Francis R. Simon

Renal cortical brush-border and basolateral membranes: Cholesterol and phospholipid Composition and relative turnover

SummaryA new procedure for the rapid isolation of renal cortical brush-border and basolateral membranes from the same homogenate is described. Brush-border membranes isolated using Mg2+-EGTA precipitation were enriched 18-fold for leucine aminopeptidase and had a recovery of 32.5%. Basolateral membrane fractions were isolated using a discontinuous sucrose gradient and showed an enrichment of 10.7-fold and recovery of 12.8% using (Na+, K+)-ATPase as a marker enzyme. Lipid analysis using two-dimensional TLC separation of phospholipids and gas liquid chromatography for cholesterol showed marked differences in the lipid composition of the brush-border and basolateral membranes. The brush-border membrane had increased sphingomyelin, phosphatidylserine, ethanolamine plasmalogens, and an increased cholesterol-to-phospholipid and sphingomyelin-to-phosphatidylcholine ratio compared to the basolateral membrane. The relative turnover of total membrane and individual phospholipid species using a double isotope ratio method was carried out. Phospholipids were labeled with either phosphorus 32 and 33 or acetate (3H, 1-14C). The relative turnover of phospholipid species and cholesterol differed strikingly. Phosphatidylcholine showed a high turnover, phosphatidylethanolamine and phosphatidylinositol had intermediate values and sphingomyelin, phosphatidylserine and cholesterol had low relative turnover rates. The order of phospholipid class relative turnover was independent of the labeled precursor used. The brush-border membrane had a significantly reduced relative turnover rate for total membrane phospholipids, sphingomyelin and cholesterol compared to the basolateral membrane. These data show marked differences in the lipid composition and relative turnover rates of the phospholipid species of the brush-border and basolateral membranes. They provide a biochemical basis for the recently reported differences in brush-border and basolateral membrane fluidity and suggest independent cellular regulation of brush-border and basolateral membrane lipids.

Ischemia induces partial loss of surface membrane polarity and accumulation of putative calcium ionophores.

To determine if ischemia induces alterations in renal proximal tubule surface membranes, brush border (BBM) and basolateral membranes (BLM) were isolated simultaneously from the same cortical homogenate after 50 min of renal pedicle clamping. Ischemia caused a selective decrease in the specific activity of BBM marker enzymes leucine aminopeptidase and alkaline phosphatase, but did not effect enrichment (15 times). Neither specific activity nor enrichment (10 times) of BLM NaK-ATPase was altered by ischemia. Contamination of BBM by intracellular organelles was also unchanged, but there was an increase in the specific activity (41.1 vs. 60.0, P less than 0.01) and enrichment (2.3 vs. 4.3, P less than 0.01) of NaK-ATPase in the ischemic BBM fraction. Ischemia increased BLM lysophosphatidylcholine (1.3 vs. 2.5%, P less than 0.05) and phosphatidic acid (0.4 vs. 1.3%, P less than 0.05). Ischemia also decreased BBM sphingomyelin (38.5 vs. 29.6%, P less than 0.01) and phosphatidylserine (16.1 vs. 11.4%, P less than 0.01), and increased phosphatidylcholine (17.2 vs. 29.7%, P less than 0.01), phosphatidylinositol (1.8 vs. 4.6%, P less than 0.01), and lysophosphatidylcholine (1.0 vs. 1.8%, P less than 0.05). The large changes in BBM phospholipids did not result from new phospholipid synthesis, since the specific activity (32P dpm/nmol Pi) of prelabeled individual and total phospholipids was unaltered by ischemia. We next evaluated if these changes were due to inability of ischemic cells to maintain surface membrane polarity. Cytochemical evaluation showed that while NaK-ATPase could be detected only in control BLM, specific deposits of reaction product were present in the BBM of ischemic kidneys. Furthermore, using continuous sucrose gradients, the enzymatic profile of ischemic BBM NaK-ATPase shifted away from ischemic BLM NaK-ATPase and toward the BBM enzymatic marker leucine aminopeptidase. Taken together, these data suggest that NaK-ATPase activity determined enzymatically and cytochemically was located within ischemic BBM. We propose that ischemia impairs the ability of cells to maintain surface membrane polarity, and also results in the accumulation of putative calcium ionophores.

