Journal of Neurochemistry

Leli, Ubaldo; Cataldo, Anne; Shea, Thomas B.; Nixon, Ralph A.; Hauser, George

doi: 10.1111/j.1471-4159.1992.tb11328.xpmid: 1548459

Abstract: Certain biological actions of phorbol esters cannot be duplicated by diacylglycerol (DAG). Thus, the human neuroblastoma cell line SH‐SY5Y differentiates when exposed to 12‐tetradecanoyl‐13‐acetyl‐β‐phorbol (TPA) and protein kinase C (PKC) inhibitors, but not when exposed to DAG. To investigate the specific features of the phorbol diester molecule that might be responsible for these effects, we examined the extension of neurites, expression of neuron‐specific enolase, and appearance and localization of phosphorylated high molecular weight neurofilament subunits (NF‐H). TPA, 12‐deoxy‐13‐tetradecanoyl‐β‐phorbol, and staurosporine, but not DAG or 4‐O‐methyl‐TPA, caused neurite outgrowth. Neuron‐specific enolase was expressed in cells treated with TPA and 12‐deoxy‐13‐tetradecanoyl‐β‐phorbol but not with DAG, staurosporine, or 4‐O‐methyl‐TPA. NF‐H increased in the perikarya of cells treated with DAG and 4‐O‐methyl‐TPA, in processes and to varying degrees in perikarya of TPA‐ and 12‐deoxy‐13‐tetradecanoyl‐β‐phorbol‐treated cells, but much more in the processes than in the perikarya of staurosporine‐differentiated cells. These findings and additional differences between the differentiation induced by TPA (a PKC activator) and staurosporine (a PKC inhibitor), including distinct morphology of the cell body and processes and time of appearance of the morphological phenotype, suggest that activators and inhibitors of PKC induce differentiation of SH‐SY5Y cells by different mechanisms, and that the five‐membered/seven‐membered terpene ring region present in TPA must be intact for the induction of morphological differentiation.

Copani, A.; Genazzani, A. A.; Aleppo, G.; Casabona, G.; Canonico, P. L.; Scapagnini, U.; Nicoletti, F.

doi: 10.1111/j.1471-4159.1992.tb11329.xpmid: 1372342

Abstract: Micromolar concentrations of piracetam, aniracetam, and oxiracetam enhanced α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐stimulated 45Ca2+ influx in primary cultures of cerebellar granule cells. Nootropic drugs increased the efficacy but not the potency of AMPA and their action persisted in the presence of the voltage‐sensitive calcium channel blocker nifedipine. Potentiation by oxiracetam was specific for AMPA receptor‐mediated signal transduction, as the drug changed neither the stimulation of 45Ca2+ influx by kainate or N‐methyl‐d‐aspartate nor the activation of inositol phospholipid hydrolysis elicited by quisqualate or (±)‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid. Piracetam, aniracetam, and oxiracetam increased the maximal density of the specific binding sites for (3H)AMPA in synaptic membranes from rat cerebral cortex. Taken collectively, these results support the view that nootropic drugs act as positive modulators of AMPA‐sensitive glutamate receptors in neurons.

Crews, F. T.; McElhaney, R.; Freund, G.; Ballinger, W. E.; Raizada, Mohan K.

doi: 10.1111/j.1471-4159.1992.tb11330.xpmid: 1312569

Abstract: Patients with chronic alcoholism and/or Alzheimer's disease show degenerative changes in the cerebral cortex and hippocampus. To investigate possible changes in insulin‐like growth factor I receptor binding sites in brain tissue of patients with these pathological conditions, the number of 125I‐insulin‐like growth factor I binding sites was determined in tissues obtained from control patients and those with Alzheimer's and/or with a history of alcoholism. The four experimental groups examined consisted of patients from similar age groups. Postmortem histology and a clinical history were used for the diagnosis of Alzheimer's disease and alcoholism, respectively. Careful clinical records were kept concerning other variables such as immediate cause of death and medications administered before death. Specific binding of 125I‐insulin‐like growth factor I to homogenates prepared from cerebral cortex of Alzheimer's, alcoholic, alcoholic Alzheimer's, and age‐matched control patients was similar, although Alzheimer's patients tended to have slightly higher binding values. No significant differences in insulin‐like growth factor I binding in cerebral cortex were found with regard to age of patients, the interval between death and autopsy, and CNS‐active medications. No statistical differences in 125I‐insulin‐like growth factor I binding were noted in hippocampal tissue from the four patient groups. Thus, human insulin‐like growth factor I binding sites in cerebral cortex and hippocampus appear unaffected by several variables.

Hicks, Barry W.; Parsons, Stanley M.

doi: 10.1111/j.1471-4159.1992.tb11331.xpmid: 1312570

Abstract: Both phosphointermediate‐ and vacuolar‐type (P‐and V‐type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P‐type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P‐type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 μM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110‐kDa subunits. The V‐type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 μM. The V‐type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V‐type ATPase revealed two Michaelis constants for MgATP of ∼ 26 μM and 2.0 mM. The V‐type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V‐type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+, K+‐ATPase in the synaptic vesicle preparation is demonstrated.

Ploeg, Ingeborg; Parkinson, Fiona E.; Fredholm, Bertil B.

doi: 10.1111/j.1471-4159.1992.tb11332.xpmid: 1548460

Abstract: In a previous study we showed that in vivo treatment with pertussis toxin could inhibit some, but not all, effects of adenosine in the rat hippocampus. In this study we investigated the effect of pertussis toxin on the binding of adenosine analogues to A1 receptors in rat brain. Intraventricular injection of pertussis toxin (10 μg into the lateral ventricle) did not affect A1 receptor binding in any brain region studied, as evaluated by autoradiography. In vitro treatment of brain sections (10 μm) with pertussis toxin for 5 h, under conditions when >80% of the G proteins were ADP ribosylated, did not alter radioligand binding to adenosine A1 receptors. GTP (10 μM) virtually abolished the high‐affinity agonist binding to the A1 receptor. On the other hand, in solubilized cortical membrane preparations, pertussis toxin pretreatment induced a complete shift of the A1 receptors to the low‐affinity state. This suggests that the ability of pertussis toxin to affect G proteins coupled to A1 receptors in brain depends not only on the distribution of the toxin but also on the configuration of receptors and G proteins.

Liao, Jian; Heider, Harald; Sun, Man‐Chi; Brodbeck, Urs

doi: 10.1111/j.1471-4159.1992.tb11333.xpmid: 1548461

Abstract: Acetylcholinesterases (EC 3.1.1.7, AChE) have varying amounts of carbohydrates attached to the core protein. Sequence analysis of the known primary structures gives evidence for several asparagine‐linked carbohydrates. From the differences in molecular mass determined on sodium dodecyl sulfate‐polyacrylamide gel before and after deglycosylation with N‐glycosidase F (EC 3.2.2.18), it is seen that dimeric AChE from red cell membranes is more heavily glycosylated than the tetrameric brain enzyme. Furthermore, dimeric and tetrameric forms of bovine AChE are more heavily glycosylated than the corresponding human enzymes. Monoclonal antibodies 2E6, 1H11, and 2G8 raised against detergent‐soluble AChE from electric organs of Torpedo nacline timilei as well as Elec‐39 raised against AChE from Electrophorus electricus cross‐reacted with AChE from bovine and human brain but not with AChE from erythrocytes. Treatment of the enzyme with N‐glycosidase F abolished binding of monoclonal antibodies, suggesting that the epitope, or part of it, consists of N‐linked carbohydrates. Analysis of N‐acetylglucosamine sugars revealed the presence of N‐acetylglucosamine in all forms of cholinesterases investigated, giving evidence for N‐linked glycosylation. On the other hand, N‐acetylgalactosamine was not found in AChE from human and bovine brain or in butyrylcholinesterase (EC 3.1.1.8) from human serum, indicating that these forms of cholinesterase did not contain O‐linked carbohydrates. Despite the notion that within one species, the different forms of AChE arise from one gene by different splicing, our present results show that dimeric erythrocyte and tetrameric brain AChE must undergo different postsynthetic modifications leading to differences in their glycosylation patterns.

Taniyama, Kohtaro; Niwa, Masami; Kataoka, Yasufumi; Yamashita, Kimihiro

doi: 10.1111/j.1471-4159.1992.tb11334.xpmid: 1312571

Abstract: Modulation of the γ‐aminobutyric acidB (GABAB) receptor‐mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation‐evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12‐O‐Tetradecanoylphorbol 13‐acetate (TPA) potentiated the high K+‐evoked Ca2+‐dependent release of NA and ACh, but not the ouabain‐evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic‐vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K+‐evoked release of NA and ACh, but not the ouabain‐evoked Ca2+‐independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor‐mediated inhibition of high K+‐evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation‐evoked Ca2+‐dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor‐mediated response.

