Hepatocyte Plasma Membrane Research Articles

Molecular characterization, expression and functional analysis of yak IFITM3 gene

IFITM3 is interferon-induced transmembrane 3, which plays an extremely key role in anti-proliferation, anti-virus and anti-tumor diseases.In this study, the yak (Bos grunniens) IFITM3 (BgIFITM3) gene contained a 5′-untranslated region (UTR) (25 bp), a coding region (441 bp), and a 3′-UTR (115 bp). The expression of BgIFITM3 gene in liver was significantly higher than that in heart, spleen, lung and kidney (P < 0.01). BgIFITM3 protein was localized on the yak hepatocyte plasma membrane, and its expression was significantly different between 1 day and 15 months of age (P < 0.05). Moreover, the prokaryotic expression vector of BgIFITM3 protein was constructed and expressed successfully, with a molecular weight of 19.5 kDa. The activities of yak hepatocyte were significantly inhibited after treating with BgIFITM3 protein (10 and 20 μg/mL) (P < 0.01). The expression levels of ERBB-2, IRS-1, PI3KR-1, AKT-1 and MAPK-3 were significantly lower after treating with 20 μg/mL BgIFITM3 protein (P < 0.05). Besides, the activities of HepG2 cells were significantly inhibited after treating with BgIFITM3 protein (1, 10 and 20 μg/mL) (P < 0.05). While, the cloning ability and migration ability of HepG2 cells were significantly inhibited after treating with 10 μg/mL BgIFITM3 protein (P < 0.05). Finally, the mitochondria of HepG2 cells was concentrated, cristae widened, and the double film density of mitochondria was increased after treating with 10 μg/mL BgIFITM3 protein. After 10 μg/mL BgIFITM3 protein treating, the expression levels of VDAC-2, VDAC-3 and p53 genes were significantly increased, but the expression level of GPX-4 gene was significantly decreased (P < 0.01).Taken together, the BgIFITM3 protein could inhibit the proliferations of yak hepatocyte and HepG2 cells by regulating the PI3K/Akt pathway or ferroptosis-related genes, respectively. These results benefit for further study of the function of BgIFITM3 protein.

Constitutive oxidants from hepatocytes of male iPLA2β-null mice increases the externalization of phosphatidylethanolamine on plasma membrane

We have found that group VIA calcium-independent phospholipase A2 (iPLA2β) has specificity for hydrolysis of phosphatidylethanolamine (PE) in mouse livers. Phospholipids (PLs) are transported to plasma membrane and some PLs including PE are externalized to maintain membrane PL asymmetry. Here we demonstrated that hepatocytes of iPLA2β-null (KO) mice showed an increase in PE containing palmitate and oleate. We aimed to examine whether externalization of PE on the outer leaflets could be affected by iPLA2β deficiency and its modulation by reactive oxygen species (ROS) or apoptosis. As duramycin has high affinity to PE, we used duramycin conjugated with biotin (DLB) and streptavidin 488 as a probe for detection of externalized PE. Compared to WT, naïve KO hepatocytes showed an increase in both PE externalization and ROS generation. These events were observed in male but not in female KO mice. Hydrogen peroxide or menadione treatment enhanced PE externalization to the same extent for both male/female WT and KO hepatocytes. By indirect immunofluorescence, DLB-streptavidin staining was observed as small punctuated spots on the cell surface of menadione-treated KO hepatocytes. Unlike the reported PS externalization, CD95/FasL treatment did not lead to any increase in PE externalization, and iPLA2β deficiency-dependent PE externalization was also not correlated with apoptosis. Thus, constitutive (but not induced) ROS generation in iPLA2β-deficient hepatocytes leads to PE externalization observed only in male mice. Such PE externalization may imply detrimental effects regarding further oxidation of PE fatty acids and the binding with pathogens on the outer leaflets of hepatocyte plasma membrane.

Hypolipidemic drugs inhibiting the proprotein convertase of subtilisin/kexin type 9 (PCSK9): monoclonal antibodies, antisense oligonucleotides, small interfering ribonucleic acids

Hypolipidemic therapy is one of the essential components for the management of patients with cardiovascular diseases (CVD). In this regard, the main task of modern research is to find new targets for creating additional effective groups of hypolipidemic drugs. In 2003, canadian and french research groups led by N. Seidah and M. Abifadel discovered a new enzyme proprotein convertase subtilisin/kexin type 9 (PCSK9), which later turned out to play an important role in lipid metabolism. The main mechanism of action of PCSK9 is to regulate the density of low-density lipoprotein receptors (LDLR) in the cell membrane of hepatocytes. Increased activity of PCSK9 significantly accelerates the degradation of LDL and leads to an increase in the concentration of atherogenic classes of lipoproteins-low-density lipoproteins (LDL). In contrast, reduced PCSK9 activity is accompanied by a decrease in LDL concentrations and a reduced risk of developing atherosclerosis and CVD. The second of the recently discovered and less studied mechanism of PCSK9 protearogenic action is an increase in inflammatory processes in the atherosclerotic plaque. Given this adverse contribution of PCSK9 to the development and progression of atherosclerosis and CVD, the main task of the researchers was to develop drugs that inhibit THIS enzyme. To date, several new groups of drugs have been developed that target the stages of biosynthesis and the function of PCSK9. In this article, we will focus in detail on discussing the mechanisms of action and effectiveness of the following groups of hypolipidemic drugs: anti-PCSK9 monoclonal antibodies (alirocumab, evolocumab), small interfering ribonucleic acids (incliciran), and antisense nucleotides.

New groups of hypolipidemic medications based on inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9). Part 1

Hypolipidemic therapy is one of the essential components for the management of patients with cardiovascular diseases (CVD). In this regard, the main task of modern research is to find new targets for creating additional effective groups of hypolipidemic medications. Canadian and French research groups led by N. Seidah and M. Abifadel discovered a new enzyme — proprotein convertase subtilisin-kexin type 9 (PCSK9) in 2003. It turned out to play an important role in lipid metabolism later. The main mechanism of action of PCSK9 is to regulate the density of low-density lipoprotein receptors (LDLR) in the cell membrane of hepatocytes. Increased activity of PCSK9 accelerates the degradation of LDL significantly, and leads to an increase in the concentration of atherogenic classes of lipoproteins — low-density lipoproteins (LDL). In contrast, reduced PCSK9 activity is accompanied by a decrease in LDL concentrations and a reduced risk of developing atherosclerosis and CVD. The second of the recently discovered and less studied mechanism of PCSK9 protearogenic action is an increase in inflammatory processes in the atherosclerotic plaque. Considering this adverse contribution of PCSK9 to the development and progression of atherosclerosis and CVD, the main task of the researchers was to develop medications that inhibit THIS enzyme. Several new groups of medications that target the stages of biosynthesis and the function of PCSK9 have been developed by now. In this article, we will focus on details discussing the mechanisms of action and effectiveness of the following groups of hypolipidemic medications: anti-PCSK9 monoclonal antibodies (alirocumab, evolocumab), small interfering ribonucleic acids (incliciran), and antisense nucleotides.

New groups of hypolipidemic drugs based on inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9). Part 1

The hypolipidemic therapy is one of the essential components for the management of patients with cardiovascular diseases (CVD). In this regard, the main task of modern research is to find new targets for creating additional effective groups of lipid-lowering drugs. In 2003, a Canadian and French research team led by N. Seidah and M. Abifadel discovered a new enzyme, proprotein convertase subtilisin-kexin type 9 (PCSK9), which plays an important role in lipid metabolism.&#x0D; The main mechanism of action of PCSK9 is to regulate the density of low-density lipoprotein receptors (LDLR) in the cell membrane of hepatocytes. The increased activity of PCSK9 significantly accelerates the degradation of LDLR and leads to an increase in the concentration of atherogenic classes of lipoproteins the low-density lipoproteins (LDL). A reduced activity of PCSK9, on the contrary, is accompanied by a decrease in the concentration of LDL and a decrease in the risk of developing atherosclerosis and CVD. The second, recently discovered and less studied, mechanism of the protearogenic action of PCSK9 is the enhancement of inflammatory processes in the atherosclerotic plaque. Given this unfavorable contribution of PCSK9 to the development and progression of atherosclerosis and CVD, the main task of the researchers was to develop drugs that inhibit this enzyme. To date, several new drug groups have been developed that target the biosynthesis steps and the function of PCSK9.&#x0D; In this article, we will focus in detail on the discussion of the mechanisms of action and effectiveness of the following groups of lipid-lowering drugs: anti-PCSK9 monoclonal antibodies (alirocumab, evolocumab), small interfering ribonucleic acids (incliciran) and antisense nucleotides.

Zinc dyshomeostasis in azoxymethane-induced colonic precancerous and cancerous lesions in Fischer rats.

Zinc is an essential micronutrient involved in various biological processes. It is also argued that tumors need zinc for maintenance and proliferation and tumor cell apoptosis. Zinc homeostasis is regulated by the gastrointestinal tract and involves interplay of host, dietary, environmental and social factors such as alcohol consumption. The DNA alkylation agent azoxymethane (AOM), which is primarily activated in the liver, induces a high incidence of initiation and promotion steps of precancerous lesions in the colon of rats. The altered expression of hepatic zinc transporters by AOM may lead to zinc dyshomeostasis in liver. Decreased serum zinc concentration, despite increased liver zinc also indicates altered liver zinc mobilization and failure to regulate zinc homeostasis. During the transformation from normal colonic mucosa to colonic epithelial hyperplasia and aberrant crypt formation, a reduction in zinc concentration is observed. It will be interesting to study further if the same trend continues throughout tumor progression towards adenocarcinomas. Lowered local zinc concentrations in the colon epithelium may not just reflect a bystander effect, but may induce cell proliferation and compromise DNA integrity due to impairment of zinc-containing proteins. In congruence with the tissue zinc concentrations, metallothionein levels were found to be less induced in AOM -administered colon compared to normal healthy colon. Lowered tissue zinc levels in small and large intestine were also associated with increased expression of mRNA and protein ZnT1. In this regard, the mode of zinc responsiveness to ZnT1 mimics that of metallothionein, albeit at a lower level for ZnT1.

Lectin recognition and hepatocyte endocytosis of GalNAc-decorated nanostructured lipid carriers

Liver is the main organ for metabolism but is also subject to various pathologies, from viral, genetic, cancer or metabolic origin. There is thus a crucial need to develop efficient liver-targeted drug delivery strategies. Asialoglycoprotein receptor (ASGPR) is a C-type lectin expressed in the hepatocyte plasma membrane that efficiently endocytoses glycoproteins exposing galactose (Gal) or N-acetylgalactosamine (GalNAc). Its targeting has been successfully used to drive the uptake of small molecules decorated with three or four GalNAc, thanks to an optimisation of their spatial arrangement. Herein, we assessed the biological properties of highly stable nanostructured lipid carriers (NLC) made of FDA-approved ingredients and formulated with increasing amounts of GalNAc. Cellular studies showed that a high density of GalNAc was required to favour hepatocyte internalisation via the ASGPR pathway. Interaction studies using surface plasmon resonance and the macrophage galactose-lectin as GalNAc-recognising lectin confirmed the need of high GalNAc density for specific recognition of these NLC. This work is the first step for the development of efficient nanocarriers for prolonged liver delivery of active compounds.

MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcohol-associated fatty liver and steatohepatitis

Autophagy maintains cellular homeostasis and plays a critical role in the development of non-alcoholic fatty liver and steatohepatitis. The pseudokinase mixed lineage kinase domain-like (MLKL) is a key downstream effector of receptor interacting protein kinase 3 (RIP3) in the necroptotic pathway of programmed cell death. However, recent data reveal that MLKL also regulates autophagy. Herein, we tested the hypothesis that MLKL contributes to the progression of Western diet-induced liver injury in mice by regulating autophagy. Rip3+/+, Rip3-/-, Mlkl+/+ and Mlkl-/- mice were fed a Western diet (FFC diet, high in fat, fructose and cholesterol) or chow for 12 weeks. AML12 and primary mouse hepatocytes were exposed to palmitic acid (PA). The FFC diet increased expression, phosphorylation and oligomerization of MLKL in the liver. Mlkl, but not Rip3, deficiency protected mice from FFC diet-induced liver injury. The FFC dietalso induced accumulation of p62 and LC3-II, as well as markers of endoplasmic reticulum stress, in Mlkl+/+ but not Mlkl-/- mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor, leupeptin. Using an mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression and translocation, first to autophagosomes and then to the plasma membrane, independently of Rip3. Mlkl, but not Rip3, deficiency prevented inhibition of autophagy in PA-treated hepatocytes. Overexpression of Mlkl blocked autophagy independently of PA. Additionally, pharmacologic inhibition of autophagy induced MLKL expression and translocation to the plasma membrane in hepatocytes. Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury by inhibiting autophagy. Autophagy is a regulated process that maintains cellular homeostasis. Impaired autophagy contributes to cell injury and death, thus playing a critical role in the pathogenesis of a number of diseases, including non-alcohol-associated fatty liver and steatohepatitis. Herein, we show that Mlkl-dependent, but Rip3-independent, signaling contributed to diet-induced liver injury and inflammatory responses by inhibiting autophagy. These data identify a novel co-regulatory mechanism between necroptotic and autophagic signaling pathways in non-alcoholic fatty liver disease.

Down-regulation of ER-α36 mRNA in serum exosomes of the patients with hepatocellular carcinoma.

Down-regulation of ER-α36 mRNA in serum exosomes of the patients with hepatocellular carcinoma.

Probing the effects of hexavalent chromium exposure on histology and fatty acid metabolism in liver of Bufo gargarizans tadpoles.

Hexavalent chromium is one of the major detrimental heavy metal pollutants. B.gargarizans tadpoles were treated with different concentrations of Cr6+ (0, 13, 52, 104, 208, and 416μg Cr6+ L-1) from Gs 2 to Gs 42. The effect of Cr6+ on histopathological alterations and transcript levels of fatty acid metabolism-related genes as well as fatty acids composition and content in liver were examined. Histopathological changes were observed in liver at 52, 104, 208, and 416μg Cr6+ L-1. Moreover, RT-qPCR analyses showed the downregulated mRNA levels of the genes related to fatty acid synthesis (SCD, MECR, TECR and ELOVL1) and fatty acid β-oxidation (ACOT1, PPT1, HADH and ACAA2) at 416μg Cr6+ L-1. However, the mRNA expression of fatty acid β-oxidation-related genes (ECHS1, HADHA and ACAA1) were significantly upregulated at 13, 52, 104, 208 and 416μg Cr6+ L-1. In situ hybridization revealed BSEP was expressed in hepatocyte nucleus and plasma membrane, and HSD17B12 was abundantly expressed in the plasma membrane. The HSD17B12 mRNA levels were significantly upregulated in tadpoles exposed to all Cr6+ treatment groups, while the BSEP mRNA levels were downregulated at 104, 208 and 416μg Cr6+ L-1 groups compared to control. In addition, an increase in polyunsaturated fatty acids and a decrease in monounsaturated fatty acids were found in 52, 104 and 416μg Cr6+ L-1 groups. Overall, chronic exposure to Cr6+ may suppress fatty acid synthesis, disturb fatty acid β-oxidation, aggravate disorders of hepatic function and induce hepatic impairment in B.gargarizans tadpoles.

Quantitative Evaluation of Liver Function with Gd-EOB-DTPA-enhanced MR Imaging

The quantitative evaluation of liver function is important, not only for monitoring, but also for the preoperative evaluation of the functional hepatic reserve. The indocyanine green (ICG) clearance has been used as a reliable quantitative liver function test; however, ICG clearance cannot evaluate segmental liver function. Gd-EOB-DTPA is a bolus-injectable paramagnetic contrast media taken up by hepatocytes via organic anion transporting peptide, specifically expressed on cell membrane of hepatocytes; therefore quantitative evaluation of segmental liver function with use of Gd-EOBD-TPA-enhanced MR imaging is feasible. Methods for quantitative evaluation of liver function using Gd-EOB-DTPA-enhanced MR imaging can be divided into the following two major categories : dynamic and static methods. In the dynamic method, the time-intensity curve obtained from dynamic contrast enhanced MR imaging is analyzed using various pharmacokinetic models. The compartment model analysis is a representative method, categorized as a dynamic model-dependent analysis. In the static method, post contrast enhanced images, usually during the hepatobiliary phase, are analyzed using relaxation time or signal intensity of the liver. The static methods are easy to apply in clinical practice. The hepatocellular uptake index (HUI) is a static signal intensity-based method, which is obtained from the signal intensity and volume of the liver and spleen on Gd-EOB-DTPA-enhanced MR images during the hepatobiliary phase. HUI has been accepted as the most reliable predictor of segmental liver function. In this article, pharmacokinetics of Gd-EOB-DTPA is first described using the compartment model. Subsequently, derivation of 2-in-liner-1-uptake-1-out-2-compartment model and HUI are shown as examples of quantitative evaluation of the liver function with the use of Gd-EOB-DTPA-enhanced MR imaging.

Reciprocity between muscarinic and nicotinic cholinoreactive structures of liver tissue in conditions, in substrates and in products of free radical oxidation of liver lipids in 5-day period of cooling in rats

The effects of administration of cholinotropic agents (neostigmine, hexamethonium, pilocarpine, atropine, nicotine) on liver tissue after 5-day period of cooling of rats were compared. The analysis of the conditions inducing lipid peroxidation (LPO), the analysis of the substrate components of the LPO in liver, and the evaluation of the LPO products content in 5-day period of cold loads were made. The data obtained indicate on the contradirectory effects when pilocarpine and nicotine were administered to animals, as well as after administration of atropine and hexamethonium to animals. There was the similar effects after administration of both pilocarpine and hexamethonium and both nicotine and atropine in conditions of activated LPO assessed by substrate components of LPO in the liver after 5-day cooling. On the basis of these results, a hypothesis of reciprocity between muscarinic and nicotinic cholinoreceptors located on the plasma membrane of hepatocytes in the LPO processes of the liver during the period of cold loads has been formulated.

Cholinergic mechanisms of regulation of free radical oxidation of the liver lipids in cold adaptation in rats

The effects of administration of cholinotropic agents (neostigmine, hexamethonium, pilocarpine, atropine, nicotine) on liver tissue after the 5 days period of cooling of rats were compared. The analysis of the conditions inducing lipid peroxidation (LPO), the analysis of the substrate components of the LPO in liver, and the evaluation of the LPO products content in the 5 days period of cold loads were made. The data obtained indicate on the contradirectory effects when pilocarpine and nicotine were administered to animals, as well as after administration of atropine and hexamethonium to animals. There was the similar effects after administration of both pilocarpine and hexamethonium and both nicotine and atropine in conditions of activated LPO assessed by substrate components of LPO in the liver after 5 days cooling. On the basis of these results, a hypothesis of reciprocity between muscarinic and nicotinic cholinoreceptors located on the plasma membrane of hepatocytes in the LPO processes of the liver during the period of cold loads has been formulated.

Rat Organic Anion Transport Protein 1A1 Interacts Directly With Organic Anion Transport Protein 1A4 Facilitating Its Maturation and Trafficking to the Hepatocyte Plasma Membrane.

