fed either a control or pioglitazone-supplemented diet, there was no correlation between PIP 2-phospholipase C activity in isolated liver membranes and either glucose or insulin concentrations in the circulation. These data indicate that an alteration in PIP 2-phospholipase C activity of liver plasma membranes is neither a cause nor an obligatory consequence of insulin resistance in KKA y mice or its amelioration by pioglitazone. Alterations of liver membrane phospholipase C activity in NIDDM, therefor, may reflect diabetic pathology other than the insulin resistance associated with this disease.

Effect of acute alcohol administration on TNF-alpha binding to neutrophils and isolated liver plasma membranes.

The mechanisms underlying the effects of alcohol (ethanol, ETOH) on host defense are poorly understood. ETOH modulation of the cytokine regulatory network is one possible way by which ETOH could alter nonspecific immune function. In this study we examined the ability of acute alcohol intoxication to alter lipopolysaccharide (LPS)-induced changes in tumor necrosis factor (TNF)-alpha binding to neutrophils and isolated liver plasma membranes. Rats were injected intravenously with a primed constant infusion of ETOH for 7 hr to maintain blood ETOH concentration at approximately 35 mM. Four hours after the start of ETOH infusion, the animals received intravenously either sterile saline or LPS (100 micrograms/100 g body weight) and were sacrificed at the end of ETOH infusion. Blood neutrophils and liver plasma membranes were isolated, and TNF-alpha binding characteristics determined using recombinant human [125I]TNF-alpha. ETOH treatment alone induced a significant decrease (51%) of neutrophil Bmax for TNF-alpha, without affecting the cytokine binding to plasma membranes. LPS, with or without ETOH, significantly decreased (61%) neutrophil Bmax for TNF-alpha and increased (115%) its binding to liver plasma membranes. The KD values of binding to either neutrophils or liver plasma membranes were not altered by ETOH or LPS treatment of animals. By decreasing the cytokine binding to neutrophils, ETOH may impair the control exerted by TNF-alpha on cell function, thus damaging host defense.

Reduced insulin binding to liver plasma membranes in inherently obese diabetic CBA/CA mice

Insulin binding to isolated liver plasma membranes was measured in mice from C57BL and LACG strains, and in normal and obese diabetic mice from the Bristol CBA/Ca colony. A simple and rapid three-step method for the preparation and purification of liver plasma membranes, using Percoll density gradient centrifugation was used. Both high and low affinity binding was detected in membranes from all four groups of mice. The K d values for binding were similar in all groups, but the insulin binding capacity ( B max) at low and high affinity was significantly reduced in obese CBA mice compared to age-matched lean controls. It is proposed that insulin receptor down-regulation may account for the insulin resistance observed in spontaneously obese diabetic CBA mice.

Molecular analysis of the tight junction

The tight junction (zonula occludens) constitutes a selectively permeable barrier in the paracellular pathway of most epithelia. It is also thought to play a role in the maintenance of the cell surface compositional asymmetry characteristic of epithelial cells. The identification of ZO-1 and cingulin, the first two proteins found to be exclusively associated with the tight junction, permits novel investigations of this important epithelial cell structure at the biochemical level.ZO-1 is a high molecular weight polypeptide (&gt;200 kD) found at the tight junctions of a variety of epithelia as well as endothelia, and ultrastructural localization studies on isolated liver plasma membranes indicate that this molecule is clustered at the points of membrane contact on the cytoplasmic surface of the junction. Physical analysis demonstrates ZO-1 to be an elongated, monomeric, phosphorylated protein, peripherally associated with the junctional membrane. Cingulin was originally isolated from chicken intestine, and, like ZO-1, is a peripheral membrane component of the junction which exhibits an elongated shape. Antibodies directed against this molecule show two primary bands at 140 kD and 108 kD on immunoblots of several epithelial tissues, although the relationship between these two elements remains undefined. Little information currently exists regarding the relationship of ZO-1 and cingulin to each other or to tight junction structure or function. We report here a comparison of the immunocytochemical properties of these junctional components as well as an examination of the phosphorylation state of ZO-1.

Dynamic determination of kinetic parameters for the interaction between polypeptide hormones and cell-surface receptors in the perfused rat liver by the multiple-indicator dilution method.

