Selective tonicity-induced expression of the neutral amino-acid transporter SNAT2 in oligodendrocytes in rat brain following systemic hypertonicity.

Sodium-coupled neutral amino-acid transporter member 2 (SNAT2) belongs to the family of neutral amino-acid transporters. SNAT2 is encoded by the gene Slc38a2, whose expression was reported to increase in vitro in fibroblasts, endothelial and renal cells exposed to a hypertonic medium. SNAT2 tonicity-induced expression brings about cellular accumulation of amino-acid, which contributes to osmoadaptation to hypertonicity. Since brain osmoadaptation is observed in relationship to neurological disorders resulting from pathological osmotic imbalances in blood plasma, we have investigated, through immunocytochemistry, SNAT2 expression in brain of rats subjected to systemic hypertonicity. Following prolonged systemic hypertonicity (24 h), small, strongly immunolabeled elements were observed that were not present in sham-treated animals. They were evenly distributed in the gray matter, with a lower density in the forebrain and a higher density in the brain stem. However the highest density by far was observed in white matter, where they were frequently aligned in chain-like rows. These observations suggested an oligodendrocyte location that was further established by double immunofluorescent labeling, using the oligodendrocyte phenotypic markers 2'-3'-cyclic nucleotide 3'phosphodiesterase and carbonic anhydrase II. SNAT2-positive elements were found associated with oligodendrocyte cell bodies, while oligodendrocyte processes were devoid of labeling. A quantitative analysis performed in the cerebral cortex indicated that virtually all SNAT2-positive elements were associated with oligodendrocyte cell bodies and conversely that the overwhelming majority of oligodendrocytes showed SNAT2 immunolabeling. The tonicity-induced expression of SNAT2 was not observed following acute systemic hypertonicity (6 h). Our results suggest that the osmoadaptation of brain oligodendrocytes to hypertonicity relies upon amino-acid accumulation through the tonicity-induced expression of SNAT2. The possible significance of these findings in relationship to the selective loss of oligodendrocytes observed in osmotic demyelination syndrome is discussed.

Large discrepancies in cellular distribution of the tonicity-induced expression of osmoprotective genes and their regulatory transcription factor TonEBP in rat brain.

Osmoprotective genes are tonicity-activated genes involved in cellular osmoadaptation to hypertonicity and considered to be regulated by a specific transcription factor called tonicity-responsive enhancer-binding protein (TonEBP). In the brain we had previously established that TonEBP was expressed and tonicity-induced in neurons only. Here we have compared in various brain regions of rats subjected to systemic hypertonicity, the cellular expression of TonEBP through immunocytochemistry and the cellular expression of osmoprotective genes, namely aldose reductase (AR), sodium-dependent myo-inositol transporter (SMIT), betaine/GABA transporter (BGT1) and taurine transporter (TauT), by in situ hybridization using non-radioactive digoxigenin-labeled riboprobes. In neurons where TonEBP was strongly tonicity-induced, AR-mRNA labeling was strongly increased in some subsets (e.g. hippocampus pyramidal cells, cerebellar Purkinje cells and neurons of the hypothalamic magnocellular nuclei) but remained undetectable in some other subsets (e.g. neurons in cerebral cortex). Tonicity-induced AR-mRNA labeling was observed only several hours after the tonicity-induced expression of TonEBP. SMIT-mRNA labeling was tonicity-induced as densely and evenly distributed dots in neuron poor regions (e.g. cerebral cortex layer I and hippocampus stratum lacunosum-moleculare). The tonicity-induced expression of SMIT-mRNA may thus occur in non-neuronal cells, presumably astrocytes, where TonEBP is neither significantly expressed, nor tonicity-induced. In neurons showing a strong tonicity-induced expression of TonEBP, no SMIT-mRNA labeling was observed. BGT1-mRNA and TauT-mRNA labeling could not be detected, even after systemic hypertonicity. The present work reveals large discrepancies between the cellular distribution of the tonicity-induced expression of osmoprotective genes and that of their regulatory transactivator TonEBP. Depending on the cell subsets and the osmoprotective genes, TonEBP may appear insufficient or conversely unnecessary for the tonicity-induced activation of an osmoprotective gene. Altogether our results show that brain cells, even from the same class, activate distinct osmoprotective genes through distinct activation processes to adapt to hypertonicity.

