ABSTRACT
Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1-5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.
Subject(s)
GTP Phosphohydrolases/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Calcium/metabolism , Cell Movement , Cell Proliferation , Endoplasmic Reticulum/metabolism , HeLa Cells , Humans , Lysophospholipids , Mitochondria/pathology , Signal Transduction , Sphingosine/analogs & derivatives , Sphingosine-1-Phosphate ReceptorsABSTRACT
AIM: Diabetes is a result of an interplay between genetic, environmental and lifestyle factors. Keratin intermediate filaments are stress proteins in epithelial cells, and keratin mutations predispose to several human diseases. However, the involvement of keratins in diabetes is not well known. K8 and its partner K18 are the main ß-cell keratins, and knockout of K8 (K8-/- ) in mice causes mislocalization of glucose transporter 2, mitochondrial defects, reduced insulin content and altered systemic glucose/insulin control. We hypothesize that K8/K18 offer protection during ß-cell stress and that decreased K8 levels contribute to diabetes susceptibility. METHODS: K8-heterozygous knockout (K8+/- ) and wild-type (K8+/+ ) mice were used to evaluate the influence of keratin levels on endocrine pancreatic function and diabetes development under basal conditions and after T1D streptozotocin (STZ)-induced ß-cell stress and T2D high-fat diet (HFD). RESULTS: Murine K8+/- endocrine islets express ~50% less K8/K18 compared with K8+/+ . The decreased keratin levels have little impact on basal systemic glucose/insulin regulation, ß-cell health or insulin levels. Diabetes incidence and blood glucose levels are significantly higher in K8+/- mice after low-dose/chronic STZ treatment, and STZ causes more ß-cell damage and polyuria in K8+/- compared with K8+/+ . K8 appears upregulated 5 weeks after STZ treatment in K8+/+ islets but not in K8+/- . K8+/- mice showed no major susceptibility risk to HFD compared to K8+/+ . CONCLUSION: Partial K8 deficiency reduces ß-cell stress tolerance and aggravates diabetes development in response to STZ, while there is no major susceptibility to HFD.
Subject(s)
Diabetes Mellitus, Experimental , Keratin-8/metabolism , Animals , Diabetes Mellitus, Type 2 , Diet, High-Fat/adverse effects , Heterozygote , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Keratin-8/genetics , Mice , Mice, KnockoutABSTRACT
AIMS/HYPOTHESIS: Dietary and microbial factors and the gut immune system are important in autoimmune diabetes. We evaluated inflammatory activity in the whole gut in prediabetic NOD mice using ex vivo imaging of reactive oxygen and nitrogen species (RONS), and correlated this with the above-mentioned factors. METHODS: NOD mice were fed a normal diet or an anti-diabetogenic casein hydrolysate (CH) diet. RONS activity was detected by chemiluminescence imaging of the whole gut. Proinflammatory and T cell cytokines were studied in the gut and islets, and dietary effects on gut microbiota and short-chain fatty acids were determined. RESULTS: Prediabetic NOD mice displayed high RONS activity in the epithelial cells of the distal small intestine, in conjunction with a proinflammatory cytokine profile. RONS production was effectively reduced by the CH diet, which also controlled (1) the expression of proinflammatory cytokines and colonisation-dependent RegIIIγ (also known as Reg3g) in ileum; (2) intestinal T cell activation; and (3) islet cytokines. The CH diet diminished microbial colonisation, increased the Bacteroidetes:Firmicutes ratio, and reduced lactic acid and butyric acid production in the gut. CONCLUSIONS/INTERPRETATION: Epithelial RONS production and proinflammatory T cell activation appears in the ileum of NOD mice after weaning to normal laboratory chow, but not after weaning to an anti-diabetogenic CH diet. Our data suggest a link between dietary factors, microbial colonisation and mucosal immune activation in NOD mice.
Subject(s)
Caseins/pharmacology , Intestines/immunology , Intestines/microbiology , Lymphocyte Activation/physiology , Animals , Diet , Female , Flow Cytometry , Free Radicals/metabolism , Intestines/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Reactive Nitrogen Species/metabolism , Reactive Oxygen Species/metabolismABSTRACT
BACKGROUND AND PURPOSE: Muscarinic toxins (MTs) are snake venom peptides named for their ability to interfere with ligand binding to muscarinic acetylcholine receptors (mAChRs). Recent data infer that these toxins may have other G-protein-coupled receptor targets than the mAChRs. The purpose of this study was to systematically investigate the interactions of MTs with the adrenoceptor family members. EXPERIMENTAL APPROACH: We studied the interaction of four common MTs, MT1, MT3, MT7 and MTα, with cloned receptors expressed in insect cells by radioligand binding. Toxins showing modest to high-affinity interactions with adrenoceptors were additionally tested for effects on functional receptor responses by way of inhibition of agonist-induced Ca²âº increases. KEY RESULTS: All MTs behaved non-competitively in radioligand displacement binding. MT1 displayed higher binding affinity for the human α(2B)-adrenoceptor (IC50 = 2.3 nM) as compared with muscarinic receptors (IC50 ≥ 100 nM). MT3 appeared to have a broad spectrum of targets showing high-affinity binding (IC50 = 1-10 nM) to M4 mAChR, α(1A)-, α(1D)- and α(2A)-adrenoceptors and lower affinity binding (IC50 ≥ 25 nM) to α(1B)- and α(2C)-adrenoceptors and M1 mAChR. MT7 did not detectably bind to other receptors than M1, and MTα was specific for the α(2B)-adrenoceptor. None of the toxins showed effects on ß1- or ß2-adrenoceptors. CONCLUSIONS AND IMPLICATIONS: Some of the MTs previously found to interact predominantly with mAChRs were shown to bind with high affinity to selected adrenoceptor subtypes. This renders these peptide toxins useful for engineering selective ligands to target various adrenoceptors.
Subject(s)
Receptors, Adrenergic/metabolism , Receptors, Muscarinic/metabolism , Snake Venoms/pharmacology , Animals , Calcium/metabolism , Cells, Cultured , Elapidae , Female , Humans , Inhibitory Concentration 50 , Muscarinic Agonists/metabolism , Muscarinic Agonists/pharmacology , Protein Binding , Radioligand Assay/methods , Rats , Rats, Sprague-Dawley , Receptors, Adrenergic/genetics , Receptors, Muscarinic/genetics , Snake Venoms/chemistry , SpodopteraABSTRACT
Intermediate filament (IF) proteins and heat shock proteins (HSPs) are large multimember families that share several features, including protein abundance, significant upregulation in response to a variety of stresses, cytoprotective functions, and the phenocopying of several human diseases after IF protein or HSP mutation. We are now coming to understand that these common elements point to IFs as important cellular stress proteins with some roles akin to those already well-characterized for HSPs. Unique functional roles for IFs include protection from mechanical stress, whereas HSPs are characteristically involved in protein folding and as chaperones. Shared IF and HSP cytoprotective roles include inhibition of apoptosis, organelle homeostasis, and scaffolding. In this report, we review data that corroborate the view that IFs function as highly specialized cytoskeletal stress proteins that promote cellular organization and homeostasis.
Subject(s)
Cytoskeleton/metabolism , Heat-Shock Proteins/metabolism , Intermediate Filament Proteins/metabolism , Stress, Physiological/physiology , Animals , Apoptosis/physiology , Cytoskeleton/genetics , Cytoskeleton/ultrastructure , Heat-Shock Proteins/genetics , Homeostasis/physiology , Humans , Intermediate Filament Proteins/genetics , Organelles/metabolism , Organelles/ultrastructure , Protein Folding , Stress, MechanicalABSTRACT
Keratins 18 and 19 (K18/K19) are epithelial-specific intermediate filament proteins. Apoptosis induces caspase cleavage at the highly conserved K18 or K19 Asp237, which in K18 is preceded by cleavage at Asp396. We characterized the keratin N-terminal fragments that are generated upon caspase digestion of K18/K19 at Asp237 in order to study keratin dynamics during apoptosis. This was carried out by generating and characterizing antibodies selective to K18/K19 Asp237. K18 or K19 peptides that expose Asp237 in 234VEVD were used for rabbit immunization. The generated antibodies recognized cleaved but not intact K18/K19, exclusively, as determined by blotting or immunofluorescence staining of apoptotic human HT29 cells or livers isolated from Fas-Ab-injected mice. Antibodies to K18/K19 Asp237 recognized the common VEVD-motif as determined by immunoblotting of cells transfected with K18, K19 or K20. The K18/K19 VEVD-directed antibodies demonstrated sequential Asp396 then Asp237 K18 cleavage during apoptosis. Specific-keratin selectivity of the anti-Asp237 antibodies was confirmed by their inability to recognize K14 after UV-induced apoptosis in transfected cells. The Asp237-containing apoptotic keratin fragments are secreted into the medium of cultured HT29 cells and are stable up to 96 h after inducing apoptosis. Furthermore, the generated antibodies recognize keratin apoptotic fragments in sera of mice undergoing hepatocyte apoptosis and sera of patients with cirrhosis, and also recognize apoptotic cells in various epithelial human tumours. Therefore, the N-terminal caspase-generated K18 fragment is stable in tissues and biological fluids. The Asp237-directed antibodies provide a powerful tool to study apoptosis in human and mouse tissues, cells and serum, using a broad range of detection modalities.
