摘要:

Abstract:With the advent of cloning, sequencing, and patchclamping techniques, knowledge of the postsynaptic actions of amino acid neurotransmitters has undergone a dramatic advance. The primary sequences of the inhibitory receptors for γ‐aminobutyric acid (GABA) (Schofield et al., 1987) and glycine (Grenningloh et al., 1987) are now established, and patch‐clamp analysis has elucidated many of the factors that regulate the opening of their ion channels. The excitatory glutamate receptors are being extensively characterized at both the pharmacological (reviewed by Foster and Fagg, 1984) and the electrophysiological (reviewed by Cull‐Candy and Usowicz, 1987) level. In this climate, it is perhaps surprising that the fundamental presynaptic release mechanism for the amino acid neurotransmitters remains controv

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb09126.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Sorbitol dehydrogenase (EC 1.1.1.14) was isolated from bovine brain and purified 3,000‐fold to apparent homogeneity, as judged by polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 36 units/mg of protein; a molecular weight of 39,000 for each of the four identical subunits and 155,000 for the intact enzyme were determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and gel exclusion chromatography, respectively. The presence of one Zn2+per subunit was confirmed by atom absorption spectroscopy; inactivation of the enzyme by metal‐chelating agents points to the essential role that Zn2+plays in the catalytically competent enzyme. The enzyme is also inactivated by thiol‐blocking reagents; with respect to inactivation by sodium pyrophosphate, sorbitol dehydrogenase is different from closely related alcohol dehydr

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb09127.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:[3H]Tetrodotoxin binds to a single class of receptor sites in homogenates of human brain with aKDof 9.1 nMat 0°C and a maximal binding capacity of 5.9 pmol/mg of protein. This tetrodotoxin receptor has been solubilized, and several parameters influencing the efficiency of this critical step have been studied. Treatment of brain membranes with 2% (wt/vol) Nonidet P‐40 solubilizes up to 38% of the tetrodotoxin receptor sites. The duration of this solubilization step must not exceed 15 min at an optimal pH of 6.8. The binding activity is most stable when exogenous phosphatidylcholine is added to the soluble receptor with a phosphatidylcholine/detergent ratio of 1

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb09128.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:To assess the functions of Cl−‐dependent glutamate “binding” (Cl−‐dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 γg/ml), 10 μMaconitine, 56 mMK+, and 50 μMmonensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60–85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+but not on Ca2‐. The bindings of γ‐[3H]aminobutyric acid and 5‐[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without afecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl−‐dependent glutamate uptake sites. Na+‐dependent glutamate binding (Na+‐dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl−‐dependent glutamate sequestration system in synaptic membranes is

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb09129.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:The nature of the opiate modulation of adenylate cyclase following acute and chronic agonist exposure has been investigated in rat spinal cord. Using membranes of both adult rat spinal cord and spinal cord‐dorsal root ganglion cocultures, we found thatK‐opiate receptors are negatively coupled to adenylate cyclase. TheK‐opiate agonists (e.g., U50488) inhibit significantly and dose‐dependently the basal and the forskolin‐stimulated cyclase activities, whereas μ and δ agonists are ineffective. The regulatory action is stereospecific and requires the presence of GTP. EGTA treatment of the plasma membranes abolished the effect ofK‐opiate agonists on the basal cyclase activity, and this inhibitory effect could not be restored by subsequent addition of Ca2+. The EGTA treatment did not affect theKagonist inhibition of the forolin‐stimulated cyclase. The results also show that following chronic exposure of cultured cells to etorphine or U50488, there is a loss ofKagonist inhibition of the cyclase. Moreover, this desensitization process appears to be heterologous, because α2‐adrenergic agonists (e.g., clonidine or norepinephrine) and the muscarinic agonist (carbachol) exhibited significantly lower potency for inhibiting cyclase activity when compared to untreated cultures. This pattern of heterologous desensitization suggests that chronic exposure toKopiates leads to alterations in postreceptor regulatory components, possibly GT