Hepatitis C virus NS5A protein binds TBP and p53, inhibiting their DNA binding and p53 interactions with TBP and ERCC3.

Among the hepatotropic viruses, hepatitis C virus (HCV) is considered to be the leading cause of liver disease in humans, affecting approximately 2% of the world population. HCV-encoded nonstructural protein 5A (NS5A) is a 56-58-kDa phosphoprotein, which is produced from the processing of viral polyprotein. The potential mechanism(s) by which NS5A is able to influence key cellular processes are largely unknown. In this study, we investigated the functional properties of NS5A. In vivo co-immunoprecipitation and pull-down assays demonstrated that NS5A forms a heteromeric complex with TATA box binding protein (TBP) and tumor suppressor protein p53. Mutants of TBP and p53 showed reduced binding to NS5A. To determine the functional relevance of these associations, we found that NS5A inhibits the binding of both p53 and TBP to their DNA consensus binding sequences in vitro. NS5A also inhibited the p53-TBP and p53-excision repair cross complementing factor 3 (ERCC3) protein-protein complex formation. Furthermore, NS5A repressed the p53 regulated p21 (WAF1) promoter and a synthetic promoter containing multiple p53 responsive DNA elements binding sites in HCT116 p53(+/+) cell line. p53-mediated transcriptional activation from both promoters was reduced approximately 3-5-fold following expression of NS5A. Taken together, these results suggest that NS5A may exert its influence on key cellular processes by functional associations with p53 and TBP. This could explain one of the possible mechanism(s) by which NS5A is able to exert its effect on cellular gene expression and cell growth regulation.

Effect of estrogens on the liver

Although the liver is not generally regarded as a target organ for estrogens, several clinical observations and a number of physiological studies suggest that it is. The identification of estrogen receptors in liver cytosol and nuclei support this conclusion. 1, 2 We shall discuss several of the more important physiological and clinical effects of estrogens upon the liver (table 1) and shall illustrate them by brief case reports.

Characterization of the mechanisms involved in the gender differences in hepatic taurocholate uptake

Gender differences in the hepatic transport of organic anions is well established. Although uptake of many organic anions is greater in females, sodium-dependent taurocholate uptake is greater in hepatocytes from male rats. We examined the hypothesis that endogenous estrogens alter the number of sinusoidal bile acid transporters and/or decrease membrane lipid fluidity. The initial sodium-dependent uptake of [3H]taurocholate was 75% greater in hepatocytes from males than from either intact or oophorectomized females rats. Taurocholate maximal uptake was increased twofold ( P < 0.03) without a significant change in the Michaelis-Menten constant. Sinusoidal membrane fractions were isolated from male and female rat livers with equal specific activities and enrichments of Na+-K+-ATPase. Males had a significant ( P < 0.05) increase in cholesterol esters and phosphatidylethanolamine-to-phosphatidylcholine ratio. Fluorescence polarization indicated decreased lipid fluidity in females. In females, expression of the sodium-dependent taurocholate peptide (Ntcp) and mRNA were selectively decreased to 46 ± 9 and 54 ± 4% ( P < 0.01), respectively, and the organic anion transporter peptide (Oatp) and Na+-K+-ATPase α-subunit were not significantly different. Nuclear run-on analysis indicated a 47% ( P < 0.05) decrease in Ntcp transcription, without a significant change in Oatp. In conclusion, these studies demonstrated that decreased sodium-dependent bile salt uptake in female hepatocytes was due to decreased membrane lipid fluidity and a selective decrease in Ntcp.Gender differences in the hepatic transport of organic anions is well established. Although uptake of many organic anions is greater in females, sodium-dependent taurocholate uptake is greater in hepatocytes from male rats. We examined the hypothesis that endogenous estrogens alter the number of sinusoidal bile acid transporters and/or decrease membrane lipid fluidity. The initial sodium-dependent uptake of [3H]taurocholate was 75% greater in hepatocytes from males than from either intact or oophorectomized females rats. Taurocholate maximal uptake was increased twofold (P < 0.03) without a significant change in the Michaelis-Menten constant. Sinusoidal membrane fractions were isolated from male and female rat livers with equal specific activities and enrichments of Na+-K+-ATPase. Males had a significant (P < 0.05) increase in cholesterol esters and phosphatidylethanolamine-to-phosphatidylcholine ratio. Fluorescence polarization indicated decreased lipid fluidity in females. In females, expression of the sodium-dependent taurocholate peptide (Ntcp) and mRNA were selectively decreased to 46 +/- 9 and 54 +/- 4% (P < 0.01), respectively, and the organic anion transporter peptide (Oatp) and Na+-K+-ATPase alpha-subunit were not significantly different. Nuclear run-on analysis indicated a 47% (P < 0.05) decrease in Ntcp transcription, without a significant change in Oatp. In conclusion, these studies demonstrated that decreased sodium-dependent bile salt uptake in female hepatocytes was due to decreased membrane lipid fluidity and a selective decrease in Ntcp.

