Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Effect of intracellular ATP on Cl- current (ICl) mediated by the GABA (gamma-aminobutyric acid) receptor subtype, GABAA, was studied in dissociated nucleus tractus solitarii (NTS) neurones using the whole-cell mode of patch clamp. A concentration-jump technique termed 'Y tube' was used to rapidly apply agents externally. Dissociated neurones were obtained from 1- to 3-week-old rats. 2. When the patch-pipette solution contained 2 mM-ATP, the amplitude of ICl elicited by 10(-5) M-GABA did not show any time-dependent decrease (apparent run-down), for more than 60 min after the initial recording. In the presence of ATP, the half-maximum concentration (KD) and Hill coefficient calculated from the GABA concentration-response curve were 9.12 microM and 1.47, respectively. 3. In the absence of intracellular ATP, the amplitude of GABA-induced ICl decreased with time. The relative peak amplitudes after 20 and 60 min from the initial recording were 0.40 +/- 0.09 (n = 11) and 0.16 +/- 0.05 (n = 8) with respect to the initial response. 4. Removal of Mg2+ from the internal solution induced run-down of the GABA response even in the presence of 2 mM-intracellular ATP, suggesting that both intracellular ATP (2 mM or more) and Mg2+ are necessary to prevent run-down of the GABA response. 5. Activation of dephosphorylation processes by alkaline phosphatase (100-200 microM) did not affect the GABA response in neurones perfused with internal solution containing 2 mM-ATP and 3 mM-Mg2+. Blocking the dephosphorylation process by okadaic acid, a phosphatase inhibitor, did not prevent the run-down of the GABA response. 6. Calcium influxes passing through both the voltage-dependent L-type Ca2+ channel and the glutamate receptor-operated cation channel did not affect ICl induced by GABA. 7. GABA-induced ICl was also maintained by adding 2 mM-ADP or ATP gamma S (adenosine-5'-O-3-thiotriphosphate) to the internal solution containing Mg2+. Addition of 2 mM-adenosine, AMP, cyclic AMP, AMP-PNP (adenylimido-diphosphate) or ADP beta S (adenosine-5'-O-2-thiodiphosphate) to the internal solution did not prevent the run-down of the GABA response even in the presence of 3 mM-intracellular Mg2+. Based on the chemical specificity of these ATP analogues, it is suggested that there is an ATP-sensitive binding site (ATP receptor) in the cytoplasmic side of the cell membrane.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Direct modulation of GABAA receptor by intracellular ATP in dissociated nucleus tractus solitarii neurones of rat. 138 52

In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed.
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PMID:Antibodies against neuroactive amino acids and neuropeptides. II. Simultaneous immunoenzymatic double staining with labeled primary antibodies of the same species and a combination of the ABC method and the hapten-anti-hapten bridge (HAB) technique. 170 57

1. Current mediated by GABAA receptors was examined in pyramidal cells acutely dissociated from the hippocampus of mature guinea-pigs. Current responses were measured using whole-cell voltage-clamp recordings. An internal perfusion technique was used to change the intracellular contents during recording. 2. Application of GABA (100-300 microM) by short duration pressure pulses produced outward current responses at a holding potential of -10 mV. When recordings were made with intracellular solutions which did not contain Mg-ATP, GABA responses progressively decreased to less than 10% of their initial values after 10 min. This 'run-down' of the GABA response could not be accounted for by desensitization since the rate of run-down was not dependent upon agonist application. 3. The run-down of the GABAA response was reversed when Mg2+ (4 mM) and ATP (2 mM) were introduced into the intracellular perfusate. In addition to the presence of Mg-ATP, buffering of Ca2+ in the intracellular solution to low levels (approximately 10(-8) M) was also necessary to stabilize the GABAA response. 4. The role of a phosphorylation process in regulating the GABAA receptor was tested. After the GABA response stabilized, introduction of alkaline phosphatase (100 micrograms/ml) to the intracellular perfusate caused a complete run-down of the GABA response. 5. Stable GABA responses were obtained when ATP was replaced by ATP-gamma-S (adenosine 5'-O-(thiotriphosphate), an analogue of ATP that donates a thiophosphate group resulting in a product that is more resistant to hydrolysis. Following such treatment GABA responses declined more slowly after the introduction of intracellular alkaline phosphatase. 6. Run-down of GABA responses accelerated when intracellular Ca2+ concentration ([Ca2+]i) was elevated to about 5 x 10(-4) M. The run-down caused by elevated [Ca2+]i could be stopped and reversed by reducing [Ca2+]i to about 10(-8) M. 7. The introduction of ATP-gamma-S to the intracellular medium retarded the run-down of GABA responses caused by elevation of [Ca2+]i. 8. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), a calmodulin inhibitor, reduced the rate of run-down induced by elevated [Ca2+]i. 9. These results suggest that the function of the GABAA receptor is maintained by phosphorylation of the receptor or some closely associated regulatory molecule. Elevation of [Ca2+]i destabilizes the function of the GABAA receptor, probably by activating a Ca2+/calmodulin-dependent phosphatase.
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PMID:GABAA receptor function is regulated by phosphorylation in acutely dissociated guinea-pig hippocampal neurones. 215 38

An investigation of several neurochemical consequences of exposure of the rat to 3/4 of the estimated single injection LD50 quantity of trimethyltin chloride (TMT) indicated that a significant elevation in the levels of glutamine (Gln) and 5-hydroxyindole acetic acid (5-HIAA) occurred at post-dosing day 7 in each examined region of the brain; elevated Gln persisted in the hippocampus through day 14 and returned to control levels at day 28. At post-dosing day 7, levels of glutamate were decreased in the hippocampus, while levels of GABA were decreased in hippocampus and frontal cortex, but not in corpus striatum; hippocampal glutamate and GABA returned to control levels by post-dosing day 14. Decreased levels of taurine (Tau) occurred on day 7 in both hippocampus and frontal cortex; hippocampal Tau remained below control levels through post-dosing day 28. Levels of other amino acids and of amines and amine metabolites were not altered by TMT in the 7 to 28 day post-dosing interval. At day 7, TMT treatment did not alter brain regional activities of glutamine synthetase; however, plasma ammonia was elevated 100% above the control value. Alterations in several serum enzymes (esp., alkaline phosphatase and aspartate aminotransferase) revealed several other peripheral consequences of TMT exposure which persist through post-dosing day 28. The more prominent and wide-spread neurochemical alterations resulting from TMT exposure appear to reflect consequences of hyperammonemia resulting from a peripheral effect of the organotin compound.
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PMID:Trimethyltin-induced alterations in brain amino acids, amines and amine metabolites: relationship to hyperammonemia. 243 91

Placental transport of taurine was studied in isolated brush border microvillous plasma membrane vesicles by a rapid filtration technique. Brush border microvillous plasma membrane vesicles were prepared from syncytio trophoblast of human term placenta by a method of differential centrifugation and calcium precipitation. The specific activities of alkaline phosphatase, 5' nucleotidase and gamma-GTP in the membrane preparation were enriched to 13-14 times, 12-13 times, and 5-6 times respectively, as high as those in the homogenate. The membrane vesicles exhibit uptake of 3H-labeled taurine into an osmotically reactive intravesicular space. Taurine uptake by vesicles was stimulated specifically by an inward sodium gradient, and replacement of NaCl in the transport medium by KCl, LiCl, and choline chloride had no effect on the transport activity of the vesicles. Taurine transport is inhibited competitively by the presence of beta alanine and GABA. The initial rate of transport followed saturation kinetics with respect to the taurine concentration: An apparent Km of 0.22mM and Vmax of 67 pmol/mg protein were calculated in 20 seconds. These results indicate that transport of taurine across the placental brush border membrane is sodium dependent and carrier mediated.
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PMID:[Placental transport of taurine in brush border microvilli]. 608 91

We have studied a large Mennonite kindred in which 20 members were affected with Hirschsprung disease (HSCR), 5 of whom had one or more manifestations of Waardenburg syndrome (WS) type II (WS2). Eleven additional relatives had signs of WS2 without HSCR. Since HSCR and WS2 each represent perturbations of neural crest migration/differentiation, this large pedigree with apparent cosegregation of HSCR and WS2 offered an opportunity to search for linkage between these loci, candidate genes, and random DNA markers, particularly in view of recent discoveries of genes for Waardenburg syndrome type I (WS1) and Hirschsprung disease (c-ret). We have examined the following possible linked markers in 69 relatives in this family: the c-ret gene (HSCR); the human PAX3 gene (HuP2) on chromosome 2q (WS1) and placental alkaline phosphatase (ALPP) on chromosome 2q (linked to WS1); argininosuccinate synthetase (ASS) on chromosome 9q, close to ABO blood groups which have shown weak linkage to WS; and the beta 1 GABA receptor gene (GABARB1) on chromosome 4q13-11, close to c-kit, deletions of which cause piebaldism. Linkage between any of these loci and HSCR/WS in this kindred was excluded, demonstrating that there is at least one further locus for HSCR other than c-ret.
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PMID:Second locus for Hirschsprung disease/Waardenburg syndrome in a large Mennonite kindred. 780 41

1. Endosulfan insecticide is a polychlorinated compound used for controlling a variety of insects; it is practically water-insoluble, but readily adheres to clay particles and persists in soil and water for several years. Its mode of action involves repetitive nerve-discharges positively correlated to increase in temperature. This compound is extremely toxic to most fish and can cause massive mortalities. In fish, it causes marked changes in Na and K concentrations, decrease in blood Ca(2+) and Mg levels and inhibits Na, K and Mg-dependent ATPase (in brain). 2. Bioaccumulation of endosulfan is reported for marine animals; however, freshwater animals (e.g., crayfish) accumulate it to some extent, but they lose the compound rapidly during depuration. Endosulfan is generally less toxic to aquatic invertebrates than fish. However, it causes decreases in adenylate energy charge, oxygen consumption, hemolymph amino acids, succinate dehydrogenase, heart-beat (mussel) and altered osmoregulation. 3. Generally, mammals are less susceptible to endosulfan's toxicity than aquatic animals. The majority of studies conducted on laboratory mammals can be summarized. (a) Neurotoxicity: male rats are more sensitive than females to endosulfan, which decreases brain and plasma acetylcholinesterase activity. Endosulfan I (a metabolite) causes a significant change in norepinephrine, 5-HT and GABA. (b) Renal toxicity: inhibition of MFOs activity was noticed in rats; other effects included changes in proximal convoluted tubules and necrosis of the tubular epithelium. (c) Hepatotoxicity: chemically-induced aminopyrine N-demethylase and aniline hydrolase were found in rat liver, and reduction in the glycogen level occurred. (d) Hematologic toxicity: endosulfan exposure resulted in a significant decrease in the level occurred. (d) Hematologic toxicity: endosulfan exposure resulted in a significant decrease in the erythrocyte glutathione reductase, hemoglobin amount, RBC number and mean corpuscular volume. 4. Respiratory toxicity: involved dyspnea, acute emphysema, cyanosis and hemorrhages in teh interalveolar portions of rat's lungs. 5. Biochemical: in rats, endosulfan caused increased glucose-6-phosphate dehydrogenase activity, blood glucose level, phospholipid contents of the microsomal and surfactant system, and profoundly induced the activity of alcohol dehydrogenase and cytosolic glutathione S-transferases. It also decreased significantly Na+, K+ and Mg(2+) ATPases, plasma calcium level and alkaline phosphatase in the intestinal epithelium. 6. Immunologic toxicity: rat serum antibody titer to tetanus toxin, IgG, IgM and gammaglobulins were significantly reduced. 7. Reproductive toxicity: degenerative changes in the seminiferous epithelium, induction of the rate-limiting enzyme in testosterone production (3beta-hydroxysteroid transferase and 17 beta-hydroxysteroid transferase), histological changes in reproductive organs, testicular atrophy and the occurrence of ovarian cysts were noticed in rat. Reduction in the weight of secondary sex organ was also observed.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Bioaccumulative potential and toxicity of endosulfan insecticide to non-target animals. 790 Sep 59

