RESUMEN
Our previous work has correlated permanent alterations in the rat neurosecretory machinery with epileptogenesis. Such findings highlighted the need for a greater understanding of the molecular mechanisms underlying epilepsy so that novel therapeutic regimens can be designed. To this end, we examined kindling in transgenic mice with a defined reduction of a key element of the neurosecretory machinery: the v-SNARE (vesicle-bound SNAP [soluble NSF attachment protein] receptor), synaptobrevin/vesicle-associated membrane protein 2 (VAMP2). Initial analysis of biochemical markers, which previously displayed kindling-dependent alterations in rat hippocampal synaptosomes, showed similar trends in both wild-type and VAMP2(+/-) mice, demonstrating that kindled rat and mouse models are comparable. This report focuses on the effects that a ~50% reduction of synaptosomal VAMP2 has on the progression of electrical kindling and on glutamate release in hippocampal subregions. Our studies show that epileptogenesis is dramatically attenuated in VAMP2(+/-) mice, requiring both higher current and more stimulations to reach a fully kindled state (two successive Racine stage 5 seizures). Progression through the five identifiable Racine stages was slower and more variable in the VAMP2(+/-) animals compared with the almost linear progression seen in wild-type littermates. Consistent with the expected effects of reducing a major neuronal v-SNARE, glutamate-selective, microelectrode array (MEA) measurements in specific hippocampal subregions of VAMP2(+/-) mice showed significant reductions in potassium-evoked glutamate release. Taken together these studies demonstrate that manipulating the levels of the neurosecretory machinery not only affects neurotransmitter release but also mitigates kindling-induced epileptogenesis.
Asunto(s)
Epilepsia/fisiopatología , Excitación Neurológica/genética , Excitación Neurológica/fisiología , Proteína 2 de Membrana Asociada a Vesículas/biosíntesis , Proteína 2 de Membrana Asociada a Vesículas/fisiología , Animales , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Región CA2 Hipocampal/efectos de los fármacos , Región CA2 Hipocampal/metabolismo , Interpretación Estadística de Datos , Giro Dentado/efectos de los fármacos , Giro Dentado/metabolismo , Estimulación Eléctrica , Ácido Glutámico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microelectrodos , Proteínas SNARE/fisiología , Proteína 2 de Membrana Asociada a Vesículas/genéticaRESUMEN
Critical to SNARE protein function in neurotransmission are the accessory proteins, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP), and NSF, that play a role in activation of the SNAREs for membrane fusion. In this report, we demonstrate the depolarization-induced, calcium-dependent phosphorylation of NSF in rat synaptosomes. Phosphorylation of NSF is coincident with neurotransmitter release and requires an influx of external calcium. Phosphoamino acid analysis of the radiolabeled NSF indicates a role for a serine/threonine-specific kinase. Synaptosomal phosphorylation of NSF is stimulated by phorbol esters and is inhibited by staurosporine, chelerythrine, bisindolylmaleimide I, calphostin C, and Ro31-8220 but not the calmodulin kinase II inhibitor, Kn-93, suggesting a role for protein kinase C (PKC). Indeed, NSF is phosphorylated by PKC in vitro at Ser-237 of the catalytic D1 domain. Mutation of this residue to glutamic acid or to alanine eliminates in vitro phosphorylation. Molecular modeling studies suggest that Ser-237 is adjacent to an inter-subunit interface at a position where its phosphorylation could affect NSF activity. Consistently, mutation of Ser-237 to Glu, to mimic phosphorylation, results in a hexameric form of NSF that does not bind to SNAP-SNARE complexes, whereas the S237A mutant does form complex. These data suggest a negative regulatory role for PKC phosphorylation of NSF.
Asunto(s)
Proteínas Portadoras/metabolismo , Neurotransmisores/metabolismo , Sinaptosomas/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Potenciales de la Membrana , Datos de Secuencia Molecular , Proteínas Sensibles a N-Etilmaleimida , Fosforilación , Proteína Quinasa C/metabolismo , Ratas , Serina/metabolismo , Sinaptosomas/fisiologíaRESUMEN
L-type voltage-sensitive Ca2+ channels (VSCCs) preferentially modulate several neuronal processes that are thought to be important in epileptogenesis, including the slow afterhyperpolarization (AHP), LTP, and trophic factor gene expression. However, little is yet known about the roles of L-type VSCCs in the epileptogenic process. Here, we used cell-attached patch recording techniques and single cell mRNA analyses to study L-type VSCCs in CA1 neurons from partially dissociated (zipper) hippocampal slices from entorhinally-kindled rats. L-type Ca2+-channel activity was reduced by >50% at 1.5-3 months after kindling. Following recording, the same single neurons were extracted and collected for mRNA analysis using a recently developed method that does not amputate major dendritic processes. Therefore, neurons contained essentially full complements of mRNA. For each collected neuron, mRNA contents for the L-type pore-forming alpha1D/Ca(v)1.3-subunit and for calmodulin were then analyzed by semiquantitative kinetic RT-PCR. L-type alpha1D-subunit mRNA was correlated with L-type Ca2+-channel activity across single cells, whereas calmodulin mRNA was not. Thus, these results appear to provide the first direct evidence at the single channel and gene expression levels that chronic expression of an identified Ca2+-channel type is modulated by epileptiform activity. Moreover, the present data suggest the hypothesis that down regulation of alpha1D-gene expression by kindling may contribute to the long-term maintenance of epileptiform activity, possibly through reduced Ca2+-dependent AHP and/or altered expression of other relevant genes.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Excitación Neurológica/fisiología , Células Piramidales/metabolismo , ARN Mensajero/metabolismo , Animales , Epilepsia/metabolismo , Hipocampo/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
We have previously shown that inhibition of expression of the plasma membrane Ca(2+)-ATPase isoform 1 in PC6 cells leads to loss of nerve growth factor-mediated neurite extension (Brandt, P.C., Sisken, J.E., Neve, R.L., and Vanaman, T.C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 13843-13848). Cells lacking plasma membrane Ca(2+)-ATPase 1 did not attach to collagen-coated plates as tightly as controls, suggesting that a defect in adhesion might be underlying the inability to extend neurites. We report here that cell lines lacking plasma membrane Ca(2+)-ATPase 1 do not produce alpha(1) integrin, which is required for both collagen adherence and neurite extension. Because alpha(1) integrin gene transcription can be down-regulated by glucocorticoids, the response of cells to glucocorticoids was investigated. Cortisol-dependent transactivation from the mouse mammary tumor virus promoter in cells lacking plasma membrane Ca(2+)-ATPase 1 was stimulated 145-216-fold over untreated cells compared with 15-26-fold for controls. This increase was not due to increased binding affinity of the receptor for cortisol, an increased number of cortisol-binding sites, or increased translocation of the receptor to the nucleus. Expression of additional glucocorticoid receptor-dependent genes required for neurite extension must also be altered in cells missing the plasma membrane Ca(2+)-ATPase 1 because constitutive expression of alpha(1) integrin did not restore their nerve growth factor-mediated neurite extension capability. The impact of plasma membrane Ca(2+)-ATPase isoform 1 on other signaling systems and the resultant profound yet subtle effects on PC6 cells strongly suggests that it plays an important role in modulating signal transduction pathways downstream of Ca(2+)-mediated signals.
Asunto(s)
Antígenos CD/genética , ATPasas Transportadoras de Calcio/metabolismo , Regulación de la Expresión Génica , Receptores de Glucocorticoides/fisiología , Animales , ATPasas Transportadoras de Calcio/genética , Proteínas de Transporte de Catión , Membrana Celular/enzimología , Núcleo Celular/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Hidrocortisona/farmacología , Integrina alfa1 , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Virus del Tumor Mamario del Ratón/genética , Ratones , Células PC12 , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Regiones Promotoras Genéticas , Ratas , Receptores de Glucocorticoides/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Activación Transcripcional/efectos de los fármacos , Triamcinolona/farmacocinéticaRESUMEN
BACKGROUND: Many inhalation anesthetics at clinically relevant concentrations inhibit plasma membrane Ca2+-adenosine triphosphatase (PMCA) ion pumping in brain synaptic membranes and in cultured cells of neural origin. In this study, the authors investigated the effect of inhalation anesthetics on cytosolic calcium homeostasis in cortical neurons maintained at physiologic and room temperatures and on cortical neurons and pheochromocytoma cells with antisense blockade of specific PMCA isoforms. METHODS: Using Ca2+-specific confocal microfluorimetry, the anesthetic effects on Ca2+ dynamics were examined in mouse embryonic cortical neurons in association with ligand-stimulated Ca2+ influx. Studies were done at 21 degrees C and 37 degrees C. Mouse embryonic cortical neurons with oligodeoxyribonucleotide blockade of PMCA2 expression and transfected rat pheochromocytoma cells with blocked expression of PMCA1 were also examined. RESULTS: Baseline and poststimulation peak cytosolic calcium concentrations ([Ca2+]i) were increased, and Ca2+ clearance was delayed in cells exposed at 37 degrees C, but not at 21 degrees C, to concentrations < or = 1 minimum alveolar concentration (MAC)-equivalent of halothane, isoflurane, and sevoflurane. Neurons exposed to xenon solutions < or = 0.4, 0.6, and 0.8 MAC showed dose-related perturbations of cytosolic Ca2+. Calcium dynamics were altered in neural cells with blocked PMCA isoform production, but at much lower halothane concentrations: 0.5 MAC for cortical neurons and 0.1 MAC for pheochromocytoma cells. CONCLUSIONS: By extruding Ca2+ through the plasma membrane, PMCA maintains resting neuronal [Ca2+]i at low levels and clears physiologic loads of Ca2+ after influx through calcium channels. Inhalation anesthetics perturb this process and thus may interfere with neurotransmitter release, altering interneuronal signaling.
Asunto(s)
Anestésicos por Inhalación/farmacología , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Halotano/farmacología , Isoflurano/farmacología , Éteres Metílicos/farmacología , Neuronas/fisiología , Animales , Membrana Celular/metabolismo , Células Cultivadas , Electrofisiología , Transporte Iónico/efectos de los fármacos , Ratones , Neuronas/ultraestructura , Ratas , Sevoflurano , TemperaturaRESUMEN
As a consequence of its central role in the regulation of calcium metabolism in the platelet, the plasma membrane Ca2+-ATPase (PMCA) was assessed for cAMP-dependent and tyrosine phosphorylation. Addition of forskolin or prostaglandin E1, agents known to elevate platelet cAMP and calcium efflux, to platelets pre-labeled with [32P]PO4 resulted in the direct phosphorylation of platelet PMCA. Similarly, addition of the catalytic subunit of protein kinase A to platelet plasma membranes resulted in a 1.4-fold stimulation of activity. Thus, the previously reported inhibition of platelet activation by elevated intracellular cAMP may be accomplished in part by stimulation of PMCA, likely resulting in a decrease in intracellular calcium. Treatment with thrombin evoked tyrosine phosphorylation of platelet PMCA, while PMCA from resting platelets exhibited little tyrosine phosphorylation. Phosphorylation of platelet plasma membranes by pp60(src) resulted in 75% inhibition of PMCA activity within 15 min. Similarly, membranes isolated from thrombin-treated platelets exhibited 40% lower PMCA activity than those from resting platelets. Phosphorylation of erythrocyte ghosts and purified PMCA by pp60(src) also resulted in up to 75% inhibition of Ca2+-ATPase activity, and inhibition was correlated with tyrosine phosphorylation. Sequencing of a peptide obtained after 32P labeling of purified erythrocyte PMCA in vitro showed that tyrosine 1176 of PMCA4b is phosphorylated by pp60(src). These results indicate that tyrosine phosphorylation of platelet PMCA may serve as positive feedback to inhibit PMCA and increase intracellular calcium during platelet activation.
Asunto(s)
Plaquetas/enzimología , ATPasas Transportadoras de Calcio/metabolismo , AMP Cíclico/metabolismo , Proteínas de la Membrana/metabolismo , Tirosina/metabolismo , Membrana Celular/enzimología , Humanos , Fosforilación , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismoRESUMEN
It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Transformación Celular Viral , Fibroblastos/enzimología , Isoenzimas/metabolismo , Virus 40 de los Simios , Animales , ATPasas Transportadoras de Calcio/genética , Proteínas de Transporte de Catión , Línea Celular , Línea Celular Transformada , Fibroblastos/citología , Prueba de Complementación Genética , Humanos , Isoenzimas/genética , Pulmón/citología , Pulmón/enzimología , ATPasas Transportadoras de Calcio de la Membrana Plasmática , Reacción en Cadena de la Polimerasa , ARN Mensajero , Piel/citología , Piel/enzimologíaRESUMEN
Numerous lines of evidence indicate that calcium signaling is essential for nerve growth factor (NGF)-directed neuronal cell differentiation. We report here that blocking production of the plasma membrane Ca(2+)-ATPase isoform 1 (PMCA1) in PC6 cells with antisense RNA impairs their ability to extend normal neurites in response to NGF. This result does not appear to be due to loss in NGF signaling as NGF-dependent tyrosine phosphorylation of erk1 and erk2, as well as expression of the NGF-inducible immediate early gene, NGFI-A, was observed in these cells. Resting cytosolic calcium levels did not appear to be altered in the antisense transfectants and release of calcium from internal bradykinin-sensitive calcium pools was unchanged. However, the rate of removal of free cytosolic calcium following this release was reduced in the antisense-transfected cells compared with controls. It is concluded that PMCA1 is involved in neurite extension and/or stabilization either through moderation of local calcium levels, or by some other mechanism.
Asunto(s)
ATPasas Transportadoras de Calcio/biosíntesis , Isoenzimas/biosíntesis , Factores de Crecimiento Nervioso/farmacología , Neuritas/fisiología , ARN sin Sentido/farmacología , Receptores de Factor de Crecimiento Nervioso/fisiología , Neoplasias de las Glándulas Suprarrenales , Animales , Secuencia de Bases , Bradiquinina/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Membrana Celular/enzimología , Células Clonales , Cartilla de ADN , Isoenzimas/antagonistas & inhibidores , Neuritas/efectos de los fármacos , Células PC12 , Feocromocitoma , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Ratas , Receptores de Factor de Crecimiento Nervioso/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , TransfecciónAsunto(s)
ATPasas Transportadoras de Calcio/fisiología , Adhesión Celular/fisiología , Membrana Celular/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Catión , Modelos Biológicos , Datos de Secuencia Molecular , ATPasas Transportadoras de Calcio de la Membrana Plasmática , RatasRESUMEN
The structures of three nitro-substituted phenothiazines [1,3,4-trifluoro-2-nitrophenothiazine, 10-(4-chlorobutyl)-1,3,4-trifluoro-2-nitrophenothiazine and 10-(4-chlorobutyl)-3-nitrophenothiazine] have been determined. The first of these red compounds forms infinite stacks in the solid state, in which donor and acceptor regions of the approximately planar molecules alternate. The molecules of the other two compounds, which have folded, or 'butterfly', conformations in the solid state, do not form stacks, presumably because the bulky chlorobutyl substituents cannot be accommodated. The very dark color of solid 3-nitrophenothiazine suggests the presence of extended molecular stacks, but crystals suitable for a structure determination could not be obtained.
Asunto(s)
Fenotiazinas/química , Cristalización , Cristalografía por Rayos X , Conformación Molecular , Estructura MolecularRESUMEN
Various photoactive phenothiazines were synthesized that possessed a 2-azido, 3-azido, 2-benzoyl, or 1,3,4-trifluoro-2-azido functionality in combination with various modifications of the N-alkyl side chain. These phenothiazines were evaluated for their ability to inhibit the calmodulin-mediated activation of phosphodiesterase (PDE). All were active in inhibiting the action of calmodulin (CaM), but those possessing either a 3-azido and a 4-(4-methyl-1-piperazinyl)butyl side chain or a 2-benzoyl group and 3-(dimethylamino)propyl side chain proved to be most active (I50 = 14 +/- 3 microM and 7 +/- 1 microM, respectively) when compared to the known inhibitor, chlorpromazine (CPZ, I50 = 30 microM). Calmodulin was photolabeled with ca. 35% efficiency in a light- and calcium-dependent fashion using a radiolabeled analog, 3-azido-10-(4-(4-[14C]methyl-1-piperazinyl)butyl)phenothiazine, of one of these compounds. Competition studies using this radiolabeled analog and CPZ were consistent with binding to one or both of the hydrophobic binding pockets of CaM.
Asunto(s)
Calmodulina/química , Fenotiazinas/síntesis química , Marcadores de Afinidad/síntesis química , Marcadores de Afinidad/química , Animales , Sitios de Unión , Unión Competitiva , Bovinos , Reactivos de Enlaces Cruzados , Activación Enzimática , Técnicas In Vitro , Estructura Molecular , Fenotiazinas/química , Fenotiazinas/efectos de la radiación , Hidrolasas Diéster Fosfóricas/metabolismo , FotoquímicaRESUMEN
The biotinylated probe, 3-azido-10-(4-(4-biotinyl-1-piperazinyl)butyl)phenothiazine, was used to examine the phenothiazine binding domains in calmodulin (CaM) by photolabeling. This phenothiazine, synthesized from 3-azido-10-(4-(1-piperazinyl)butyl)phenothiazine and d-biotinyl tosylate, inhibited the CaM-mediated activation of phosphodiesterase (PDE) with an I50 of 12.5 (+/- 2.8) microM. Photolabeling of CaM produced covalent adducts in excellent yield (32%) in a light- and Ca2+-dependent manner. Studies performed over a range of drug concentrations suggested a 2:1 stoichiometry for the binding of the phenothiazine probe to CaM. Limited trypsin digestion and purification of the resulting fragments by either SDS-PAGE or HPLC provided two principal phenothiazine-containing peptides. Amino acid composition and sequence analyses performed on these two peptides established that both the N- and C-terminal domains in CaM, particularly the regions amino terminal to Ca2+-binding loops 1 and 3, were modified by the photoactivated phenothiazine derivative. These data, particularly for the C-terminal domain, are in excellent agreement with the X-ray structure analysis of a 1:1 CaM-trifluoperazine complex.
Asunto(s)
Marcadores de Afinidad/síntesis química , Calmodulina/química , Fenotiazinas/síntesis química , Marcadores de Afinidad/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biotina/química , Calmodulina/genética , Calmodulina/metabolismo , Bovinos , Activación Enzimática , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Fenotiazinas/química , Hidrolasas Diéster Fosfóricas/metabolismo , FotoquímicaRESUMEN
The development of targeted, bidentate photoaffinity reagents for mapping the interacting domains of calmodulin (CaM) with the enzymes that it regulates required the synthesis and evaluation of the binding affinity of various phenothiazines. These photoaffinity reagents would possess a photoactive 3-azidophenothiazine group for cross-linking the hydrophobic binding domain of CaM, a second photoactive benzophenone group that would be activated at a different wavelength than the 3-azidophenothiazine group, and a suitable radiolabel. Difficulties were encountered in identifying those structural features that would be compatible with the introduction of a benzophenone group, with the solubility of these benzophenone-substituted phenothiazines, and with the ability of these phenothiazines to inhibit the calmodulin-mediated activation of phosphodiesterase. Solutions to this problem involved the preparation of phenothiazines possessing a quaternary ammonium salt, a zwitterionic amino acid, or a carbohydrate moiety. The phenothiazines that possessed photoactive 3-azido and benzophenone groups and in which one of the piperazine nitrogens in the side chain was converted to a quaternary, N-methylammonium iodide inhibited the calmodulin-mediated activation of phosphodiesterase at a level comparable to that of chlorpromazine.
Asunto(s)
Marcadores de Afinidad/síntesis química , Calmodulina/química , Fenotiazinas/síntesis química , Marcadores de Afinidad/química , Animales , Sitios de Unión , Calmodulina/metabolismo , Bovinos , Técnicas In Vitro , Estructura Molecular , Fenotiazinas/química , Hidrolasas Diéster Fosfóricas/metabolismo , FotoquímicaRESUMEN
Protein phosphatase 2A (PP2A) isolated from whole rat brain homogenate supernatants has been compared with that extracted from rat synaptosomal membranes. Both purified enzymes are comprised of the three known PP2A polypeptide chains of 65 (A subunit), 55 (B/B' subunit), and 38 (C subunit) kDa and have okadaic acid inhibition curves (Ki = 0.05 nM) nearly identical to that reported for skeletal muscle PP2A. The isolated 38-kDa subunit of rat brain PP2A appears to contain phosphotyrosine based on cross-reactivity with a specific monoclonal antibody (PY-20). Amino acid compositions and sequences of peptides isolated from the 65- and 38-kDa species correspond to regions of the cDNA-deduced sequences of the regulatory and catalytic subunits of protein phosphatase 2A from several sources. Studies reported here also demonstrate that autophosphorylated protein kinases, particularly Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), are excellent substrates for brain PP2A. Furthermore, Ca(2+)-dependent K(+)-depolarization of hippocampal synaptosomes was accompanied by a sequential increase, then decrease, in CaM kinase II phosphorylation level over a 45-s time course. The decrease was blocked by 1 nM okadaic acid. These data demonstrate that the type 2A protein phosphatase is present at the synapses of CNS neurons where its localization could alter the functions of phosphoproteins involved in synaptic plasticity.
Asunto(s)
Encéfalo/enzimología , Fosfoproteínas Fosfatasas/análisis , Fosfoproteínas Fosfatasas/fisiología , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Citosol/enzimología , Citosol/metabolismo , Citosol/ultraestructura , Éteres Cíclicos/farmacología , Hipocampo/enzimología , Hipocampo/metabolismo , Hipocampo/ultraestructura , Masculino , Datos de Secuencia Molecular , Ácido Ocadaico , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Proteínas Quinasas/análisis , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-Dawley , Membranas Sinápticas/enzimología , Membranas Sinápticas/ultraestructura , Sinaptosomas/enzimología , Sinaptosomas/metabolismo , Sinaptosomas/ultraestructuraRESUMEN
The regulation of intracellular calcium is essential for proper muscle function. Muscle cells have several mechanisms for dealing with the rapid and large changes in cytosolic calcium level that occur during contraction. Among these is the plasma membrane Ca(2+)-ATPase (PMCA), which pumps calcium from the cytosol to the extracellular space. We have previously shown that in human fetal muscle the PMCA1 isoforms present are PMCA1a-d, with PMCA1b and c predominating. Alternative splicing of mRNAs encoding proteins involved in muscle contraction is common in developing muscle. Therefore, we examined the expression of muscle-specific PMCA mRNAs in pre- and postfusion mouse C2 myoblasts. The housekeeping form of the CA(2+)-ATPase, PMCA1b, was found at all times and under all conditions. However, the other predominating isoform found in muscle, PMCA1c, was expressed on myotube formation. Simple cell-cell contact was not sufficient to induce PMCA1c expression, as cells plated at confluence but harvested before myotubule formation did not express PMCA1c. The induction of this muscle-specific Ca(2+)-ATPase at myotube formation suggests that it may play an important role in muscle function.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Isoenzimas/metabolismo , Músculos/enzimología , Empalme del ARN , Animales , Secuencia de Bases , Fusión Celular/fisiología , Línea Celular , Membrana Celular/enzimología , Precursores Enzimáticos/genética , Ratones , Sondas Moleculares/genética , Datos de Secuencia Molecular , Músculos/citología , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismoRESUMEN
The function of proteases in brain tumor invasion is currently not well established. For tumors of epithelial and fibromatous origin collagenase production can enhance the invasive capacity of cells to penetrate basement membranes. We showed previously that a c-Ha-ras transformed glial cell line (CxT24neo3) invaded hamster brain tissue in vivo. These cells were also capable of invading reconstituted basement membrane and embryonic chick hearts in vitro. Since the histopathology of CxT24neo3 tumors mimics that of glioblastoma multiforme in humans, CxT24neo3 was used as the model in vitro for this type of brain tumor. Presently, we detected by zymogram analysis a gelatinase that was secreted by CxT24neo3 and that had an apparent molecular mass of 62 kD. To verify whether gelatinase affected invasion in vitro of these glial cells we determined the efficacy of a substrate specific collagenase inhibitor on invasion in vitro. GM6001 is a synthetic polypeptide that specifically occupies the substrate binding sites of metalloprotease. Since this drug did not show cytotoxicity, its specificity for metalloprotease is a valuable tool to evaluate the physiological function of these enzymes on invasion. We found that treatment of CxT24neo3 with GM6001 reduced the fraction of invading CxT24neo3 cells through reconstituted basement membrane. These data suggest that metalloproteases can stimulate brain tumor invasion.
Asunto(s)
Amidas/farmacología , Dipéptidos/farmacología , Gelatinasas/antagonistas & inhibidores , Invasividad Neoplásica , Proteínas de Neoplasias/antagonistas & inhibidores , Neuroglía/patología , Inhibidores de Proteasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Tirosina/análogos & derivados , Animales , Membrana Basal/patología , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Embrión de Pollo , Colágeno , Cricetinae , Medios de Cultivo Condicionados/química , Medio de Cultivo Libre de Suero , Combinación de Medicamentos , Gelatinasas/metabolismo , Corazón/embriología , Laminina , Mesocricetus , Miocardio/patología , Proteínas de Neoplasias/metabolismo , Neuroglía/enzimología , Proteoglicanos , Proteínas Recombinantes de Fusión , Transfección , Tirosina/farmacologíaRESUMEN
The relationship of structural and functional moieties on calmodulin is important in all venues of cell activity. In this study, we investigate the effect of lysine modification on calmodulin function. Azidosalicylate reagents containing different "linker arm" lengths, between the photoactive terminus and an amine-reactive N-hydroxysuccinimidyl ester moiety were used to modify calmodulin lysines at three different positions in a calcium-dependent manner. The short cross-linker, (ASNE-2 (where ASNE represents azidosalicylate N-hydroxysuccinimidyl ester), modifies Lys-75, whereas the longer reagent, ASNE-6, modifies lysines 21, 75, and 94. The modification of these different lysines is shown to be calcium-dependent. At 1-100 microM levels of calcium, only Lys-94 is modified, suggesting that modification of this residue is directed by both the binding of calcium to calcium-binding loops III and IV and the hydrophobic pocket exposed between these two loops as a result of calcium binding. At higher calcium concentrations (> 200 microM), where sites I and II become filled, modification of Lys-21 or Lys-75 also was observed. All the modified calmodulins were able to stimulate 3',5'-cyclic-nucleotide phosphodiesterase fully although the Kact for the Lys-75 and Lys-21 derivatives increased 10- and 50-fold, respectively. None of the modifications affected the activation of erythrocyte plasma membrane Ca(2+)-ATPase. Only the ASNE-6 Lys-75 derivative showed efficient (40%) photocross-linking to the Ca(2+)-ATPase. The ASNE-2 Lys-75 derivative as well as the ASNE-6 Lys-21 and Lys-94 derivatives did not show efficient calcium-dependent photocross-linking to this enzyme.
Asunto(s)
Calcio/metabolismo , Calmodulina/química , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Secuencia de Aminoácidos , Animales , Azidas/química , Sitios de Unión , ATPasas Transportadoras de Calcio/metabolismo , Calmodulina/metabolismo , Bovinos , Reactivos de Enlaces Cruzados , Cristalografía , Activación Enzimática , Membrana Eritrocítica/enzimología , Técnicas In Vitro , Lisina/química , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Estructura Terciaria de Proteína , Relación Estructura-Actividad , PorcinosRESUMEN
The plasma membrane Ca(2+)-pumping ATPase (Ca(2+)-ATPase) mRNAs are encoded on four different genes designated PMCA1-PMCA4. The primary transcripts from some of these genes are known to be alternately spliced in the region encoding the regulatory domains of the enzymes. The known alternately spliced forms of these Ca(2+)-ATPase mRNAs and a new spliced variant of PMCA4 (PMCA4b), presented here, represent at least nine different mRNAs encoding the Ca(2+)-ATPases. In this report, the examination of the tissue-specific distribution of these alternately spliced mRNAs using polymerase chain reaction amplification of cDNA coupled with Southern blotting revealed that each spliced variant had a unique tissue distribution. PMCA1b and PMCA4a were present in all tissues examined. PMCA1a, PMCA1b, and PMCA4b were expressed in excitable tissues, whereas PMCA1d was expressed only in muscle tissues. PMCA2 was found in liver, adrenal gland, spinal cord, and brain. PMCA3a was present in spinal cord, and PMCA3b in thymus, adrenal gland, spinal cord, and brain. The mRNA for a new spliced variant of PMCA4 (PMCA4b) was detected in this study. Complementary DNAs for this isoform were isolated and characterized from human and bovine brain. This alternately spliced form of the PMCA4 mRNA contained an exon inserted at the splice junction immediately following the sequence encoding the calmodulin-binding domain. As has also been shown for PMCA1a, this insertion produced a shift in the reading frame at the 3'-end of the PMCA4 mRNA that yielded a sequence encoding a Ca(2+)-ATPase lacking a large portion of the C-terminal regulatory domain. When the human PMCA4 gene spanning this region of variable exon splicing was sequenced, it confirmed the intron-exon boundaries where alternate splicing occurs to produce PMCA4a and PMCA4b.
Asunto(s)
ATPasas Transportadoras de Calcio/genética , ADN/genética , Proteínas de la Membrana/genética , Empalme del ARN , ARN Mensajero/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Membrana Celular/enzimología , Exones , Feto/metabolismo , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido Nucleico , Distribución TisularRESUMEN
Purified porcine erythrocyte membrane Ca(2+)-ATPase and 3':5'-cyclic nucleotide phosphodiesterase were stimulated in a dose-dependent, saturable manner with the vitamin D-dependent calcium binding protein from rat kidney, calbindin-D28k (CaBP-D28k). The concentration of CaBP-D28k required for half-maximal activation (K0.5 act.) of the Ca(2+)-ATPase was 28 nM compared to 2.2 nM for calmodulin (CaM), with maximal activation equivalent upon addition of either excess CaM or CaBP-D28k. 3':5'-Cyclic nucleotide phosphodiesterase (PDE) also showed equivalent maximum saturable activation by calbindin (K0.5 act. = 90 nM) or calmodulin (K0.5 act. = 1.2 nM). CaBP-D28k was shown to effectively compete with CaM-Sepharose for PDE binding. Immunoprecipitation with CaBP-D28k antiserum completely inhibited calbindin-mediated activation of PDE but had no effect on calmodulin's ability to activate PDE. While the physiological significance of these results remains to be established, they do suggest that CaBP-D28k can activate enzymes and may be a regulator of yet to be identified target enzymes in certain tissues.