Hermenegildo, Carlos; Felipo, Vicente; Miñana, María‐Dolores; Grisolía, Santiago

doi: 10.1111/j.1471-4159.1992.tb11335.xpmid: 1312572

Abstract: We have tested if inhibition of protein kinase C is able to prevent and/or to restore the decrease of Na+, K+‐ATPase activity in the sciatic nerve of alloxan‐induced diabetic mice. Mice were made diabetic by subcutaneous injection of 200 mg of alloxan/kg of body weight. The activity of Na+, K+‐ATPase decreased rapidly (43% after 3 days) and slightly thereafter (58% at 11 days). We show that intraperitoneal injection of 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H7), an inhibitor of protein kinase C, prevents completely the loss of Na+, K+‐ATPase activity produced by alloxan. Also, H7 injected into diabetic mice, 4–9 days after the injection of alloxan, restores the activity of the enzyme. The amount of activity recovered depends on the dose of H7 administered; complete recovery was reached with injection of 15 mg of H7/kg of body weight. The effect of H7 is transient, with a half‐life of ∼ 1 h.

Marini, A. M.; Lipsky, R. H.; Schwartz, J. P.; Kopin, I. J.

doi: 10.1111/j.1471-4159.1992.tb11336.xpmid: 1548462

Abstract: Cultured cerebellar astrocytes rapidly accumulate 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) from the incubation medium, reaching a plateau within 10 min, whereas within that time negligible amounts of 1‐methyl‐4‐phenylpyridinium (MPP+) have entered the astrocytes. MPTP accumulation is essentially independent of temperature and is proportional to extracellular concentration at steady state: The steady‐state concentration achieved within these cells is about 50‐fold higher at relatively low extracellular concentrations. MPTP appears to accumulate intracellularly within lysosomes, because lysosomotropic agents such as ammonium chloride and chloroquine markedly diminish the accumulation. Moreover, a proton gradient is required, because MPTP accumulation is abolished by the hydrogen ion antiporter monensin. Over an interval of several days, MPTP is converted to MPP+ intracellularly, with a concomitant decrease in medium MPTP and increase in medium MPP+. A constant, small but significant amount of MPP+ is retained intracellularly over a 72‐h interval. Increasing the medium MPTP concentrations results in increased conversion of MPTP and enhanced intracellular retention of MPTP and MPP+. Neither MPTP nor MPP+ is neurotoxic to cultured cerebellar astrocytes as determined by cell counts and rate of conversion of MPTP to MPP+.

Beaman‐Hall, Carol M.; Hozza, Mark J.; Vallano, Mary Lou

doi: 10.1111/j.1471-4159.1992.tb11337.xpmid: 1312573

Abstract: A modification of the polymerase chain reaction (PCR) was used to amplify nucleotide sequences encoding the 50‐kDa (α) or 58‐ to 60‐kDa (β′,β) subunits of a brain‐specific type II calcium/calmodulin‐dependent protein kinase (CaM kinase II). Rat brain RNA from different regions and at different postnatal ages was purified, and reverse transcriptase was used to produce cDNA templates. Oligonucleotide primer pairs flanking a unique sequence in the coding region of the β′,β subunit‐specific cDNA or a unique sequence in the 3′ noncoding region of the α subunit‐specific cDNA were used to amplify sequences encoding portions of these subunits by PCR. Adult rat forebrain contained approximately three times as much α subunit mRNA as β′,β subunit mRNA, whereas adult rat cerebellum contained a molar ratio of 1 α: 5 β′,β. Intermediate levels of α and β′,β subunit mRNAs were observed in adult pons/medulla, and in 4‐ and 8‐day neonatal forebrain. This amplification assay was also used to demonstrate the presence of α subunit mRNA in cerebellar granule cells and 4‐day neonatal forebrain, which was reported to be undetectable by other methods. Cerebellar granule cells contained less α subunit RNA relative to whole cerebellum, suggesting that this cell type expresses an isoform of CaM kinase II containing less α subunit protein in the holoenzyme. The observed levels of subunit‐specific mRNAs were shown to parallel the levels of expressed protein subunits, suggesting that expression of kinase isoforms is transcriptionally regulated. The data also indicate that the conditions used for amplification of CaM kinase II mRNAs are semi‐quantitative.