Organic anion transport proteins (OATPs) on the basolateral surface of hepatocytes mediate uptake of a number of drugs and endogenous compounds. Previous studies showed that rat OATP1A1 (rOATP1A1) has a postsynaptic density protein, drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) consensus binding motif at its C-terminus and binds to PDZ domain containing 1 (PDZK1), which is required for its cell-surface localization. PDZK1 associates with rOATP1A1-containing endocytic vesicles within cells, mediating recruitment of motor proteins required for microtubule-based trafficking to the plasma membrane. rOATP1A4 also traffics to the plasma membrane, although it lacks a PDZ binding consensus sequence. The current study was designed to test the hypothesis that trafficking of rOATP1A4 to the plasma membrane requires its direct interaction with rOATP1A1 resulting in a complex that traffics through the cell in common subcellular vesicles in which the cytosolic tail of rOATP1A1 is bound to PDZK1. We found that 74% of rOATP1A4-containing rat liver endocytic vesicles (n=12,044) also contained rOATP1A1. Studies in transfected HEK293 cells showed surface localization of rOATP1A1 only when coexpressed with PDZK1 whereas rOATP1A4 required coexpression with rOATP1A1 and PDZK1. Studies in stably transfected HeLa cells that constitutively expressed PDZK1 showed that coexpression of rOATP1A4 with rOATP1A1 resulted in more rapid appearance of rOATP1A4 on the plasma membrane and faster maturation to its fully glycosylated form. Similar results were observed on immunofluorescence analysis of single cells. Immunoprecipitation of rat liver or transfected HeLa cell lysates with rOATP1A1 antibody specifically co-immunoprecipitated rOATP1A4 as determined by western blotting. Conclusion: These studies indicate that optimal rOATP1A4 trafficking to the cell surface is dependent upon coexpression and interaction with rOATP1A1. As rOATP1A1 binds to the chaperone protein, PDZK1, rOATP1A4 functionally hitchhikes through the cell with this complex.

Clearance of Indocyanine Green in Severe Pediatric Burns.

Clearance of indocyanine green dye (ICGc) reflects sinusoidal perfusion and hepatocyte cell membrane function. Thus, ICGc is a reflection of the functional reserve of intact hepatocytes. The purpose of this study was to identify predictors of ICGc in severely burned children during the acute hospitalization and at the time of discharge from the intensive care unit (ICU). A secondary aim was to determine the relationship between liver size and patient ICGc. Twenty-six children (0.8-17 years old) with 35% or greater total body surface area burned (%TBSA-B) were included. Assessment of ICGc (in milliliters per minute per meter squared) was done during the acute hospitalization (median: 6 days after admission, median: 14 days postburn) and at the time of discharge from the ICU (median: 19 days after admission, median: 27 days postburn). Age, TBSA-B, % third-degree burns, inhalation injury, preexisting chronic malnutrition, hematocrit, liver dysfunction, and time from burn injury were incorporated in multiple linear regressions as predictive variables of ICGc. Only variables with p < 0.05 were retained in the final models. Time from injury and age were the strongest predictors of ICGc during the acute admission but not at the time of discharge from the ICU. Time from injury was negatively associated with ICGc, whereas age was positively associated. At the time of discharge from the ICU, ICGc was increased in proportion to the %TBSA-B, whereas inhalation injury and preexisting chronic malnutrition were associated with lower ICGc. There was no correlation between change-to-predicted liver length and ICGc. The intrinsic ability of the liver to extract ICG from plasma was lower in younger burned patients during the acute admission and in those with preexisting chronic malnutrition and inhalation injury at the time of discharge from the ICU. Prognostic/Epidemiologic, level III.

Functional properties of mouse ferroportin, a cellular iron‐export protein

Iron homeostasis is a topic of great public‐health importance because of the high prevalence of iron‐deficiency anemia and of hereditary disorders that cause iron overload. Ferroportin (Fpn)—the only known mammalian cellular iron exporter—is expressed on the plasma membrane of macrophages, enterocytes, and hepatocytes. Fpn, under the control of the iron‐regulatory hormone hepcidin, serves a critical role in the regulation of systemic iron. Whereas we have previously characterized the functional properties of human Fpn, a great deal of research in iron homeostasis utilizes mouse models, highlighting the need to study mouse Fpn in vitro. For example, the flatiron mouse model of classical ferroportin disease bears the mutation H32R in mFpn and is characterized by systemic iron deficiency and macrophage iron loading. The flatiron mouse also appears to exhibit a manganese phenotype [Seo YA and Wessling‐Resnick M (2015) FASEB J 28, 2726–2733], raising the possibility that mouse Fpn (mFpn) serves a role in Mn metabolism, although Mn is not a substrate of human Fpn. We therefore tested the hypothesis that mouse but not human Fpn can transport Mn. We characterized the functional properties of mFpn by using radiotracer assays in RNA‐injected Xenopus oocytes expressing mouse or human Fpn. We found that mFpn can export several transition metals, with selectivity Fe &gt; Co &gt; Ni, but not Cd. Whereas the H32R mutant abolished mFpn‐mediated Fe efflux relative to wildtype, neither wildtype nor mutant mFpn could transport Mn. mFpn‐mediated Fe efflux was pH‐dependent with estimated optimal pH of 8.2 ± (SEM) 0.2 when [Ca2+]o = 2 mM. mFpn‐mediated Fe efflux was activated by extracellular Ca2+, proceeded independently of extracellular Na+, and was inhibited 51% ± 1% by 10 μM hepcidin (30‐min preincubation). Our data (i) reveal that human and mouse Fpn share similar functional properties and (ii) do not support the notion that Fpn can transport Mn.Support or Funding InformationNIH grants R01 DK107309, P30 DK078392, and R25 HL115473, and the American Physiological SocietyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Cell Junctional Protein Network Associated with Connexin-26.

GJB2 mutations are the leading cause of non-syndromic inherited hearing loss. GJB2 encodes connexin-26 (CX26), which is a connexin (CX) family protein expressed in cochlea, skin, liver, and brain, displaying short cytoplasmic N-termini and C-termini. We searched for CX26 C-terminus binding partners by affinity capture and identified 12 unique proteins associated with cell junctions or cytoskeleton (CGN, DAAM1, FLNB, GAPDH, HOMER2, MAP7, MAPRE2 (EB2), JUP, PTK2B, RAI14, TJP1, and VCL) by using mass spectrometry. We show that, similar to other CX family members, CX26 co-fractionates with TJP1, VCL, and EB2 (EB1 paralogue) as well as the membrane-associated protein ASS1. The adaptor protein CGN (cingulin) co-immuno-precipitates with CX26, ASS1, and TJP1. In addition, CGN co-immunoprecipitation with CX30, CX31, and CX43 indicates that CX association is independent on the CX C-terminus length or sequence. CX26, CGN, FLNB, and DAMM1 were shown to distribute to the organ of Corti and hepatocyte plasma membrane. In the mouse liver, CX26 and TJP1 co-localized at the plasma membrane. In conclusion, CX26 associates with components of other membrane junctions that integrate with the cytoskeleton.

Oxidative stress promotes redistribution of TRPM2 channels to the plasma membrane in hepatocytes

Transient Receptor Potential Melastatin (TRPM) 2 is a non-selective Ca2+ permeable cation channel and a member of the Transient Receptor Potential (TRP) channel family. TRPM2 has unique gating properties; it is activated by intracellular ADP-ribose (ADPR), whereas Ca2+ plays a role of an important co-factor in channel activation, increasing TRPM2 sensitivity to ADPR. TRPM2 is highly expressed in rat and mouse hepatocytes, where it has been shown to contribute to oxidative stress-induced cell death and liver damage due to paracetamol-overdose. The mechanisms regulating the activity of TRPM2 channels in hepatocytes, however, are not well understood. In this paper, we investigate the localisation of TRPM2 protein in hepatocytes. The presented results demonstrate that in rat hepatocytes under normal conditions, most of the TRPM2 protein is localised intracellularly. This was determined by confocal microscopy using TRPM2-and plasma membrane (PM)-specific antibodies and immunofluorescence, and biotinylation studies followed by western blotting. Interestingly, in hepatocytes treated with either H2O2 or paracetamol, the amount of TRPM2 co-localised with PM is significantly increased, compared to the untreated cells. It is concluded that trafficking of TRPM2 to the PM could potentially contribute to a positive feedback mechanism mediating Ca2+ overload in hepatocytes under conditions of oxidative stress.

Evidence for the interaction of peroxiredoxin-4 with the store-operated calcium channel activator STIM1 in liver cells

Ca2+ entry through store-operated Ca2+ channels (SOCs) in the plasma membrane (PM) of hepatocytes plays a central role in the hormonal regulation of liver metabolism. SOCs are composed of Orai1, the channel pore protein, and STIM1, the activator protein, and are regulated by hormones and reactive oxygen species (ROS). In addition to Orai1, STIM1 also interacts with several other intracellular proteins. Most previous studies of the cellular functions of Orai1 and STIM1 have employed immortalised cells in culture expressing ectopic proteins tagged with a fluorescent polypeptide such as GFP. Little is known about the intracellular distributions of endogenous Orai1 and STIM1. The aims are to determine the intracellular distribution of endogenous Orai1 and STIM1 in hepatocytes and to identify novel STIM1 binding proteins. Subcellular fractions of rat liver were prepared by homogenisation and differential centrifugation. Orai1 and STIM1 were identified and quantified by western blot. Orai1 was found in the PM (0.03%), heavy (44%) and light (27%) microsomal fractions, and STIM1 in the PM (0.09%), and heavy (85%) and light (13%) microsomal fractions. Immunoprecipitation of STIM1 followed by LC/MS or western blot identified peroxiredoxin-4 (Prx-4) as a potential STIM1 binding protein. Prx-4 was found principally in the heavy microsomal fraction. Knockdown of Prx-4 using siRNA, or inhibition of Prx-4 using conoidin A, did not affect Ca2+ entry through SOCs but rendered SOCs susceptible to inhibition by H2O2. It is concluded that, in hepatocytes, a considerable proportion of endogenous Orai1 and STIM1 is located in the rough ER. In the rough ER, STIM1 interacts with Prx-4, and this interaction may contribute to the regulation by ROS of STIM1 and SOCs.

Вплив питної води з різним вмістом стеаратів калію і натрію на вільнорадикальні процеси в організмі щурів

Introduction One of the reasons for the lack of quality drinking water is the poor quality of natural water, which is constantly polluted by waste water from industrial and municipal enterprises, surface runoff from the fields and territories of settlements with which it enters a large amount of toxic substances. Among them, surface-active substances such as potassium and sodium stearate are not the last.The aim of the study to determine the specifics of lipid peroxidation and the state of the antioxidant protection under the influence of potassium and sodium stearates on the body of the experimental rats when they enter with drinking water.Research Methods. White rats were divided into 7 groups, which consumed water with stearates in quantities equal to the maximum non-active dose of the substance (MND), 1/2 MND and 1/4 MND for 25 days. The content of TBC-active lipid peroxidation products, diene conjugates and the activity of superoxide dismutase and catalase in liver homogenate were determined.Results and Discussion. In the homogenate of the liver of animals of all groups, there was a significant increase in the level of diene conjugates and TBK-active products in proportion to the concentration of the substance. It was also found that in animals that consumed water with different concentrations of potassium stearate, inhibition of antioxidant enzymes was observed. Consumption of water with different concentrations of sodium stearate caused the activation of these enzymes.Conclusions. Prolonged use by test animals of aqueous solution with concentrations of potassium and sodium stearate in MND and 1/2 MND negatively affects the state of hepatocyte cell membranes due to the activation of the lipid peroxidation processes, namely TBK-active products and diene conjugates, the amount of which depended on the concentration of stearates in drinking water. The use of drinking water by test animals with maintenance of potassium and sodium stearates at various doses caused changes in the content of antioxidant enzymes: water with potassium stearate inhibits the activity of these indicators, and causes sodium activation with sodium stearate.