Biomedical subjects

H Wolosker

Publications and source records attributed to H Wolosker.

At least 19 recordsLinked to original sources

PubMed2002

Naturally occurring free D-aspartate is a nuclear component of cells in the mammalian hypothalamo-neurohypophyseal system.

It is generally believed that only L-amino acids have a physiological role in species other than bacteria. Recently, the existence of some D-amino acids, particularly D-aspartate, in various organs of several higher animals has been reported. Here we demonstrate that naturally occurring free D-aspartate is localized subcellularly to the heterochromatin in the nucleoli (but not in either the dendrites or axonal terminals) of magnocellular neurosecretory neurons in the rat hypothalamus, and also of microglia and pericytes in the posterior pituitary. Our results imply that naturally occurring free D-aspartate might have a physiological role in nuclear function in mammals. The findings provide new insight for the biological function of D-stereoisomers of amino acids as well as the organization of the nucleus of at least some eukaryotic cells.

Animals↗

PubMed2001

A new strategy to decrease N-methyl-D-aspartate (NMDA) receptor coactivation: inhibition of D-serine synthesis by converting serine racemase into an eliminase.

Serine racemase is a brain-enriched enzyme that synthesizes d-serine, an endogenous modulator of the glycine site of N-methyl-d-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of l-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of l-serine is inhibited. Likewise, l-serine O-sulfate inhibits the synthesis of d-serine in primary astrocyte cultures. We conclude that the synthetic compound l-serine O-sulfate is a better substrate than l-serine as well as an inhibitor of d-serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.

Ammonia↗

PubMed2000

Human serine racemase: moleular cloning, genomic organization and functional analysis.

High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.

Amino Acid Sequence↗

PubMed2000

Chloride-dependent inhibition of vesicular glutamate uptake by alpha-keto acids accumulated in maple syrup urine disease.

Maple syrup urine disease is a metabolic disorder caused by mutations of the branched chain keto acid dehydrogenase complex, leading to accumulation of alpha-keto acids and their amino acid precursors in the brain. We now report that alpha-ketoisovaleric, alpha-keto-beta-methyl-n-valeric and alpha-ketoisocaproic acids accumulated in the disease inhibit glutamate uptake into rat brain synaptic vesicles. The alpha-keto acids did not affect the electrochemical proton gradient across the membrane, suggesting that they interact directly with the vesicular glutamate carrier. Chloride anions have a biphasic effect on glutamate uptake. Low concentrations activate the uptake (0.2 to 8 mM), while higher concentrations are inhibitory. The alpha-keto acids inhibited glutamate uptake by a new mechanism, involving a change in the chloride dependence for the activation of glutamate uptake. The activation of glutamate uptake by low chloride concentrations was shifted toward higher concentrations of the anion in the presence of alpha-keto acids. Inhibition by alpha-keto acids was abolished at high chloride concentrations (20 to 80 mM), indicating that alpha-keto acids specifically change the stimulatory effect of low chloride concentrations. High glutamate concentrations also reduced the inhibition by alpha-keto acids, an effect observed both in the absence and in the presence of low chloride concentrations. The results suggest that in addition to their possible pathophysiological role in maple syrup urine disease, alpha-keto acids are valuable tools to study the mechanism of vesicular transport of glutamate.

ATP-Binding Cassette Transporters↗

PubMed2000

A mammalian iron ATPase induced by iron.

While molecular mechanisms for iron entry and storage within cells have been elucidated, no system to mediate iron efflux has been heretofore identified. We now describe an ATP requiring iron transporter in mammalian cells. (55)Fe is transported into microsomal vesicles in a Mg-ATP-dependent fashion. The transporter is specific for ferrous iron, is temperature- and time-dependent, and detected only with hydrolyzable nucleotides. It differs from all known ATPases and appears to be a P-type ATPase. The Fe-ATPase is localized together with heme oxygenase-1 to microsomal membranes with both proteins greatly enriched in the spleen. Iron treatment markedly induces ATP-dependent iron transport in RAW 264.7 macrophage cells with an initial phase that is resistant to cycloheximide and actinomycin D and a later phase that is inhibited by these agents. Iron release, elicited in intact rats by glycerol-induced rhabdomyolysis, induces ATP-dependent iron transport in the kidney. Mice with genomic deletion of heme oxygenase-1 have selective tissue iron accumulation and display augmented ATP-dependent iron transport in those tissues that accumulate iron.

Adenosine Triphosphatases↗

PubMed2000

D-serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor.

Functional activity of N-methyl-D-aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although D-serine is a more potent agonist. Localizations of D-serine and it biosynthetic enzyme serine racemase approximate the distribution of NMDA receptors more closely than glycine. We now show that selective degradation of d-serine with D-amino acid oxidase greatly attenuates NMDA receptor-mediated neurotransmission as assessed by using whole-cell patch-clamp recordings or indirectly by using biochemical assays of the sequelae of NMDA receptor-mediated calcium flux. The inhibitory effects of the enzyme are fully reversed by exogenously applied D-serine, which by itself did not potentiate NMDA receptor-mediated synaptic responses. Thus, D-serine is an endogenous modulator of the glycine site of NMDA receptors and fully occupies this site at some functional synapses.

Aging↗

PubMed2000

D-aspartate disposition in neuronal and endocrine tissues: ontogeny, biosynthesis and release.

High levels of D-aspartate occur in the brain and endocrine glands, such as pineal, adrenal and pituitary. In the brain, D-aspartate levels are highest in embryonic and early postnatal stages. Notably high levels occur in the early postnatal cortical plate and subventricular zone of the cerebral cortical cultures, implying a role in development. In embryonic neuronal primary culture cells, we detected high levels of endogenous D-aspartate and demonstrated biosynthesis of [14C]D-aspartate using [14C]L-aspartate as precursor. Synthesis of D-aspartate in cell cultures is inhibited by amino-oxyacetic acid, an inhibitor of pyridoxal phosphate-dependent enzymes. In the rat adrenal medulla, D-aspartate is depleted by treatment of the animals with intraperitoneal nicotine injections. In adrenal slices, D-aspartate is released by depolarization with KCl or acetylcholine, implying physiological release by activation of the cholinergic innervation of the adrenal. Our characterization of D-aspartate ontogeny, biosynthesis and depolarization-induced release implies specific physiological roles for this amino acid.

Acetylcholine↗

PubMed2000

Amyloid precursor proteins inhibit heme oxygenase activity and augment neurotoxicity in Alzheimer's disease.

Amyloid precursor protein (APP) generates the beta-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimer's disease. We report that APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimer's disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP-HO interactions may contribute to neuronal cell death in Alzheimer's disease.

Alzheimer Disease↗

PubMed2000

Regulation of rat magnocellular neurosecretory system by D-aspartate: evidence for biological role(s) of a naturally occurring free D-amino acid in mammals.

Little evidence is available for the physiological function of D-amino acids in species other than bacteria. Here we demonstrate that naturally occurring freed -aspartate (D-Asp) is present in all magnocellular neurons of rat hypothalamus. The levels of this naturally occurring D-amino acid were elevated during lactation and returned to normal thereafter in the magnocellular neurosecretory system, which produces oxytocin, a hormone responsible for milk ejection during lactation. Intraperitoneal injections of D-Asp reproducibly increased oxytocin gene expression and decreased the concentration of circulating oxytocin in vivo. Similar changes were observed in the vasopressin system. These results provide evidence for the role(s) of naturally occurring free D-Asp in mammalian physiology. The findings argue against the conventional concept that only L-stereoisomers of amino acids are functional in higher species.

Animals↗

PubMed1999

Serine racemase: a glial enzyme synthesizing D-serine to regulate glutamate-N-methyl-D-aspartate neurotransmission.

Although D amino acids are prominent in bacteria, they generally are thought not to occur in mammals. Recently, high levels of D-serine have been found in mammalian brain where it activates glutamate/N-methyl-D-aspartate receptors by interacting with the "glycine site" of the receptor. Because amino acid racemases are thought to be restricted to bacteria and insects, the origin of D-serine in mammals has been puzzling. We now report cloning and expression of serine racemase, an enzyme catalyzing the formation of D-serine from L-serine. Serine racemase is a protein representing an additional family of pyridoxal-5' phosphate-dependent enzymes in eukaryotes. The enzyme is enriched in rat brain where it occurs in glial cells that possess high levels of D-serine in vivo. Occurrence of serine racemase in the brain demonstrates the conservation of D-amino acid metabolism in mammals with implications for the regulation of N-methyl-D-aspartate neurotransmission through glia-neuronal interactions.

Amino Acid Sequence↗

PubMed1999

Purification of serine racemase: biosynthesis of the neuromodulator D-serine.

High levels of D-serine occur in mammalian brain, where it appears to be an endogenous ligand of the glycine site of N-methyl-D-aspartate receptors. In glial cultures of rat cerebral cortex, D-serine is enriched in type II astrocytes and is released upon stimulation with agonists of non-N-methyl-D-aspartate glutamate receptors. The high levels of D-serine in discrete areas of rat brain imply the existence of a biosynthetic pathway. We have purified from rat brain a soluble enzyme that catalyzes the direct racemization of L-serine to D-serine. Purified serine racemase has a molecular mass of 37 kDa and requires pyridoxal 5'-phosphate for its activity. The enzyme is highly selective toward L-serine, failing to racemize any other amino acid tested. Properties such as pH optimum, Km values, and the requirement for pyridoxal phosphate resemble those of bacterial racemases, suggesting that the biosynthetic pathway for D-amino acids is conserved from bacteria to mammalian brain.

Amino Acid Isomerases↗

PubMed1999

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid.

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP<-->Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.

ATP Synthetase Complexes↗

PubMed1999

Haem oxygenase-1 prevents cell death by regulating cellular iron.

Haem oxygenase-1 (HO1) is a heat-shock protein that is induced by stressful stimuli. Here we demonstrate a cytoprotective role for HO1: cell death produced by serum deprivation, staurosporine or etoposide is markedly accentuated in cells from mice with a targeted deletion of the HO1 gene, and greatly reduced in cells that overexpress HO1. Iron efflux from cells is augmented by HO1 transfection and reduced in HO1-deficient fibroblasts. Iron accumulation in HO1-deficient cells explains their death: iron chelators protect HO1-deficient fibroblasts from cell death. Thus, cytoprotection by HO1 is attributable to its augmentation of iron efflux, reflecting a role for HO1 in modulating intracellular iron levels and regulating cell viability.

Animals↗

PubMed1998

Cain, a novel physiologic protein inhibitor of calcineurin.

Calcineurin is a widely distributed protein phosphatase regulated by calcium and calmodulin. It mediates the immunosuppressive actions of drugs such as cyclosporin and FK506, and has been implicated in a number of calcium-sensitive pathways in the nervous system, including regulation of neurotransmitter release and modulation of long-term changes in synaptic plasticity. Calcineurin associates physiologically with other proteins, including calmodulin, FKBP12 (FK506-binding protein), the ryanodine receptor, and the inositol 1,4,5-trisphosphate receptor. We now report the identification, molecular cloning, and functional characterization of a novel protein, cain (calcineurin inhibitor), that interacts with and inhibits calcineurin. The full-length cain cDNA predicts a 240-kDa protein with no significant homology to any known protein. Cain associates with calcineurin both in vitro and in vivo, leading to a non-competitive inhibition of calcineurin activity. The putative calcineurin-binding domain of cain, a 38-amino acid region defined by mutational analysis, is highly basic. Like calcineurin, cain has a prominent neuronal expression and a wide tissue distribution. Cain's expression pattern in the brain closely resembles that of calcineurin, indicating a physiologic association between the two proteins.

Amino Acid Sequence↗

PubMed1998

Molecularly cloned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity.

Glucosamine-6-phosphate deaminase (GNPDA) catalyzes the conversion of glucosamine-6-phosphate to fructose-6-phosphate, a reaction that under physiological conditions proceeds to the formation of fructose-6-phosphate. Though first identified in mammalian tissues in 1956, the enzyme has not previously been molecularly characterized in mammalian tissues, although a bacterial GNPDA has been cloned. Recently, a protein displaying similarity to bacterial GNPDA was purified and cloned from sperm extract. It was proposed that this protein was the factor, found in sperm extracts, that causes calcium oscillations in cells; thus, the protein was named 'oscillin.' We demonstrate that oscillin is the mammalian form of glucosamine 6-phosphate deaminase by showing that cloned oscillin has a robust GNPDA activity and can account for all such activity in mammalian tissues extracts. In situ hybridization and immunohistochemistry localize GNPDA selectively to tissues with high energy requirements such as the apical zone of transporting epithelia in the proximal convoluted tubules of the kidney and the small intestine; to neurons (but not glia) and especially to nerve terminals in the brain; and to motile sperm. Recombinant GNPDA and GNPDA purified to homogeneity from hamster sperm fail to elevate intracellular calcium when injected into mouse eggs over a wide range of concentrations under conditions in which sperm extracts elicit pronounced calcium oscillations. Thus, the calcium-releasing or oscillin activity of sperm extracts is due to a substance other than GNPDA. Since GNPDA is the sole enzyme linking hexosamine systems with glycolytic pathways, we propose that it provides a source of energy in the form of phosphosugar derived from the catabolism of hexosamines found in glycoproteins, glycolipids, and sialic acid-containing macromolecules. Evidence that GNPDA can regulate hexosamine stores comes from our observation that transfection of GNPDA into HEK-293 cells reduces cellular levels of sialic acid.

Aldose-Ketose Isomerases↗

PubMed1997

Sarco/endoplasmic reticulum Ca2+-ATPase isoforms: diverse responses to acidosis.

The effects of acidic pH on the kinetics of Ca2+-ATPase isoforms from intracellular membranes of skeletal muscle, cardiac muscle, cerebellum and blood platelets were studied. At neutral pH, all four Ca2+-ATPase isoforms exhibited similar Ca2+-concentration requirements for half-maximal rates of Ca2+ uptake and ATP hydrolysis. A decrease in the pH from 7.0 to 6.0 promoted a decrease in both the apparent affinity for Ca2+ [increasing half-maximal activation (K0.5)] and the maximal velocity (Vmax) of Ca2+ uptake. With skeletal muscle vesicles these effect were 5 to 10 times smaller than those observed with all the other isoforms. Acidification of the medium from pH 7.0 to 6.5 caused the release of Ca2+ from loaded vesicles and a decrease in the amount of Ca2+ retained by the vesicles at the steady state. With the vesicles derived from skeletal muscle these effects were smaller than for vesicles derived from other tissues. The rate of passive Ca2+ efflux from skeletal and cardiac muscle vesicles, loaded with Ca2+ and diluted in a medium containing none of the ligands of Ca2+-ATPase, was the same at pH 7.0 and 6.0. In contrast, the rate of Ca2+ efflux from cerebellar and platelet vesicles increased 2-fold after acidification of the medium. The effects of DMSO, Mg2+ with Pi and arsenate on the rate of Ca2+ efflux varied among the different preparations tested. The differences became more pronounced when the pH of the medium was decreased from 7.0 to 6.0. It is proposed that the kinetic differences among the Ca2+-ATPase isoforms may reflect different adaptations to cellular acidosis, such as that which occurs during ischaemia.

Acidosis↗

PubMed1996

Effects of Hg2+ and CH3Hg+ on Ca2+ fluxes in rat brain microsomes.

A permanent increase in cytosolic Ca2+ levels seems to be associated with various pathological situations which may result in cell death. Hg2+ and CH3Hg+ are potent neurotoxic agents, but the precise molecular mechanism(s) underlying their effects are not sufficiently understood. In the present study we investigated the potential role of Ca(2+)-ATPase located in the endoplasmic reticulum as a molecular target for mercury. Hg2+ and CH3Hg+ inhibited Ca(2+)-ATPase and Ca2+ uptake by brain microsomes with similar potencies. However, the inhibitory potency of Hg2+ was higher than that of CH3Hg+, probably reflecting differences in the affinity for the sulfhydryl groups of these compounds. Passive or unidirectional Ca2+ efflux (measured in the absences of Ca(2+)-ATPase ligands) was increased significantly by CH3Hg+ and Hg2+. Again, the potency of Hg2+ was higher than that of CH3Hg+. Blockers of Ca2+ channels (ruthenium red, procaine, heparin) did not affect the increase in passive Ca2+ efflux induced by mercury compounds, possibly indicating that Ca2+ release occurs through Ca(2+)-ATPase. Addition of physiological concentrations of glutathione (GSH) simultaneously with mercury abolished the inhibitory effects of both forms of Hg on ca(2+)-transport. However, if the enzyme was first inhibited with Hg2+ or CH3Hg+ and subsequently treated with GSH, the reversal of inhibition was about 50%, suggesting that part of the cysteinyl residues involved in the inhibitory actions of mercury in Ca(2+)-transport bind to mercury with an extremely high affinity.

Animals↗

PubMed1996

Inhibition of creatine kinase by S-nitrosoglutathione.

The sarcoplasmic reticulum-bound creatine kinase from rabbit skeletal muscle was inhibited by the nitric oxide donor S-nitrosoglutathione (GSNO). This led to a decrease in Ca2+ uptake in sarcoplasmic reticulum vesicles when the transport was driven by ATP generated from phosphocreatine and ADP. In contrast, the Ca 2+ transport measured using 2 mM ATP as substrate was unaffected by GSNO up to 200 microM. GSNO (5-20 microM) inhibited the activity of both soluble and membrane-bound creatine kinase. Oxyhemoglobin (15-40 microM) protected creatine kinase against inactivation by GSNO. The inhibition by 10 microM GSNO was reversed by the addition of dithiothreitol (2 mM). The results indicate that nitric oxide (NO, including NO+, NO and NO-) inactivates creatine kinase in vitro by promoting nitrosylation of critical sulphydryl groups of the enzyme.

Adenosine Triphosphate↗