Differential cellular distribution of tonicity-induced expression of transcription factor TonEBP in the rat brain following prolonged systemic hypertonicity.

In a previous work performed on cerebral cortex and hippocampus we reported that tonicity-responsive enhancer binding protein (TonEBP), originally identified as a transactivator of osmoprotective genes involved in osmoadaptation of renal cells, was induced in neurons only, but to varying levels, following acute systemic hypertonicity. Whether or not this cellular specificity reflected a unique ability of neurons or a differential time course among brain cells for tonicity-induction of TonEBP was investigated throughout the brain in this study by subjecting the animals to prolonged systemic hypertonicity. In normal rats, TonEBP immunolabeling and TonEBP-mRNA in situ hybridization labeling showed a widespread, uneven and parallel distribution. TonEBP was expressed primarily in the cell nuclei of neurons, where it was heterogeneously distributed in a nucleoplasmic and a granular pool. In rats subjected to prolonged systemic hypertonicity, TonEBP labeling increased in the cell nuclei of neurons only. The tonicity-induced expression of TonEBP for a given cell group of neurons was rather uniform but varied greatly among neuronal cell groups and was positively correlated with the average size of the cell nuclei, as determined by quantitative analysis of digitized images. The detailed distribution of tonicity-induced expression of TonEBP is reported throughout the brain. In normal rats, a very minor proportion of non-neuronal cells, identified as a subset of astrocytes and possibly oligodendrocytes, showed faint nuclear immunolabeling, which however did not increase in hypertonic animals. Ependymocytes, capillary endothelial cells, and microglial cells showed no TonEBP labeling, even in hypertonic animals. Altogether our data indicate that neurons, albeit possibly to a varying extent, are the only brain cells able to use TonEBP-mediated processes for adaptation to a systemic hyperosmotic unbalance.

Taurine biosynthetic enzymes and taurine transporter: molecular identification and regulations.

Many biological effects of taurine rely upon its cellular concentration, which is primarily controlled by taurine biosynthetic enzymes cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD) and taurine transporter (TauT). The cloning of CDO, CSD and TauT in various species provided first-hand information on these proteins, as well as molecular tools to investigate their regulations. CDO upregulation in hepatocytes in response to high sulfur amino acids appears clearly as the most spectacular among the regulations of the biosynthetic enzymes. Downregulation of TauT activity by activation of PKC appears particularly well documented. A unique serine residue could be identified as a phosphorylation site that leads to an inactive form of TauT. The previously revealed downregulation of TauT expression by taurine and hypertonicity-induced upregulation of TauT expression were shown to result from a modified transcription rate of TauT gene, but the precise molecular mechanisms are not yet formally established. Other regulations of taurine transporter expression were more recently reported, which involve glucose, tumor suppressor protein p53, tumor necrosis factor-alpha, and nitric oxide. This review reports the experimental models and data that support these various regulations but also points out the aspects that remain poorly understood or unknown concerning their molecular basis and physiological significance.

Transcription factor tonicity-responsive enhancer-binding protein (TonEBP) which transactivates osmoprotective genes is expressed and upregulated following acute systemic hypertonicity in neurons in brain.

Tonicity-responsive enhancer-binding protein (TonEBP) was initially identified as a transcription factor involved in adaptation of renal cells to hypertonicity by activation of osmoprotective genes encoding proteins for accumulation of compatible osmolytes. Since brain osmoadaptation is observed in relationship to neurological disorders resulting from pathological osmotic disbalances of blood plasma we have investigated through immunocytochemistry TonEBP expression in cerebral cortex and hippocampus of normal rat and rats submitted to an acute systemic hypertonicity or to a prolonged systemic hypotonicity. TonEBP-expressing cells were identified using double immunofluorescence and appropriate cell type markers. Their relative proportion was determined by quantitative image analysis. In normal rats TonEBP expressed primarily in neurons where it was strictly located in the cell nucleus but heterogeneously distributed into a nucleoplasmic pool and a granular pool. In animals made acutely hypertonic TonEBP labeling increased dramatically exclusively in the nuclei of neurons and reached a maximum within 1 h. In hypertonic animals TonEBP labeling covered the whole cell nucleus of virtually all neurons, appeared finely punctuated but was no more granular. Optical density of the labeling as determined by image analysis correlated linearly with the increased plasma osmolality. In animals made hypotonic for several days no conspicuous decrease of TonEBP labeling was observed. In normal animals a very minor proportion of non-neuronal cells showed a faint TonEBP nuclear labeling. This proportion increased slightly in hypertonic animals. Nevertheless these non-neuronal TonEBP-positive nuclei which belonged to oligodendrocytes and to a small subpopulation of astrocytes remained always very weakly labeled when compared with neuron nuclei. Brain capillary endothelial cells as well as microglial cells showed no TonEBP-labeling even in hypertonic animals. Our data demonstrate that in brain TonEBP is significantly expressed and tonicity-overexpressed in neurons and accordingly suggest that neurons only among brain cells accumulate compatible osmolytes through TonEBP-mediated activation of osmoprotective genes to adapt to acute systemic hypertonicity.

Gene expression of the taurine transporter and taurine biosynthetic enzymes in rat kidney after antidiuresis and salt loading.

Taurine is thought to be an osmolyte in the kidney medulla. We have investigated the gene expression of the taurine transporter (TauT) and the enzymes of taurine biosynthesis, cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD). We achieved this by measuring their mRNA levels using reverse transcriptase polymerase chain reaction (RT-PCR) in five kidney regions of rats in various hydration states; namely, normal hydration, after 2 days of antidiuresis following chronic diuresis and finally after acute salt loading. The mRNA levels of the well-established tonicity-sensitive genes coding for the aldose reductase (AR), the sodium myo-inositol transporter (SMIT) and the betaine transporter (BGT1) were also determined for the sake of comparison. In normally hydrated rats, TauT-, CDO-, and CSD-mRNA were enriched in the outer stripe of the outer medulla (OS). Following antidiuresis, the mRNA levels of TauT, CDO, CSD, SMIT, BGT1 and AR were all similarly increased in the papilla when compared with levels in rats submitted to a chronic diuresis. After acute salt loading, the mRNA level of TauT, like that of SMIT and BGT1, was overexpressed in OS whereas the mRNA levels of CDO and CSD remained unchanged. Like SMIT, BGT1 and AR genes, TauT, CDO and CSD genes appear to be tonicity-sensitive genes which can be activated in vivo by hypertonicity in the rat kidney. However, tonicity-induced activation of the TauT gene is more sensitive than that of CDO and CSD genes.

Regional expression and histological localization of cysteine sulfinate decarboxylase mRNA in the rat kidney.

Cysteine sulfinate decarboxylase (CSD) is the rate-limiting biosynthetic enzyme of the pathway that forms taurine, a putative osmolyte in the kidney, which was previously localized in various segments of the nephron. Although CSD is known to be expressed in whole kidney extracts, no information on CSD mRNA regional expression and histological localization is yet available. Western blotting and Northern blotting were performed in four dissected regions of the kidney using an antiserum against recombinant CSD and a [(32)P]-dCTP-labeled CSD cDNA probe, respectively. In situ hybridization was carried out using a [(35)S]-CTP-labeled CSD RNA probe. A single protein (53 kD) and a single mRNA (2.5 kb) were detected, both of which appeared to be most enriched in the outer stripe of the outer medulla. In situ hybridization of CSD mRNA showed strong labeling of the thick tubules in the outer stripe of the outer medulla and in cortical medullary rays that corresponded to the proximal straight tubules. The significance of this restricted expression of CSD is discussed in relationship to the data previously reported on the location of taurine and the location of the taurine transporter along the nephron.

Gene expression of the transporters and biosynthetic enzymes of the osmolytes in astrocyte primary cultures exposed to hyperosmotic conditions.

Sorbitol, myo-inositol, betaine, and taurine are held as organic osmolytes. When cells are exposed to a hyperosmotic medium, they accumulate these organic compounds and thus achieve osmotic equilibrium with the medium while maintaining their volume. In astrocyte primary cultures adapted to a chemically defined medium and then exposed to a medium made 30% hyperosmotic by adding sodium chloride or raffinose, we have comparatively investigated the expression of the genes encoding the proteins that control the cellular accumulation of these osmolytes, namely sorbitol biosynthetic enzyme, aldose reductase (AR), taurine biosynthetic enzymes, cysteine dioxygenase (CDO), and cysteine sulfinic acid decarboxylase (CSD), and the transporters of taurine (TauT), myo-inositol (SMIT), and betaine (BGT1) by assaying the corresponding mRNA levels through relative quantitative RT-PCR. When exposed to the hyperosmotic medium the astrocytes shrank rapidly and then slowly regained their initial volume after several hours. CDO- and CSD-mRNA remained unchanged, whereas AR-mRNA appeared increased only with the medium made hyperosmotic with sodium chloride. The mRNA levels of the transporters only showed significant and comparable increases in both hyperosmotic conditions. They were all significantly higher after 4-h exposure and back or close to normal values after 24-h exposure. The maximum level occurred at around 4 h (SMIT), 8 h (BGT1), and 12 h (TauT). The amplitude of BGT1-mRNA increase was much larger. When taurine was added to the hyperosmotic medium the cell volume recovery was greatly accelerated and the osmo-induced overexpression of TauT-, SMIT-, and BGT1-mRNA was fully prevented. The activation of the genes encoding the osmolyte transporters appears to be triggered when the cell shrinks below a certain volume threshold and prolonged once the cell volume has regained this threshold value most likely as a result of a marked inertia of the transducing pathway. Since the upregulation pattern of the transporters of the different osmolytes notably differs, we speculate that the activation threshold varies from one gene to another.

Taurine down-regulates basal and osmolarity-induced gene expression of its transporter, but not the gene expression of its biosynthetic enzymes, in astrocyte primary cultures.

Taurine content of astrocytes is primarily regulated by transport from the extracellular medium and endogenous biosynthesis from cysteine. We have investigated the gene expression of the taurine transporter (TauT) and the taurine biosynthetic enzymes, cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD), in astrocyte primary cultures in relationship to cell taurine content. TauT, CDO, and CSD mRNA levels were determined through quantitative RT-PCR. Cell taurine content was depleted by adapting the cells to a taurine-free chemically defined medium and increased by incubating the cells in the same medium containing exogenous taurine. With increased cell taurine content the level of TauT mRNA decreased, whereas the levels of CDO and CSD mRNA remained unchanged. In astrocytes exposed to a hyperosmotic medium the TauT mRNA level increased, whereas the CDO and CSD mRNA levels were not significantly altered. The osmolarity-induced up-regulation of TauT mRNA expression was fully prevented by increasing cell taurine content. Thus, the gene expression of the taurine transporter, but not that of the taurine biosynthetic enzymes, appears to be under the control of two antagonistic regulations, namely, a taurine-induced down-regulation and an osmolarity-induced up-regulation.

Gene expression of taurine transporter and taurine biosynthetic enzymes in brain of rats with acute or chronic hyperosmotic plasma. A comparative study with gene expression of myo-inositol transporter, betaine transporter and sorbitol biosynthetic enzyme.

Cells exposed to hyperosmotic conditions maintain their volume by accumulating organic osmolytes. Taurine is considered as an osmolyte in brain cells. Accumulation of other osmolytes (sorbitol, myo-inositol and betaine), was shown in renal cells to result from an upregulation of the expression of the genes regulating osmolyte cell content. We have investigated the gene expression of the taurine transporter (TauT) and of the taurine biosynthetic enzymes, cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD) by measuring their mRNA levels in brain of salt-loaded rats. mRNA levels of genes previously identified as osmosensitive, namely aldose reductase (AR), myo-inositol transporter (SMIT) and betaine transporter (BGT1) were also determined. In whole brain, TauT-, SMIT- and BGT1-mRNA levels were significantly increased following acute salt-loading but SMIT-mRNA levels only remained elevated following chronic salt-loading while CDO-, CSD- and AR-mRNA levels remained unchanged in both conditions. Following acute salt-loading, mRNA levels of TauT, CDO, CSD, SMIT, BGT1 and AR were increased in cerebral cortex while SMIT- and BGT1-mRNA levels only were increased in striatum and habenula.TauT, CDO and CSD genes may be upregulated in brain of salt-loaded rats but the upregulation of the TauT gene appears more widespread. TauT, CDO and CSD are thus putative osmosensitive genes. However the actual pattern (amplitude, time course and regional occurrence) of the upregulation of each of the putative (TauT, CDO and CSD) and established (AR, SMIT and BGT1) osmosensitive genes differs markedly. This indicates that there exist other factors in brain cells which can selectively prevent the upregulation of these genes by hyperosmolarity.

Characterization of the cDNA coding for rat brain cysteine sulfinate decarboxylase: brain and liver enzymes are identical proteins encoded by two distinct mRNAs.

Cysteine sulfinate decarboxylase (CSD) is considered as the rate-limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT-PCR and RACE-PCR products of rat brain mRNAs, a 2,396-bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, approximately 2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5' untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR-amplified products of genomic DNA suggests that the brain form results from the use of a 3' alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT-PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue-specific regulation of CSD genomic expression.

Immunocytochemical localization of cysteine sulfinate decarboxylase in astrocytes in the cerebellum and hippocampus: a quantitative double immunofluorescence study with glial fibrillary acidic protein and S-100 protein.

Immunocytochemistry of cysteine sulfinate decarboxylase was performed with a new rabbit antiserum that we have recently produced and characterized using as antigen an 11,000-fold purified fraction isolated from rat liver. This antiserum precipitated cysteine sulfinate decarboxylase enzymatic activity, labeled one band (mol. wt 51,000) on immunoblots of crude tissue extracts and did not stain any cells in peripheral tissues devoid of cysteine sulfinate decarboxylase. According to these criteria, this antiserum appeared to be specific for cysteine sulfinate decarboxylase. Numerous cells were immunolabeled in the cerebellum and the hippocampus. Most notable was the labeling of the small cells surrounding the Purkinje cells and sending radial fibers up to the pial surface of the cerebellar cortex or the staining of small star-shaped cells with thin immunolabeled processes abutting on blood vessels. Identified nerve cells such as the Purkinje cells and granule cells in the cerebellum or the pyramidal and granule cells in the hippocampus were devoid of any immunoreactivity. simultaneous double immunofluorescence was carried out using anti-glial fibrillary acidic protein or anti-S-100 monoclonal antibodies. Cysteine sulfinate decarboxylase as well as glial fibrillary acidic protein- or S-100-immunopositive cells were plotted independently for the same section. Quantitative analysis of the maps indicated that the overwhelming majority of cysteine sulfinate decarboxylase-immunolabeled cells were positive for the established astrocytes markers, glial fibrillary acidic protein or S-100. Between 82 and 98% of cysteine sulfinate decarboxylase-immunolabeled cells were also glial fibrillary acidic protein-positive, depending upon the layer. Cysteine sulfinate decarboxylase immunostaining was localized within the cytoplasm, while that of glial fibrillary acidic protein was linked to the cytoskeleton. Since both labels could not be fully superposed, some double immunolabeled cells may have escaped our analysis. More than 94% up to 99% of cysteine sulfinate decarboxylase-immunolabeled cells were simultaneously S-100-immunopositive. Our quantitative data establish that cysteine sulfinate decarboxylase is strictly localized in astrocytes in the cerebellum and in the hippocampus. This finding suggests that taurine is synthesized by astrocytes in the brain and accordingly may play a role in relation to glial function, possibly within the framework of glial-neuronal interactions.

Localization of gamma-aminobutyric acid and glutamic acid decarboxylase in the pancreas of the nonobese diabetic mouse.

Glutamic acid decarboxylase (GAD), among other potential autoantigens, is thought to play a crucial role in type I diabetes, particularly in a spontaneous model of the disease, the nonobese diabetic (NOD) mouse. In the pancreas, the presence of GAD and gamma-aminobutyric acid (GABA), the decarboxylation product of GAD and a putative neurotransmitter in the islets of Langerhans, is well documented in the beta-cells. This is particularly true in rats, in which another GABAergic structure exists near the islets, the neuronal bodies. In this study, first the GABA content was measured in isolated islets from NOD and C57BL/6 mice (controls), and a decrease was found in NOD females as their insulitis progressed. Second, for the first time in mice, confocal analysis of immunofluorescent-labeled pancreatic sections revealed near the islets neuronal structures in which GAD and neuropeptide Y were colocalized, as they are in the brain. These structures were always observed in the pancreata of both sexes of C57BL/6 mice at the various ages investigated. In NOD mice, however, these neuronal structures were only detected in young females ( < 10 weeks old) and in males until an intermediate age. Moreover, patches of T cells surrounding GAD-containing fibers were seen in the vicinity of the islets with incipient periinsulitis.

Molecular cloning and sequence analysis of the cDNA encoding rat liver cysteine sulfinate decarboxylase (CSD).

The taurine biosynthesis enzyme, cysteine sulfinate decarboxylase (CSD), was purified to homogeneity from rat liver. Three CSD peptides generated by tryptic cleavage were isolated and partially sequenced. Two of them showed a marked homology with glutamate decarboxylase and their respective position on the CSD amino acid sequence was postulated accordingly. Using appropriate degenerated primers derived from these two peptides, a PCR amplified DNA fragment was generated from liver poly(A)+ mRNA, cloned and used as a probe to screen a rat liver cDNA library. Three cDNAs, length around 1800 bp, were isolated which all contained an open reading frame (ORF) encoding a 493 amino acid protein with a calculated molecular mass of 55.2 kDa close to the experimental values for CSD. The encoded protein contained the sequence of the three peptides isolated from homogenous liver CSD. Our data confirm and significantly extend those recently published (Kaisaki et al. (1995) Biochim. Biophys. Acta 1262, 79-82). Indeed, an additional base pair found 1371 bp downstream from the initiation codon led to a shift in the open reading frame which extended the carboxy-terminal end by 15 amino acid residues and altogether modified 36 amino acids. The validity of this correction is supported by the finding that the corrected reading frame encoded a peptide issued from CSD tryptic cleavage that was not encoded anywhere in the CSD sequence previously reported.

Specificity of cysteine sulfinate decarboxylase (CSD) for sulfur-containing amino-acids.

Cysteine sulfinate decarboxylase (CSD) which decarboxylates cysteine sulfinic acid (CSA) to form hypotaurine is thought to be involved in the biosynthesis of taurine. It was recently localized in astrocytes in the cerebellum and hippocampus by immunocytochemistry. Another sulfur-containing amino-acid (SCAA), homocysteic acid (HCA), was also found in astrocytes in these regions. We therefore investigated the specificity of CSD vs CSA and HCA as well as the related analogs homocysteine sulfinic acid (HCSA) and cysteic acid (CA). CSD was immunotrapped from brain and liver tissue supernatant using a specific CSD antiserum and Protein-A Sepharose. It was then incubated with the L-form of the various SCAA. Reaction products were identified and quantified by pre-column o-phthalaldehyde derivatization HPLC. CA and HCA from 2.5 to 25 mM inhibited the formation of hypotaurine from CSA (0.25 mM). Moreover, the inhibition curves were parallel for liver and brain CSD. CA or HCA (25 mM) elicited a near-total inhibition. HCSA did not produce a significant inhibition up to 25 mM. Incubation with 25 mM CSA or CA led to the formation of hypotaurine and taurine, respectively. The ratio of formation of taurine to that of hypotaurine was similar for CSD from liver and brain. In contrast no homotaurine, the decarboxylated reaction product of HCA, could be detected following incubation with 25 mM HCA. According to the sensitivity of the HPLC analysis this indicates that the decarboxylation of HCA, if any, was 130-fold and 50-fold less than that of CSA by CSD from liver and brain, respectively, in our experimental conditions. Similarly, following incubation with HCSA, no new peak appeared on the chromatogram when compared to a blank sample. These results show that CSD from either brain or liver has a high specificity for CSA and CA, which are the SCAA involved in the biosynthesis of taurine. HCA is an inhibitor of CSD but does not appear to be a substrate for CSD in vitro. HCSA is neither a substrate nor an inhibitor of CSD in vitro. Accordingly, CSD is unlikely to play a role in the metabolism of HCA or HCSA in vivo.

Evidence that activation of the hypothalamo-pituitary-adrenal axis by electrical stimulation of the noradrenergic A1 group is not mediated by noradrenaline.

The paraventricular nucleus (PVN) of the hypothalamus, where the CRF-containing neurosecretory cells controlling the hypothalamo-pituitary-adrenal (HPA) axis are located, receives a dense noradrenergic innervation from the A1 group of the caudal ventrolateral medulla. In the present study we studied the relationship between release of noradrenaline (NA) in the PVN and activation of the HPA axis in response to electrical stimulation of the A1 region. In the urethane-anesthetized male rat, extracellular NA in the PVN was monitored on line by electrochemical recording while the activity of the HPA axis was estimated by measurement of ACTH in blood samples. A 1 min, 10 Hz stimulation evoked a significant increase of extracellular NA in the PVN as well as an ACTH surge in blood. The NA and ACTH response evoked by stimulation in the 3- to 14-Hz range were found to be frequency dependent. However, whilst the NA response increased in an exponential manner with respect to frequency, the ACTH response appeared to plateau between 10 and 14 Hz. Specific lesions of the noradrenergic terminals in the PVN, by bilateral local administration of 6-hydroxydopamine, markedly reduced the ACTH response to stimulation. Intracerebroventricular injection of desmethylimipramine, a NA uptake inhibitor, enhanced the increase in extracellular NA evoked by submaximal stimulation about 2.5-fold but did not modify the corresponding ACTH response. Combined intracerebroventricular injection of alpha- and beta-adrenergic antagonists, phentolamine and propanolol respectively, did not prevent the ACTH response evoked by stimulation. Following stimulation of the caudal ventrolateral medulla, the ACTH response thus appears to result from the stimulation of the A1 noradrenergic group projecting to the PVN. However, the inability of pharmacological manipulations which enhance or block central noradrenergic transmission to influence the ACTH response suggests that the noradrenergic endings in the PVN originating from the A1 group use a transmitter other than NA to activate the HPA axis at the PVN level.

[Demonstration of cysteine sulfinate decarboxylase (EC 4.1.1.29) in cultured oviduct epithelial cells in cows and goats]. / Mise en évidence de la cystéine sulfinate décarboyxlase (EC 4.1.1.29) dans les cellules épithéliales tubaires de vache et de chèvre en culture.

Tubal fluid contains high amounts of hypotaurine and taurine. These amino-acids are important for gametes and embryo survival. They are synthesized and secreted by oviduct epithelial cells in vitro. Cysteine sulfinate decarboxylase (EC 4.1.1.29) activity was identified by selective immuno-trapping using a specific antiserum in cow and goat oviduct epithelial monolayers. This result suggests that cysteine is converted to hypotaurine and taurine via cysteine sulfinic acid in these cells.

Production and characterization of a new specific antiserum against the taurine putative biosynthetic enzyme cysteine sulfinate decarboxylase.

We have shown previously that cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme of taurine in the brain, is identical to the liver enzyme according to biochemical, kinetic, and immunochemical criteria. In the present work, CSD was purified in its native form from rat liver. The purification was performed in eight steps, which included conventional chromatography (diethylaminoethyl cellulose, hydroxylapatite), followed by HPLC (hydrophobic, adsorption, and ion-exchange HPLC). The purification factor was 11,000, and the final yield was around 2%. The procedure led to the enrichment of a protein, the molecular mass of which was 51,000 daltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final fraction was more than 90% homogeneous. By using this fraction as the antigen, an antiserum was raised in rabbit that (a) quantitatively immunoprecipitated CSD activity from liver and brain extract, and (b) immunolabeled one band (51,000 daltons) on immunoblots of partially purified fractions from liver. Enrichment of CSD specific activity and that of the protein immunolabeled by the antiserum for a given step, e.g., hydrophobic HPLC, were consistently parallel. The antiserum was used to carry out CSD immunocytochemistry in cerebellum. Numerous small cells were labeled in the Purkinje cell layer, the granular layer, and the white matter. In the molecular layer, Bergmann radial fibers were immunostained. The Purkinje and stellate cells were devoid of any labeling at the cell body and terminal levels. The antiserum appears to be specific for CSD and suitable for immunocytochemical visualization of CSD in the brain.