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb09130.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Botulinum C2 toxin is known to ADP‐ribosylate actin. The toxin effect was studied on [3H]noradrenaline secretion of PC12 cells. [3H]Noradrenaline release was stimulated five‐ to 15‐fold by carbachol (100 μM) or K+(50 mM) and 10–30‐fold by the ionophore A23187 (5 μM). Pretreatment of PC12 cells with botulinum C2 toxin for 4–8 h at 20°C, increased carbachol‐, K+‐, and A23187‐induced, but not basal, [3H]noradrenaline release maximally 1.5‐ to threefold, whereas ≥75% of the cellular actin pool was ADP‐ribosylated. Treatment of PC12 cells with botulinum C2 toxin for up to 1 h at 37°C also increased stimulated [3H]noradrenaline secretion, whereas toxin treatment for>1 h decreased the enhanced [3H]noradrenaline release stimulated by carbachol and K+but not by A23187. Concomitantly with toxin‐induced stimulation of secretion, 20–50% of the cellular actin was ADP‐ribosylated, whereas>60% of actin was modified when exocytosis was attenuated. The data indicate that ADP‐ribosylation of actin by botulinum C2 toxin largely modulates stimulation of [3H]noradrenaline release. Moreover, the biphasic toxin effects suggest that distinct mechanisms are involve

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb09131.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Previous studies that demonstrated that mouse brain accumulated significantly more radioactivity from subcutaneously administered 5‐methyltetrahydrofolate labelled in the methyl group compared to the label in the folate moiety are open to two interpretations. The methyl group could have been transferred to another compound (probably methionine) prior to its transport into the brain. Alternatively, if plasma 5‐methyltetrahydrofolate per se is significantly involved in the provision of methyl groups to brain and nerve tissue it would be expected that the folate moiety would be returned to the plasma to complete the cycle and thus would appear not to have been taken up. In this article, using competition experiments that exploit the differences in the mechanism of transport of methionine and 5‐methyltetrahydrofolate into brain and nerve, evidence is presented that in the rat the methyl group of 5‐methyltetrahydrofolate is transported after its conversion to met

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb09132.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:Polyunsaturated fatty acid (PUFA) levels (an index of the amount of substrate available for lipid peroxidation) were measured in several brain regions from patients who died with Parkinson's disease and age‐matched control human postmortem brains. PUFA levels were reduced in parkinsonian substantia nigra compared to other brain regions and to control tissue. However, basal malondialdehyde (MDA; an intermediate in the lipid peroxidation process) levels were increased in parkinsonian nigra compared with other parkinsonian brain regions and control tissue. Expressing basal MDA levels in terms of PUFA content, the difference between parkinsonian and control substantia nigra was even more pronounced. Stimulating MDA production by incubating tissue with FeSO4plus ascorbic acid, FeSO4plus H2O2, or air alone produced lower MDA levels in the parkinsonian substantia nigra, probably reflecting the lower PUFA content. These results may indicate that an increased level of lipid peroxidation continues to occur in the parkinsonian nigra up to the time of death, perhaps because of continued exposure to excess free radicals derived from some endogenous or exogenous neurotoxic specie

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb09133.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:The synthesis and transport of slowly transported polypeptides in sciatic nerves of rats was investigated by [35S]methionine pulse labeling and gel electrophoresis in control, diabetic, and insulin‐treated diabetic rats. To detect very early changes diabetes was induced by streptozocin only 5 days prior to the labeling of the dorsal root ganglion cells. Fourteen days were allowed for axonal transport. In this experimental system, the neurofilament triplet is transported at an apparent velocity of 1.1 ± 0.1 mm/day (mean ± SD). The actin‐related complex, including actin and two polypeptides of 87 kilodaltons and 37 kilodaltons, was transported at a velocity of 2.6 ± 0.2 mm/day. For α‐ and β‐tubulin we found an apparent transport velocity of 2.2 ± 0.1 mm/day, placing it between actin and the neurofilament triplet. The diabetic rats had a selective 32% decrease in the amount of the heaviest neurofilament subunit: 0.47 ± 0.19% of trichloroacetic acid‐insoluble radioactivity versus 0.69 ± 0.17% in controls; 2p<0.05. This decrease was associated with a proximal accumulation of the two lighter neurofilament sub‐units. Insulin treatment of a diabetic group failed to normalize the changes of axonal transport and additional changes suggesting a hypoglycemic i

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb09134.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Abstract:The mechanisms of carbachol‐induced muscarinic acetylcholine receptor (mAChR) down‐regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma X glioma hybrid NG108‐15 cell line by specific ligand binding assays.N‐[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mMcarbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by ∼80%. Bacitracin (1–4 mg/ml) and methyl‐amine (1–15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist‐induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1–10 μM) and colchicine (1–10 μM) prevented carbachol‐induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist‐induced mAChR down‐regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 μM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular tra

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1989.tb09135.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY