摘要:

Abstract:The biochemistry and functional neurochemistry of the synaptosomal plasma membrane phosphoprotein B‐50 (GAP‐43) are reviewed. The protein is putatively involved in seemingly diverse functions within the nervous system, including neuronal development and regeneration, synaptic plasticity, and formation of memory and other higher cognitive behaviors. There is a considerable amount of information concerning the spatial and temporal localization of B‐50 (GAP‐43) in adult, fetal, and regenerating nervous tissue but far less is known about the physical chemistry and biochemistry of the protein. Still less information is available about posttranslational modifications of B‐50 (GAP‐43) that may be the basis of neurochemical mechanisms that could subsequently permit a variety of physiological functions. Hence, consideration is given to several plausible roles for B‐50 (GAP‐43) in vivo, which are discussed in the context of the cellular localization of the protein, significant posttranslational enzymes, and regulatory proteins, including protein kinases, phosphoinositides, calmodulin

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb11398.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:In the brain, 5′‐deiodinase (5′‐D) is responsible for the metabolic activation of thyroxine (T4) into 3,5,3′‐triiodothyronine (T3) and 5‐deiodinase (5‐D) deiodinates T4and T3into inactive metabolites. This study examines the effects of factors known to induce astroglial 5′‐D activity on the 5‐D activity in cultured rat astroglial cells. The potencies of these factors were compared after 8 h of incubation, when stimulations by these factors near their maximal effects. 12‐O‐Tetradecanoylphorbol 13‐acetate (TPA) at 10−7Mwas a potent inducer of 5‐D activity, producing a 30‐ to 80‐fold increase after 8 h. The maximal effect of TPA was observed after about 14 h. The TPA stimulation of 5‐D activity was not dependent on glucocorticoids, unlike 5′‐D activity. In comparison with TPA, 8‐bromo‐cyclic AMP (10−3M) was a poor inducer of 5‐D activity whereas it is an excellent inducer of 5′‐D activity. It produced a 2‐ to 20‐fold increase in 5‐D activity after 8 h. Natural acidic fibroblast growth factor (20 ng/ml) produced a degree of stimulation similar to that of TPA after 8 h. The maximal effect of acidic fibroblast growth factor was observed after about 16 h (until a 120‐fold increase). Recombinant acidic fibroblast growth factor also induced 5‐D activity. Basic fibroblast growth factor was less potent than acidic fibroblast growth factor for increasing 5‐D activity (maximal increase by 40‐ to 50‐fold after 8 h). Platelet‐derived growth factor (20 ng/ml) and epidermal growth factor (100 ng/ml) were poor inducers of 5‐D activity (8‐ to 12‐fold increase at 8 h); insulin (10−6M) was without effect. The 5‐D activity induced by TPA and acidic fibroblast growth factor manifested the characteristics of type III 5‐D (Kmfor T30.5–0.8 nM, thiol‐dependent, 6‐n‐propyl‐2‐thiouracil‐insensitive). The results demonstrate that, like 5′‐D activity, 5‐D activity is induced by multiple pathways. The relative potencies of TPA, fibroblast growth factors, and cyclic AMP on 5‐D and 5′‐D activities were different, as were the time courses of their actions. These data indicate that 5‐ and 5′‐D are distinct enzymes and support the view th

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb11399.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:The simultaneous measurement of the accumulation of cyclic AMP and inositol phosphates in rat cerebral cortical slices is described. After stimulation, the separation of cyclic AMP and inositol phosphates was achieved using ion‐exchange chromatography and their concentrations were determined by means of a double‐labeling technique, the substrates adenine and inositol being labeled with14C and3H, respectively. The recoveries were 70–80% for inositol phosphates and 40–50% for cyclic AMP. To test the applicability of the method, norepinephrine was chosen as an agonist, because it is known to stimulate the production of these two second messengers by interacting with α‐ and β‐adrenergic receptors. This procedure is an improvement over existing methods, because we obtained the simultaneous formation of3H‐inositol phosphates and [14C]cyclic AMP in a concentration‐dependent process. EC50values were similar for the two, 8.5 ± 3.9 μMfor3H‐inositol phosphates and 20.2 ± 6.3 μMfor [14C]cyclic AMP, and close to the values obtained when each process was studied alone. The action of adrenergic antagonists was also tested. Propranolol blocked the norepinephrine stimulation of [14C]cyclic AMP, but did not inhibit the norepinephrine stimulation of3H‐inositol phosphates. The opposite results were observed with prazosin. Our results suggest that this method could be a useful tool to examine the interaction between these two r

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb11400.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:Quantitative autoradiography was used to examine the distribution of [3H]phorbol 12,13 dibutyrate ([3HIPDBu) binding to protein kinase C in the middle frontal and temporal cortices and the hippocampal region of nine control and nine elderly subjects with Alzheimer's disease (AD). AD patients had a clinical diagnosis of the disease that was confirmed neuropathologically by the presence of numerous plaques in the hippocampus and cerebral cortex. Choline acetyltransferase (ChAT) activity was significantly reduced in the middle frontal and temporal cortex and in the hippocampus of AD subjects, with the deficit being>60% of control values. Quantitative autoradiographic analysis of [3H]PDBu binding to protein kinase C revealed a heterogeneous pattern in control brain, being particularly high in superficial layers of the cortex and CAI of the hippocampus. There were no significant differences between control and AD sections in all areas examined within the middle frontal cortex, e.g., layers 1–11 control, 491 ± 46 versus AD, 537 ± 39 pmol/g of tissue; middle temporal cortex, e.g., layers 1–11 control, 565 ± 68 versus AD, 465 ± 72 pmol/g of tissue; and hippocampal formation, e.g., CAI control, 51 ± 28 versus AD, 498 ± 25 pmol/g of tissue. In a parallel study, [3H]PDBu binding to homogenate preparations of control and AD brain confirmed that there was no significant difference in [3H]PDBu binding in either the particulate or the cytosolic fraction. We have demonstrated in a well‐defined population of AD patients that [3H]PDBu binding to protein kinase C remains preserved in brain regions that are severely affected by the neuropathological and neurochemical corre

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb11401.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:D1and D2receptor densities in human substantia nigra were examined by use of the specific binding of, respectively, [3H]SCH 23390 [R(+)‐7‐chloro‐8‐hydroxy‐3‐[3H]methyl‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine] and [3H]spiperone. A unilateral loss of striato‐ and pallidonigral pathways by an infarction (n = 4) had no effect on the ipsilateral nigral D2receptors, but reduced the ipsilateral nigral D1receptors by 48–60% compared with the intact side. These data suggest that a substantial fraction of D1receptors in human substantia nigra is located on terminals of striato‐and/or pallidonigral neurons, whereas D2receptors are confined to intrinsic nigral cells. We also examined the effect of aging on the D1and D2receptors in substantia nigra obtained from 25 postmortem human brains (age range 19–88 years). The densities of both receptor types were not affected by the aging process. Since nigrostriatal dopaminergic neurons degenerate with aging, these results suggest either that the nigral D2receptors are up‐regulated in response to a progressive depletion of dopamine in the substantia nigra or that, in contrast to the rat, they are

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb11402.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:Studies were conducted to ascertain the temporal and dose‐dependent effects of nicotinic ligand exposure on functional activity of different nicotinic acetylcholine receptor (nAChR) subtypes, as expressed by cells of the PC12 rat pheochromocytoma (ganglia‐type nAChR) or the TE671/RD human (muscle‐type nAChR) clonal line. Chronic (3–72‐h) agonist (nicotine or carbamylcholine) treatment of cells led to a complete (TE671) or nearly complete (PC12) loss of functional nAChR responses, which is referred to as “functional inactivation.”Some inactivation of nAChR function was also observed for the nicotinic ligandsd‐tubocurarine (d‐TC), mecamylamine, and decamethonium. Half‐maximal inactivation of nAChR function was observed within 3 min for TE671 cells and within 10 min for PC12 cells treated with inactivating ligands. Functional inactivation occurred with dose dependencies that could not always be reconciled with those obtained for acute agonist activation of nAChR function or for acute inhibition of those responses byd‐TC, decamethonium, or mecamylamine. Treatment of TE671 or PC12 cells with the nicotinic antagonist pancuronium or alcuronium alone had no effect on levels of expression of functional nAChRs. However, evidence was obtained that either of these antagonists protected TE671 cell muscle‐type nAChRs or PC12 cell ganglia‐type nAChRs from functional inactivation on long‐term treatment with agonists. Recovery of TE671 cell nAChR function following treatment with carbamylcholine, nicotine, ord‐TC occurred with half‐times of 1–3 days whether cells were maintained in situ or harvested and replated after removal of ligand. By contrast, 50% recovery of functional nAChRs on PC12 cells occurred within 2–6 h after drug removal. In either case the time course for recovery from nAChR functional inactivation is much slower than recovery from nAChR “functional desensitization,”which is a reversible process that occurs on shorter‐term (0–5‐min) agonist exposure of cells. These results indicate that ganglia‐type and muscle‐type nAChRs are similar in their sensitivities to functional inactivation by nicotinic ligands but differ in their rates of recovery from and onset of those effects. The ability of drugs such as the agonistsd‐TC, decamethonium, and mecamylamine to induce functional inactivation may relate to their activities as partial/full agonists, channel blockers, and/or allosteric regulators. Effects of drugs such as pancuronium and alcuronium are likely to reflect simple competitive inhibition of primary ligand binding at functional activation sites. Agonist‐induced functional inactivation of TE671 cell nAChRs occurs under conditions previously shown to induce increases in numbers of nAChR ligand binding sites expressed on the cell surface and in total membrane pools and contrasts with coordinate down‐regulation of nAChR ligand binding and function following chronic ag

