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1.
To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant. 相似文献
2.
To examine the factor affecting LH-induced progesterone production in ovine luteal slices, an experimental procedure was employed wherein each slice served as its own control. The role of microfilaments in steroidogenesis was studied in luteal slices treated with cytochalasin B (an inhibitor of microfilament function). Cytochalasin B treatment resulted in significant reduction of progesterone production by luteal slices in response to LH and the addition of serum to the medium did not alter this effect. The ability of luteal slices to respond to LH with increased progesterone secretion was restored when cytochalasin B was removed from the medium. Further studies indicated that inhibition of LH-induced progesterone production by treatment with cytochalasin B was not a result of a change in: 1) cyclic adenosine 3'-5'-monophosphate production in response to LH; 2) mitochondrial membrane permeability to cholesterol; or 3) activity of 3β-hydroxysteroid dehydrogenase, Δ5,Δ4-isomerase enzyme complex.The possibility existed that microfilaments were necessary for cholesterol transport to mitochondria in response to LH stimulation. However, mitochondrial cholesterol content was unchanged in response to LH in the presence or absence of aminoglutethimide (an inhibitor of cholesterol side-chain cleavage enzyme activity) as determined by uptake of 3H-cholesterol or total content determined by gas-liguid chromatography. Further, treatment with cytochalasin B had no effect on mitochondrial cholesterol content. These results suggest a role for microfilaments in LH-induced progesterone production at a point prior to the conversion of cholesterol to pregnenolone. 相似文献
3.
When the root-phloem slices ofDaucus carota cv. Hokkaidô-gosun were cultured on a Murashige and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (2,4-D medium) and cyclic AMP or its analogues, tracheary elements were formed in the dark, while they were not formed on the medium containing only 2,4-D in the dark. The number of tracheary elements induced by cyclic AMP was far less than that induced by cytokinin or 8-bromo-cyclic AMP. But when theophylline, an inhibitor of cyclic AMP phosphodiesterase, was used in combination with cyclic AMP in the culture, the number of tracheary elements was significantly increased. A remarkable increase in cytokinin activity was found in the hydrolyzate of soluble RNA extracted from the slices cultured on the 2,4-D medium containing 8-bromo-cyclic AMP, but only negligible cytokinin activity was detected in the hydrolyzate of soluble RNA extracted from the slices cultured on the 2,4-D medium without 8-bromo-cyclic AMP. Since cytokinin production occurred in the slices cultured in the light, it was supposed that light irradiation might induce cyclic AMP production. The mechanism of cytokinin production leading to tracheary element formation mediated by cyclic AMP level is discussed. 相似文献
4.
The rate of -aminobutyric acid (GABA) synthesis in rat-brain slices was determined by inhibiting GABA transaminase with 20-M gabaculine and measuring the increase of GABA. Added 500-M glutamine increased the rate of GABA synthesis by 50%, indicating that glutamate decarboxylase is not saturated in brain slices. The stimulation of GABA synthesis with added glutamine in brain slices was much less than that reported for synaptosomes. The lower stimulation in slices was attributable to astrocytic glutamine production, as the rate of GABA synthesis decreased by 44% when glutamine production was inhibited with methionine sulfoximine. Added glutamine restored the rate to the maximal value observed in brain slices. The rate of GABA synthesis was decreased by 65% in slices pretreated with an inhibitor of glutaminase, and added glutamine did not reverse this effect. These results suggest that glutamine produced by astrocytes is a quantitatively important precursor of GABA synthesis in cortical slices. 相似文献
5.
Pinto MC Mourão FA Binda NS Leite HR Gomez MV Massensini AR Gomez RS 《Neurochemistry international》2012,61(5):713-720
Brain ischemic tolerance is a protective mechanism induced by a preconditioning stimulus, which prepare the tissue against harmful insults. Preconditioning with N-methyl-d-aspartate (NMDA) agonists induces brain tolerance and protects it against glutamate excitotoxicity. Recently, the glycine transporters type 1 (GlyT-1) have been shown to potentiate glutamate neurotransmission through NMDA receptors suggesting an alternative strategy to protect against glutamate excitotoxicity. Here, we evaluated the preconditioning effect of sarcosine pre-treatment, a GlyT-1 inhibitor, in rat hippocampal slices exposed to ischemic insult. Sarcosine (300mg/kg per day, i.p.) was administered during seven consecutive days before induction of ischemia in hippocampus by oxygen/glucose deprivation (OGD). To access the damage caused by an ischemic insult, we evaluated cells viability, glutamate release, nitric oxide (NO) production, lactate dehydrogenase (LDH) levels, production of reactive oxygen species (ROS), and antioxidant enzymes as well as the impact of oxidative stress in the tissue. We observed that sarcosine reduced cell death in hippocampus submitted to OGD, which was confirmed by reduction on LDH levels in the supernatant. Cell death, glutamate release, LDH levels and NO production were reduced in sarcosine hippocampal slices submitted to OGD when compared to OGD controls (without sarcosine). ROS production was reduced in sarcosine hippocampal slices exposed to OGD, although no changes were found in antioxidant enzymes activities. This study demonstrates that preconditioning with sarcosine induces ischemic tolerance in rat hippocampal slices submitted to OGD. 相似文献
6.
