BBA - Molecular Basis of Disease (v.1535, #1)
Microsomal fatty aldehyde dehydrogenase catalyzes the oxidation of aliphatic aldehyde derived from ether glycerolipid catabolism: implications for Sjögren–Larsson syndrome by William B. Rizzo; Eric Heinz; Marcia Simon; Debra A. Craft (1-9).
The enzyme that catalyzes the oxidation of fatty aldehyde derived from ether glycerolipid catabolism has not been identified. To determine whether microsomal fatty aldehyde dehydrogenase (FALDH) is responsible, we investigated the metabolism of 1-O-[9,10-3H-octadecyl]-glycerol ([3H]OG) in FALDH-deficient cultured cells from patients with Sjögren–Larsson syndrome (SLS) and in mutant Chinese hamster ovary (CHO) cells. Intact fibroblasts from SLS patients incubated with [3H]OG showed a selective deficiency (38±7% of normal) in the incorporation of radioactivity into fatty acid, but no decrease in incorporation of radioactivity into fatty alcohol, total lipids and phosphatidylethanolamine (PE). Consistent with fatty aldehyde accumulation, incorporation of radioactivity into N-alkyl-phosphatidylethanolamine, which is derived from Schiff base formation of free aldehyde with PE, was 4-fold higher in SLS fibroblasts compared to normal controls. Similar results were seen with SLS keratinocytes, whereas FALDH-deficient CHO cells showed a more profound reduction in radioactive fatty acid to 12±2% of normal. These results implicate FALDH in the oxidation of ether-derived fatty aldehyde in human and rodent cells. Metabolism of ether glycerolipids is a previously unrecognized source of fatty aldehyde that may contribute to the pathogenesis of SLS.
Keywords: Ether lipid; Fatty aldehyde; Aldehyde dehydrogenase; Metabolism; Plasmalogen;
Influence of diabetes on cardiac nitric oxide synthase expression and activity by K. Stockklauser-Färber; Th. Ballhausen; A. Laufer; P. Rösen (10-20).
There is some evidence that the endothelium dependent vasodilatation of coronary arteries is impaired in both types of diabetes. The underlying mechanisms are not yet clear, in particular whether this defect is caused by a direct effect of diabetes on the activity and the expression of nitric oxide synthases (NOS) or indirectly by an enhanced inactivation of nitric oxide. Methods: To study this question we determined the activity (conversion of L-arginine to citrulline) and the mRNAs encoding the isoforms of NOS (using polymerase chain reaction after reverse transcription of the mRNAs into cDNAs by reverse transcriptase) in hearts of streptozotocin diabetic rats and in rat heart endothelial cells (RHEC). The formation of reactive oxygen intermediates (ROI) was measured by the dichloro-dihydro-fluorescein method. Results: The activity of total NOS and the amounts of mRNAs encoding ecNOS and iNOS were dependent on the duration of diabetes. After a diabetes duration of 4 to 6 weeks both the total activity as well as the mRNAs encoding ecNOS and iNOS were elevated. A reduction of NOS activity and the amounts of mRNAs of ecNOS and iNOS was only seen after a diabetes duration longer than 20 weeks, a time at which a loss of endothelium has been described. In RHEC, high glucose (22 mM) and H2O2 (100 μM) were able to increase the mRNA encoding ecNOS, but not iNOS. This increase in ecNOS mRNA was inhibited by lipoic acid (1 μM). In addition, high glucose (22 and 30 mM) led to an enhanced formation of ROI and to activation of the transcription NFκB. Conclusion: These observations suggest that diabetes causes a temporary increase in NOS activity and ecNOS mRNA in the rat heart which is presumably the consequence of an enhanced oxidative stress exerted by hyperglycaemia. Together with previously published observations, our data suggest that the impairment of endothelium dependent vasodilatation in rat heart is not the consequence of a reduced activity and expression of NOS, but is caused by an enhanced inactivation of nitric oxide by ROI.