Subject(s)
Caspases/metabolism , Epithelial Cells/metabolism , Keratins/metabolism , Animals , Apoptosis , Biomarkers/analysis , Enzyme Activation , Epithelial Cells/pathology , Fluorescent Antibody Technique , HT29 Cells , Humans , Keratin-18/metabolism , Mice , Peptide Fragments/metabolism , Transfection/methodsABSTRACT
The major pathways for protein degradation are the proteasomal and lysosomal systems. Derangement of protein degradation causes the formation of intracellular inclusions, and apoptosis and is associated with several diseases. We utilized hepatocyte-derived cell lines to examine the consequences of the cytoplasmic hepatocyte Mallory-Denk body-like inclusions on organelle organization, autophagy and apoptosis, and tested the hypothesis that autophagy affects inclusion turnover. Proteasome inhibitors (PIs) generate keratin-containing Mallory-Denk body-like inclusions in cultured cells and cause reorganization of mitochondria and other organelles, autophagy and apoptosis. In cultured hepatoma cells, caspase inhibition blocks PI-induced apoptosis but not inclusion formation or autophagy activation. Autophagy induction by rapamycin decreases the extent of PI-induced inclusions and apoptosis in Huh7 and OUMS29 cells. Surprisingly, blocking of autophagy sequestration by 3 methyl adenine or beclin 1 siRNA, but not bafilomycin A1 inhibition of autophagic degradation, also inhibits inclusion formation in the tested cells. Therefore, autophagy can be upstream of apoptosis and may promote or alleviate inclusion formation in cell culture in a context-dependent manner via putative autophagy-associated molecular triggers. Manipulation of autophagy may offer a strategy to address the importance of inclusion formation and its significance in inclusion-associated diseases.
Subject(s)
Apoptosis , Autophagy , Inclusion Bodies/ultrastructure , Keratins/analysis , Cell Line , Cycloheximide/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Humans , Inclusion Bodies/chemistry , Leupeptins/pharmacology , Organelles/ultrastructure , Proteasome Inhibitors , Protein Synthesis Inhibitors/pharmacologyABSTRACT
Simple epithelial tissues such as liver and pancreas express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins. K8 and K18 null mice and transgenic mice that express mutant K18 (K18C) manifest several hepatocyte abnormalities and demonstrate that K8/18 are important in maintaining liver tissue and cell integrity, although other potential functions remain uncharacterized. Here, we report an additional abnormal liver phenotype, which is similar in K8 null, K18 null, and K18C mouse models. Liver histologic examination showed large polynuclear areas that lacked cell membranes, desmosomal structures, and filamentous actin. Similar, but less prominent, areas were observed in the pancreas. The parenchyma outside the polynuclear areas displayed irregular sinusoidal structures and markedly enlarged nuclei. Most K8 null hepatocytes were positive for the proliferating cell nuclear antigen (PCNA) with a doubled DNA content in comparison with the predominantly PCNA-negative wild-type hepatocytes. The distribution of the 14-3-3zeta protein was also altered in K8 null mice. Taken together, our results indicate that absence of keratin filaments causes disturbances in cell-cycle regulation, driving cells into the S-G2 phase and causing aberrant cytokinesis. These effects could stem from disturbed functions of K8/18-dependent cell-cycle regulators, such as the signaling integrator, 14-3-3.
