RESUMEN

The agonist stimulation of a variety of cells results in the induction of specific lipid metabolism in nuclear membranes, supporting the hypothesis of an important role of the lipids in nuclear signal transduction. While the existence of a phosphatidylinositol cycle has been reported in cellular nuclei, little attention has been given to the metabolism of phosphatidylcholine in nuclear signaling. In the present study the metabolism of phosphatidylcholine in the nuclei of neuroblastoma cells LA-N-1 was investigated. The incubation of LA-N-1 nuclei with radioactive choline, phosphocholine or CDP-choline led to the production of labelled phosphatidylcholine. The incorporation of choline and phosphocholine but not CDP-choline was enhanced in nuclei of TPA treated cells. Moreover the presence of choline kinase, phosphocholine cytidylyltransferase and phosphocholine transferase activities were detected in the nuclei and the TPA treatment of the cells stimulated the activity of the phosphocholine cytidylyltransferase. When cells prelabelled with [3H]palmitic acid were stimulated with TPA in the presence of ethanol, an increase of labelled diacylglycerol and phosphatidylethanol in the nuclei was observed. Similarly, an increase of labelled diacylglycerol and phosphatidic acid but not of phosphatidylethanol occurred in [3H]palmitic acid prelabelled nuclei stimulated with TPA in the presence of ethanol. However the production of phosphatidylethanol was observed when the nuclei were treated with TPA in the presence of ATP and GTPgammaS. The stimulation of [3H]choline prelabelled nuclei with TPA also generated the release of free choline and phosphocholine. The results indicate the presence of PLD and probably PLC activities in LA-N-1 nuclei and the involvement of phosphatidylcholine in the production of nuclear lipid second messengers upon TPA stimulation of LA-N-1 cells. The correlation of the disappearance of phosphatidylcholine, the production of diacylglycerol and phosphatidic acid with the stimulation of phosphatidylcholine synthesis in nuclei of TPA treated LA-N-1 suggests the existence of a phosphatidylcholine cycle in these nuclei.

Asunto(s)

Núcleo Celular/metabolismo , Fosfatidilcolinas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Radioisótopos de Carbono , Núcleo Celular/efectos de los fármacos , Colina/metabolismo , Colina Quinasa/metabolismo , Citidina Difosfato Colina/metabolismo , Humanos , Cinética , Neuroblastoma , Ácido Palmítico/metabolismo , Fosforilcolina/metabolismo , Tritio , Células Tumorales Cultivadas

RESUMEN

Experimental studies have indicated that the mechanisms offered for explaining the neurotoxicity of amyloid beta peptide (AbetaP) are diverse, and include altered enzyme activities, disrupted calcium homeostasis, and increased free radical formation. AbetaP appears to interact at the cell membrane with a multitude of receptor sites and also inserts physically into the membrane matrix. This membrane insertion affects the membrane fluidity and potentially influences the function of resident membrane proteins. We propose a unifying hypothesis to explain the experimental observations of the diverse cellular responses to AbetaP. The indiscriminate physical insertion of AbetaP into the cell membrane unspecifically activates a host of membrane processes by perturbation of the membrane proteins. This recurrent activation of membrane processes eventually culminates in neuronal cell death. We recommend that successful therapeutic interventions should be directed at reducing or preventing the interaction of AbetaP with neuronal cell membranes.

Asunto(s)

Péptidos beta-Amiloides/farmacología , Péptidos beta-Amiloides/fisiología , Membrana Celular/fisiología , Proteínas de la Membrana/fisiología , Neuronas/fisiología , Animales , Membrana Celular/efectos de los fármacos , Humanos , Fluidez de la Membrana/fisiología , Proteínas de la Membrana/efectos de los fármacos , Modelos Neurológicos , Neuronas/efectos de los fármacos , Neurotoxinas