Keywords: Diabetes; Nitric oxide; Coronary artery;
Modification of the composition of polycystin-1 multiprotein complexes by calcium and tyrosine phosphorylation by Lin Geng; Christopher R. Burrow; Hsi-Ping Li; Patricia D. Wilson (21-35).
Mutations in the PKD1 gene are responsible for >85% of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1, polycystin-1, is a large, modular membrane protein, with putative ligand-binding motifs in the extracelluar N-terminal portion, 9–11 transmembrane domains and an intracellular C-terminal portion with phosphorylation sites. A role for polycystin-1 as a cell surface receptor involved in cell–matrix and cell–cell interactions has been proposed. In this study, we have analyzed polycystin-1 and associated protein distribution in normal human epithelial cells and examined the role of cell–matrix versus cell–cell interactions in regulation of the assembly of polycystin-1 multiprotein complexes. Immunocytochemistry, sucrose density gradient sedimentation, co-immunoprecipitation analyses and in vitro binding assays have shown that polycystin-1 associates with the focal adhesion proteins talin, vinculin, p130Cas, FAK, α-actinin, paxillin and pp60c-src in subconfluent normal human fetal collecting tubule (HFCT) epithelia when cell–matrix interactions predominate. Polycystin-1 also forms higher S value complexes with the cell–cell adherens junction proteins E-cadherin, β- and γ-catenins in confluent cultures when cell–cell interactions are predominant. Polycystin-1 multiprotein complexes can be disrupted by cytochalasin D but not by colchicine, suggesting involvement of the actin cytoskeleton. Although inhibition of tyrosine phosphorylation by tyrphostin inhibits polycystin-1–FAK interactions, E-cadherin interactions are enhanced. High calcium treatment also increases polycystin-1–E-cadherin interactions.
Keywords: Focal adhesion; Focal adhesion kinase; Actin binding protein; β-Catenin; Tyrosine phosphorylation; Calcium;
Sustained elevation of norepinephrine depresses hepatocellular function by Ping Wang; Stephen M Tait; Irshad H Chaudry (36-44).
The sympathetic-adrenal system is rapidly activated in response to sepsis or hemorrhagic shock, resulting in an increase in circulating levels of catecholamines. Although it has been shown that the occurrence of hepatocellular dysfunction under such conditions is associated with an early and sustained increase in plasma norepinephrine (NE), it remains unknown whether the increased NE per se plays any role in producing hepatocellular dysfunction. To study this, exogenous NE was administered, by implantation of a peritoneal mini-osmotic pump (consistently releasing NE), to produce a plasma level of NE similar to that observed after sepsis or hemorrhage. At 24 h after the pump implantation, cardiac output was determined by dye dilution technique and hepatocellular function [i.e., the maximal velocity (V max) and the efficiency of the transport (K m) of in vivo indocyanine green clearance) was assessed without blood sampling. In addition, tissue perfusion in various organs was determined using laser Doppler flowmetry. Plasma levels of interleukin-6 were measured by bioassay and liver enzymes were assayed enzymatically. The results indicate that sustained (24 h) elevation of plasma levels of NE caused a decrease in cardiac output and microvascular blood flow in the liver, spleen, and small intestine. In addition, the increase in plasma NE produced significant depression in hepatocellular function as evidenced by reduced V max and K m. This was associated with elevated plasma levels of liver enzymes, indicating hepatocyte injury. In addition, plasma levels of interleukin-6 increased significantly. These findings suggest that sustained elevated levels of NE, observed after sepsis or hemorrhagic shock, may play an important role in producing hepatocellular dysfunction and altered hepatocyte integrity.
Keywords: Polymicrobial sepsis; Hemorrhagic shock; Indocyanine green clearance; Hemodynamics; Interleukin-6;
A role for linoleic acid in erythrocytes infected with plasmodium berghei by Coy D Fitch; Guang-zuan Cai; James D Shoemaker (45-49).
Unesterified fatty acids were measured in mouse erythrocytes infected either with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) lines of Plasmodium berghei. This work was undertaken to identify candidates for the lipid involved in ferriprotoporphyrin IX (FP) polymerization. Linoleic, oleic, palmitic, and stearic acids were quantified by gas chromatography/mass spectrometry. In total, they increased 4-fold with CS infections and 6-fold with CR infections. Treating infected mice with chloroquine did not affect the amounts of unesterified fatty acids in erythrocytes. Of the four fatty acids, only linoleic acid increased disproportionately to the total. It increased 16-fold for the CS line and 35-fold for the CR line. The method could detect monoglycerides but they were below the limit of detection. It could not detect diglycerides, triglycerides or phospholipids. Triglycerides and phospholipids have been tested previously, however, and found to be ineffective at promoting FP polymerization. Therefore, other than linoleic acid, the lipids most likely to be involved in FP polymerization are diglycerides. We tested dilinoleolyglycerol in the present work and found it to be an effective promoter of FP polymerization. These results suggest that linoleic acid or a diglyceride containing it has the critical role of promoting FP polymerization in malaria parasites.
Keywords: Malaria; Unsaturated fatty acid; Diglyceride; Lipid; Ferriprotoporphyrin IX polymerization; (Plasmodium berghei);
CINC blockade prevents neutrophil Ca2+ signaling upregulation and gut bacterial translocation in thermal injury by Nadeem Fazal; Muhammad Shamim; John Zagorski; Mashkoor A. Choudhry; T. Ravindranath; Mohammed M. Sayeed (50-59).
In this study, we have evaluated the role of cytokine-induced neutrophil chemoattractant (CINC), in the upregulation of neutrophil Ca2+ signaling in neutrophils from thermally injured rats treated with anti-CINC antibody. Additionally, we have determined the effect of the treatment with CINC antibody on the accumulation of activated neutrophils in the intestinal wall, and the effect of such accumulation on gut bacterial translocation. Measurements of myeloperoxidase (MPO) activity and immunohistochemical localization of neutrophils determined neutrophil sequestration in the rat intestine. Agar culture analyses and a specific Escherichia coli β-galactosidase gene polymerase chain reaction was carried out to detect gut indigenous bacterial invasion into intestinal wall and extraintestinal mesenteric lymph nodes (MLN). The results showed that pretreatment of rats with anti-CINC antibody attenuated the thermal injury-induced enhancement in [Ca2+]i responses in neutrophils both in the basal and Formyl-Met-Leu-Phe stimulated conditions. Moreover, treatment with the CINC antibody decreased neutrophil infiltration into the gut and attenuated thermal injury-caused translocation of bacteria into the MLN.
Keywords: β-Galactosidase; Mucosal barrier; Tissue injury; Intestinal dysfunction; Mesenteric lymph node; Escherichia coli;
Hippocampal level of neural specific adenylyl cyclase type I is decreased in Alzheimer’s disease by Megumi Yamamoto; Mario E. Götz; Hiroki Ozawa; Christian Luckhaus; Toshikazu Saito; Michael Rösler; Peter Riederer (60-68).
Previous studies reported disruption of adenylyl cyclase (AC)–cyclic AMP (cAMP) signal transduction in brain of Alzheimer’s disease (AD). We also demonstrated that basal and stimulated AC activities in the presence of calcium and calmodulin (Ca2+/CaM) were significantly decreased in AD parietal cortex. In the present study, we examined the amounts of Ca2+/CaM-sensitive types I and VIII AC, and Ca2+/CaM-insensitive type VII AC in the postmortem hippocampi from AD patients and age-matched controls using immunoblotting. The specificities of the anti-type VII and VIII AC antibodies were confirmed by preabsorption with their specific blocking peptides. We observed a significant decrease in the level of type I AC and a tendency to decrease in the level of type VIII AC in AD hippocampus. On the other hand, the level of type VII AC showed no alteration between AD and controls. A body of evidence from the studies with invertebrates and vertebrates suggests that types I and VIII AC may play an essential role in learning and memory. Our finding thus firstly demonstrated that a specific disruption of the Ca2+/CaM-sensitive AC isoforms is likely involved in the pathophysiology in AD hippocampus.
Keywords: Alzheimer’s disease; Type I adenylyl cyclase; Type VIII adenylyl cyclase; Immunoblotting; Cyclic AMP signal transduction system; Postmortem human brain;
Does ethanol metabolism affect erythrocyte hemolysis? by Olga V. Tyulina; Matthew J. Huentelman; Valentina D. Prokopieva; Alexander A. Boldyrev; Peter Johnson (69-77).
The effects of ethanol and acetaldehyde on the hemolytic stability of rabbit erythrocytes have been compared. Incubation of normal erythrocytes with ethanol facilitated both acidic and oxidative hemolysis and increased the percentages of cells that were hemolysed at maximal rate. Acetaldehyde exerted a similar destabilizing effect on erythrocytes only in the case of oxidative hemolysis. The destabilizing effect of ethanol was observed in catalase-inactivated erythrocytes under acidic, but not oxidative, hemolysis conditions. It is concluded that the destabilizing effect of unmetabolized ethanol occurs under conditions of acidic hemolysis, whereas the destabilizing effect of the oxidation of ethanol to acetaldehyde takes place only under the conditions of oxidative hemolysis.
Keywords: Ethanol; Acetaldehyde; Erythrocyte; Catalase; Hemolysis;
Stimulation of cannabinoid receptor agonist 2-arachidonylglycerol by chronic ethanol and its modulation by specific neuromodulators in cerebellar granule neurons by Balapal S. Basavarajappa; Mariko Saito; Thomas B. Cooper; Basalingappa L. Hungund (78-86).
In an earlier study, we reported that chronic ethanol (EtOH) stimulates the formation of anandamide in human SK-N-SH cells. In the present study, we investigated the effect of chronic EtOH on the formation of yet another cannabinoid receptor (CB1) agonist, 2-arachidonylglycerol (2-AG), in cerebellar granule neurons (CGNs). The formation of 2-[3H]AG without any stimulation was more pronounced in the older cultures than in younger cultures. Exposure of CGNs to EtOH led to a significant increase in the level of 2-[3H]AG (P<0.05). Incubation with the anandamidehydrolase inhibitor phenylmethylsulfonyl fluoride and EtOH did result in an additive increase in 2-[3H]AG, but did not with E-6-(bromomethylene)tetrahydro-3-(1-naphthelenyl)-2H-pyran-2-one. The formation of 2-[3H]AG was enhanced by ionomycin in both the control and EtOH-exposed CGNs, and the ionomycin-stimulated 2-[3H]AG synthesis was inhibited by the intracellular chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. Further, glutamate increased the formation of 2-[3H]AG only in control CGNs. MK-801 inhibited the EtOH-induced 2-[3H]AG synthesis, suggesting the participation of intracellular Ca2+ in EtOH-induced 2-[3H]AG synthesis. The dopamine receptor (D2) agonist did not modify the 2-AG synthesis in either the control or EtOH-exposed CGNs. However, the D2 receptor antagonist inhibited the EtOH-induced formation of 2-[3H]AG. The EtOH-induced 2-[3H]AG formation was inhibited by SR141716A and pertussis toxin, suggesting the CB1 receptor- and Gi/o-protein-mediated regulation of 2-AG. The observed increase in 2-AG level in CGNs is possibly a mechanism for neuronal adaptation to the continuous presence of EtOH. These findings indicate that some of the pharmacological actions of EtOH may involve alterations in the endocannabinoid signaling system.
Keywords: Cerebellar granule neuron; Ethanol; 2-Arachidonylglycerol; Endocannabinoid;