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1.
Abstract— The effects of hyperosmolal superfusion upon the release of preloaded, radio-labeled GABA has been studied, using both first cortical and first pontine brain slices. GABA release was stimulated with either hyperosmolal Na+ or sucrose superfusion in cortical slices. This stimulated release of radio-labeled GABA was partially Ca2+-dependent in cortical slices. When barium ions replaced Ca2+ in hyperosmolal medium, a similar effect was seen. High concentration of magnesium in Ca2+ -free hyperosmolal medium did not induce stimulation. The increased release of α-aminoisobutyric acid (AIBA), a non-metabolized amino acid induced by hyperosmolality, was not Ca2+-dependent.
GABA release was also stimulated with hyperosmolal sucrose superfusion in pontine slices. The effect of pre-treatment of cortical and pontine slices with β-alanine or L-2,4-diaminobutyric acid (DABA) was used to study the source of exogenous GABA release induced by hyperosmolality. In cortical slices, β-alanine blocked the hyperosmolal release of GABA and also slightly inhibited GABA uptake. DABA did not change hyperosmolal GABA release, although it inhibited GABA uptake. In pontine slices, both DABA and β-alanine inhibited GABA uptake, but were unable to inhibit the hyperosmolal release of GABA.
The data suggest that hyperosmolality causes increased release of GABA from neurons, analogous to that seen with K+-depolarization. AIBA, unlike GABA, is released from brain cells as a non-Ca2+ -dependent response to osmotic equilibration. The observation that pre-treatment with β-alanine inhibits the hyperosmolal release of GABA suggests that hyperosmolality alters glial cell function.  相似文献   

2.
Abstract: The effects of nitric oxide (NO)-generating agents on 45Ca2+ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na+-Ca2+ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca2+ uptake in the slices, although it did not affect high K+-stimulated Ca2+ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca2+ uptake, but it did not affect high K+-stimulated Ca2+ uptake. 8-Bromocyclic GMP, but not SNP, increased Na+-dependent Ca2+ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca2+ uptake in the presence of ouabain and monensin, which were required for the Ca2+ uptake in the cells. These findings suggest that NO stimulates the Na+-Ca2+ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.  相似文献   

3.
Experiments were conducted to test the possibility that organic amines inhibit ethylene production by inhibiting transport of the ethylene precursor, 1-aminocyclopro-pane-1-carboxylic acid (ACC), into the vacuole. α-Aminoisobutyric acid (αAIB) was used as a model substrate to study ACC uptake into the vacuole in relationship to ethylene production in pericarp slices of Lycopersicon esculentum Mill. cv. Liberty treated with and without organic amines and related substances. Organic amines (polyamines and other basic amines) inhibited αAIB uptake into the vacuole. These amines also enhanced ACC accumulation in the tissue and reduced the passive efflux of αAIB from the vacuole. Overall, ethylene production was inhibited. The inhibition of αAIB transport and of ethylene production followed a polyvalent cationic progression in the order polyamines > diamines> basic 1-amino acids. Ca2+, but not Mg2+, strongly stimulated αAIB uptake into the vacuole and ethylene production. At equal concentrations, Ca2+ counteracted the inhibitory effects of polyamines on both αAIB uptake and ethylene production. Competitive and irreversible inhibitors of polyamine biosynthesis stimulated αAIB uptake into the vacuole and ethylene production. The results indicate an apparent relationship between polyamines, ACC uptake into the vacuole and ethylene production.  相似文献   

4.
Thapsigargin (Tg), an inhibitor of microsomal Ca2+ ATPase, is used as a tool to study the changes in Ca2+ sequestration in sea urchin eggs and their relationship to embryonic development. Micromolar amounts of Tg inhibit ATP-dependent Ca2+ sequestration in a dose-dependent and non-reversible manner, depending on the bulk of biological material used. IC5O values are 1 nmol/L and 1–10μmol/L, respectively, in the cortical Ca2+ stores (isolated cortices preparation) and in digitonin-permeabilized eggs, a preparation giving access to the deeper reticulum compartment. Micromolar Tg does not induce Ca2+ release from 45Ca pre-loaded cortices but leads to a loss of 25% of the total Ca2+ content from the cortical area. Using microspectrofluorimetry of fura-2-loaded eggs, we found that 10 μmol/L Tg induced a moderate rise in cytosolic Ca2+ activity as compared with the fertilization-induced Ca2+ transient whether eggs were fertilized or not. Early events related to fertilization as, for example, elevation of the fertilization envelope, proton excretion and sustained increase of amino acid uptake, are triggered by 10μmol/L Tg but with a delayed onset relative to sperm-induced effects. The present findings indicate that although it triggers most fertilization-related events, Tg cannot be considered as a true mitotic agent in sea urchin eggs. When added after fertilization, Tg affects cleavage and the further embryonic development giving rise to abnormalities comparable to the animalized larvae obtained with other compounds responsible for the inhibition of reticular Ca2+ sequestration.  相似文献   

5.
Abstract: The uptake of Ca2+ by a K+-depolarized rat brain cerebral cortical crude synaptosomal preparation (P2 fraction) was investigated. The characteristics of the Ca2+ uptake system are similar to those observed by other investigators. The preparation is also a suitable model with which to study the effects of adenosine on Ca2+ uptake and neurotransmitter release, as it is generally accepted that K+-evoked Ca2+ uptake is intimately related to depolarization-induced release of neurotransmitters. We have demonstrated that an extracellular receptor is involved in mediating the adenosine-evoked inhibition of K+-evoked Ca2+ uptake. The pharmacological properties of the receptor suggest that it may be similar in some respects to the A2-receptor associated with adenylate cyclase. The adenosine uptake inhibitor, dipyridamole, potentiated the action of adenosine, suggesting that re-uptake is important in controlling the extracellular adenosine concentration and thus in the regulation of the adenosine receptor. The adenosine receptor antagonist theophylline inhibited the effects of adenosine. Calmodulin inhibited K+- evoked uptake of Ca2+ by the synaptosomal fraction.  相似文献   

6.
UPTAKE AND RELEASE OF TAURINE FROM RAT BRAIN SLICES   总被引:13,自引:8,他引:5  
Abstract— Rapid efflux of [35S]taurine from rat brain slices was observed on electrical stimulation. Slower release resulted when the Ca2+ content of the perfusion medium was replaced with Mg2+. Uptake of [35S]taurine into rat cortical slices was unaffected by GABA, glutamic acid, glycine and leucine but was inhibited by alanine, ouabain, KCN and 2,4-dinitrophenol. Of a number of analogues of taurine, 2-aminoethylsulphinic acid was the most potent in inhibiting the uptake of [35S]taurine. The rate of uptake was found to be decreased by lowering the incubation temperature. The possibility that taurine may be a neurotransmitter is discussed.  相似文献   

7.
Abstract— Saxitoxin and tetrodotoxin at low concentrations (10−7-10−8 M) exerted similar inhibitory effects on the increase in lactate production and the redistrjbution of Na+ and K+ that normally accompany electrical stimulation of rat cerebral cortical slices. In contrast, the toxins exerted dissimilar effects on the production of lactate in response to low concentrations of Ca2+ in the medium. Inhibition by tetrodotoxin occurred at a higher concentration of Ca2+ and was significantly greater than that produced by saxitoxin at concentrations of Ca2+ below 0.75 mM. These differences were not related to differential effects on the redistribution of Na+ and K+ under such conditions. The toxins had different effects on Ca2+ influx. Tetrodotoxin, but not saxitoxin, inhibited the influx of Ca2+ in the absence of electrical stimulation. The influx of Ca2+ increased when electrical pulses were applied and tetrodotoxin inhibited this increase, whereas saxitoxin potentiated influx of Ca2+ during stimulation. Our results suggest that metabolic responses to conditions that increase excitability are not governed solely by changes in the distribution of Na+ and K+. The differential effects of the toxins on Ca2+ fluxes suggest that one site of Ca2+ entry during electrical stimulation may be functionally independent of Na+ entry.  相似文献   

8.
Abstract— The rate of efflux of 45Ca2+ from slices of rat cerebral cortex was resolved into two exponential curves which were attributed to an extracellular component and an intracellular or bound component. Electrical stimulation increased efflux of 45Ca2+ from the more stable pool and the time course for the redistribution of Na+ and K+ paralleled that for the increased efflux of Ca2+. This effect of stimulationwas dependent on the presence of Na+ in the incubation medium. Lack of Na+ in the medium during loading of the slices with 45Ca2+ increased uptake but on subsequent transfer to a medium containing Na+, electrical pulses failed to increase the rate of efflux of 45Ca2+. In unstimulated slices, the rate of efflux of 45Ca2+ was dependent upon the concentration ratio of Na+ to Ca2+ in the incubation medium. Saxitoxin and tetrodotoxin inhibited the increased efflux of 45Ca2+ that occurred during electrical stimulation but exerted no effect on Ca2+-Ca2+ exchange. Our results suggest that there is a Na+-dependent turnover of Ca2+ in brain slices which may involve changes in affinity at a common binding site. The possible involvement of such a Na+-Ca2+ interaction in the regulation of neurotransmitter function is discussed.  相似文献   

