ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01882.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Guinea‐pig cerebral cortical synaptosomes were preincubated for 60 min with 100 μMd‐aspartate,l‐aspartate, orl‐glutamate. The totald‐ plusl‐aspartate content of the synaptosomal fraction increased to 235%, 195%, or 164%, respectively, of the control. Despite this no increase was seen in the very low KCl evoked, Ca2+‐dependent release of aspartate. Preincubation with the three amino acids changed the synaptosomal glutamate content to 78% (d‐aspartate), 149% (l‐aspartate), or 168% (l‐glutamate) of control. However there was no statistically significant effect of these preincubations on the extent of Ca2+‐dependent glutamate release. Thus the Ca2+‐dependent release of aspartate and glutamate is not determined by the total synaptosomal content of these amino acids. The addition of 0.1–0.5 mMglutamine to the incubation caused a massive appearance of glutamate in the extrasynaptosomal medium. Analysis of specific activities showed that glutamine was hydrolysed directly by an extra‐synaptosomal glutaminase, and that intrasynaptosomal glutamate was predominantly labelled by uptake of this glutaminase‐derived glutamate. No increase was seen in the extent of Ca2+‐dependent release of glutamate (by fluorimetry) either after preincubation with glutamine or in the continued presence of glutamine. Thus we are unable to confirm reports that glutamine expands the transmitter pool of glutamate. The extrasynaptosomal glutaminase activity in the synaptosomal preparation was inhibited by Ca2+and activated by phosphate. Identical kinetics were obtained with “free'’brain mitochondria, confirming the origi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01883.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:An on‐line microdialysis system was developed which monitored the 3,4‐dihydroxyphenylalanine (DOPA) formation in the striatum during infusion of a submicromolar concentration of anl‐aromatic amino‐acid decarboxylase inhibitor (NSD 1015). The absence of DOPA in dialysates of 6‐hydroxydopamine‐pretreated rats and the disappearance of DOPA after administration of α‐methyl‐p‐tyrosine indicated that the dialyzed DOPA was derived from dopaminergic nerve terminals. Next we investigated whether the steady‐state DOPA concentration in striatal dialysates could be considered as an index of tyrosine hydroxylase activity. The increase in DOPA output after intraperitoneal administration of haloperidol or γ‐butyrolactone and the decrease in DOPA output after intraperitoneal administration of apomorphine are in excellent agreement with results of postmortem studies, in which a decarboxylase inhibitor was used to measure the activity of tyrosine hydroxylase. The effect of haloperidol on DOPA formation was not visible when a U‐shaped cannula (0.80 mm o.d.) was used. Some methodological problems related to microdialysis of the haloperidol‐induced increase in DOPA formation are discussed. We concluded that the proposed model is a powerful and reliable in vivo method to monitor tyrosine hydroxylase activity in the brain. The method is of special interest for investigating the effect of compounds which are not able to pass the blood‐brain barrier. As an application of the method in the latter situation, we report the effect of infusion the neurotoxin 1‐methyl‐4‐phenylpyridinium ion (10 mmol/L infused over 20 min) on the activi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01884.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mMthymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W‐7 [N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin‐dependent kinases, gave the same concentration‐dependent inhibition profile of sialyltransferase as desipramine, whereas H‐7 [1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide‐dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01885.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:A human malignant melanoma cell line, Melur, secretes several glycoproteins that contain a unique carbohydrate epitope shared by neural cell adhesion molecules and recognized by the monoclonal antibodies HNK‐1, L2, and 10C5. In this report, we present evidence that one of the major melanoma glycoproteins containing the HNK‐1/10C5 epitope is the cell adhesion molecule, fibronectin, or a fibronectin‐like molecule. Melanoma‐derived fibronectin was isolated from serum‐free conditioned medium by gelatin‐Sepharose affinity adsorption and shown to react with monoclonal antibodies HNK‐1 and 10C5 in Western blot analysis. HNK‐1‐containing fibronectin was purified on a gelatin‐Sepharose column followed by an affinity column using a monoclonal antibody against the HNK‐1 carbohydrate. The purified HNK‐1‐fibronectin then could be incorporated into the extracellular matrix of hamster fibroblasts in vitro, and such a matrix was detectable using the HNK‐1 monoclonal antibody in an immunofluorescence assay. Of the seven neuroectoderm‐derived tumor cell lines tested, only the Melur melanoma cell secreted fibronectin containing the HNK‐1 carbohydrate. Identification of human neuroectoderm‐derived fibronectin as a potential carrier of the HNK‐1 carbohydrate suggests a new role for fibronectin in neural development and regeneration, and represents a new model for studying the function of this carbohyd

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01886.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The increase in hormone‐stimulated cyclic AMP accumulation observed in a variety of intact cells after chronic pretreatment with drugs that inhibit adenylate cyclase activity has been attributed to an increase in adenylate cyclase activity following withdrawal of the inhibitory drug. In NG 108–15 mouse neuroblastoma X rat glioma hybrid cells (NG cells) chronically treated with the muscarinic cholinergic agonist carbachol, we have found a significant decrease in the apparent degradation rate constant for cyclic AMP, in addition to an increase in the prostaglandin E1(PGE1)‐stimulated cyclic AMP synthesis rate in intact cells. In carbachol‐pretreated NG cells that were stimulated with a maximally effective dose of PGE1, and that accumulated steady‐state cyclic AMP concentrations fourfold or more higher than in control cells, the apparent rate constant for degradation was about 53% lower than the value for control cells. In carbachol‐pretreated cells stimulated with a submaximal dose of PGE1to yield a steady‐state cyclic AMP concentration comparable to control cells, the apparent rate constant was 31% lower than the value for control cells. In S49 mouse lymphoma cells (S49 cells) chronically treated with an analog of the inhibitory agonist somatostatin, the first‐order rate constant for cyclic AMP degradation in intact cells following isoproterenol stimulation was 29% lower than the value for control cells. Despite these changes in the kinetics of cyclic AMP degradation in intact NG cells and S49 cells, there was either no change or a minimal change (less than 10%) in phosphodiesterase activities assayed in extracts of cells chronically exposed to inhibitory drugs. These data suggest that an alteration in cyclic AMP degradation as well as in cyclic AMP synthesis is an important factor in the withdrawal response following chronic treatment of cells with drugs that inhibit ad

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01887.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The previous report that PC12 (pheochromocytoma) cells have a K+‐induced, as well as a tyramine‐induced, catecholamine release mechanism has been confirmed. Selective monoamine oxidase (MAO)‐A (clorgyline and moclobemide) and not MAO‐B inhibitors (l‐deprenyl, AGN 1135, and Ro 16–6491) potentiate the catecholamine‐releasing action of tyramine significantly more than that of K+. The potentiation of tyramine‐induced [3H]noradrenaline release from PC 12 cells by MAO‐A inhibitors has been linked to the presence of MAO‐A in these cells, for which tyramine and noradrenaline are substrates. In the above respects, it is the PC 12 cell that resembles more closely the peripheral adrenergic neuron, rather than the chromaffin cell, which is endowed with MAO‐B and lacks the tyramine‐releasable

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01888.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:The neutral and phospholipid compositions of various regions of the human brain were analyzed using autopsy material covering the life period between 33 and 92 years of age. The protein content was also measured and, on a weight basis, this content is unchanged in the cerebellum, pons, and medulla oblongata, whereas in the 90‐year‐old group it decreases in the hippocampus, gray matter, and nucleus caudatus. In white matter, the protein content decreases continuously with age. The phospholipid composition is characteristic of the region investigated, but remains unchanged during aging. The total phospholipid content exhibits only a 5–10% decrease in the oldest age group. The content of dolichol and its polyisoprenoid pattern are also characteristic of the region analyzed. Between 33 and 92 years of age, the amount of dolichol in all portions of the brain increases three‐to fourfold, but the isoprenoid pattern remains constant. The level of dolichyl‐P varies between different regions, but only a moderate increase is seen with age. Ubiquinone content is highest in the nucleus caudatus, gray matter, and hippocampus, and in all areas this content is decreased to a great extent in the oldest age groups. All regions of the human brain are rich in cholesterol, but alterations in the amount of this lipid are highly variable during aging, ranging from no change to a 40%