Subject(s)
Keratins/physiology , Liver/pathology , Actins/deficiency , Animals , Cell Cycle/physiology , Cell Cycle Proteins/metabolism , Desmosomes/pathology , Keratins/deficiency , Keratins/genetics , Mice , Mice, Knockout/genetics , Mice, Transgenic/genetics , Mutation/physiology , Pancreas/pathologyABSTRACT
Pancreatic acinar cells express keratins 8 and 18 (K8/18), which form cytoplasmic filament (CF) and apicolateral filament (ALF) pools. Hepatocyte K8/18 CF provide important protection from environmental stresses, but disruption of acinar cell CF has no significant impact. We asked whether acinar cell ALF are important in providing cytoprotective roles by studying keratin filaments in pancreata of K8- and K18-null mice. K8-null pancreas lacks both keratin pools, but K18-null pancreas lacks only CF. Mouse but not human acinar cells also express apicolateral keratin 19 (K19), which explains the presence of apicolateral keratins in K18-null pancreas. K8- and K18-null pancreata are histologically normal, and their acini respond similarly to stimulated secretion, although K8-null acini viability is reduced. Absence of total filaments (K8-null) or CF (K18-null) does not increase susceptibility to pancreatitis induced by caerulein or a choline-deficient diet. In normal and K18-null acini, K19 is upregulated after caerulein injury and, unexpectedly, forms CF. As in hepatocytes, acinar injury is also associated with keratin hyperphosphorylation. Hence, K19 forms ALF in mouse acinar cells and helps define two distinct ALF and CF pools. On injury, K19 forms CF that revert to ALF after healing. Acinar keratins appear to be dispensable for cytoprotection, in contrast to hepatocyte keratins, despite similar hyperphosphorylation patterns after injury.
Subject(s)
Keratins/physiology , Pancreatitis/pathology , Actins/metabolism , Animals , Cell Survival , Electrophoresis, Polyacrylamide Gel , Epithelial Cells/metabolism , Female , Humans , Male , Mice , Pancreatitis/metabolism , PhosphorylationABSTRACT
Disruption or absence of hepatocyte keratins 8 and 18 is associated with chronic hepatitis, marked hepatocyte fragility, and a significant predisposition to stress-induced liver injury. In contrast, pancreatic keratin disruption in transgenic mice that express keratin 18 Arg89 --> Cys (K18C) is not associated with an obvious pancreatic pathology. We compared the effects of keratin filament disruption on pancreatic acini or acinar cell viability, and on cholecystokinin (CCK)-stimulated secretion, in transgenic mice that overexpress wild-type keratin 18 and harbor normal extended keratin filaments (TG2) and K18C mice. We also compared the response of these mice to pancreatitis induced by a choline-deficient ethionine-supplemented diet or by caerulein. Despite extensive cytoplasmic keratin filament disruption, the apicolateral keratin filament bundles appear intact in the acinar pancreas of K18C mice, as determined ultrastructurally and by light microscopy. No significant pancreatitis-associated histologic, serologic, or F-actin/keratin apicolateral redistribution differences were noted between TG2 and K18C mice. Acinar cell viability and yield after collagenase digestion were lower in K18C than in TG2 mice, but the yields of intact acini and their (125)I-CCK uptake and responses to CCK-stimulated secretion were similar. Our results indicate that keratin filament reorganization is a normal physiologic response to pancreatic cell injury, but an intact keratin cytoplasmic filament network is not as essential in protection from cell injury as in the liver. These findings raise the possibility that the abundant apicolateral acinar keratin filaments, which are not as evident in hepatocytes, may play the cytoprotective role that is seen in liver and other tissues. Alternatively, identical keratins may function differently in different tissues.
Subject(s)
Keratins/physiology , Pancreas/physiology , Actin Cytoskeleton , Amino Acid Substitution , Amylases/metabolism , Animals , Cholecystokinin/pharmacology , Humans , Lipase/metabolism , Mice , Mice, Inbred Strains , Mice, Transgenic , Pancreas/pathology , Pancreas/ultrastructure , Point MutationABSTRACT
The mammalian cell cytoskeleton consists of a diverse group of fibrillar elements that play a pivotal role in mediating a number of digestive and nondigestive cell functions, including secretion, absorption, motility, mechanical integrity, and mitosis. The cytoskeleton of higher-eukaryotic cells consists of three highly abundant major protein families: microfilaments (MF), microtubules (MT), and intermediate filaments (IF), as well as a growing number of associated proteins. Within digestive epithelia, the prototype members of these three protein families are actins, tubulins, and keratins, respectively. This review highlights the important structural, regulatory, functional, and unique features of the three major cytoskeletal protein groups in digestive epithelia. The emerging exciting biological aspects of these protein groups are their involvement in cell signaling via direct or indirect interaction with a growing list of associated proteins (MF, MT, IF), the identification of several disease-causing mutations (IF, MF), the functional role that they play in protection from environmental stresses (IF), and their functional integration via several linker proteins that bridge two or potentially all three of these groups together. The use of agents that target specific cytoskeletal elements as therapeutic modalities for digestive diseases offers potential unique areas of intervention that remain to be fully explored.
Subject(s)
Cytoskeleton/physiology , Digestive System Physiological Phenomena , Digestive System/cytology , Epithelial Cells/physiology , Gastrointestinal Diseases/physiopathology , Animals , Epithelial Cells/ultrastructure , HumansABSTRACT
Many natural toxins act by modifying key functions of the phosphorylation-based signalling machinery. Microcystins comprise a good example of highly specific, signalling-targeted toxicants. These liver-specific cyanobacterial peptide toxins act as potent inhibitors of serine/threonine (ser/thr) protein phosphatases, in particular type-1 (PP1) and type-2A (PP2A). PP1 and PP2A regulate the phosphorylation of a large number of key elements in various signalling processes. Furthermore, they are crucial in maintaining cytoskeletal integrity. Consequently, microcystins disrupt the liver structure by abrogating cytoskeletal regulation. Microcystin-induced protein phosphatase inhibition in liver cells leads to rapid reorganization of all three major cytoskeletal components, microfilaments, microtubules and intermediate filaments (IFs). The inhibited dephosphorylation induces an especially marked phosphorylation of the liver IF proteins, keratins 8 and 18. The elevated phosphorylation of these proteins causes disassembly and reorganization of keratin filaments, indicating that their assembly state in vivo is regulated by a continuous phosphate turnover. In this review on microcystin-induced cellular effects, we attempt to illustrate the potentially grave consequences when phosphorylation processes are disturbed by toxicants. The aim is also to show how such signalling-targeted toxicants can be used as biochemical tools to establish the biological roles of specific signalling or regulatory processes.
Subject(s)
Cytoskeletal Proteins/metabolism , Intracellular Signaling Peptides and Proteins , Phosphoprotein Phosphatases/metabolism , Protein Kinases/metabolism , Animals , Antibodies , Carrier Proteins/pharmacology , Cell Line , Chromatography, High Pressure Liquid , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique , Keratins/metabolism , Mutagenesis, Site-Directed , Organophosphorus Compounds/pharmacology , Peptide Mapping , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation , Precipitin Tests , Protein Kinase Inhibitors , Sequence Analysis , Solubility , Transfection , Vimentin/metabolismABSTRACT
The function and regulation of keratin 8 (K8) and 18 (K18), intermediate filament (IF) proteins of the liver, are not fully understood. We employed the liver damage induced by microcystin-LR (MC-LR), a liver-specific inhibitor of type-1 and type-2A protein phosphatases, in normal and in keratin assembly-incompetent mouse strains as a model to elucidate the roles of IF phosphorylation in situ. The mouse strains used were wild-type (wt) mice and mice with abnormal filament assembly, caused by a targeted null mutation of the K8 gene or caused by expression of a point-mutated dominant negative human K18. In vivo 32P-labeled wt mice, subsequently injected with a lethal dose of MC-LR, showed hyperphosphorylation, disassembly, and reorganization of K8/K18, in particular K18, indicating high phosphate turnover on liver keratins in situ. At lethal doses, the keratin assembly-incompetent mice displayed liver lesions faster than wt mice, as indicated histopathologically and by liver-specific plasma enzyme elevations. The histological changes included centrilobular hemorrhage in all mouse strains. The assembly-incompetent mice showed a marked vacuolization of periportal hepatocytes. Indistinguishable MC-LR-induced reorganization of microfilaments was observed in all mice, indicating that this effect on microfilaments is not dependent on the presence of functional K8/K18 networks. At sublethal doses of MC-LR, all animals had the same potential to recover from the liver damage. Our study shows that K8/K18 filament assembly is regulated in vivo by serine phosphorylation. The absence or occurrence of defective K8/K18 filaments render animals more prone to liver damage, which supports the previously suggested roles of keratin IFs in maintenance of structural integrity.
Subject(s)
Intermediate Filament Proteins/physiology , Keratins/genetics , Keratins/physiology , Liver/physiology , Mutation , Phosphoprotein Phosphatases/antagonists & inhibitors , Animals , Enzyme Inhibitors/pharmacology , Female , Humans , Liver/cytology , Liver/drug effects , Male , Marine Toxins , Mice , Mice, Inbred BALB C , Mice, Transgenic/genetics , Microcystins , Peptides, Cyclic/pharmacology , Phosphorylation/drug effects , Reference ValuesABSTRACT
The importance of protein phosphatases in the maintenance of cytoskeletal structure is supported by the serious liver injury caused by microcystin-LR, a hepatotoxic inhibitor of type-1 and type-2A serine/threonine protein phosphatases. We used the microcystin-LR-induced cell injury as a model to study the roles of protein dephosphorylation in maintaining cytoskeletal structure and cellular interactions in primary rat hepatocyte cultures. Confocal microscopy revealed that the first visible effect of microcystin-LR is disruption of desmoplakin organization at the cell surface, indicating dissociation of desmosomes. This effect is followed by a dramatic reorganization of both the intermediate filament (keratins 8 and 18) and microfilament networks, resulting in a merged structure in which the intermediate filaments are organized around a condensed actin core. Keratin 8, keratin 18 and desmoplakin I/II are the major cytoskeleton-associated targets for microcystin-LR-induced phosphorylation. Hyperphosphorylation of keratin 8 and 18 is accompanied by an increased keratin solubility, which correlates with the observed morphological effects. Phosphopeptide mapping shows that four specific tryptic phosphopeptides are highly phosphorylated predominantly in the soluble pool of keratin 18, whereas keratin 8 shows no indications of such assembly state-specific sites. Phosphopeptide maps of keratins phosphorylated in vivo and in vitro indicate that Ca2+/calmodulin-dependent kinase may be involved in regulating the serine-specific phosphorylation of both keratin 8 and keratin 18, while cAMP-dependent protein kinase does not seem to play a major role in this context. Taken together, our results show that the interactions between keratin intermediate filaments and desmosomes as well as the assembly states of their main constituent proteins, are directly regulated by serine/threonine kinase/phosphatase equilibria.
Subject(s)
Cytoskeleton/physiology , Desmosomes/physiology , Keratins/physiology , Liver/physiology , Phosphoprotein Phosphatases/metabolism , Actin Cytoskeleton/physiology , Actin Cytoskeleton/ultrastructure , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/ultrastructure , Desmoplakins , Desmosomes/ultrastructure , Enzyme Inhibitors/pharmacology , Intermediate Filaments/physiology , Intermediate Filaments/ultrastructure , Keratins/ultrastructure , Liver/cytology , Male , Marine Toxins , Microcystins , Peptide Mapping , Peptides, Cyclic/pharmacology , Phosphopeptides/analysis , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphorylation , Protein Binding , Protein Phosphatase 1 , Rats , Rats, WistarABSTRACT
The liver-specific toxin microcystin-LR (MC-LR) is a potent inhibitor of type 1 (PP1) and type 2A (PP2A) protein phosphatases. A tritiated form of the toxin, [3H]dihydromicrocystin-LR ([3H]DMC-LR), was used to identify target proteins in cellular fractions prepared from rat liver homogenates. About 80% of the [3H]DMC-LR bound to proteins was in the cytosolic fraction, which contained essentially all of the PP2A. In contrast, much of the PP1 was found in particulate fractions, each with only a few percent of the total protein-bound [3]HDMC-LR. Protein-bound [3H]DMC-LR in the cytosol co-eluted with PP2A, but not with PP-1 from a DEAE-Sepharose column. Native forms of liver cytoplasmic PP2A and PP1 separated by aminohexyl-Sepharose adsorption showed similar sensitivity to inhibition by MC-LR, and bound [3H]DMC-LR proportional to the amount of phosphatase activity. The results indicate that [3H]DMC-LR can bind both PP2A and PP1 in the liver which must be important for microcystin-induced toxicity, but is recovered mainly bound to PP2A in the cytosol.
Subject(s)
Liver/enzymology , Peptides, Cyclic/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Animals , Blotting, Western , Cell Fractionation , Chromatography, DEAE-Cellulose , Cytosol/enzymology , Electrophoresis, Polyacrylamide Gel , Liver/ultrastructure , Macromolecular Substances , Marine Toxins , Microcystins , Peptides, Cyclic/metabolism , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 2 , Rats , Rats, Sprague-Dawley , TritiumABSTRACT
In this study we have examined the effects of variations of the plasma membrane phospholipid and cholesterol content on the metabolic functions of the adenylyl cyclase complex in intact cells. Exposure of cells to 0.1 U/ml of sphingomyelinase led to the degradation of 75, 55 and 40% of the cellular total sphingomyelin mass in human skin fibroblasts (HSF), Chinese hamster lung fibroblasts (CHLF) and rat liver hepatocytes (RLH), respectively. Degradation of sphingomyelin in native cells led in turn to a reduction (within 60 min) of the plasma membrane cholesterol content (by 25, 15 and 10%, respectively). This manipulation of the plasma membrane lipid content did not affect the forskolin or prostaglandin E1-induced activation of adenylyl cyclase (as measured from the conversion of [3H]adenine via [3H]ATP to [3H]cAMP). These manipulations did, however, increase the basal rate of [3H]cAMP formation in rat liver hepatocytes (but not in the fibroblast cell types). With Chinese hamster lung fibroblasts, transfected to express an alpha 2-adrenergic receptor, it was observed that the alpha 2-adrenergic receptor-induced inhibition of adenylyl cyclase activity was slightly (but significantly) diminished in sphingomyelin and cholesterol-depleted cells. With isolated rat liver hepatocytes it was observed that the glucagon (receptor) mediated activation of adenylyl cyclase was also reduced in sphingomyelinase-treated cells. In another set of experiments, CHLF and RLH cells were exposed for 2 h to vesicles prepared from dilauroylphosphatidylcholine, to increase the lateral packing density in the outer leaflet of the plasma membrane. In such treated cells, the receptor-coupling to adenylyl cyclase was markedly reduced both in CHLF (the alpha 2-adrenergic receptor) and RLH (the glucagon-receptor) cells. We conclude that the direct activation of adenylyl cyclase (i.e., by forskolin) is not markedly affected by manipulations outer leaflet phospholipid composition (either reduction of sphingomyelin or increase of phosphatidylcholine), whereas receptor-coupled events clearly are.
Subject(s)
Adenylyl Cyclases/metabolism , Cell Membrane/metabolism , Phospholipids/metabolism , Adenylyl Cyclase Inhibitors , Alprostadil/pharmacology , Animals , Cell Line/metabolism , Cholesterol/metabolism , Colforsin/pharmacology , Cricetinae , Cricetulus , Enzyme Activation/drug effects , Humans , Phosphatidylcholines , Receptors, Adrenergic, alpha/metabolism , Receptors, Gastrointestinal Hormone/metabolism , Receptors, Glucagon , Sphingomyelins/metabolismABSTRACT
The effects of dehydroabietic acid (DHAA), a dominant resin acid in pulp and paper mill effluents, on membrane-connected events were studied in human erythrocytes. Fifty percent haemolysis was achieved by 252 microM DHAA after 1 h of incubation at +37 degrees C. At sublytic concentrations, DHAA protected erythrocytes against hypotonic haemolysis, with maximum protection occurring at 125 microM. In the lower range of sublytic concentrations, DHAA induced a slight echinocytosis; at higher sublytic concentrations erythrocytes were transformed to sphero-echinocytes and a release of acetylcholinesterase (exovesicles) occurred. Furthermore, at sublytic concentrations DHAA increased potassium efflux and passive potassium influx, while active potassium influx ((Na(+)-K+)-pump activity) and phosphate efflux were decreased. Our study indicates that DHAA acts on human erythrocytes in a way typical for amphiphilic compounds. It is proposed that DHAA by intercalating into the lipid bilayer of the membrane, affects the dynamics of the bilayer which in turn alters the permeability of the bilayer and the function of ion transporting membrane proteins.