9.
Abstract: The activation of phosphoinositide hydrolysis by ibotenate (IBO) in brain slices and the binding of N -[3H]acetylaspartyl-L-glutamate (NAAG) to brain membranes are biochemical parameters previously shown to be selectively inhibited by 2-amino-4-phosphonobutyrate (AP4). We have examined whether the binding of [3H]NAAG and stimulation of phosphoinositide hydrolysis by IBO are indexing the same or different populations of AP4-sensitive excitatory amino acid sites in brain. L-AP4 and D,L-2-amino-3-phosphono-propionate (D,L-AP3) were found to be about equipotent inhibitors of IBO-stimulated phosphoinositide hydrolysis. L-AP4 and D,L-AP3 did not inhibit stimulation of phosphoinositide hydrolysis by the cholinoceptor agonist carbachol. The L-isomers of serine- O -phosphate and α-aminoadipate were selective inhibitors of IBO-stimulated phosphoinositide hydrolysis, but were less potent than L-AP4 or D,L-AP3. When these compounds were examined for their ability to inhibit [3H]NAAG binding to membranes of rat forebrain, the relative order of potency was L-α-aminoadipate = D-α-aminoadipate < L-AP4 < L-serine- O -phosphate < D-AP4 < D,L-AP3. Concentrations of NAAG up to 10−2 M did not stimulate phosphoinositide hydrolysis. Thus, although both assays are sensitive to L-AP4 inhibition, they appear to represent disparate excitatory amino acid sites in brain. Furthermore, D,L-AP3 appears to be a more selective inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis than L-AP4, and might be a more useful pharmacological tool to define the function of these receptor sites in brain.  相似文献   

10.
Abstract: In rat hippocampal slices and in neurons in primary culture, K+-induced depolarization increased markedly and rapidly tyrosine phosphorylation of a 110-kDa protein (pp110) and, to a lesser degree, of a 120-kDa protein (pp120), in a calcium-dependent fashion. Qlutamate, 1-aminocyclopentane- trans -1,3-dicarboxylic acid (an agonist of metabotropic glutamate receptors), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (an agonist of ionotropic glutamate receptors) stimulated also tyrosine phosphorylation of pp110 and pp120. These effects were not observed in astrocytes in primary culture. In hippocampal slices tyrosine phosphorylation of pp110 and pp120 was stimulated by Ca2+-ionophores and by phorbol esters and antagonized by a chelator of intracellular Ca2+and by drugs that inhibit protein kinase C. Stimulation of muscarinic and α1,-adrenergic receptors increased also tyrosine phosphorylation of pp110 and pp120. These results demonstrate that membrane depolarization and stimulation of neurotransmitter receptors activate a tyrosine phosphorylation pathway in neurons. This pathway involves an increase in intracellular Ca2+ concentrations and the activation of protein kinase C. It may provide a biochemical basis for some neurotrophic effects of electrical activity and neurotransmitters and may contribute to the role of tyrosine phosphorylation in long-term potentiation.  相似文献   

11.
Mitochondria play a central role in cell homeostasis. Amongst others, one of the important functions of mitochondria is to integrate its metabolic response with one of the major signaling pathways - the Ca2+ signaling. Mitochondria are capable to sense the levels of cytosolic Ca2+ and generate mitochondrial Ca2+ responses. Specific mechanisms for both Ca2+ uptake and Ca2+ release exist in the mitochondrial membranes. In turn, the mitochondrial Ca2+ signals are able to produce changes in the mitochondrial function and metabolism, which provide the required level of functional integration. This essay reviews briefly the current available information regarding the mitochondrial Ca2+ transport systems and some of the functional consequences of mitochondrial Ca2+ uptake  相似文献   