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01889.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Past work established a cell‐free assay for a nerve growth factor (NGF)‐activated protein kinase activity (designated N‐kinase) that utilizes tyrosine hydroxylase and histone H1 as substrates and that is distinct from a variety of well‐characterized kinases. This study explores the specificity and mechanistic pathway(s) by which N‐kinase activity is regulated in PC12 rat pheochromocytoma cells. N‐kinase is rapidly activated in these cells by treatment with NGF, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), phorbol ester, or dibutyryl cyclic AMP. Our data indicate that the stimulated activity is the same for each agent by several criteria: It exhibits the same characteristic biphasic elution pattern by Mono S fast protein liquid chromatography (FPLC), except for the case of dibutyryl cyclic AMP in which one of the activity peaks is somewhat shifted; it shows the same elution pattern by FPLC on a Superose 12 column; it possesses identical substrate specificity; and, except in the case of dibutyryl cyclic AMP, it does not show additivity when each agent is added simultaneously with NGF. The multiple forms of N‐kinase are interconvertible in that rechromatography on a Mono S column yields a single peak of activity. Also, when NGF and dibutyryl cyclic AMP are simultaneously presented to cells, the chromatographic profile resembles that with NGF alone. Activation occurs through several independent initial pathways. Down‐regulation of protein kinase C by phorbol ester pretreatment prevents N‐kinase activation by phorbol ester, but not by the other agents. A PC12 cell‐derived line deficient in cyclic AMP‐dependent protein kinase II activity exhibits N‐kinase activation by all treatments except dibutyryl cyclic AMP. The properties of N‐kinase suggests that it is similar or identical to the ribosomal S6 protein kinase described by Blenis and Erikson. Additional experiments revealed that N‐kinase activity can be stimulated in several cell lines in addition to PC12 cells. These findings indicate that the N‐kinase can be activated via multiple second‐messenger pathways and that it could therefore potentially play a significant role in mediating shared intracellular responses t

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01890.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY

摘要:

Abstract:Synenkephalin (SYN), the nonopioid amino‐terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63–70)‐octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63–70)‐octapeptide as standard, and125I‐[Tyr63]SYN(63–70)‐octapeptide as tracer, the IC50was ∼51 fmol/100‐μl sample at equilibrium or 12 fmol/100 μl in disequilibrium, and the sensitivity was ∼3 fmol/100 μl. Cross‐reactivity of the assay was 100% with [Cys63]SYN(63–70)‐octapeptide and with bovine adrenal 8.6‐kilodalton peptide digested with trypsin and carboxypeptidase B, but<0.1% with transforming growth factor‐alpha, 2 × 10‐6with Leu‐Leu‐Ala [SYN(68–70)‐tripeptide], and ≪ 10‐6with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63–70)‐octapeptide, we refer to the RIA as an assay for SYN(63–70). Tissue extracts were made in 1Macetic acid, dried, reconstituted in Tris‐CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63–70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63–70) in striatum by a factor of 1.5–2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63–70) (after sequential digestion) was ∼6:1. At least 90% of the immunoreactive SYN(63–70) in extracts of bovine caudate nucleus eluted from Sephadex G‐100 with an apparent molecular weight equal to that of bovine PRO(1–77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C‐terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1990.tb01891.x

出版商:Blackwell Publishing Ltd    

年代:1990

数据来源: WILEY