The cellular co-expression of adenosine A2a receptor mRNA and preproenkephalin A (PPE A) mRNA and A2a receptor mRNA and prosomatostatin (pSRIF) mRNA in rat striatum was studied using a combination of radioactive and non-radioactive in situ hybridization techniques. Cells containing adenosine A2a receptor mRNA were visualised using an 35S-labelled oligonucleotide whilst those containing PPE A mRNA and pSRIF mRNA were detected using alkaline phosphatase-labelled antisense oligonucleotides; both radioactive and non-radioactive hybridization signals were visualized on the same tissue section. Bright field examination of striatal sections hybridized with both the [35S]adenosine A2a receptor probe and the alkaline phosphatase-labelled PPE A probe revealed dense clusters of silver grains overlying cells containing alkaline phosphatase reaction product demonstrating that the two gene transcripts were expressed by the same medium-sized nerve cells. The cellular expression of the two mRNAs was consistently found to be concordant demonstrating that adenosine A2a receptor mRNA is expressed by medium-sized striatal enkephalin cells. In contrast, clusters of silver grains were never detected overlying striatal cells containing pSRIF mRNA indicating that this population of interneurones do not express the adenosine A2a receptor sub-type. The expression of adenosine A2a receptors by enkephalin cells in striatum suggests that adenosine may play a role in modulating the activity of GABA/enkephalin striatopallidal neurones through interaction with A2a receptors.
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PMID:Adenosine A2a receptor mRNA is expressed by enkephalin cells but not by somatostatin cells in rat striatum: a co-expression study. 791 1

Pharmacological evidence suggests that GABA may play an important role in regulating the secretory and synthetic activity of the hypothalamic periventricular somatostatin (SOM) neurones controlling growth hormone secretion. In this study, we have utilized a dual labelling in situ hybridization technique to examine whether the alpha 2 sub-unit of the GABAA receptor, which is abundant in this region, is expressed by periventricular SOM neurones. Neurones expressing SOM were detected using an alkaline phosphatase-labelled antisense oligonucleotide and the alpha 2 sub-unit with an 35S-labelled antisense oligonucleotide. Hybridization experiments with the alpha 2 sub-unit probe alone confirmed the high level of mRNA expression for this sub-unit in the rat periventricular region and simultaneous hybridization experiments with both probes revealed that > 90% (93 +/- 2%) of periventricular SOM neurones express the alpha 2 sub-unit of the GABAA receptor. These results provide the first direct evidence that periventricular SOM cells possess GABAA receptors and suggest that the great majority of these neurones synthesize a GABAA receptor containing the alpha 2 sub-unit.
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PMID:Expression of GABAA receptor alpha 2 sub-unit mRNA by periventricular somatostatin neurones in the rat hypothalamus. 793 31

Gamma-aminobutyric acid (GABA) is known to inhibit the electrical and secretory activity of oxytocin and vasopressin neurones located in the supraoptic and paraventricular nuclei following osmotic, cardiovascular or suckling stimuli. To understand fully the nature of GABA actions on these magnocellular neurones it is important to define the heteropentameric GABAA receptor proteins they express. In the present study, single and dual labelling in situ hybridisation and immunocytochemical experiments were undertaken to define the GABAA receptor gamma subunits expressed by these cells. In situ hybridisation with 35S-labelled antisense oligonucleotides showed that all magnocellular neurones in the supraoptic and paraventricular nuclei of the female rat expressed mRNA encoding the gamma 2 subunit of the GABAA receptor but not the gamma 1 or gamma 3 subunits. Immunocytochemical experiments using a specific polyclonal rabbit antibody directed against the gamma 2 subunit of the GABAA receptor showed that all hypothalamic magnocellular neurones were strongly immunoreactive for gamma 2 subunit protein. Dual in situ hybridisation experiments using the gamma 2 subunit 35 S-labelled oligonucleotide with alkaline phosphatase-labelled antisense oligonucleotides specific for either oxytocin or vasopressin revealed that essentially all oxytocin and vasopressin neurones in both the supraoptic and paraventricular nuclei expressed the gamma 2 subunit of the GABAA receptor. Similarly, sequential double immunoperoxidase staining revealed that all oxytocin and vasopressin neurones in both magnocellular nuclei of the hypothalamus were immunoreactive for the gamma 2 subunit. This study shows that only the gamma 2 subunit of the GABAA receptor gamma subunit family is expressed by hypothalamic oxytocin and vasopressin neurones. In conjunction with our previous results, these findings indicate that individual magnocellular neurones express a complement of alpha 1, alpha 2, beta 2, beta 3 and gamma 2 subunits of the GABAA receptor. The observation of strong gamma 2 subunit expression by neurones known to also express alpha 1 and alpha 2 subunit proteins suggests that these magnocellular cells may express GABAA receptors with both benzodiazepine type-1 and type-2 pharmacology.
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PMID:Characterisation of GABAA receptor gamma subunit expression by magnocellular neurones in rat hypothalamus. 875 Aug 60


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