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PubMed1979

Disorders of chloriuretic hormone secretion.

Experimental evidence supports the existence of a circulating substance, natriuretic hormone, which augments electrolyte excretion. Because such a hormone probably acts by inhibiting chloride reabsorption in the thick, ascending limb of the loop of Henle it would more accurately be called chloriuretic hormone. Chloriuretic hormone must have an action which resembles that of loop diuretics such as frusemide and ethacrynic acid. An excess of chloriuretic hormone could explain all the manifestations of Bartter's syndrome, whereas a deficiency could account for Gordon's syndrome. Hyporeninaemic hypoaldosteronism may develop in subjects who are unable to increase chloriuretic hormone concentrations appropriately in response to progressive impairment of renal function.

Aldosterone↗
PubMed2001

Brain energy metabolism in Alzheimer's disease: 99mTc-HMPAO SPECT imaging during verbal fluency and role of astrocytes in the cellular mechanism of 99mTc-HMPAO retention.

The central hypothesis of the study which has been carried out as part of the NRP38 program, is that perturbations of brain energy metabolism are critically involved in the neurodegeneration occurring in Alzheimer's disease (AD) and that they may correlate with early cognitive dysfunctioning. In the present multidisciplinary study we set out to monitor brain energy metabolism using FDG-PET and HMPAO-SPECT imaging in a cohort of individuals over 65 years of age, drawn from the general population. HMPAO-SPECT imaging, which is a simpler and more widely accessible imaging procedure than FDG-PET, was performed under basal conditions and during the performance of a cognitive task (verbal fluency test). Three groups were studied. Two groups (groups I and II) included individuals age 65 or more, with no cognitive impairment and carrying an APOE4 positive or APOE4 negative phenotype, respectively; a third group (group III) included patients with clinical signs of AD. Each subject entering the study underwent an FDG-PET, an HMPAO-SPECT and an extensive battery of neuropsychological tests which assess various aspects of cognitive functioning, with a strong emphasis on working memory, divided attention and executive functions. A total of 101 participants were submitted to brain imaging and neuropsychological testing. Among these, 60 participants received the same set of imaging and neuropsychological tasks 24-36 months after the first set (phase II). In this article, we present a preliminary analysis performed on ten subjects from groups I and II and nine subjects from group III: activation (verbal fluency task) induced a specific pattern of increase in HMPAO retention (including BA 9/10, BA 18 bilaterally and right BA 17). In contrast to controls, in nine AD subjects no significant differences in HMPAO retention were observed when comparing activation and basal conditions. The cellular and molecular mechanisms that underlie the retention of HMPAO, the tracer used for single photon emission computed tomography (SPECT) imaging, has been studied in vitro in purified preparations of neurons and astrocytes with the aim of investigating the contribution of different cell types to hexamethyl-propyleneamineoxime labeled with technetium-99m (99mTc-HMPAO) retention in vitro. Results show that 99mTc-HMPAO retention predominates in astrocytes over neurons by a factor of approximately 2.5. Diethyl maleate, ethacrynic acid and buthionine sulfoximine, three agents which significantly reduce glutathione levels, also decreased 99mTc-HMPAO retention in both astrocytes and in neurons. Decrease did not always correlate with glutathione levels however, thus suggesting that other factors could be involved. The data presented indicate that astrocytes might constitute a prominent site of 99mTc-HMPAO retention and most likely contribute significantly to the SPECT signal. In addition, they also suggest that specific alterations in glial cell metabolism could explain flow-independent changes in 99mTc-HMPAO retention in the brain as observed by SPECT in certain pathologies (including Alzheimer's disease). In particular, these observations suggest a key role of astrocytes in the signal detected with the imaging procedure, which is altered in the Alzheimer's cohort subjected to the verbal fluency activation task.

Aged↗
PubMed2001

Decreased glutathione S-transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads (Ameiurus nebulosus).

A number of freshwater lakes and reclaimed agricultural sites in Central Florida have been the receiving waters for agrochemical and municipal runoff. One of these sites, Lake Apopka, is also a eutrophic system that has been the focus of several case studies reporting altered reproductive activity linked to bioaccumulation of persistent organochlorine chemicals in aquatic species. The present study was initiated to determine if brown bullheads (Ameiurus nebulosus) from the north marsh of Lake Apopka (Lake Apopka Marsh) exhibit an altered capacity to detoxify environmental chemicals through hepatic glutathione S-transferase (GST)-mediated conjugation as compared with bullheads from a nearby reference site (Lake Woodruff). We also compared plasma sex hormone concentrations (testosterone, 17-beta estradiol, and 11 keto-testosterone) in bullheads from the two sites. Female bullheads from Lake Apopka had 40% lower initial rate GST conjugative activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 50% lower activity towards p-nitrobutyl chloride (NBC), 33% lower activity toward ethacrynic acid (ECA), and 43% lower activity toward Delta5-androstene-3,17-dione (Delta(5)-ADI), as compared with female bullheads from Lake Woodruff. Enzyme kinetic analyses demonstrated that female bullheads from Lake Apopka had lower GST-catalyzed CDNB clearance than did female Lake Woodruff bullheads. Western blotting studies of bullhead liver cytosolic proteins demonstrated that the reduced GST catalytic activities in female Lake Apopka bullheads were accompanied by lower expression of hepatic GST protein. No site differences were observed with respect to GST activities or GST protein expression in male bullheads. Female Lake Apopka bullheads also had elevated concentrations of plasma androgens (testosterone and 11-ketotestosterone) as compared with females from Lake Woodruff. In contrast, male Lake Apopka bullheads had elevated levels of plasma estrogen but similar levels of androgens as compared with male bullheads from Lake Woodruff. Collectively, our studies indicate the presence of reduced GST protein expression, reduced GST conjugative capacity and altered sex steroid homeostasis in female bullheads from a contaminated field site in Central Florida. The implications of these physiological alterations in terms of pollutant biotransformation and reproduction are discussed.

Animals↗
PubMed2000

Activities of affinity-isolated glutathione S-transferase (GST) from channel catfish whole intestine.

A glutathione S-transferase (GST) fraction was isolated from cytosol prepared from catfish intestinal mucosa by GSH-agarose affinity chromatography and its molecular weight, isoelectric points, substrate specificities and immunochemical cross-reactivity were examined. Intestinal GSTs were purified 100-fold with respect to cytosolic activity with 1-chloro-2, 4-dinitrobenzene and had high activity with ethacrynic acid, (+/-)benzo(a)pyrene-4,5-oxide, and (+/-)anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, but a low activity with 1,2-dichloro-4-nitrobenzene. SDS-polyacrylamide gel electrophoresis revealed the presence of a single band with relative molecular mass of 26700. Gel isoelectric focusing showed a major band with a pI of 8.2. A polyclonal antibody prepared against a GST pi-protein isolated from catfish proximal intestine cross-reacted well with the affinity isolated GST fraction. The catfish antibody also cross-reacted with GST from human placenta which contains predominantly pi-class GST (Mannervik, B., Guthenberg, C., 1981. Glutathione transferase (human placenta). In: Jakoby, W.B. (Ed.), Methods in Enzymology, 77. Academic Press, New York, pp. 231-235; Polidoro, G., Dillio, C., Arduini, A., Frederici, G., 1981. Molecular and catalytic properties of purified glutathione transferase from human placenta. Biochem. Med. 22, 247-259; Dao, D.D., Partridge, C.A., Kurosky, A., Awasthi, Y.C., 1982. Subunit structure of glutathione-S-transferase of human liver and placenta. IRSC Med. Sci, Lib. Compend. 10, 175; Dao, D.D., Partridge, C.A., Kurosky, A., Awasthi, Y.C., 1984. Human glutathione transferase. Characterization of the anionic forms from lung and placenta. Biochem. J. 221, 33-41), but poorly with human liver cytosol. The affinity-isolated protein fraction from whole intestine contained proteins that were immunologically related to all four major classes of human GSTs tested. N-terminal sequence analysis of the predominant band obtained by 2D electrophoresis indicated a marked homology (63-70% identical) to mammalian pi form GST isozymes and very strong similarity (80%) to a salmon hepatic GST that was designated a pi form (Dominey, R.J., Nimmo, I.A., Cronshaw, A.D., Hayes, J.D., 1991. The major glutathione S-transferase in salmonid fish livers is homologous to the mammalian pi class GST. Comp. Biochem. Physiol. (B) 100 (1), 93-98). Other bands contained insufficient protein for N-terminal analysis. Taken together, these results indicate that the predominant intestinal GST isoform is related to the pi-class enzymes, but minor GSTs related to other families are also present.

Journal Article↗
PubMed1997

Inhibitors of glutathione S-transferases as therapeutic agents.

Glutathione S-transferases (GST) are a family of phase II detoxification enzymes with broad substrate specificities. They catalyze the conjugation of glutathione with many different types of xenobiotics, rendering the compound more water soluble and thus more easily eliminated. Resistance to cancer chemotherapeutic drugs, such as the alkylating agents, has been directly correlated with the overexpression of GSTs. Subsequently, a rationale has been established to utilize agents that inhibit GST in combination with alkylating agents to circumvent this resistance. Two such agents, ethacrynic acid (EA) and Terrapin 199 (TER 199), have been examined for this purpose. EA, an inhibitor of all classes of GST isozymes, has been used clinically in combination with thiotepa. More recently, TER 199, a glutathione analog-based GST inhibitor, has been modeled specifically to inhibit GST pi, an enzyme which is commonly found at high levels in human tumor biopsies. Furthermore, a therapeutic strategy has been designed to take advantage of GST pi activation of a prodrug, TER 286. Recent studies have investigated the molecular mechanisms involved in the cellular response to GST inhibitors and have employed techniques such as differential display to examine altered gene expression as well as to identify novel genes induced by these agents. Overall, this strategy may provide further insight into the action of these agents in the cell as well as prove useful in endeavors to modulate anticancer drug resistance.

Journal Article↗
PubMedDate not supplied

Ion transport in the endolymphatic space.

The endolymph differs from all other extracellular fluids in its positive potential and in the ionic compositions in the various parts of the endolymphatic space. The endolymphatic space is surrounded by a tight neuroepithelium. The strial cells, which have a negative intracellular potential of -67 mV, generate a positive endolymphatic potential of +80 mV. The potassium ions are pumped into the endolymph by an active energy-consuming process. Intracellular ion concentrations of the marginal strial cells are 135 mM for potassium and 6 mM for sodium, which are comparable to concentrations in other epithelial cells. During anoxia or after ethacrynic acid injection potassium concentrations in the cochlear endolymph and in the marginal strial cells decrease, while sodium concentrations increase.

Animals↗
PubMed1999

Carbonyl reduction of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in cytosol of mouse liver and lung.

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a strong lung carcinogen in all species tested. To elicit its tumorigenic effects, NNK requires metabolic activation which is supposed to occur via alpha-hydroxylation by cytochrome P450 enzymes. Carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronosylation is considered to be the main detoxification pathway in humans. Therefore, NNK carbonyl reducing activity is crucial for NNK inactivation since it initiates the final excretion of this lung carcinogen. Until the present work, studies on NNK metabolism have focused exclusively on microsomal fractions, and several cytochrome P450 enzymes have been shown to be involved in alpha-hydroxylation of NNK. In addition, 11beta-hydroxysteroid dehydrogenase type 1(11beta-HSD1) which is located in the endoplasmic reticulum of the cell has been identified to catalyze the carbonyl reduction of NNK in microsomes. In this study, we provide evidence that carbonyl reduction of NNK does also take place in the cytosolic fraction of mouse liver and lung, and that cytosolic carbonyl reductase contributes to the detoxification of NNK. At a fixed substrate concentration of 1 mM NNK, the specific activity of cytosolic NNAL formation amounts to 72% (liver) and 28% (lung) compared with that in the respective microsomal fractions. Although considerable NNK carbonyl reduction occurred with NADH, the preferred cosubstrate in cytosol is either NADPH or an NADPH-regenerating system. Due to the inhibitor sensitivity to menadione, ethacrynic acid, dicoumarol and quercitrin, it is concluded that carbonyl reductase (EC 1.1.1.184) is mainly responsible for NNAL formation in liver and lung cytosol. The expression of cytosolic carbonyl reductase and microsomal 11beta-HSD1 was established on the mRNA level by reverse transcription-PCR in both liver and lung. Enzyme kinetic studies revealed a nonsaturable Michaelis-Menten kinetic of NNK carbonyl reduction in cytosol. Possibly some other cytosolic NNK carbonyl reducing enzymes are also involved in NNAL formation. In conclusion, this is the first report to show that carbonyl reduction of NNK does occur in cytosol. Further studies with purified enzyme preparations are needed to explore the detailed contribution of the cytosolic enzymes participating in the final elimination of this lung carcinogen.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed1999

Effects of some probable antioxidants on selenite-induced cataract formation and oxidative stress-related parameters in rats.

The effect of several natural and synthetic compounds on selenite-induced cataract was investigated in rat pups. Simultaneous determination of glutathione S-transferase (GST), selenium dependent glutathione peroxidase (Se-GPx), catalase (CAT), superoxide dismutase (SOD) activities and malondialdehyde (MDA) levels were carried out in the lens, erythrocyte and plasma. The results showed that propolis, diclofenac, vitamin C (Vit-C) and quercetin prevented cataract formation to the extent of 70, 60, 58.4, and 40%, respectively. Standardized extract of Ginkgo biloba (Egb 761) did not affect the cataract formation. Selenite treatment caused a significant decrease in the activity of erythrocyte SOD. This was accompanied by a simultaneous increase in the levels of MDA either in lens and in plasma. A significant increase was shown in erythrocyte GST (substrate ethacrynic acid; eaa), and GPx activities and lens GST (substrate chlorodinitro benzene; cdnb) activity. Antioxidant treatment caused significant changes in enzyme activities and MDA levels. There was no effect of selenite and antioxidants on total body weight increase during the course of the study. Blood parameters did not correlate to lens parameters following selenite treatment. Our results suggest that antioxidant supplementation following selenite exposure may prevent the cataract formation and may enhance antioxidant defence of blood and lens.

Animals↗
PubMed1998

Antiserum against Cl- pump complex recognizes 51 kDa protein, a possible catalytic unit in the rat brain.

We purified Cl- pump in the rat brain and obtained 520 or 580 kDa protein complexes which consisted of 62, 60, 55 and 51 kDa proteins. An antiserum against 520 kDa protein complex recognized 51 kDa protein in both 520 and 580 kDa complexes, and reduced both Cl(-)-ATPase and Cl(-) pump activities. Such an immunoreactive 51 kDa protein was found in the brain, spinal cord and kidney. When incubated with [gamma-(32)P]ATP, the protein complex yielded phosphorylated 51 kDa protein, the label being hydroxylamine-sensitive and increased in the presence of Cl- and/or an inhibitor of Cl- pump, ethacrynic acid. Thus, the antibody appears to recognize a possible catalytic subunit of Cl- pump, 51 kDa protein, in the rat.

Adenosine Triphosphatases↗
PubMed2000

Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii.

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis Menten kinetics with K5, = 165+/-5 microM and Vmax = 686.1+/-24.7 U mg(-1). Stimulation by potassium (K0.5 = 2.4+/-0.1 mM) and magnesium ions (K0.5 = 0.76+/-0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K0.5 = 6.0+/-0.2 mM) exhibited site site interactions (n = 1.6). Ouabain (K0.5 = 61.6+/-2.8 microM) and vanadate (K0.5 = 3.2+/-0.1 microM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium.

Animals↗
PubMed1997

Influence of non-nutrient plant components on biotransformation enzymes.

Plant foods contain numerous non-nutritive substances which exert biological activity. Most attention has been focused on the anticarcinogenic effects of these compounds. Many of the mechanisms involved include induction or inhibition of biotransformation enzymes. Each individual has its own isoenzyme pattern for the various drug-metabolizing enzymes. The multiplicity of these enzymes results in differential responses to dietary constituents. A substance may increase the level of a certain P450, and decrease the level of another. Although this complicates matters considerably, it also offers the possibility of specifically influencing biotransformation directed at a particular compound, e.g., a cytostatic agent. Using the important class of the glutathione S-transferase (GST) as an example, the various phenotypic and genetic origins of interindividual variation are described. Genetic variation is especially important for the mu and thetra class enzymes. The induction of individual isoenzymes of the GST has been studied in man rat. It was shown that the changes in the GST isoenzyme pattern induced by Brussels sprouts in rat liver and intestine were very similar to that caused by administration of ally isothiocyanate, and not to that resulting from goitrin. In man Brussels sprouts led to induction of GST alpha only. A number of naturally occurring catechols, or more likely the quinones derived from them, are effective irreversible inhibitors of GST. Eugenol, for instance, lowers GST activity in man. A second class of compounds which shows promise are alpha, beta-unsaturated aldehydes and ketones. A number of naturally occurring representatives of this class inhibit GST pi irreversibly, and ethacrynic acid, a drug with a similar reactive moiety in its structure, has already been shown to be quite useful to inhibit GST activity in cellular systems. Several approaches for future studies on the effects of dietary constituents are indicated: 1) further studies on the mechanisms of induction and inhibition of biotransformation enzymes: 2) careful studies using human volunteers, where the effects can be studied in isolation as much as possible; 3) studies of the disposition and kinetics of the dietary constituents themselves, to assess the relevance of inducing agents in food for the day-to-day human situation.

Animals↗
PubMed2004

Risk of hospitalization for upper gastrointestinal tract bleeding.

OBJECTIVE: This study evaluates the hospitalization risk for upper gastrointestinal bleeding (UGIB) with reference to the clinical characteristics of patients and drugs taken before admission. METHODS: This study is based on the GIFA (Italian Group for the Pharmacosurveillance in the Elderly) database. Cases with an ICD-9 code of esophagus, stomach or duodenum bleeding, or acute esophago-gastroduodenal disease associated with anemia have been classified as UGIB. Sex, age, year of observation, drugs taken at home, comorbidity, smoking, alcohol, and use of gastroprotectants have been also taken into account. Statistical analysis has been conducted using multivariate logistic regression models. RESULTS: 32,388 patients have been enrolled, 940 of which presented UGIB. Age, comorbidity, use of smoke and alcohol, hospitalization duration, and mortality during hospitalization were significantly higher in UGIB than nonUGIB patients. Increased UGIB risk has been found in patients taking NSAIDs (both when aspirin was included or excluded), acetaminophen, constipating agents, iron, ethacrynic acid, propranolol. Reduced UGIB risk has been found in patients taking nitrates. CONCLUSIONS: UGIB risk appears to correlate with clinical characteristics of the patient: it increases with age, comorbidity, and smoke and alcohol consumption. Among drugs, NSAIDs are associated with the highest UGIB risk, while nitrates with a reduction of risk.

Aged↗
PubMed2003

Dopamine-induced oxidative stress in neurons with glutathione deficit: implication for schizophrenia.

Glutathione (GSH) is the main non-protein antioxidant and plays a critical role in protecting cells from damage by reactive oxygen species (ROS) generated by dopamine (DA) metabolism. We reported a decrease of GSH levels ([GSH]) in CSF and in prefrontal cortex in vivo in schizophrenics [Eur. J. Neurosci. 12 (2000) 3721]. A GSH deficit may lead to membrane peroxidation and microlesions around dopaminergic terminals, resulting in loss of connectivity. To test this hypothesis, we studied the effect of DA in cultured cortical neurons with low [GSH]. DA alone decreased [GSH] by 40%. This effect appears to result from direct conjugation of DA semiquinone/quinone with GSH. Ethacrynic acid (EA) decreased [GSH] in a concentration-dependent manner. When added to EA, DA further lowers [GSH]. As this additional decrease is blocked by superoxide dismutase (SOD) or D(1)/D(2) receptor antagonists, it likely involves the generation of superoxide via activation of DA receptors. It also reduces the mitochondrial membrane potential. Most interestingly, a significant decrease in number of neuronal processes (spines analogous) was induced by 24-h application of DA only in low [GSH]. These data, compatible with our hypothesis, is consistent with the dendritic spines reduction reported in schizophrenia and could be related to abnormalities in synaptic connectivity.

Animals↗
PubMed2002

Peptidomimetic glutathione analogues as novel gammaGT stable GST inhibitors.

Elevated levels of glutathione-S-transferase (GST) isoenzymes are found in many tumor cells and are thought to play a role in the onset of multidrug resistance (MDR). To evaluate the contribution of GST to this process, inhibitors are needed. Glutathione (GSH) conjugates, although good GST inhibitors, cannot be used in vivo, because they are eliminated rapidly. In this paper, we describe the synthesis of a series of novel peptidomimetic glutathione analogues that are stabilized against peptidase mediated breakdown. The peptide bonds in GSH were replaced by isosteres, such as the 'reduced' amide (which was prepared using a novel method), N-methylamide, urethane, and methylene linkages. The in vitro evaluation of the compounds focuses on GST inhibition and stability towards gamma-glutamyl-transpeptidase (gammaGT), the main enzyme involved in GSH breakdown. The compounds were conjugated to the model electrophile ethacrynic acid (EA) to resemble GS-EA, an efficient GST inhibitor. All novel GSH-analogues were shown to inhibit rat liver cytosolic GSTs. Furthermore, peptidomimetic changes of the gamma-glutamyl-cysteine amide bond greatly improved stability towards gammaGT. These compounds may therefore be useful in the design of novel in vivo applicable GST inhibitors.

Animals↗
PubMed2001

Purification and characterization of multiple glutathione S-transferase isozymes from Chironomidae larvae.

Glutathione S-transferase (GST) has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by Chironomidae larvae. We have purified and characterized GST from cytosolic fractions of Chironomidae larvae. GST with an M(r) of 23 kDa has been purified to homogeneity from larvae by centrifugation, size exclusion chromatography on Sephadex G25, and glutathione affinity and anion exchange chromatography. The purified enzyme exhibited moderate activity towards 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, 4-nitropyridine-N-oxide, p-nitrobenzyl chloride, ethacrynic acid, and cumene hydroperoxide. The enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 5.5. The enzyme had a maximum activity at approximately pH 8 and showed activity between 30 and 40 degrees C. It became inactive at higher temperature (>50 degrees C) for 5 min. The N-terminal sequence analysis of the amino acids shows a high % of conserved regions in the enzyme. The enzyme activity was comparable to levels of metabolism observed by animal GST involved in the detoxification of xenobiotics.

Amino Acid Sequence↗
PubMed2001

Glutathione S-transferases from rainbow trout liver and freshly isolated hepatocytes: purification and characterization.

Glutathione S-transferases (GST) form an important family of biotransformation enzymes catalyzing the conjugation of glutathione to a great variety of xenobiotic compounds. The objective of this study was to compare the different characteristics of GST from freshly isolated rainbow trout hepatocytes with those corresponding to the total liver of the same fish, in order to establish the similarities. GST was purified by affinity chromatography and enzymatic activity was determined towards two substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETHA). The different isoenzymes were determined by HPLC associated with SDS-PAGE. Slight differences between the samples were obtained when the results corresponding to the enzyme activity were compared. HPLC results showed that all GST isoforms present in the total liver samples were represented in the isolated cells too, corresponding to isoforms with molecular masses of approximately 25.5 and 23.0 kDa.

Animals↗
PubMed2003

Characterization of an ectonucleoside triphosphate diphosphohydrolase 1 activity in alkaline phosphatase-depleted rat osseous plate membranes: possible functional involvement in the calcification process.

An ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) activity present in alkaline phosphatase-depleted rat osseous plate membranes, obtained 14 days after implantation of demineralized bone particles in the subcutaneous tissue of Wistar rats, was characterized. At pH 7.5, NTPDase1 hydrolyzed nucleotide triphosphates at rates 2.4-fold higher than those of nucleotide diphosphates, while the hydrolysis of nucleotide monophosphates and non-nucleotide phosphates was negligible. NTPDase 1 hydrolyzed ATP and ADP following Michaelis-Menten kinetics with V=1278.7+/-38.4 nmol Pi/min/mg and K(M)=83.3+/-2.5 microM and V=473.9+/-18.9 nmol Pi/min/mg and K(M)=150.6+/-6.0 microM, respectively, but in the absence of magnesium and calcium ions, ATP or ADP hydrolysis was negligible. The stimulation of the NTPDase1 by calcium (V=1084.7+/-32.5 nmol Pi/min/mg; and K(M)=377.8+/-11.3 microM) and magnesium (V=1367.2+/-41.0 nmol Pi/min/mg and K(M)=595.3+/-17.8 microM) ions suggested that each ion could replace the other during the catalytic cycle of the enzyme. Oligomycin, ouabain, bafilomycin A(1), theophylline, thapsigargin, ethacrynic acid, P(1),P(5)-(adenosine-5')-pentaphosphate and omeprazole had negligible effects on the hydrolysis of ATP and ADP by NTPDase1. However, suramin and sodium azide were effective inhibitors of ATP and ADP hydrolysis. To our knowledge this is the first report suggesting the presence of NTPDase1 in rat osseous plate membranes. Considering that the ectonucleoside triphosphate diphosphohydrolase family of enzymes participates in many regulatory functions, such as response to hormones, growth control, and cell differentiation, the present observations raise interesting questions about the participation of this activity in the calcification process.

Adenosine Diphosphate↗
PubMed1989

Does severe water deprivation affect the inner ear? An experimental study of the gerbilline endolymphatic sac.

Through an ultrastructural study of the endolymphatic duct (ED) and sac (ES) system, the effects of an impaired body fluid metabolism on the inner ear fluid environment in the mongolian gerbil has been evaluated. A dehydrative state has been determined depriving of water for both five and twelve days this desert animal, which is known to withstand long periods of water abstinence. The morphological changes of the ED and ES under these circumstances have been compared with those induced by ethacrynic acid intoxication. Although the cochlear partition did not show signs of damage, the ED and ES system seems to be negatively influenced by the dehydrative state and displays a marked imbibition of the subepithelial tissue, which at places shows accumulation of an organic matrix. A regulating role for the antidiuretic hormone on the inner ear fluids of the mongolian gerbil is also proposed and discussed.

Animals↗