Hepatic elimination of epidermal growth factor (EGF) via receptor-mediated endocytosis was studied by a multiple-indicator dilution method in the isolated perfused rat liver, in which cell polarity and spatial organization are maintained. In this method EGF was given with inulin, an extracellular reference, as a bolus into the portal vein, and dilution curves of both compounds in the hepatic vein effluent were analyzed. Analysis of the dilution curve for EGF, compared with that for somatostatin, which showed no specific binding to isolated liver plasma membranes, resulted as follows: (i) both extraction ratio and distribution volume of 125I-labeled EGF decreased as the injected amount of unlabeled EGF increased; (ii) the ratio plot [ln (inulin/EGF) versus time] of the dilution curve for EGF exhibited an upward straight line initially for a short period of time (approximately equal to 10 sec), whereas the ratio plot [ln (inulin/somatostatin) versus time] of somatostatin gradually decreased. The multiple-indicator dilution method was used for other peptides also. Insulin and glucagon, known to have hepatocyte receptors, behaved similarly to EGF in shape of their ratio plots. Thus, analysis of dilution curves can reveal whether or not the cell surface has receptors for certain peptides. In addition, the dilution curves for EGF at various doses (tracer approximately equal to 30 micrograms) were analyzed simultaneously based on a kinetic model incorporating the perfusion rate, the association rate constant of EGF to surface receptors (kappa on), the dissociation rate constant of EGF from the EGF-receptor complex (kappa off), and the sequestration rate constant of the complex. The kinetic parameters [the dissociation constant (Kd = kappa off/kappa on) and the number of surface receptors] calculated by this analysis were comparable with reported values obtained by in vitro direct binding measurements at equilibrium using liver homogenates. We conclude that the multiple-indicator dilution method is a good tool for analyzing the dynamics of peptide hormones--cell-surface receptor interaction under a condition in which spatial architecture of the liver is maintained.

Specificity and mechanism of influence of amino acid residues on hepatic clearance of oligopeptides.

We have investigated the influence of amino acid residues on hepatic clearance of oligopeptides by determining the rate of disappearance (nmol.(min.g liver)-1) of selective oligopeptides from the medium during isolated rat liver perfusion. (a) N terminus: the rate of disappearance of Ala-Leu was greater (p less than 0.01) than those of Gly-Leu, Phe-Leu, and Arg-Leu (208 +/- 13, 135 +/- 13, 116 +/- 12, and 127 +/- 12, respectively). (b) C terminus: the rate of disappearance of Leu-Ala (244 +/- 18) was significantly greater (p less than 0.01) than that of Leu-Gly (145 +/- 16). (c) Number of residues: with each increase in the number of alanine residues (2-4) there was a significant increase in the rate of peptide disappearance, and conversely, with each increase in the number of glycine residues (2-6) there was a significant decrease in the rate of peptide disappearance. Further studies showed no peptide transport by isolated liver plasma membrane vesicles and no significant correlation between the rates of peptide disappearance and hydrolase activities of the perfusion medium but highly significant correlation with hydrolase activity of plasma membrane. We conclude that certain amino acid residues, such as alanine, enhance hepatic clearance of oligopeptides by increasing their affinity as substrates for plasma membrane peptide hydrolases.

Alkylation of the liver plasma membrane and inhibition of the Ca 2+ ATPase by acetaminophen

Acetaminophen is activated metabolically to yield reactive species that bind covalently to liver cell macromolecules. The extent of covalent binding correlates with the occurrence and severity of hepatic necrosis. We reported previously [J. O. Tsokos-Kuhn, E. L. Todd, J. B. McMillin-Wood and J. R. Mitchell, Molec. Pharmac. 28, 56 (1985)] that active Ca 2+ accumulation of isolated liver plasma membranes is decreased 60–75% after a hepatotoxic dose of acetaminophen in vivo. We now report that the protein of isolated liver plasma membranes was substantially labeled with drug metabolites after administration of [ 3H]acetaminophen. There was no increase in passive membrane permeability that might cause diminished Ca 2+ accumulation. Intravesicular volume and relative purity of the vesicle preparations after acetaminophen were not different from controls. However, (Ca 2+,Mg 2+)-ATPase, a possible biochemical expression of the Ca 2+ pump, was decreased 31% (P < 0.025) after acetaminophen treatment. ATPase activity in both control and treated groups was enhanced by isolating membranes in the presence of 5 mM reduced glutathione (GSH), but the effects of drug treatment were not reversed. A similar effect of GSH on Ca 2+ accumulation was observed previously [J.O. Tsokos-Kuhn, E. L. Todd, J. B. McMillin-Wood and J. R. Mitchell, Molec. Pharmac. 28, 56 (1985)]. These data are consistent with a hypothesis wherein alkylation of membrane proteins by reactive acetaminophen metabolites is a factor in the onset of hepatic necrosis after acetaminophen. They are not consistent with an oxidative stress hypothesis where thiol S-thiolation of membrane components is postulated to produce altered membrane permeability or thiol-reversible alterations in membrane protein structure and enzymatic function.