RESUMEN

Heterotrimeric and small molecular mass guanine nucleotide binding (GTP-binding) proteins were found in neuronal and glial nuclei isolated from rat brain. Neuronal nuclei bound 0.213 +/- 0.055 pmoles of GTP/microg protein (n = 8) and glial nuclei bound 0.145 +/- 0.038 pmoles of GTP/microg protein (n = 8). The intrinsic GTPase activity of neuronal and glial nuclei was 0.0019 +/- 0.0005 pmoles GTP hydrolyzed/min/microg protein (n = 10) and 0.0022 +/- 0.0006 pmoles GTP hydrolyzed/min/microg protein (n = 10), respectively. Western blot analysis was carried out using a peptide-specific antibody that recognizes a common sequence in the alpha-subunit of the various heterotrimeric G-proteins. The antibody revealed the presence of a polypeptide of molecular mass of 40 kDa only in neuronal nuclei. Small molecular mass GTP-binding proteins were detected by incubating nitrocellulose blots with [alpha-32P]GTP. The results demonstrated the presence of 25-26 kDa GTP-binding proteins in both populations of nuclei. However, the binding of [alpha-32P]GTP to neuronal nuclei was approximately 3-fold greater than to the glial nuclei. Further analysis by two-dimensional polyacrylamide gel electrophoresis resolved the neuronal nuclei 26 kDa protein into three forms (a-c) with the most acidic form (c) being the major species. The neuronal 25 kDa protein was resolved into two forms that were present in approximately equal concentration. In glial nuclei, only the 26 kDa (c) and a small amount of the 25 kDa proteins were detected. However, both populations of nuclei contained the small molecular mass GTP-binding protein, ran. Differential association of non-ran small molecular mass GTP-binding proteins and heterotrimeric G-proteins with neuronal nuclei suggests a potential role for these guanine nucleotide binding proteins in the function of this cell type.

Asunto(s)

Encéfalo/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Western Blotting , Encéfalo/enzimología , Encéfalo/ultraestructura , Núcleo Celular/enzimología , Electroforesis en Gel de Poliacrilamida , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/química , Peso Molecular , Neuroglía/enzimología , Neuroglía/ultraestructura , Neuronas/enzimología , Neuronas/ultraestructura , Ratas

RESUMEN

There is a consensus that by some still to be defined mechanism amyloid beta peptide, which accumulates in Alzheimer's disease brain tissue, contributes to the characteristic neurodegeneration. We suggest that one of these mechanisms for amyloid beta peptide is the ability to activate cellular phospholipases. Excessive phospholipid hydrolysis would produce a variety of lipidic second messengers. These catabolites would then evoke unnecessary stereotypic responses. This indiscriminate activation of the phospholipases could be responsible for the increased amounts of phospholipid catabolites found in Alzheimer's disease brain tissue. Failure to maintain regeneration of the membrane components would result in a loss of essential cellular neuronal processes.

Asunto(s)

Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/fisiología , Degeneración Nerviosa/fisiopatología , Neurotoxinas/metabolismo , Fosfolipasas/fisiología , Humanos

RESUMEN

Amyloid beta protein is the major protein component of neuritic plaques found in the brain of Alzheimer's disease. The activation of phospholipase D by amyloid beta protein (25-35), quisqualate and phorbol 12, 13-dibutyrate was investigated in LA-N-2 cells by measuring phosphatidylethanol formation. The activation of phospholipase D by quisqualate and APP (25-35) was calcium-independent. The AbetaP (25-35) and quisqualate activation of phospholipase D appeared to be mediated through a pertussis toxin-sensitive GTP-binding protein. Phospholipase D activation by AbetaP (25-35), quisqualate and phorbol dibutyrate was not blunted by the protein kinase C inhibitors, staurosporine, H-7 and RO-31-8220. However, it was abolished by overnight exposure to phorbol dibutyrate. This activation of phospholipase D was prevented by the tyrosine kinase inhibitor, genistein but not by tyrophostin A. Several excitatory amino acid antagonists were tested for their ability to prevent the phospholipase D activation by quisqualate and AbetaP (25-35). Only NBQX was effective with an IC50 of 75 microM for AbetaP (25-35) and quisqualate. Activation of phospholipase D by AbetaP or quisqualate was absent in LA-N-2 cells previously desensitized by quisqualate or AbetaP (25-35), but the activation by phorbol dibutyrate was unaltered. The responsiveness to AbetaP and quisqualate in previously desensitized cells reappeared subsequent to a period of resensitization. The observations with the antagonist NBQX, and the desensitization and resensitization experiments, are consistent with a receptor occupancy mediated activation of phospholipase D by quisqualate and by AbetaP (25-35).

Asunto(s)

Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Fosfolipasa D/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Línea Celular , Activación Enzimática , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas de Unión al GTP/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Forbol 12,13-Dibutirato/farmacología , Proteínas Quinasas/metabolismo , Ácido Quiscuálico/farmacología , Receptores de Glutamato Metabotrópico/agonistas

RESUMEN

It has been established that amyloid beta peptide (AbetaP) activates phospholipase A2, phospholipase C and phospholipase D of LA-N-2 cells and other cell types. Nicotine in addition to being a cholinergic agonist, may be neuroprotective. We have investigated the ability of (-)nicotine to blunt the phospholipase activations by AbetaP in LA-N-2 cells. (-)Nicotine inhibits the AbetaP activation of phospholipase A2, with an IC50 of 76 microM and of phospholipase D with an IC50 of 252 microM. (-)Nicotine did not blunt the AbetaP activation of phospholipase C. These inhibitions of AbetaP activations were not observed with (+)nicotine or cotinine. The (-)nicotine inhibition of AbetaP activation of these two phospholipases was unaffected by hexamethonium and D-tubocurarine. There was no inhibition of the phospholipase A2 activity present in homogenates of LA-N-2 cells. Exposure of LA-N-2 cells to (-)nicotine for 2 h resulted in the blockade of phospholipase A2 activation by kainate and AbetaP but did not affect the ability of quisqualate and AbetaP to activate phospholipase D. These data suggest that if the nicotine inhibition of AbetaP activations is receptor occupancy mediated then it is by an atypical receptor type.

Asunto(s)

Péptidos beta-Amiloides/metabolismo , Ácidos Aristolóquicos , Encéfalo/enzimología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Fosfolipasa D/metabolismo , Fosfolipasas A/metabolismo , Acetofenonas/farmacología , Ácidos Araquidónicos/farmacología , Química Encefálica/efectos de los fármacos , Química Encefálica/fisiología , Células Cultivadas , Cotinina/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Hexametonio/farmacología , Ácido Kaínico/farmacología , Antagonistas Nicotínicos/farmacología , Fenantrenos/farmacología , Fosfolipasas A2 , Ácido Quiscuálico/farmacología , Tubocurarina/farmacología , Fosfolipasas de Tipo C/metabolismo

RESUMEN

We hypothesized that hydrocephalus in young animals could cause a delay in myelination. Hydrocephalus was induced in 3-week-old rats by injecting kaolin into the cisterna magna. Ventricular size was assessed by magnetic resonance imaging. After 1 to 4 weeks, rats were either sacrificed, or treated by diversionary shunting of cerebrospinal fluid and then sacrificed 3 to 4 weeks later. Samples of corpus callosum/supraventricular white matter, fimbria, medulla, and spinal cord were assayed for myelin-related enzyme activities including p-nitrophenylphosphorylcholine phosphocholine phosphodiesterase (PNPCP), glycerophosphocholine phosphocholine phosphodiesterase (GPCP), and 2',3'-cyclic neucleotide 3'-phosphodiesterase (CNPase), and the oligodendrocyte enzyme UDP-galactose, ceramide galactosyltransferase (CGa1T). Myelin basic protein (MBP) and proteolipid protein (PLP) were assayed in cerebrum by immunoblots and Northern blot. The corpus callosum was processed for electron microscopy and myelin thickness to axon diameter ratios were quantified. One week after induction of hydrocephalus, CGa1T and GPCP activity were reduced in the corpus callosum there was less MBP and PLP in the cerebrum, and myelin sheaths around axons greater than 0.4 micron in diameter were abnormally thin. With persistent hydrocephalus, the corpus callosum became thinned, axons were lost, and myelin-related enzyme activities and proteins were decreased. Treatment of hydrocephalus at 1 week largely prevented the damage while shunting at 4 weeks failed to restore the injured white matter. Early reduction in CGa1T activity in the medulla and spinal cord suggest that oligodendrocyte production of myelin was reduced, even before irreversible damage occurred in the corticospinal tracts. We conclude that hydrocephalus in the immature rat brain delays myelination, but compensatory myelination is possible if treatment is instituted prior to the development of axonal injury. Possible mechanisms of oligodendrocyte impairment are discussed.

