摘要:

Abstract:We have examined the effects of systemic kainic acid (KA) administration (9 mg/kg, i.p.) on rat behavior, brain damage, and polyamine levels and the action of the specific ornithine decarboxylase inhibitor α‐difluoromethylornithine (DFMO) on these effects. KA elicited convulsant activity in 63% of the animals. In the acute convulsant phase (1–3 h after KA), a rapid decline (−39% at 3 h) of spermidine content in frontal cortex was found. After the acute convulsant phase, levels of hippocampal spermidine and spermine were reduced (−70 and −66%, respectively, at 8 h). A dramatic increase of putrescine content (681, 1,382, and 336% at 8h, 24h, and 9 days, respectively, after KA) was found, associated with histological signs of cortical brain damage (ischemia and necrosis). There was a close relationship between the concentration of putrescine and signs of delayed toxicity (body weight losses) 24 h and 9 days after KA. DFMO partially antagonized the convulsant activity and reduced the increased putrescine levels to ∼50% of values in KA‐treated animals at 24 h but did not change the pattern of histological damage. The role of polyamines in the early and late phases of KA‐induced neurotoxic

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb02091.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:Many of the neurotransmitter systems that are altered in senile dementia of the Alzheimer type are known to mediate their effects via G proteins, yet the integrity of guanine nucleotide‐binding proteins (G proteins) in Alzheimer's diseased brains has received minimal investigation. The aim of this study was to establish whether the level of Gα subunits of five G proteins was altered in Alzheimer's disease. We used immunoblotting (Western blotting) to compare the amounts of Gi1, Gi2, GsH (heavy molecular weight), GSL (light molecular weight), and Goin the frontal cortex and hippocampus, two regions severely affected by the disease, and the cerebellum, which is less severely affected. The number of senile plaques was also quantified. We report that there was no significant difference in the level of these Gα subunits between Alzheimer's diseased and age‐matched postmortem brains. These results suggest that alterations in the amount of G protein α subunits are not a feature of Alzheimer's d

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb02092.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl‐Sepharose, and Mono Q. Gradient sodium dodecyl sulphate‐polyacrylamide gel elec‐trophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC‐β‐1 and PLC‐γ of bovine brain. With both enzymes phosphatidylinositol 4,5‐bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2‐cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC‐β‐1 and PLC‐γ on Mono Q. Purified rat brain protein kinase C phosphorylated PLO‐γ but not PLC‐β‐1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphory‐lation of

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb02093.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:The height of peak 2,h2, recorded using linear sweep voltammetry with 350‐μm‐diameter carbon paste electrodes in rat striatum was measured from the day of implantation (day 0) to 4 months after surgery. The value ofh2was at a minimum on day 0 (0.6 ± 0.2 nA; n = 20), rose sharply to a maximum on day 2 (6.3 ± 0.9 nA; n = 12), and decreased to a stable level by day 7 (3.3 ± 0.7 nA; n = 16), which lasted for 4 months (3.2 ± 0.6 nA; n = 9). These changes were shown by microinfusion of uricase to be due to variations in the concentration of extracellular uric acid, althoughh2appears to have a small baseline contribution of ∼0.3 nA from 5‐hydroxyindoleacetic acid. The stable value ofh2recorded under chronic conditions was estimated to correspond to a minimal uric acid concentration of 50 μmol/ L, which represents a 10‐fold increase in the extracellular level of this purine metabolite compared with the initial (acute) value. Very similar results were obtained using a mi‐crodialysis technique that detected uric acid directly. These estimates of striatal uric acid concentration are in marked contrast to those obtained using 40‐μm diameter carbon fiber electrodes, which showed adecreasefrom the acute preparation to<1 μmol/L under chronic conditions. Large values ofh2were also recorded with chronically implanted paste electrodes in the hippocampus and frontal cortex. The results suggest that large in vivo probes, such as carbon paste electrodes and dialysis tubes, markedly disturb the neurochemical balance in the extracellular fluid even 1 day following implantation and emphasize the need to develop further small sensors for in vivo neurochemical analysis with

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb02094.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:Following a previous report on detection of muscarinic receptors in myelin with the implied presence of G proteins, we now demonstrate by more direct means the presence of such proteins and their quantification. Using [35S]guanosine 5′‐O‐(3‐thiotriphosphate) ([35S]GTPγS) as the binding ligand, purified myelin from bovine brain was found to contain approximately half the binding activity of whole white matter (138 ± 9 vs. 271 ± 18 pmol/mg of protein). Scatchard analysis of saturation binding data revealed two slopes, a result suggesting at least two binding populations. This binding was inhibited by GTP and its analog but not by 5′‐adenylylimidodiphosphate [App(NH)p], GMP, or UTP. Following sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) of myelin proteins and blotting on nitrocellulose, [α‐32P]GTP bound to three bands in the 21–27‐kDa range in a manner inhibited by GTP and GTPγS but not App(NH)p. ADP‐ribosylation of myelin with [32P]NAD+and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholatc extract of myelin subjected to chromatography on a column of phenyl‐Sepharose gave at least three major peaks of [35S]GTPγS binding activity. SDS‐PAGE and immunoblot analyses of peak I indicated the presence of Goα, Giα, and Gsα. Further fractionation of peak II by diethyl‐aminoethyl‐Sephacel chromatography gave one [35S]GTPγS binding peak with the low‐molecular‐mass (21–27 kDa) proteins and a second showing two major protein bands of 36 and 40 kDa on SDS‐PAGE. Immunoblot analysis of this material identified the 36‐kDa protein as the β subunit, whereas the fraction containing 40‐kDa polypeptides reacted with specific antibodies to Goα and Giα. Thus, purified myelin from bovine brain has been shown to contain several GTP‐binding proteins resembling in broad outline the G proteins of whole brain and potentially able to transduce signals received by mus

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb02095.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:TheN‐methyl‐d‐aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK‐801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small‐molecular‐weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50values (in μM): glycinc, 0.31;d‐serine, 0.20;d‐cycloserine, 1.46; (+)‐HA‐966, 4.06; and 7‐chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK‐801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 μM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK‐801 binding approached equilibrium, their ability to enhance [3H]MK‐801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK‐801 binding under both equilibrium and nonequilibrium conditions, although the high‐affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specifc [3H]MK‐801 binding under nonequilibrium conditions with an IC50of 4 μM, and this value was unaltered when [3H] MK‐801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines, and desipramine are integral c

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb02096.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:The effects of light, 2‐amino‐4‐phosphonobutyric acid (APB), and kainic acid on rat retinal γ‐aminobutyric acid (GABA)‐ergic transmission were studied by measuring levels of retinal GABA following subcutaneous injection of gabaculine, an irreversible inhibitor of GABA‐transaminase. Post‐gabaculine levels of retinal GABA in light‐exposed rats were significantly greater than those in rats held in darkness. The synaptic mechanism of this effect of light was examined by measuring post‐gabaculine levels of retinal GABA in rats placed into either lighted or darkened conditions after receiving unilateral intravitreal injections of APB, a glutamate analogue that selectively decreases the activity of ON synaptic pathways in the retina. APB attenuated the post‐gabaculine accumulation of GABA in rats held in the light, but not in those placed into darkness. Furthermore, the light‐dependent increment in post‐gabacu line accumulation of retinal GABA was entirely APB sensitive, and the effect of APB was entirely light dependent. In contrast to APB, kainic acid stimulated the post‐gabaculine accumulation of retinal GABA in vivo. Our findings suggest that APB and kainic acid influence GABAergic transmission at different sites in the retina and that some retinal GABAergic neurons are either O

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb02097.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:We have reported previously that tyrosine hydroxylase in the crude extract from rat striatum exists in the inactive form showing almost no activity at the physiological pH and that the inactive form is produced by the action of the end products of the enzyme, such as dopamine. The incubation of the enzyme with the end products resulted in not only the inactivation but also a remarkable itabilization of the enzyme. Catechols possessing amino groups but no negatively charged groups on the side chains (catecholamine‐type catechols) were effective at a concentration as low as 10‐7Mboth the inactivation and stabilization of the enzyme. In contrast, catechols not possessing positively or negatively charged side chains (3,4‐dihydroxyphenylethylene‐glycol‐type catechols) were ineffective at a concentration of 10‐7Mbut effective at a concentration of 10‐6Mfor both the inactivation and stabilization. Catechols possessing negatively charged groups (3,4‐dihydroxyphenylacetic acid‐type catechols) were ineffective even at a concentration of 1 0‐6M.Thus, the end products of tyrosine hydroxylase appear to serve to keep the enzyme inactive and stable. The reaction mechanism of the conversion of the enzyme from the active/ labile form to the inactive/stable form by dopamine wa

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb02098.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb02099.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:Different membrane‐associated isoforms of the Neurol cell adhesion molecules have been described. One of them, N‐CAM120, has been shown to be anchored to the membranes by a complex glycan‐phosphatidylinositol group and to be releasabie, under soluble form, by the bacterial enzyme phosphatidylinositol‐phospholipase C. We used the C6 rat astrocytoma cell line expressing both N‐CAM120 and the transmembrane isoform N‐CAM 140 as a model system. We investigated whether artificial depletion of cell membrane N‐CAM 120 influences the synthesis and the messenger RNA transcript levels of the isoforms of the Neurol cell adhesion molecules. Our results showed an increase in the rate of N‐CAM120 protein synthesis, whereas the expression of N‐CAM140 decreased. Additionally, perturbations in the levels of the 6.7‐kb messenger RNA encoding for N‐CAM 140 were observed, whereas the 2.7‐kb transcript encoding for N‐CAM120 remained stable. Examination of the time course for the reexpression of N‐CAM 120 showed that control levels were recovered after 24 h. We provide evidence that: N‐CAM 120 spontaneously released in the culture medium is not incorporated into the extracellular matrix; however, its concentration is important because, if the medium was changed, cell

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb02100.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY