RESUMEN
Presenilins are involved in the proteolytic production of Alzheimer's amyloid peptides, but are also known to regulate Ca(2+) homeostasis in various cells types. In the present study, we examined intracellular Ca(2+) stores coupled to muscarinic receptors and capacitative Ca(2+) entry (CCE) in the human neuroblastoma SH-SY5Y cell line, and how these were modulated by over-expression of either wild-type presenilin 1 (PS1wt) or a mutant form of presenilin 1 (PS1 deltaE9) which predisposes to early-onset Alzheimer's disease. Ca(2+) stores discharged by application of 100 microM muscarine (in Ca(2+)-free perfusate) in PS1wt and PS1 DeltaE9 cells were significantly larger than those in control cells, as determined using Fura-2 microfluorimetry. Subsequent CCE, observed in the absence of muscarine when Ca(2+) was re-admitted to the perfusate, was unaffected in PS1wt cells, but significantly suppressed in PS1 deltaE9 cells. However, when Ca(2+) stores were fully depleted with thapsigargin, CCE was similar in all three cell groups. Western blots confirmed increased levels of PS1 in the transfected cells, but also demonstrated that the proportion of intact PS1 in the PS1 deltaE9 cells was far greater than in the other two cell groups. This study represents the first report of modulation of both Ca(2+) stores and CCE in a human, neurone-derived cell line, and indicates a distinct effect of the PS1 mutation deltaE9 over wild-type PS1.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Calcio/metabolismo , Proteínas de la Membrana/genética , Mutación , Neuroblastoma/metabolismo , Enfermedad de Alzheimer/genética , Western Blotting , Técnicas de Cultivo de Célula , Exones , Humanos , Proteínas de la Membrana/metabolismo , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Neuroblastoma/genética , Neuroblastoma/patología , Presenilina-1 , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Factores de TiempoRESUMEN
The effects of amyloid beta protein on voltage-gated K(+) channel currents were studied using the whole-cell patch-clamp technique. The 1-40 amino acid form of amyloid beta protein was applied to primary cultures of rat cerebellar granule and cortical neurones for 24 h. Both the unaggregated and aggregated forms of the peptide, which have differing biological activities, were used. In cerebellar granule neurones, 24-h pre-incubation with 1 microM unaggregated amyloid beta protein resulted in a 60% increase in the 'A'-type component of K(+) current. Increased delayed rectifier activity was Cd(2+)-sensitive and was presumed to be secondary to an increase in voltage-gated Ca(2+) channel current activity. Unaggregated amyloid beta protein had no effect on any component of the K(+) channel current in cortical neurones. One micromolar of aggregated amyloid beta protein had no effect on K(+) channel current in either cell type but reduced cell survival within 24 h as measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assays. The unaggregated form of amyloid beta protein had no neurotoxic effects when applied to either neurone type for up to 72 h. These data indicate that the unaggregated, non-pathological form of amyloid beta protein causes changes in the ion channel function of neurones, possibly reflecting a physiological role for the peptide.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Activación del Canal Iónico/efectos de los fármacos , Neuronas/fisiología , Fragmentos de Péptidos/toxicidad , Canales de Potasio/fisiología , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/química , Animales , Cloruro de Cadmio/farmacología , Células Cultivadas , Cerebelo/citología , Corteza Cerebral/citología , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Fragmentos de Péptidos/química , Potasio/metabolismo , Ratas , SolubilidadRESUMEN
Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.
Asunto(s)
Calcio/metabolismo , Neuronas/metabolismo , Oxígeno/metabolismo , Enfermedad de Alzheimer/metabolismo , Western Blotting , Hipoxia de la Célula , Homeostasis , Humanos , Neuroblastoma , Células Tumorales CultivadasRESUMEN
The effects of chronic hypoxia (2.5% O(2), 24 h) on [3H]noradrenaline ([3H]NA) release evoked from human neuroblastoma SH-SY5Y cells by depolarisation and by activation of muscarinic receptors was investigated. Depolarization of cells with 100 mM K(+) evoked [3H]NA release, and chronic hypoxia enhanced this release significantly. In fluorimetric studies, the K(+)-evoked rises of [Ca(2+)](i) observed in response to 100 mM K(+) were also significantly enhanced. Muscarine-evoked [3H]NA release was also dramatically enhanced by chronic hypoxia. However, muscarine-induced release of Ca(2+) from intracellular stores and subsequent capacitative Ca(2+) entry was unaffected. The protein kinase C inhibitors GF 109 203X and RO-31-8220 did not prevent the enhancement of muscarine-evoked release caused by chronic hypoxia. These findings indicate that chronic hypoxia increases release of [3H]NA from human neuroblastoma SH-SY5Y cells. Enhancement of K(+)-evoked release was attributable to an enhancement of depolarisation-mediated Ca(2+) influx. In contrast, the larger enhancement of muscarine-evoked [3H]NA release was not due to greater release of Ca(2+) from internal stores, nor due to enhanced Ca(2+) influx. Furthermore, it was not attributable to activation of protein kinase C. These findings suggest that enhancement of sympathetic output, known to occur following prolonged hypoxia, may be mediated in part by enhancement of exocytosis.
Asunto(s)
Hipoxia Encefálica/metabolismo , Neuronas/metabolismo , Norepinefrina/farmacocinética , Calcio/metabolismo , Hipoxia de la Célula/fisiología , Enfermedad Crónica , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Humanos , Indoles/farmacología , Maleimidas/farmacología , Potenciales de la Membrana/fisiología , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Neuroblastoma , Neuronas/citología , Potasio/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Receptores Muscarínicos/metabolismo , Estimulación Química , Tritio , Células Tumorales CultivadasRESUMEN
The aim of this study was to investigate a possible role of the myristoylated alanine-rich C kinase substrate (MARCKS) in the mechanism of noradrenaline uptake and release in the human neuroblastoma cell line SH-SY5Y. A stable cell line showing a twofold overexpression of MARCKS was prepared by transfecting SH-SY5Y with pCEP4 containing MARCKS cDNA in the sense orientation. This cell line showed no changes in the expression of neurofilaments or markers of noradrenergic large dense-cored vesicles compared with both untransfected SH-SY5Y and SH-SY5Y transfected with pCEP4 only (mock transfected). Similarly, no differences in the rate of cell growth could be detected between these three cell lines. In contrast, specific uptake and depolarization-evoked (100 mM K(+)) release of noradrenaline from the cell line overexpressing MARCKS was inhibited by approximately 50% compared with mock-transfected SH-SY5Y. K(+)-evoked noradrenaline release enhanced by pretreatment with 12-O-tetradecanoylphorbol 13-acetate (100 nM) was also inhibited by 50%. In contrast, carbachol-evoked noradrenaline release was unaffected. Thus, in SH-SY5Y cells, overexpression of MARCKS leads to a decrease in the K(+)-evoked noradrenaline release possibly by increased actin cross-linking preventing the movement of noradrenaline containing large dense-cored vesicles to the plasma membrane in response to depolarization.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Norepinefrina/metabolismo , Potasio/farmacología , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Células Tumorales Cultivadas/metabolismo , Proteína de Unión al GTP cdc42 , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , Exocitosis/fisiología , Reguladores de Proteínas de Unión al GTP , Vectores Genéticos/fisiología , Humanos , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Neuroblastoma , Norepinefrina/farmacocinética , Proteínas de Unión al ARN , Transfección/métodos , Tritio/farmacocinética , Células Tumorales Cultivadas/efectos de los fármacos , Proteínas de Unión al GTP rhoRESUMEN
The aim of this study was to obtain further understanding of the mechanism by which activation of muscarinic M(1) receptors inhibits K(+)-evoked noradrenaline (NA) release in the human neuroblastoma SH-SY5Y. Previous studies have found that muscarinic M(1) and M(3) receptors couple to the activation of phospholipase C in SH-SY5Y cells leading to an increase in (a) intracellular calcium ([Ca(2+)](i)) and (b) activation of protein kinase C (PKC). This study used specific inhibitors of PKC and conditions which deplete Ca(2+)(i) stores to examine the role of protein kinase C and changes in [Ca(2+)](i) in mediating the inhibition of K(+)-evoked NA release by muscarine. Our data show that pretreatment of SH-SY5Y cell layers with bisindolylmaleimide I (BIM-I) (i) failed to reverse inhibition of K(+)-evoked NA release by muscarine but (ii) did overcome the attenuation of muscarine inhibition following pretreatment with TPA. Furthermore pretreating cell layers with Ca(2+)-free Hepes buffered saline in the presence of thapsigargin, conditions which prevented muscarine induced increases in [Ca(2+)](i), failed to prevent inhibition of K(+)-evoked NA release by muscarine. The effect of muscarine on K(+)-evoked uptake of Ca(2+)(e) was examined in SH-SY5Y cells loaded with Fura-2. Muscarine inhibited Ca(2+)(e)-uptake by decreasing the rate at which Ca(2+) entered SH-SY5Y cells via voltage sensitive Ca(2+)-channels. Thus this study shows that muscarine inhibits depolarisation-evoked NA release by a mechanism which is not dependent on activation of PKC or release of Ca(2+) from internal stores.
Asunto(s)
Calcio/metabolismo , Neuronas/metabolismo , Norepinefrina/farmacocinética , Receptores Muscarínicos/metabolismo , Venenos Elapídicos/farmacología , Estrenos/farmacología , Humanos , Potenciales de la Membrana/fisiología , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Neuroblastoma , Forboles/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Potasio/farmacología , Proteína Quinasa C/metabolismo , Pirrolidinonas/farmacología , Receptor Muscarínico M1 , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/fisiología , Estimulación Química , Tritio , Células Tumorales CultivadasAsunto(s)
Células Cromafines/metabolismo , Fusión de Membrana , Neuronas/metabolismo , Animales , Axones/metabolismo , Endocitosis , Exocitosis , HumanosRESUMEN
Our previous studies have shown that noradrenaline release is enhanced by activation of protein kinase Calpha in SH-SY5Y cells. In the present study, we report that activation of protein kinase Calpha leads to (a) partial redistribution of the F-actin cytoskeleton and (b) a 2.5-fold increase in the number of large dense-cored vesicles within 100 nm of the plasma membrane. This redistribution can be prevented by down-regulation of protein kinase Calpha by up to 48 h exposure to phorbol dibutyrate. Treatment with the secretagogues 100 mM KCl, the Ca2+ ionophore A23187 (20 microM) and 1 mM carbachol also leads to a partial disassembly of the F-actin cytoskeleton. This is accompanied by an increase in the number of large dense cored vesicles at the plasma membrane following exposure to KCl and A23187 but not following exposure to carbachol. These results are discussed in relation to the hypothesis that a key step in the enhancement of noradrenaline release following activation of protein kinase Calpha and elevation of intracellular calcium is the movement of large dense cored vesicles to the plasma membrane following partial disassembly of the F-actin cytoskeleton.
Asunto(s)
Actinas/análisis , Isoenzimas/metabolismo , Neuroblastoma/metabolismo , Proteína Quinasa C/metabolismo , Calcimicina/farmacología , Carbacol/farmacología , Membrana Celular/ultraestructura , Regulación hacia Abajo , Activación Enzimática , Humanos , Ionóforos/farmacología , Liposomas , Neuroblastoma/patología , Potasio/farmacología , Proteína Quinasa C-alfa , Tasa de Secreción , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Sucrose gradient centrifugation combined with electron microscopy revealed that undifferentiated SH-SY5Y cells contain predominantly one population of noradrenaline containing vesicles, i.e. large dense cored vesicles. These vesicles have been purified approximately twenty times using sucrose/D2O gradients. Electron microscopy of sucrose/D2O fractions confirms that large dense cored vesicles are enriched in the fractions containing predominantly dopamine- -hydroxylase, chromogranin A, noradrenaline and neuropeptide Y. The membranes of these vesicles contain the typical large dense cored vesicle markers dopamine- -hydroxylase, synaptotagmin, cytochrome b561 and rab 3. Stimulation of SH-SY5Y cells with carbachol and KCl shows that noradrenaline and neuropeptide Y are released in the same proportion as stored in the large dense cored vesicles. The immuno-blot pattern and intensity of chromogranin A and chromogranin B present in large dense cored vesicles and in the released material were definitely the same. This suggests that noradrenaline and the proteins/peptides are released in the same molar stoichiometry as they are stored in large dense cored vesicles. These data provide for the first time experimental evidence that the neuroblastoma cell line SH-SY5Y contains functionally active large dense cored vesicles similar to those of sympathetic neurons and indicate that this cell line is a suitable experimental cell model to study the exocytotic pathway of large dense cored vesicles.
Asunto(s)
Cromograninas/metabolismo , Neuronas/metabolismo , Neuropéptido Y/metabolismo , Norepinefrina/metabolismo , Diferenciación Celular , Endocitosis , Exocitosis , Humanos , Modelos Biológicos , Neuroblastoma , Neuronas/citología , Células Madre/citología , Células Madre/metabolismo , Células Tumorales CultivadasRESUMEN
We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K+]o (100 mM) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [3H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K+-evoked release was unaffected by hypoxia (PO2 approximately 30-38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca2+o with 1 mM EGTA abolished NA release. K+-evoked release was also dramatically reduced in the presence of 200 microM Cd2+ to block voltage-gated Ca2+ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd2+-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca2+ influx through the nAChR pore, or by activating an unidentified Cd2+-resistant Ca2+-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.
Asunto(s)
Neuroblastoma/metabolismo , Norepinefrina/metabolismo , Receptores Nicotínicos/metabolismo , Calcio/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Humanos , Agonistas Nicotínicos/farmacología , Potasio/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Tritio , Células Tumorales CultivadasAsunto(s)
Citocalasina D/farmacología , Demecolcina/farmacología , Ganglios/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Norepinefrina/metabolismo , Activación Enzimática , Ganglios/metabolismo , Humanos , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
The effect of protein kinase C (PKC) on the release of neurotransmitters from a number preparations, including sympathetic nerve endings, brain slices, synaptosomes, and neuronally derived cell lines, is considered. A comparison is drawn between effects of activation of PKC on neurotransmitter release from small synaptic vesicles and large dense-cored vesicles. The enhancement of neurotransmitter release is discussed in relation to the effect of PKC on: 1. Rearrangement of the F-actin-based cytoskeleton, including the possible role of MARCKS in this process, to allow access of large dense-cored vesicles to release sites on the plasma membrane. 2. Phosphorylation of key components in the SNAP/SNARE complex associated with the docking and fusion of vesicles at site of secretion. 3. Ion channel activity, particularly Ca2+ channels.
Asunto(s)
Encéfalo/fisiología , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Neuronas/fisiología , Neurotransmisores/metabolismo , Proteína Quinasa C/metabolismo , Sistema Nervioso Simpático/fisiología , Sinaptosomas/fisiología , Animales , Línea Celular , Citoesqueleto/fisiología , Homeostasis , Humanos , Canales Iónicos/fisiología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Proteínas/metabolismoRESUMEN
The ability of angiotensin II (AII) to regulate [Ca++]i in human neuroblastoma (SH-SY5Y) cells stably expressing recombinant rat AT1A receptors was investigated using microfluorimetric methods, and compared to responses obtained by stimulation of native muscarinic receptors. Applications of AII or carbachol produced biphasic rises of [Ca++]i, but in Ca++-free solutions (containing 1 mM ethylene glycol-bis (beta-aminoethyl ether)N,N,N,'N'-tetraacetic acid), both agonists produced only transient monophasic rises of [Ca++]i, and second applications were without effect. Application of Ca++(o) (2.5 mM) to cells after exposure to either agonist produced a Ni2+-sensitive rise of [Ca++]i in the absence of agonist ("capacitative Ca++ influx"). After removal of Ca++(o), both AII and carbachol elicited a second rise of [Ca++]i. Thapsigargin (1 microM) prevented these second rises of [Ca++]i. During capacitative Ca++ influx, application of AII failed to produce a further rise of [Ca++]i. In contrast, carbachol produced a further rise of [Ca++]i, attributable to activation of both nicotinic and muscarinic receptors, because it was reduced (but not abolished) by mecamylamine (1 microM) and was observed when muscarine was used as the agonist. Thus, activation of recombinant AT1A and muscarinic receptors in SH-SY5Y cells leads to mobilization of Ca++ from a common intracellular pool, and stimulates capacitative Ca++ influx. Muscarinic (but not AII) receptor occupancy is capable of stimulating an additional Ca++ influx pathway.
Asunto(s)
Angiotensina II/farmacología , Calcio/metabolismo , Carbacol/farmacología , Neuroblastoma/metabolismo , Receptores de Angiotensina/efectos de los fármacos , Animales , Células Cultivadas/efectos de los fármacos , Humanos , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
Western blot analysis showed that the human neuroblastoma SH-SY5Y expresses the proteins synaptotagmin l, synaptobrevin, synapsin-l, rab3a, syntaxin, SNAP-25, NSF, alpha-SNAP, and munc-18, which have been implicated in the movement, docking, and fusion of vesicles during exocytosis from other neuroendocrine cells. The subcellular localization of secretogranins I and II, synaptotagmin l, neuropeptide Y, rab3a, synaptobrevin, synaptophysin, and syntaxin was investigated by immunofluorescence microscopy and revealed punctate staining patterns characteristic of secretory vesicles. The comigration of noradrenaline, secretogranin II, and dopamine-beta-hydroxylase on sucrose-D2O gradient fractions indicates the presence of a population of noradrenaline-containing large dense-cored vesicles (LDCVs). In addition, a lighter vesicle population is also present that does not appear to be noradrenergic and contains a 48-kDa synaptophysin antigen absent from the large dense-cored vesicles. Immunocytochemical experiments show that not all of the vesicles that express synaptotagmin l contain secretogranin II. Thus, our studies suggest that two types of vesicle are present in SH-SY5Y cells, one of which, the LDCVs, contains noradrenaline. These findings confirm our previous studies suggesting that depolarization-evoked release of noradrenaline from SH-SY5Y occurs by LDCV exocytosis. This enhances the value of SH-SY5Y as a cell line in which to study the mechanism by which noradrenaline release is regulated.
Asunto(s)
Proteínas de Unión al Calcio , Gránulos Citoplasmáticos/química , Neuroblastoma , Proteínas de Transporte Vesicular , Especificidad de Anticuerpos , Proteínas Portadoras/análisis , Proteínas Portadoras/inmunología , Cromograninas , Gránulos Citoplasmáticos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Glicoproteínas de Membrana/análisis , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Proteínas Sensibles a N-Etilmaleimida , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/inmunología , Neuropéptidos/análisis , Norepinefrina/análisis , Proteínas/análisis , Proteínas Qa-SNARE , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Fracciones Subcelulares/química , Proteína 25 Asociada a Sinaptosomas , Sinaptotagminas , Tritio , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/ultraestructuraRESUMEN
The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the alpha-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-epsilon from cytosolic and membrane-associated to cytoskeleton- and membrane-associated. TPA had no effect on the cytosolic location of PKC-zeta. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 microM). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 nM) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-alpha (but not -epsilon or -zeta). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-alpha.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Neuroblastoma/metabolismo , Norepinefrina/metabolismo , Ésteres del Forbol/farmacología , Fosfotransferasas/metabolismo , Proteína Quinasa C/metabolismo , Translocación Genética , Alanina/metabolismo , Activación Enzimática , Humanos , Isoenzimas/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Neuroblastoma/patología , Forbol 12,13-Dibutirato/farmacología , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteínas/metabolismo , Fracciones Subcelulares/metabolismo , Especificidad por Sustrato , Acetato de Tetradecanoilforbol/farmacología , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Long-term (8-48 h) treatment of SH-SY5Y neuroblastoma cells with phorbol-12,13-dibutyrate (PDBu; 100 nM) promotes the down-regulation of protein kinase C (PKC) subtypes alpha and epsilon and reduces by up to 60% noradrenaline (NA) release evoked via both PKC-dependent (M3-muscarinic receptor activation) and PKC-independent (depolarization) pathways, over similar time courses. A similar effect on release is observed following long-term (16-48 h) incubation with the PKC inhibitor Ro 31-7549 (10 microM), even after removal of the inhibitor, indicating a mechanism which is not rapidly reversible. Evidence is presented which suggests that long-term treatment with PDBu does not (1) affect calcium entry, (2) modulate levels of proteins important in the secretory mechanism or (3) reduce the number of secretory vesicles. Thus, the decrease in NA release in SH-SYSY cells following down-regulation of PKC appears to be the result of a sustained reduction in PKC activity acting on a component of the secretory pathway not involved in the regulation of calcium entry or vesicle number.
Asunto(s)
Norepinefrina/farmacocinética , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Carcinógenos/farmacología , Regulación hacia Abajo , Humanos , Indoles/farmacología , Maleimidas/farmacología , Neuroblastoma , Forbol 12,13-Dibutirato/farmacología , Células Tumorales CultivadasRESUMEN
Exposure of human neuroblastoma SH-SY5Y cells to 300 nM neuropeptide Y (NPY) or 1 microM muscarine separately failed to evoke release of [3H]noradrenaline ([3H]NA). Both agonists, however, induced a modest rise in [Ca2+]i. When NPY and muscarine were applied simultaneously, the rise in [Ca2+]i was greater than the sum of the rises of either agonist applied alone, and also evoked [3H]NA release, NPY evoked a rise in [Ca2+]i when applied during prolonged exposure to muscarine, although the peak level of [Ca2+]i was significantly lower (p < 0.05) than that reached following simultaneous application, and [3H]NA release was not stimulated. Simultaneous exposure of SH-SY5Y cells to muscarine and NPY thus induces a greater than additive rise in [Ca2+]i exceeding a critical level required to evoke [3H]NA release.
Asunto(s)
Calcio/metabolismo , Neuropéptido Y/farmacología , Norepinefrina/metabolismo , Línea Celular , Humanos , Muscarina/farmacología , Agonistas Muscarínicos/farmacología , Estimulación QuímicaRESUMEN
In order to stabilize the C-terminal dodecapeptide of neuropeptide Y (NPY) we replaced Leu28 and Thr32 by Lys and Glu, respectively, and subsequently linked these residues by lactamization. This peptide analog of NPY shows a more than 100-fold increase in affinity compared to the C-terminal linear dodecapeptide in receptor binding studies performed at human neuroblastoma cells SMS-KAN, which exclusively express the Y2 receptor subtype. Signal transduction was investigated by measuring Ca2+ current inhibition in human SH-SY5Y cells and cyclic [Lys28-Glu32] NPY Ac-25-36 and NPY were shown to be equipotent in this assay. Thus, this molecule is the smallest Y2 receptor selective full agonist of NPY. Using 2D-NMR experiments and molecular modelling techniques, the structures of the linear and cyclic peptides have been investigated and significant differences have been found, which may explain the improvement in biological activity. Thus, a model of the bioactive conformation of NPY at the human Y2 receptor is suggested.