12.
Abstract: Under control conditions, superfused slices of the dorsal half of the lumbar enlargement from adult rats released Met-enkephalin-like material (MELM) that behaved as authentic Met-enkephalin under two different chromatographic procedures (Bio-gel filtration, HPLC). MELM release increased markedly on exposure of slices to batrachotoxin (0.5 μ M ) or to an excess of K+ (28 and 56 m M instead of 5.6 m M ). The K + -evoked release was totally dependent on the presence of Ca2+ in the super-fusing fluid whereas the spontaneous efflux of MELM was only partially Ca2+-dependent. Further experiments performed with tissues of polyarthritic rats indicated that the increase in their MELM levels was associated with a lower fractional rate constant of MELM release, therefore suggesting that spinal Met-enkephalin turnover might be reduced in chronically suffering animals. Examination of the possible modulation of MELM release by various neuroactive compounds present within the dorsal horn revealed that cholecystokinin (10 μ M ), but not its desulphated derivative, substance P-sulphoxide (10 μ M ), and to a lesser extent substance P, enhanced the K+-evoked MELM release. In contrast, γ-aminobutyric acid (10 μ M ) and (–)-baclofen (1 μ M ) partially prevented the stimulatory effect of K+ on MELM release. Other compounds such as serotonin, somatostatin, and neurotensin altered neither the spontaneous nor the K+-evoked release of MELM.  相似文献   

13.
Abstract: 45Ca2+ uptake measurements were performed on intact and osmotically lysed synaptosomes from rat brain to study the possible influence of prostaglandins (PGs) on Ca2+ movements into and within the nerve endings. The K+-induced 45Ca2+ uptake of intact synaptosomes was not influenced by several inhibitors of PG synthesis. 45Ca2+ uptake in lysed synaptosomal preparations was promoted by ATP and seemed to be largely attributable to mitochondria, as it was inhibited by mitochondrial poisons. This Ca2+ uptake was strongly reduced by PG synthesis inhibitors but also by PG precursor fatty acids. Both PG synthesis inhibitors and precursors, according to their relative efficacy in blocking Ca2+ uptake, were able to induce Ca2+ efflux from preloaded intrasynaptosomal organelles. The PGs E2, F, D2, and thromboxane B2 were without effect on 45Ca2+ uptake in lysed synaptosomal preparations. On the basis of our results it does not seem likely that PGs influence Ca2+ availability by modulating Ca2+ fluxes into or within the nerve endings. The observed inhibitory effects of PG synthesis inhibitors and precursors on the intrasynaptosomal Ca2+ uptake might be due to unspecific impairment of mitochondrial functions.  相似文献   

14.
Uptake and Release of N-Methyl-d-Aspartate by Rat Brain Slices   总被引:2,自引:0,他引:2  
Abstract: The excitant amino acid, N -methyl- d -aspartate, was actively taken up by slices of rat cerebral cortex. This uptake was Na+ - and temperature-dependent, but was relatively inefficient (Km 3 MM, Vmax 0.07 μmol/g/min) compared with that of other acidic amino acids. The uptake of N -methyl- d -aspartate does not appear to have a rate-limiting influence on the time course of N -methyl- d -aspartate-induced excitation since potent uptake inhibitors, such as threo-3-hydroxy- l -aspartate, do not influence the excitant action of N -methyl- d -aspartate. The relatively prolonged excitant action of this acidic amino acid may be the result of relatively slow dissociation of the activated receptor complex. Reloaded N -methyl- d -aspartate can be released from rat brain slices by stimulation with K+ ions. Such K+-stimulated release appeared to be Ca2+-independent, unlike the K+-stimulated release of preloaded d -aspartate. These findings suggest that N -methyl- d -aspartate may be a weak but selective substrate for a glial acidic amino acid uptake system.  相似文献   

15.
Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P2) and slices of rat cerebral cortex were investigated. The P2 fraction had a Na+-dependent high-affinity uptake system for cysteine sulfinic acid (Km, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [14C]cysteine sulfinic acid from preloaded cortical slices or P2 fractions was examined by a superfusion method, which avoided reuptake of released [14C]cysteine sulfinic acid. High K+ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P2 fraction in a partly Ca2+-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P2 fraction.  相似文献   