Heparan sulphate-degrading endoglycosidase in liver plasma membranes

An endoglycosidase is described in isolated liver plasma membranes that brings about a rapid and selective degradation of membrane-associated heparan sulphate, pre-labelled biosynthetically with Na2(35)SO4. The enzyme attacked mainly the polysaccharide chains of a hydrophobic membrane proteoglycan and it had little effect on a proteoglycan that could be displaced from the membranes with 1.0 M-NaCl. The highest activity was measured in the pH range 7.5-8.0, and the enzyme was almost completely inhibited below pH 5.5. Breakdown of susceptible polysaccharide chains was fast, being complete in 20-30 min. The major oligosaccharide fraction (Mr approx. 6000) produced by the enzyme was considerably smaller than the intact heparan sulphate chains. Enzyme activity was retained in membranes solubilized in 1% (v/v) Triton X-100. The high pH optimum and plasma-membrane association distinguish this enzyme from other heparan sulphate-degrading endoglycosidases that have acid pH optima and may be of lysosomal origin. A plasma-membrane endoglycosidase could modulate cellular interactions mediated by heparan sulphate, and/or release biologically active fragments of the polysaccharide from the cell periphery.

Ca 2 +-mobilizing hormones elicit phosphatidylethanol accumulation via phospholipase D activation

Vasopressin, angiotensin II and epinephrine elicited the accumulation of phosphatidylethanol in rat hepatocytes exposed to ethanol and of phosphatidate in the absence of ethanol. When isolated liver plasma membranes were exposed to ethanol, GTPγS stimulated the production of phosphatidylethanol whereas phosphatidate was formed in the absence of ethanol. With increasing ethanol concentrations, phosphatidate formation declined whereas phosphatidylethanol production increased. These findings suggest that rat hepatocytes possess a hormone-dependent phospholipase D activity that can also catalyse the formation of phosphatidylethanol.

Retinoic acid inhibition of transplasmalemma diferric transferrin reductase

All trans retinoic acid inhibited diferric transferrin reduction by HeLa cells. The NADH diferric transferrin reductase activity of isolated liver plasma membranes was also inhibited by retinoic acid. Retinol and retinyl acetate had very little effect. Transplasma membrane ferricyanide reduction by HeLa cells and NADH ferricyanide reductase of liver plasma membrane was also inhibited by retinoic acid, therefore the inhibition was in the electron transport system and not at the transferrin receptor. Since the transmembrane electron transport has been shown to stimulate cell growth, the growth inhibition by retinoic acid thus may be based on inhibition of the NADH diferric transferrin reductase.

Solubilization and reconstitution of hepatic System A-mediated amino acid transport. Preparation of proteoliposomes containing glucagon-stimulated transport activity

System A-mediated amino acid transport activity from rat liver plasma membrane vesicles has been solubilized and reconstituted into proteoliposomes using a freeze-thaw-dilution technique. The presence of cholate, at a cholate to protein ratio of 1:1, during the freeze-thaw step resulted in an enhancement in recoverable transport activity. The carrier required both phosphatidylcholine and phosphatidylethanolamine for optimal activity, but the addition of cholesterol to the reconstitution procedure appeared to have no significant effect on the resulting activity. A lipid to protein ratio of 20:1 yielded maximal transport activity. Sonication of the proteoliposomes provided some improvement in the accuracy of replicate assays for a given proteoliposome preparation. Isolated liver plasma membrane vesicles prepared from rats treated in vivo with glucagon in combination with dexamethasone contained stimulated System A activity. This enhanced transport activity could be solubilized and recovered in proteoliposomes generated from these plasma membranes. The data support the proposal that hormone regulation of the hepatic System A gene results in the de novo synthesis and plasma membrane insertion of the carrier protein itself.