Asunto(s)

Animales Recién Nacidos/fisiología , Encéfalo/crecimiento & desarrollo , Hidrocefalia/fisiopatología , Caolín , Vaina de Mielina/fisiología , Animales , Apoproteínas/metabolismo , Derivaciones del Líquido Cefalorraquídeo , Cuerpo Calloso/ultraestructura , Enzimas/metabolismo , Hidrocefalia/patología , Hidrocefalia/cirugía , Proteína Básica de Mielina/metabolismo , Proteína Proteolipídica de la Mielina/metabolismo , Vaina de Mielina/ultraestructura , Ratas , Ratas Sprague-Dawley , Valores de Referencia

RESUMEN

The amyloid beta protein (25-35) stimulated appearance of 3H-inositol phosphates from [3H]inositol-prelabeled LA-N-2 cells was investigated. This stimulation was unaltered by extra- and intracellular calcium chelators in a calcium-free medium or by several protein kinase inhibitors. This phospholipase C stimulation by amyloid beta protein appeared to be pertussis toxin sensitive. It is possible that this phospholipase C stimulation by amyloid beta protein is a receptor-mediated process. This possibility is based on two related observations. The stimulation is ablated by the presence of conventional antagonists for metabotropic, adrenergic, and bombesin agonists. The IC50 values were 12 microM for propranolol, 15 microM for AP-3, and 25 nM for [Tyr4,D-Phe12]bombesin. Additional support comes from results of desensitization and resensitization experiments. Amyloid beta protein stimulation of phospholipase C was absent from LA-N-2 cells previously treated with norepinephrine, trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), bombesin, or amyloid beta peptide. In a similar manner, LA-N-2 cells previously treated with amyloid beta protein were no longer responsive to norepinephrine, t-ACPD, or bombesin. The responsiveness to amyloid beta protein returned, subsequent to a period of resensitization for the individual agonists. It is suggested that this observed amyloid beta protein stimulation of phospholipase C may be responsible for the elevated quantity of inositol seen in the brains of Alzheimer's disease patients.

Asunto(s)

Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Fosfolipasas de Tipo C/metabolismo , Agonistas Adrenérgicos/farmacología , Agonistas alfa-Adrenérgicos/farmacología , Bombesina/farmacología , Calcio/metabolismo , Quelantes/farmacología , Toxina del Cólera/farmacología , Cicloleucina/análogos & derivados , Cicloleucina/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Epinefrina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Guanosina Trifosfato/metabolismo , Humanos , Neuroblastoma , Fármacos Neuroprotectores/farmacología , Norepinefrina/farmacología , Toxina del Pertussis , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Sensibilidad y Especificidad , Tionucleótidos/farmacología , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/enzimología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Factores de Virulencia de Bordetella/farmacología

RESUMEN

We investigated the influence of ion compositions on the membrane potential in LA-N-1 human neuroblastoma cells using bisoxonol as a potential-sensitive fluorescent dye. The ability of K+, ouabain, veratridine, and maitotoxin to induce membrane depolarization was evaluated. Increasing concentrations of K+ ions from 10 to 50 mM caused a dose-dependent increase of bisoxonol fluorescence, which was completely independent on Na+ and Ca2+. Ouabain (5 mM), an inhibitor of the Na+, K(+)-ATPase, failed to induce membrane depolarization. Veratridine (40 and 100 microM), a Na+ channel activator, only in the presence of 10 micrograms of Leiurus scorpion venom reduced the membrane potential. Maitotoxin (MTX) from 3 to 10 ng/mL depolarized LA-N-1 cells in a dose-dependent manner, and produced a rapid and sustained increase of intracellular free calcium monitored by means of fluorescent probe fura-2. The MTX-induced depolarization and the increase in cytosolic free calcium concentration were dependent on extracellular Ca2+ ions. On the other hand, Na+ ions also seem to be, although only partially, implicated in the MTX effects, since both the blockade of tetrodotoxin (TTX)-sensitive voltage-operated Na+ channels and the removal of Na+ ions were able to reduce the depolarization. In conclusion, our data indicate that the depolarizing action of MTX on LA-N-1 cells is Ca(2+)- and Na(+)-dependent, although the latter only partially, and that this effect is dependent on Ca2+ influx into the cells likely through a voltage-insensitive calcium-entry system.