16.
Abstract: To gain insight into neuronal-glial signaling in brain, cerebellar Bergmann glia and granule neurons were studied in acutely isolated slices with the aid of laser scanning confocal microscopy. Both Bergmann glia and granule neurons responded to N -methyl- d -aspartate (NMDA) with a rise in [Ca2+]i. However, the glial NMDA response was frequently inhibited by tetrodotoxin, suggesting that the response depended on neuronal action potentials, rather than on direct activation of NMDA receptors on the Bergmann glia. Further experiments demonstrated that the NMDA response in Bergmann glia was not inhibited by a combination of non-NMDA glutamate receptor blockers 6-cyano-7-nitroquinoxaline-2,3-dione and α-methyl-4-carboxyphenylglycine. Bergmann glia also responded to norepinephrine and high K+, and the responses were not inhibited by tetrodotoxin. The glial norepinephrine response was blocked by phentolamine but not by the removal of external Ca2+, indicating a direct activation of α1-adrenergic receptors that mediated release of Ca2+ from intracellular stores. The KCI-induced response in both neurons and glia was dependent on external Ca2+ and was blocked by verapamil or nifedipine. In summary, our data indicate that Bergmann glia in situ recognize a signal(s) released from neurons during neuronal activity.  相似文献   

17.
Abstract: Rapid Ca2+ signals evoked by K+ depolarization of rat cerebral cortical synaptosomes were measured by dual-channel Ca2+ spectrofluorometry coupled to a stopped-flow device. Kinetic analysis of the signal rise phase at various extracellular Ca2+ concentrations revealed that the responsible voltage-dependent Ca2+ channels, previously identified as P-type Ca2+ channels, inactivate owing to the rise in intracellular Ca2+ levels. At millimolar extracellular Ca2+ concentrations the channels were inactivated very rapidly and the rate was dependent on the high influx rate of Ca2+, thus limiting the Ca2+ signal amplitudes to 500–600 n M. A slower, probably voltage-dependent regulation appears to be effective at lower Ca2+ influx rates, leading to submaximal Ca2+ signal amplitudes. The functional feedback regulation of calcium channels via a sensor for intracellular Ca2+ levels appears to be responsible for the different inhibition characteristics of Cd2+ versus ω-agatoxin IVa.  相似文献   

18.
Parkinson's disease (PD) is characterized in part by the presence of α-synuclein (α-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α-synuclein gene ( SNCA ) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type α-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of α-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the α-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+]i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of α-synuclein. However, only WT α-syn transfected cells displayed significantly impaired viability. Our findings suggest that α-syn regulates Ca2+ entry pathways and, consequently, that abnormal α-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis.  相似文献   

19.
Abstract: Superfused cortical brain slices from neonatal rats demonstrated large increases in levels of NMR-detectable lipids after sample preparation and perfusion with standard artificial CSF. These increases were reduced by an average of 58% by perfusion with buffer with low (no added) Ca2+ or by perfusion in Ca2+-free buffer. Perfusion with buffer with elevated MgSO4 (10 mmol/L) reduced the lipid changes by 47%. A reduction of 88% was observed in samples perfused in buffer with both low Ca2+ and high Mg2+, suggesting a role for Mg2+ in reducing lipolysis distinct from its known ability to block Ca2+ influx.  相似文献   

20.
Abstract: The effects of (-)-hydroxycitrate (OHC) and citrate on the concentration of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh) in the tissue and on the release of ACh into the medium were investigated in experiments on slices of rat caudate nuclei incubated in media with 6.2 or 31.2 m M K+, 0 or 2.5 mM Ca2+, and 0, 1, or 10 m M EGTA. OHC diminished the concentration of acetyl-CoA in the slices under all conditions used: in experiments with 2.5 m M OHC, the concentration of acetyl-CoA was lowered by 25-38%. Citrate, in contrast, had no effect on the level of acetyl-CoA in the tissue. Although both OHC and citrate lowered the concentration of ACh in the slices during incubations with 6.2 m M K+ and 1 m M EGTA, they had different effects on the content of ACh during incubations in the presence of Ca2+. The concentration of ACh in the slices was increased by citrate during incubations with 2.5 mM Ca2+ and 31.2 or 6.2 m M K+, but it was lowered or unchanged by OHC under the same conditions. The release of ACh into the medium was lowered or unchanged by OHC and lowered, unchanged, or increased by citrate. It is concluded that most effects of OHC on the metabolism of ACh can be explained by the inhibition of ATP-citrate lyase; with glucose as the main metabolic substrate, ATP-citrate lyase appears to provide about one-third of the acetyl-CoA used for the synthesis of ACh. Experiments with citrate indicate that an increased supply of citrate may increase the synthesis of ACh. The inhibitory effect of citrate on the synthesis of ACh, observed during incubations without Ca+2, is interpreted to be a consequence of the chelation of intracellular Ca2+; this interpretation is supported by the observation of a similar effect caused by 10 m M EGTA.  相似文献   

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