1. Sodium dichloroacetate (1mM) inhibited glucose production from L-lactate in kidney-cortex slices from fed, starved or alloxan-diabetic rates. In general gluconeogenesis from other substrates was no inhibited. 2. Sodium dichloracetate inhibited glucose production from L-lactate but no from pyruvate in perfused isolated kidneys from normal or alloxan-diabetic rats. 3. Sodium dichloroacetate is an inhibitor of the pyruvate dehydrogenase kinase reaction and it effected conversion of pyruvate dehydrogenase into its its active (dephosphorylated) form in kidney in vivo. In general, pyruvate dehydrogenase was mainly in the active form in kidneys perfused or incubated with L-lactate and the inhibitory effect of dichloroacetate on glucose production was not dependent on activation of pyruvate dehydrogenase. 4. Balance data from kidney slices showed that dichloroacetate inhibits lactate uptake, glucose and pyruvate production from lactate, but no oxidation of lactate. 5. The mechanism of this effect of dichloroactetate on glucose production from lactate has not been fully defined, but evidence suggests that it may involve a fall in tissue pyruvate concentration and inhibition of pyruvate carboxylation. 相似文献
7.
Cytosolic Proteolysis of τ by Cathepsin D in Hippocampus Following Suppression of Cathepsins B and L
Abstract: Incubation of cultured hippocampal slices with an inhibitor [ N -CBZ- l -phenylalanyl- l -alanine-diazomethyl ketone (ZPAD)] of cathepsins B and L resulted in the degradation of high molecular weight isoforms of τ protein and the production of a 29-kDa τ fragment (τ29 ). A τ antibody that is sensitive to the phosphorylated state of its epitopes did not recognize τ proteins or the τ29 fragment in slices that had been treated with a protein phosphatase inhibitor. This strongly suggests that the τ fragment was located in an extralysosomal compartment accessible to kinases and phosphatases. τ29 exhibited a significant capacity for binding to microtubules and thus has the potential for interfering with normal τ-tubulin interactions. Three lines of evidence indicated that ZPAD-induced τ proteolysis was mediated by cathepsin D: (a) slices treated with the inhibitor had markedly elevated levels of cathepsin D in both lysosomal and extralysosomal compartments; (b) co-incubation of cathepsin D and τ at neutral pH resulted in a loss of intact τ proteins and production of a 29-kDa fragment; and (c) the lysosomotropic drug chloroquine blocked ZPAD-induced increases in mature cathepsin D, and this was accompanied by a suppression of ZPAD-induced τ proteolysis. Changes in lysosomal hydrolases and cytoskeletal perturbations occur during brain aging. The present results suggest that the enzymatic and structural effects are related and, more specifically, are linked by alterations in the concentration and localization of cathepsin D. The τ fragments with microtubule binding capacity generated by cathepsin D could also be a source for the small polypeptides found in association with age-related pathological features. 相似文献
8.
A role for adrenaline and calmodulin in modulating cyclic AMP levels during the lactogenic cycle 总被引:1,自引:0,他引:1
The effect on lactose production of several external modulators of intracellular cyclic AMP was studied in rat mammary gland tissue slices and explants. Adrenaline, a beta-adrenergic receptor effector, forskolin, a direct adenylate cyclase activator and fluphenazine, a calmodulin inhibitor, all produced an increase in the intracellular level of cyclic AMP and a concomitant inhibition of lactose production. These results suggest a role for adrenaline and calmodulin in modulating cyclic AMP levels in mammary tissue during the lactogenic cycle. 相似文献
9.
Regulation of acetylcholine metabolism varied in brain slices from hippocampus and septum which have different proportions of cholinergic nerve cell bodies and nerve endings. Anoxia (0% oxygen) inhibited acetylcholine synthesis (-77%) and its calcium-dependent release (-87%) from hippocampal slices but had no effect on synthesis or release by septal slices. [1,5-14C]Citrate incorporation into acetylcholine was higher in septum than in hippocampus, which suggested that citrate metabolism differs regionally. (-)Hydroxycitrate, a specific inhibitor of ATP citrate (pro3S)-lyase (EC 4.1.3.8), reduced [U-14C]glucose incorporation into acetylcholine more in septal than in hippocampal slices. 14CO2 production from glucose or citrate was similar in control and experimental conditions in the two regions. These findings indicate that acetylcholine metabolism varies regionally, which may partially explain the selective vulnerability of certain brain areas to anoxia and other metabolic insults. 相似文献
10.
Effect of Isozyme-Selective Inhibitors of Phosphodiesterase on Histamine-Stimulated Cyclic AMP Accumulation in Guinea-Pig Hippocampus 总被引:2,自引:0,他引:2
Addition of histamine (0.1 mM) to guinea-pig hippocampal slices causes a 20- to 30-fold increase in the accumulation of cyclic AMP compared with basal levels. This accumulation represents a balance between cyclic AMP production by adenylate cyclase and cyclic AMP breakdown mediated by phosphodiesterase (PDE). However, brain tissues are known to contain several different PDE isozymes. To determine which are involved in this response to histamine, the effect of isozyme-specific PDE inhibitors on cyclic AMP accumulation was examined in the hippocampus. MB 22948 (0.1 mM), an inhibitor of PDEs I and II, had no significant effect on the response to either 1 microM or 0.1 mM histamine. SKF 94120 (0.1 mM), a PDE III inhibitor, was also without effect in the presence of 1 microM histamine, although with 0.1 mM histamine, it caused a weak (1.25-fold compared with control), but statistically significant, enhancement of cyclic AMP accumulation. However, both rolipram (0.1 mM), a PDE IV inhibitor, and 3-isobutyl-1-methylxanthine (0.1 or 1 mM), an inhibitor of all forms of PDE, significantly increased cyclic AMP accumulation (2.8- to 6.5-fold compared with controls), and the relative size of this effect decreased with increasing histamine concentration. It is concluded that PDE IV is the main PDE isozyme involved in cyclic AMP turnover in guinea-pig hippocampal slices responding to histamine. 相似文献
11.
The interaction between lipid peroxidation and prostaglandin synthesis in rabbit kidney-medulla slices. 总被引:1,自引:0,他引:1
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Lipid peroxidation induced by ascorbic acid and Fe2+ was inhibited by mepacrine (phospholipase A2 inhibitor) and aspirin (prostaglandin cyclo-oxygenase inhibitor) in rabbit kidney-medulla slices. Moreover, ascorbic acid and Fe2+ potentiated the inhibitory effect on prostaglandin E2 formation by mepacrine, but they had no influence on prostaglandin E2 production decreased by aspirin. Lipid peroxidation induced by ascorbic acid and Fe2+ appears to be affecting the activity of prostaglandin endoperoxide synthase. These results suggest that lipid peroxidation is connected closely with the prostaglandin-generating system, and it has the potential to modulate the turnover of arachidonic acid and prostaglandin synthesis. 相似文献
12.
The Relation between Photosynthesis, Respiration, and Crassulacean Acid Metabolism in Leaf Slices of Aloe arborescens Mill 总被引:6,自引:5,他引:1
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Leaves and leaf slices from Aloe arborescens Mill. were used to study the interrelations between Crassulacean acid metabolism, photosynthesis, and respiration. Oxygen exchange of leaf slices was measured polarographically. It was found that the photosynthetic utilization of stored malic acid resulted in a net evolution of oxygen. This oxygen production, and the decrease in acid content of the leaf tissue, were completely inhibited by amytal, although the rate of respiratory oxygen uptake was hardly affected by the presence of this inhibitor of mitochondrial electron transport. Other poisons of respiration (cyanide) and of the tricarboxylic acid cycle (trifluoroacetate, 2-diethyl malonate) also were effective in preventing acid-dependent oxygen evolution. It is concluded that the mobilization of stored acids during light-dependent deacidification of the leaves depends on the operation of the tricarboxylic acid cycle and of the electron transport of the mitochondria. 相似文献
13.
Adenosine, through activation of its A(1) receptors, has neuroprotective effects during hypoxia and ischemia. Recently, using transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1), we reported that nucleoside transporter-mediated release of adenosine from neurons was not a key mechanism facilitating the actions of adenosine at A(1) receptors during hypoxia/ischemia. The present study was performed to test the importance of CD73 (ecto-5'-nucleotidase) for basal and hypoxic/ischemic adenosine production. Hippocampal slice electrophysiology was performed with CD73(+/+) and CD73(-/-) mice. Adenosine and ATP had similar inhibitory effects in both genotypes, with IC(50) values of approximately 25 μM. In contrast, ATP was a less potent inhibitor (IC(50) = 100 μM) in slices from mice expressing hENT1 in neurons. The inhibitory effects of ATP in CD73(+/+) and CD73(-/-) slices were blocked by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and were enhanced by the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI), consistent with effects that are mediated by adenosine after metabolism of ATP. AMP showed a similar inhibitory effect to ATP and adenosine, indicating that the response to ATP was not mediated by P2 receptors. In comparing CD73(-/-) and CD73(+/+) slices, hypoxia and oxygen-glucose deprivation produced similar depression of synaptic transmission in both genotypes. An inhibitor of tissue non-specific alkaline phosphatase (TNAP) was found to attenuate the inhibitory effects of AMP and ATP, increase basal synaptic activity and reduce responses to oxygen-glucose deprivation selectively in slices from CD73(-/-) mice. These results do not support an important role for CD73 in the formation of adenosine in the CA1 area of the hippocampus during basal, hypoxic or ischemic conditions, but instead point to TNAP as a potential source of extracellular adenosine when CD73 is absent. 相似文献
14.
Lactate production by liver slices from fetal rats (17th--18th day of gestation) is enhanced about two fold by aminooxyacetate, an inhibitor of aspartate transaminase (EC 2.6.1.1). Such an effect is consistent with an increase of the cytosolic NAD-redox state owing to the parallel fall in the pyruvate level, whereas the glycolytic flux does not seem to be influenced appreciably. Indeed, although the inhibitor causes a marked increase of fructose 1,6-diphosphate, glucose-6-phosphate decreases only slightly. These results suggest that in fetal rat liver the malate-aspartate shuttle is operative in the reoxidation of cytosolic NADH produced during aerobic glycolysis. 相似文献
15.
Lactate production by liver slices from fetal rats (17th–18th day of gestation) is enhanced about two fold by aminooxyacetate, an inhibitor of aspartate transaminase (EC 2.6.1.1). Such an effect is consistent with an increase of the cytosolic NAD-redox state owing to the parallel fall in the pyruvate level, whereas the glycolytic flux does not seem to be influenced appreciably. Indeed, although the inhibitor causes a marked increase of fructose 1,6-diphosphate, glucose 6-phosphate decreases only slightly. These results suggest that in fetal rat liver the malate-aspartate shuttle is operative in the reoxidation of cytosolic NADH produced during aerobic glycolysis. 相似文献
16.
Glucose Deprivation Results in a Lactate Preventable Increase in Adenosine and Depression of Synaptic Transmission in Rat Hippocampal Slices 总被引:4,自引:1,他引:3
John C. Fowler 《Journal of neurochemistry》1993,60(2):572-576
Abstract: The effect of glucose deprivation on adenosine levels and on synaptic transmission was investigated in rat hippocampal slices. Incubation of hippocampal slices either in glucose-free medium or in the presence of the glucose transport inhibitor cytochalasin B (50 μ M ) increased bath adenosine levels and depressed the extracellularly recorded synaptic potential or population spike. The addition of lactate (10 m M ), a precursor for mitochondrial ATP generation, prevented the elevation in adenosine and the depression of the population spike. These results indicate that the neuroinhibitory modulator adenosine is elevated during glucose deprivation and contributes to the hypoglycemic depression of synaptic transmission. The increase in adenosine during glucose deprivation can be prevented by providing substrate for mitochondrial ATP generation. The present results indicate an interaction between lactate and adenosine such that an increase in lactate may contribute to a decline in adenosine production. 相似文献
17.
Georgios I Panoutsopoulos Christine Beedham 《Cellular physiology and biochemistry》2005,15(1-4):89-98
BACKGROUND/AIMS: The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared liver slices has not been previously reported. The present investigation compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activities in the oxidation of vanillin, isovanillin and protocatechuic aldehyde with freshly prepared liver slices. METHODS: Vanillin, isovanillin or protocatechuic aldehyde was incubated with liver slices in the presence/absence of specific inhibitors of each enzyme, followed by HPLC. RESULTS: Vanillin was rapidly converted to vanillic acid. Vanillic acid formation was completely inhibited by isovanillin (aldehyde oxidase inhibitor), whereas disulfiram (aldehyde dehydrogenase inhibitor) inhibited acid formation by 16% and allopurinol (xanthine oxidase inhibitor) had no effect. Isovanillin was rapidly converted to isovanillic acid. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. Protocatechuic aldehyde was converted to protocatechuic acid at a lower rate than that of vanillin or isovanillin. Allopurinol only slightly inhibited protocatechuic aldehyde oxidation, isovanillin had little effect, whereas disulfiram inhibited protocatechuic acid formation by 50%. CONCLUSIONS: In freshly prepared liver slices, vanillin is rapidly oxidized by aldehyde oxidase with little contribution from xanthine oxidase or aldehyde dehydrogenase. Isovanillin is not a substrate for aldehyde oxidase and therefore it is metabolized to isovanillic acid predominantly by aldehyde dehydrogenase. All three enzymes contribute to the oxidation of protocatechuic aldehyde to its acid. 相似文献
18.
G D Van Rossum 《Biochimica et biophysica acta》1976,423(1):111-121
1. In slices of rat liver, oligomycin inhibited the net transport of Na+ and K+ by a maximum of 30% and endogenous respiration by 25%. These effects were not increased by a number of modifications in the incubation conditions. 2. Mitochondria isolated from the slices after incubation showed respiratory control ratios that were somewhat less than in mitochondria from fresh liver, but state 3 respiration retained normal sensitivity to oligomycin. 3. Low concentrations of oligomycin or cyanide reduced respiration and ATP levels of the slices but did not affect ion transport unless these levels fell below a definite critical value. In contrast, ouabain and atractyloside each caused substantial degrees of transport inhibition at ATP levels which were in excess of the critical value. 4. High concentrations of cyanide and oligomycin reduced ATP contents maximally by 90% and 65%, respectively. Studies of lactate production, and of the effects of arsenite on respiration and ATP levels, suggested that substrate-level phosphorylation in the citric-acid cycle was the major source of the oligomycinresistant ATP synthesis. 5. The results suggest that oligomycin acts in the liver slices primarily as an inhibitor of oxidative phosphorylation, and that this is the cause of the partial inhibition of ion transport. The oligomycin-resistant ion-transporting activity is consistent with the persisting level of ATP synthesis. 相似文献
19.
Increasing oxygen from 5 to 95% has previously been shown to increase prostaglandin (PG) production in renal inner medullary slices. The possible role of oxidative phosphorylation in this process was investigated. The oxidative phosphorylation inhibitors, dinitrophenol (DNP), oligomycin, and cyanide were evaluted for their effects on PGE2 production and ATP levels. None of the inhibitors affected PGE2 synthesis, although they lowered ATP levels at the concentrations tested. In contrast, incubation of inner medullary tissue slices with 0% oxygen resulted in decreases both in PGE2 and ATP levels. This suggests that the effect of oxygen on prostaglandin synthesis may be due to substrate limiting effects rather than an effect on oxidative phosphorylation. When 22 mM 2-deoxyglucose was added to the incubation medium or when glucose was omitted, PGE2 levels increased. Sodium fluoride, presumably acting as a glycolytic inhibitor, increased PGE2 levels, with a maximal effect at 10 mM. ATP levels were 37% of control values with 20 mM NaF. This indicates that glucose may inhibit prostaglandin synthesis. These results indicate that oxygen (substrate) availability can limit inner medullary PGE2 production. In view of the low pO2 in the inner medulla, especially during antidiuresis, oxygen can potentially regulate prostaglandin production in this tissue. 相似文献
20.
Glycine, a photorespiratory intermediate, enhanced the in vivo reduction of nitrate in barley (Hordeum vulgare L.) leaf slices, when included in the assay medium. Isonicotinyl hydrazide, an inhibitor of glycine oxidation, partially reduced NO2− production. The enhancement caused by glycine treatment was reversed by isonicotinyl hydrazide when both were present together in the medium. Similar effects were observed when the excised leaves were preincubated with the metabolite and the inhibitor. Glycine also partially relieved the inhibition of nitrate reduction caused by malonate, an inhibitor of the tricarboxylic acid cycle. The results support the hypothesis that glycine decarboxylation activity is a source of NADH for nitrate reductase activity. 相似文献