Asunto(s)

Membrana Celular/fisiología , Neuroblastoma/metabolismo , Oxocinas , Calcio/metabolismo , Calcio/farmacología , Membrana Celular/efectos de los fármacos , Citosol/química , Citosol/efectos de los fármacos , Citosol/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Toxinas Marinas/metabolismo , Toxinas Marinas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Neuroblastoma/patología , Ouabaína/farmacología , Potasio/farmacología , Sodio/farmacología , Células Tumorales Cultivadas , Veratridina/farmacología

RESUMEN

A series of single alanine substituted analogs of amyloid beta peptide (25-35) were tested for their ability to activate the phospholipases of cultured LA-N-2 cells. Substitution of alanine for the amino acids 29-34 prevented the activation of phospholipases A2 and D. In addition substitution of alanine at 28 prevented phospholipase D but not phospholipase A2 activation. All the alanine substitutions, except for positions 33 and 35, blunted phospholipase C activations. There were no activations by scrambled amyloid beta peptide.

Asunto(s)

Alanina/metabolismo , Péptidos beta-Amiloides/farmacología , Fragmentos de Péptidos/farmacología , Fosfolipasa D/efectos de los fármacos , Fosfolipasas A/efectos de los fármacos , Fosfolipasas de Tipo C/efectos de los fármacos , Péptidos beta-Amiloides/química , Activación Enzimática , Humanos , Fragmentos de Péptidos/química , Fosfolipasa D/metabolismo , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Relación Estructura-Actividad , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/metabolismo

RESUMEN

Amyloid beta protein (25-35) stimulates the phospholipase A2, C and D activation of LA-N-2 cells. Nordihydroguaiaretic acid reduced the phospholipase D activation by 30% (P < 0.008) and indomethacin reduced the phospholipase A2 activation by 58% (P < .001). There were no reductions of the amyloid beta protein activations by acetylsalicylic acid (ASA), gentisic acid, sulindac sulfone and acetaminophen. The activation of phospholipase C by amyloid beta protein was unaffected by these compounds.

Asunto(s)

Precursor de Proteína beta-Amiloide/efectos de los fármacos , Indometacina/farmacología , Masoprocol/farmacología , Fosfolipasas/efectos de los fármacos , Neoplasias Encefálicas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Neuroblastoma/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos

RESUMEN

Because of the importance of oligodendrocytes (OL) in forming and maintaining myelin in the CNS and the fact that the remyelination in the CNS is very limited in contrast to the peripheral nervous system, we investigated the effect of a chemically defined medium OLDEM, previously characterized by the maintenance of mature myelinating OL, on oligodendroblasts (or OL progenitors) in culture. The effect of each component of this medium as well as different combinations of them were also examined. Cultures were examined at different developmental stages immunocytochemically for developmental markers, such as transferrin, sulfatides, myelin basic protein and proteolipid protein. OLDEM accelerated the appearance of developmental markers and concomitant morphological changes. Furthermore, myelin-specific enzymes such as glycerophosphorylcholine phosphodiesterase; p-nitro-phenolphosphocholine phosphodiesterase; 2'3'-cyclic nucleotide 3'-phosphodiesterase and UDP galactose: ceramide galactosyltransferase had enzymatic activities similar to values found in pure myelin, indicating that OLDEM allows the optimal expression of myelin-related genes. The effect of each OLDEM constituent was evaluated by immunocytochemistry and by measurement of enzymatic activities. With each single additive or multiple combinations, oligodendrocytes displayed different degrees of maturation. Deletion of selenium, glucose, and galactose severely affected cell survival as well as enzymes expression in young cultures. However, older cultures were more resistant to these deletions. Putrescine and insulin did not cause such effects on survival, but their absence affected cell maturation. None of the OLDEM additives individually supported survival and/or maturation. Enzyme assays performed on isolated myelin-like membranes or the cells soma revealed a redistribution of the activity between these fractions as the cell matured. The biological role of each of these constituents on the maturation of the oligodendroglial cells is discussed. These observations indicate that OLDEM constituents have a powerful effect on OL progenitor maturation, and membrane formation. This medium will be used for investigating the remyelination potential of adult OL progenitors.

Asunto(s)

Vaina de Mielina/fisiología , Oligodendroglía/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Medios de Cultivo , Inmunohistoquímica , Vaina de Mielina/metabolismo , Vaina de Mielina/ultraestructura , Neuroglía/fisiología , Oligodendroglía/enzimología , Oligodendroglía/ultraestructura , Hidrolasas Diéster Fosfóricas/metabolismo , Ratas

RESUMEN

L-Glutamate, a major excitatory amino acid, plays an important role in learning and memory. L-Glutamate uptake into synaptic vesicles is an ATP-dependent process. Exposure of neurons to high, sustained extracellular concentrations of glutamate results in excitotoxicity. Elevated levels of phosphomonoesters (PMEs), phosphodiesters (PDEs), and phosphocreatine (PCr) have been reported in Alzheimer disease (AD). In this article, the effects of selected PMEs, PDEs, and PCr on vesicular L-[3H]glutamate uptake into isolated bovine synaptic vesicles are investigated. D-myo-Inositol-1-monophosphate (I1P), D-myo-inositol-2-monophosphate (I2P), sn-glycero-3-phosphate, (alpha-GP) and PCr significantly stimulated L-[3H]glutamate uptake into synaptic vesicles. Phosphoethanolamine (PE), phosphocholine (PC), L-phosphoserine (L-PS) sn-glycero-3-phosphocholine (GPC), and sn-glycero-3-phosphoethanolamine (GPE) had little or no effect on vesicular L-glutamate uptake. These observations suggested that the vesicular uptake of glutamate can be regulated by endogenous PMEs and PCr. The mechanism of activation by I1P, I2P, and alpha-GP appears to be stimulation of Mg(2+)-ATPase activity. These effects on vesicular glutamate uptake may be important in diseases in which the levels of these metabolites are altered, as they are in AD.

Asunto(s)

Ácido Glutámico/metabolismo , Fosfatos/farmacología , Fosfocreatina/farmacología , Vesículas Sinápticas/metabolismo , Aminoácidos/metabolismo , Animales , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Bovinos , Glicerofosfatos/metabolismo , Técnicas In Vitro , Inositol/metabolismo , Cinética , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/enzimología

RESUMEN

Multiple cellular responses are regulated through the generation of lipid second messengers upon activation of phospholipases. One such response concerns the activity of a class of kinase constituting the protein kinase C family. The production of specific molecular species of lipid second messengers may be therefore of prime importance in the activation of a member of the PKC isoforms. Prompted by this possibility we investigated the production of 1,2 diacyl-sn-glycerol (DAG) and phosphatidic acid (PtdOH) in LA-N-1 neuroblastoma cells under various physiological states. 12-0-Tetradecanoylphorbol 13-acetate (TPA) stimulation activated a phospholipase D (PLD) specific for phosphatidylcholine (PtdCho) in proliferating cells and a phospholipase C (PLC) specific for phosphatidylethanolamine (PtdEtn) in retinoic acid (RA) differentiated cells. These separate activations produced different molecular species of DAG or PtdOH. PtdOH was able to stimulate the Ca2+ dependent protein kinase C (PKC) by a mechanism which differed from the action of DAG. PtdOH did not induce the translocation of the PKC to the membrane. Moreover PtdOH, in contrast to DAG, prevented PKC degradation by inhibiting the enzymatic hydrolysis by m-calpain. These observations suggest that the stimulation of cells by agonists elicited the production of specific molecular species of lipid second messengers depending on the physiological status of the cells, and probably on the nature of the stimulus. It seems therefore likely that the generation of specific lipid second messengers may activate specific PKC isoforms resulting in a specific cellular response.

Asunto(s)

Metabolismo de los Lípidos , Neuroblastoma/metabolismo , Sistemas de Mensajero Secundario , Animales , Diferenciación Celular , División Celular , Humanos , Neuroblastoma/patología , Células Tumorales Cultivadas

RESUMEN

There is evidence available suggesting that membrane alterations occur in Alzheimer's disease including the metabolism of membrane phospholipids. We have quantitated in vitro the phospholipase D activity of homogenates from Alzheimer's disease brain tissue. There was a significant increase of this enzyme activity as compared to controls. Amyloid beta protein is the predominant protein of the characteristic senile plaques found in Alzheimer's disease. Treatment of LA-N-2 cells, a human cholinergic neuroblastoma clone, with amyloid beta protein results in an activation of phospholipases A, C and D.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Fosfolípidos/metabolismo , Receptores Colinérgicos/metabolismo , Línea Celular , Humanos

RESUMEN

Phospholipase D activity of rat brain neuronal nuclei, measured with exogenous phosphatidylcholine as substrate, was characterized. The measured activity of neuronal nuclei was at least 36-fold greater than the activity in glia nuclei. The pH optimum was 6.5, and unsaturated but not saturated fatty acids stimulated the enzyme. The optimal concentration of sodium oleate for stimulation of the enzyme activity was 1.2 mM in the presence of 0.75 mM phosphatidylcholine. This phospholipase D activity was cation independent. In the absence of NaF, used as a phosphatidic acid phosphatase inhibitor, the principal product was diglyceride; whereas in the presence of NaF, the principal product was phosphatidic acid. The phospholipase D, in addition to having hydrolytic activity, was able to catalyze a transphosphatidylation reaction. Maximum phosphatidylethanol formation was seen with 0.2-0.3 M ethanol. GTPgammaS, ATPgammaS, BeF2, AIF3, phosphatidic acid, and phosphatidylethanol inhibited the neuronal nuclei phospholipase D activity. The addition of the cytosolic fraction of brain, liver, kidney, spleen, and heart to the incubation mixtures resulted in inhibition of the phospholipase D activity. Phospholipase D activity was detectable in nuclei prepared from rat kidney, spleen, heart, and liver.

Asunto(s)

Neuronas/enzimología , Fosfolipasa D/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Núcleo Celular/enzimología , Corteza Cerebral/enzimología , Corteza Cerebral/ultraestructura , Citosol/enzimología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Riñón/enzimología , Hígado/enzimología , Miocardio/enzimología , Neomicina/farmacología , Neuroglía/enzimología , Ácido Oléico , Ácidos Oléicos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipasa D/antagonistas & inhibidores , Ratas , Fluoruro de Sodio/farmacología , Bazo/enzimología

RESUMEN

The release of [3H]inositol phosphates from myo-[3H]inositol-prelabeled LA-N-2 cells was measured in the presence of beta-adrenoceptor, metabotropic glutamate and bombesin agonists. Norepinephrine and isoproterenol increased the formation of [3H]inositol phosphates in a dose-dependent manner, with an EC50 of 100 microM for norepinephrine and an EC50 of 5 microM for isoproterenol. These stimulations were abolished by propranolol, a beta-adrenoceptor antagonist, with an IC50 in the range of 50-55 microM for both norepinephrine and isoproterenol. The stimulation of [3H]inositol phosphate appearance occurred with varying concentrations of trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), a metabotropic glutamate receptor agonist. This release of [3H] inositol phosphates was blunted by its antagonist, 2-amino-3-phosphonopropionic acid (AP-3). Bombesin and neuromedin-B, a bombesin-like peptide, also increased the appearance of [3H]inositol phosphates. This was blunted by the antagonist [Tyr4, D-Phe12] bombesin. The appearance of [3H]inositol phosphates stimulated by t-ACPD was coupled through a cholera toxin-sensitive G-protein and the bombesin-stimulated appearance of [3H]inositol phosphates was coupled through a pertussis toxin-sensitive G-protein. The norepinephrine-stimulated appearance of [3H]inositol phosphates was toxin insensitive. The stimulation of the [3H]inositol phosphate appearance by these three agonists was protein kinase and Ca2+ independent.

Asunto(s)

Bombesina/farmacología , Cicloleucina/análogos & derivados , Norepinefrina/farmacología , Fosfolipasas de Tipo C/metabolismo , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Cicloleucina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Proteínas de Unión al GTP/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Isoproterenol/farmacología , Marcaje Isotópico , Neuroblastoma/enzimología , Neuroblastoma/metabolismo , Neuroquinina B/análogos & derivados , Neuroquinina B/farmacología , Neurotoxinas/farmacología , Propranolol/farmacología , Receptores de Bombesina/agonistas , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Tritio , Células Tumorales Cultivadas

RESUMEN

ATP-dependent uptake of glutamate into synaptic vesicles has been well documented. Stimulation of glutamate uptake into synaptic vesicles by other high-energy phosphates has not been described. In this paper, we examine the stimulation of phosphocreatine (PCr)-induced glutamate uptake and determine whether this stimulation is secondary to conversion of PCr to ATP. We found the following. 1) PCr stimulates glutamate uptake into synaptic vesicles in the absence of added ATP. 2) At a glutamate concentration of 50 microM, no concentration of added ATP could produce the degree of stimulation seen in the presence of PCr. 3) 0.5 mM iodoacetamide completely inhibits synaptic vesicle creatine kinase activity but does not inhibit PCr-stimulated glutamate uptake. 4) PCr-dependent glutamate uptake, unlike ATP-dependent uptake, is not magnesium- or chloride-dependent. 5) 0.5 mM N-ethylmaleimide, a selective H+-ATPase inhibitor, completely inhibits ATP-dependent glutamate uptake but only slightly inhibits PCr-dependent glutamate uptake. 6) PCr-dependent glutamate uptake is sensitive to valinomycin, a K+/H+ translocator, whereas the ATP-dependent uptake is not. Therefore, it appears that in addition to the well-known ATP-dependent glutamate uptake system, there is a previously unreported PCr-dependent glutamate uptake system in synaptic vesicles. The total glutamate uptake by synaptic vesicles is likely the sum of both ATP- and PCr-dependent glutamate uptake.

Asunto(s)

Adenosina Trifosfato/metabolismo , Ácido Glutámico/metabolismo , Fosfocreatina/metabolismo , Vesículas Sinápticas/metabolismo , Adenosina Trifosfato/farmacología , Animales , Transporte Biológico Activo/efectos de los fármacos , Encéfalo/metabolismo , Bovinos , Técnicas In Vitro , Cinética , Fosfocreatina/farmacología , Vesículas Sinápticas/efectos de los fármacos

RESUMEN

An oleate dependent form of phospholipase D is present in rat brain neuronal nuclei and both the hydrolytic and transphosphatidylation activities measured. Several acidic phospholipids were found to inhibit this activity in a dose dependent manner. The IC50 values varied from 3.5 microM for PIP2 to 200 microM for phosphatidic acid. The hydrolysis of PIP2 by phospholipase C would be expected to result in the disinhibition of the oleate dependent phospholipase D activity.

Asunto(s)

Encéfalo/efectos de los fármacos , Glicerofosfolípidos , Neuronas/efectos de los fármacos , Fosfolipasa D/antagonistas & inhibidores , Fosfolípidos/farmacología , Adenosina Trifosfato/farmacología , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Cardiolipinas/farmacología , Núcleo Celular/enzimología , Guanosina Trifosfato/farmacología , Isoenzimas/antagonistas & inhibidores , Neuronas/enzimología , Ácido Oléico , Ácidos Oléicos/farmacología , Ácidos Fosfatidicos/metabolismo , Ácidos Fosfatidicos/farmacología , Fosfatidilgliceroles/farmacología , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/farmacología , Fosfatidilserinas/farmacología , Ratas , Fosfolipasas de Tipo C/metabolismo

RESUMEN

Phosphatidic acid (PA), a hydrolytic product of phospholipase D activity, stimulated cytosolic protein kinase C (PKC) activity when LA-N-1 neuroblastoma cells in culture were treated with PA, without translocating the enzyme to the membrane. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) translocated and activated PKC in a dogmatic manner. Partially purified PKC activity derived from LA-N-1 neuroblastoma cells was stimulated by PA alone or in the presence of phosphatidylserine or TPA, without affecting [3H]phorbol dibutyrate binding, indicating that the site of action of PA was different from the phorbol ester or diacylglycerol binding site. These results suggest an unorthodox pattern of PKC stimulation mediated by PA which appears to be yet another mode of PA signal transduction.

Asunto(s)

Neuroblastoma/metabolismo , Ácidos Fosfatidicos/farmacología , Proteína Quinasa C/efectos de los fármacos , Unión Competitiva , Calcio/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Factores de Tiempo