Hepatocyte Nuclear Factor (HNF) 1 and HNF4 Mediate Hepatic Multidrug Resistance Protein 2 Up-Regulation during Hepatitis C Virus Gene Expression

Hepatitis C virus (HCV) is known to induce hepatic oxidative stress that is implicated in the up-regulation of multidrug resistance proteins (MRPs). The relationship between increased prooxidant production, MRPs, and HCV has not been investigated. Here, we report that a homeodomain-containing transcription factor, hepatocyte nuclear factor (HNF) 1, plays a central role in liver gene regulation during HCV gene expression and/or subgenome replication. MRP2 protein and mRNA expression were increased and MRP2 promoter activity was increased 7-fold. Mutations within the putative HNF1 binding site of the human MRP2 promoter abrogated HCV-induced activation, implicating HNF1 in the induction of MRP2 by HCV. The mechanism by which HNF1-mediated activation occurs seems to be transcriptional, because the regulated expression of HNF4, which is known to control HNF1 expression, was also increased. Consistent with this finding, HNF1 mRNA was increased 10-fold. A promoter-luciferase construct of the human HNF1 gene was activated in an HNF4-dependent manner, and a mutant construct lacking the HNF4 binding site was not activated in HCV-positive cells. Consistent with this hypothesis, HNF4 protein and mRNA levels as well as HNF4 promoter activity and DNA binding activity were increased. The expression of HNF1 seems to play a critical role in the induction of hepatic MRP2 secondary to HCV subgenomic replication. The ability of HCV to induce HNF1 and HNF4 is attributed to 1) increased oxidative stress and 2) direct protein-protein interactions between HCV nonstructural component (NS) 5A and HNF1, leading to enhanced HNF1 DNA binding. In conclusion, we describe a novel mechanism by which HCV gene expression may induce adaptive responses involving MRP2 via HNF1 activation. This may constitute, in part, the cellular detoxification task force during HCV infection.

Effects of Ethanol on Membrane Order: Fluorescence Studies

The close correlation of membrane solubility with intoxicating potency for a variety of alcohols and other intoxicant-anesthetics indicates a membrane site of action for alcohol and related drugs (e.g., REF. 1). One theory is that alcohol alters the functional properties of cell membranes because it alters their physical properties. A detailed discussion of this concept is beyond the scope of this paper but is the topic of several recent This paper will discuss the use of fluorescent probes in the study of effects of ethanol on membrane physical properties. Ethanol is often said to alter membrane “fluidity.” Fluidity does not have a precise definition and is usually taken to mean the relative motional freedom of membrane constituents, particularly lipids. In a more rigorous sense, fluidity consists of rotational diffusion, which is related to microviscosity and hindered anisotropic rotations, which are related to lipid order!36 One focus of this paper is how fluorescence measurements can be used to distinguish between effects of alcohol on these two propertiesmicroviscosity and lipid order.

Hepatic Alkaline Phosphatase Isoenzymes: Isolation, Characterization and Differential Alteration

Although it is generally believed that hepatic alkaline phosphatase is localized to liver plasma membranes, 63% is present in the cytosol fraction after ultracentrifugation of rat liver homogenates. Divalent cation requirements, heat inactivation, pH optima, Km and chemical inhibition characteristics of partially purified alkaline phosphatase enzymes prepared from membrane and cytosol fractions suggested different structural forms. Furthermore, bile duct obstruction and ethinyl estradiol administration preferentially increased membrane-bound alkaline phosphatase activity, while cytosol activity was unaltered. In contrast, phenobarbital treatment decreased membrane-bound alkaline phosphatase and increased cytosol activity. These studies support the presence of two forms of hepatic alkaline phosphatase in rat liver which are regulated by different control mechanisms.

Qualitative alteration in hepatic microsomal cytochrome P-450 apoproteins associated with bile duct ligation, and the administration of ethinyl estradiol, phenobarbital and 3-methylcholanthrene

Abstract We have investigated in rat liver whether different forms of cytochrome P-450 are altered in hepatic disorders associated with impaired drug metabolism. Total hepatic cytochrome P-450 is decreased after either bile duct ligation or the administration of ethinyl estradiol. In contrast, phenobarbital administered alone increases hepatic content of cytochrome P-450, and when administered with ethinyl estradiol the reduction in cytochrome P-450 was prevented. Microsomal ethylmorphine N -demethylase activities paralleled changes in cytochrome P-450 content, except in bile duct ligation. where it is diminished to a greater extent. Four forms of microsomal cytochrome P-450 apoproteins. ranging in molecular weight from 50,000 to 58,000, were tentatively identified in a sodium dodecyl sulfate (SDS)-6 M urea polyacrylamide gel electrophoresis system by their responsiveness to pharmacological agents, turnover and benzidine peroxidase staining. Phenobarbital administration increased primarily band IV (50,000 daltons); in contrast only band III (53,000 daltons) was responsive to 3-methyl-cholanthene. Bile duct ligation was associated with a marked reduction in bands I, III and IV while bands I and III were decreased with ethinyl estradiol administration. Simultaneous administration of phenobarbital and ethinyl estradiol demonstrated a return of band I and an increase in density of bands II and IV. The mechanism of this reversal by phenobarbital was determined by the double-isotope technique and demonstrates that phenobarbital increases the relative synthesis rates of P-450 apoproteins in ethinyl estradiol-treated rats. These sludies support the hypothesis that mulliple forms of cytochrome P-450 are present in liver microsomal membranes and that alterations in specific apoproteins may be associated with an increase or a decrease in the functional properties of cytochrome P-450.

Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2).

Multidrug resistance-associated protein 2 (MRP2) (ABCC2) is an ATP-binding cassette membrane protein located primarily on apical surface of hepatocytes that mediates transport of conjugated xenobiotics and endogenous compounds into bile. MRP2 is highly expressed in hepatocytes, and at lower levels in small intestines, stomach and kidney. Previous reports have characterized mammalian MRP2 promoters, but none have established the molecular mechanism(s) involved in liver enriched expression. This study aims to investigate the mechanism of hepatic MRP2 regulation. A 2130 bp of MRP2 promoter was cloned from PAC-1 clone P108G1-7, to identify putative liver specific/hormone responsive functional DNA binding sites. Using deletion analysis, site specific mutagenesis and co-transfection studies, liver specific expression was determined. MRP2 promoter-LUC constructs were highly expressed in liver cell lines compared to non-liver cells. The region extending from -3 to+458 bp of MRP2 promoter starting from AUG contained the potential binding sites for CAATT box enhancer binding protein (C/EBP), hepatocytes nuclear factor 1, 3 and 4 (HNF1, HNF3, and HNF4. Only HNF1 and HNF4 co-transfection with MRP2 luciferase increased expression. Site specific mutational analysis of HNF1 binding site indicated an important role for HNF1alpha. HNF4alpha induction of MRP2 was independent of HNF1 binding site. C/EBP, HNF3, and HNF6 inhibited HNF1alpha while HNF4alpha induced MRP2 luciferase expression and glucocorticoids stimulated MRP2 expression. This study emphasizes the complex regulation of MRP2 with HNF1alpha and HNF4alpha playing a central role. The coordinated regulation of xenobiotic transporters and oxidative conjugation may determine the adaptive responses to cellular detoxification processes.