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb11403.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:We studied the molecular mechanism of noradrenaline release from the presynaptic terminal and the involvement of the protein kinase C substrate B‐50 (GAP‐43) in this process. To gain access to the interior of the presynaptic terminal, we searched for conditions to permeate rat brain synaptosomes by the bacterial toxin streptolysin O. A crude synaptosomal/mitochondrial preparation was preloaded with [3H]noradrenaline. After permeation with 0.8 IU/ml streptolysin O, noradrenaline efflux could be induced in a concentration‐dependent manner by elevating the free Ca2+concentration from 10−8to 10−5M.Efflux of the cytosolic marker protein lactate dehydrogenase was not affected by this increase in Ca2+. Ca2+‐induced efflux of noradrenaline was largely dependent on the presence of exogenous ATP. Changing the Na+/K+ratio in the buffer did not affect Ca2+‐induced noradrenaline release. Release of noradrenaline could also be evoked by phorbol esters, indicating the involvement of protein kinase C. Ca2+‐ and phorbol ester‐induced release were not additive at higher phorbol ester concentrations (>10−7M). We compared the sensitivities of Ca2+‐ and phorbol ester‐induced release of noradrenaline to the protein kinase inhibitors H‐7 and polymyxin B and to antibodies raised against synaptic protein kinase C substrate B‐50. Ca2+‐induced release was inhibited by B‐50 antibodies and polymyxin B, but not by H‐7; phorbol ester‐induced release was inhibited by polymyxin B and by H‐7, but only marginally by antibodies to B‐50. We suggest that phorbol esters and Ca2+stimulate noradrenaline release through different mechanisms and that the essential role of B‐50 in Ca2+‐induced noradrenaline release may involve other properties of B‐50 besi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb11404.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:The activity of protein tyrosine kinases was determined in extracts from Alzheimer's disease brains and age‐and postmortem time‐matched control brains at autopsy using the synthetic peptide substrate poly(Glu4Tyr1). The specific activity of protein tyrosine kinases in the particulate fraction decreased roughly twofold (p<0.02) in Alzheimer's disease frontal cortex relative to unaffected control cortex. Cytosolic protein tyrosine kinase activity in Alzheimer's disease tissue was not significantly different from that in control tissue. In contrast to reduced particulate protein tyrosine kinase activity, analysis of Western blots of cytosolic and particulate fractions revealed increases in cytosolic antiphosphotyrosine immunoreactive polypeptides with molecular masses of 55 and 60 kDa. Quantitative immunohistochemistry and morphometry of frontal cortex sections with the antiphosphotyrosine antibody indicated increased antiphosphotyrosine staining in the neurons, although the number of antiphosphotyrosine‐positive neurons per square millimeter decreased. Also, increased antiphosphotyrosine staining was observed in the hippocampal neurons. These results suggest that altered protein tyrosine kinases and protein tyrosine phosphorylation are involved in the pathology of Alzheimer's di

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb11405.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:Among the drug‐metabolizing enzymes present in the rat brain, one form of UDP‐glucuronyltransferase catalyzes the formation of the polar metabolite 1‐naphthyl‐β‐D‐glucuronide from 1‐naphthol. We measured the activity of this isoform in different brain regions and showed its heterogeneous distribution. Conjugation activities were found to be the highest in the olfactory bulbs (25.4 nmol/h/mg protein) and lowest in the cerebellum (4.5 nmol/h/mg protein). As the blood–brain barrier prevents the passage of hydrosoluble molecules, we studied in vivo the characteristics of the efflux of labeled 1‐naphthyl‐β‐D‐glucuronide injected into the lateral ventricle and the cortex tissue, using tritiated water and labeled inulin as reference compounds. The results reported here indicate that intracerebrally formed glucuronide is cleared from brain tissue by both diffusion and a

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb11406.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Abstract:Biochemical changes in the rat brain cholinergic system during and after 60 min of ischemia were studied using a four‐vessel occlusion model. Extracellular acetylcholine (ACh) concentrations in the unanesthetized rat hippocampus markedly increased during ischemia and reached a peak (about 13.5 times baseline levels) at 5–10 min after the onset of ischemia. At 2–5 h after reperfusion, extracellular ACh concentrations were reduced to 64–72% of the levels of controls. ACh levels in the hippocampus, striatum, and cortex decreased significantly during ischemia and exceeded their control values just after reperfusion. A significant increase in hippocampal ACh level after 2 days of reperfusion and a decrease in [14C]ACh synthesis from [14C]glucose in hippocampal slices excised at 2 days after reperfusion were observed. The extracellular concentrations and tissue levels of choline markedly increased after ischemia. These results show that ACh is markedly released into the extracellular space in the hippocampus during ischemia, and they suggest that ACh synthesis is activated just after reperfusion and that cholinergic activity is reduced after 2–48 h of reperfusion in the hi

ISSN:0022-3042    

DOI:10.1111/j.1471-4159.1991.tb11407.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY