RESUMEN
Ca2+ influx and activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) during nonlethal ischemic preconditioning have been implicated in the protection of the heart against subsequent lethal ischemic injury. Thus, we determined if Ca2+ influx, PKC and MAPK also mediate ischemic preconditioning-induced protection in neurons. Preconditioning by exposure of E18 rat cortical cultures to 90 min of nonlethal oxygen-glucose deprivation (OGD) 24 h prior to 180-240 min of lethal OGD was neuroprotective. Exposure to nominally free Ca2+, or blockade of the alpha-amino-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptor with CNQX did not eliminate protection. MAPK activity did not change and PKC activity decreased by 50% relative to normal baseline levels at 0 and 24 h following preconditioning. The sustained decrease in PKC activity was not due to a loss of enzyme as determined from immunoblots using pan and epsilon-, beta- and zeta-specific PKC antibodies. Neuroprotection was maintained with pharmacological inhibition of PKC activity by staurosporine, chelerythrine and calphostin C and MAPK activity by PD 98059 during preconditioning, indicating that activation of these enzymes during preconditioning was not necessary for protection. Therefore, in contrast to cardiac tissue, ischemic preconditioning of neurons does not require activation of PKC and MAP kinase, and protection is maintained with substantial removal of extracellular Ca2+ or blockade of the AMPA receptor.
Asunto(s)
Isquemia Encefálica/metabolismo , Precondicionamiento Isquémico , Isquemia Miocárdica/metabolismo , Neuronas/citología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Células Cultivadas , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/citología , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Flavonoides/farmacología , Glucosa/farmacología , Miocardio/citología , Miocardio/enzimología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Oxígeno/farmacología , Proteína Quinasa C/metabolismo , RatasRESUMEN
Treating SH-SY5Y human neuroblastoma cells with 1 microM staurosporine resulted in a three- to fourfold higher DNA-dependent protein kinase (DNA-PK) activity compared with untreated cells. Time course studies revealed a biphasic effect of staurosporine on DNA-PK activity: an initial increase that peaked by 4 h and a rapid decline that reached approximately 5-10% that of untreated cells by 24 h of treatment. Staurosporine induced apoptosis in these cells as determined by the appearance of internucleosomal DNA fragmentation and punctate nuclear morphology. The maximal stimulation of DNA-PK activity preceded significant morphological changes that occurred between 4 and 8 h (40% of total number of cells) and increased with time, reaching 70% by 48 h. Staurosporine had no effect on caspase-1 activity but stimulated caspase-3 activity by 10-15-fold in a time-dependent manner, similar to morphological changes. Similar time-dependent changes in DNA-PK activity, morphology, and DNA fragmentation occurred when the cells were exposed to either 100 microM ceramide or UV radiation. In all these cases the increase in DNA-PK activity preceded the appearance of apoptotic markers, whereas the loss in activity was coincident with cell death. A cell-permeable inhibitor of DNA-PK, OK-1035, significantly reduced staurosporine-induced punctate nuclear morphology and DNA fragmentation. Collectively, these results suggest an intriguing possibility that activation of DNA-PK may be involved with the induction of apoptotic cell death.
Asunto(s)
Apoptosis/fisiología , Neoplasias Encefálicas/enzimología , Proteínas de Unión al ADN , Neuroblastoma/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias Encefálicas/patología , Caspasas/metabolismo , Fragmentación del ADN , Proteína Quinasa Activada por ADN , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Hidrazonas/farmacología , Immunoblotting , Neuroblastoma/patología , Proteínas Nucleares , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridonas/farmacología , Estaurosporina/antagonistas & inhibidores , Estaurosporina/toxicidad , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
Changes in protein kinase C (PKC) were compared in primary cortical neurons exposed to glutamate and in the CA-1 hippocampal region of rats subjected to transient cerebral ischaemia. After a 15-min exposure of cortical neurons to excitotoxic levels of glutamate, a 50-60% loss of membrane PKC activity but only about a 20% loss in the amount of enzyme was observed, suggesting that in addition to enzyme loss other mechanisms also contributed to the overall loss of membrane PKC activity. Glutamate induced a 25-40% decrease in immunodetectable levels of PKC alpha, beta, gamma, and lambda but no detectable changes in PCK epsilon and zeta. The loss of PKC activity coincided with a shift in electrophoretic mobility of PKC gamma, epsilon, and lambda, but not of PKC alpha, beta, or zeta, suggesting post-translational modification of some PKC isoforms. By comparison, in rats subjected to transient (15-min) global ischaemia, a similar 50-60% decrease in membrane PKC activity, a 20-25% loss in the amount of PKC, and a shift in PKC mobility were observed in CA-1 neurons 6 h post-reperfusion. In both the in vivo and the in vitro "ischaemic" models, administration of the AMPA receptor antagonist NBQX prevented the loss of PKC activity. These results indicate that the loss of PKC observed in in vivo ischaemia is likely to be due to excitotoxic damage and that this event can be closely mirrored in primary neuronal cultures damaged by glutamate.
Asunto(s)
Isquemia Encefálica/enzimología , Corteza Cerebral/enzimología , Ácido Glutámico/toxicidad , Neuronas/enzimología , Proteína Quinasa C/biosíntesis , Proteína Quinasa C/metabolismo , Animales , Isquemia Encefálica/fisiopatología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/farmacología , Masculino , Neuronas/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
The effects of glucose, K+, and cholecystokinin octapeptide (CCK-8) on intracellular free Ca2+ concentration ([Ca2+]i) and membrane-associated protein kinase C (PKC) activity were examined in cultured islet cells from neonatal rats. Raising the glucose concentration from 2.8 to 22.2 mM or external K+ (from 5 to 45 mM), or adding CCK-8 (200 nM) all triggered a [Ca2+]i surge that peaked between 3 and 10 min afterward, depending on the stimulus, and then declined, either to a suprabasal plateau (glucose and K+) or to basal levels (CCK-8). These same manipulations triggered a burst of membrane-associated PKC activity that peaked between 5 and 10 min and then variously declined. Incubation in Ca(2+)-free medium abolished both the effects of glucose and K+ on [Ca2+]i and the stimulation of membrane-associated PKC activity. The K(+)-triggered stimulation of PKC activity was also inhibited by pretreating the cells with the general Ca2+ entry blocker lanthanum (1 mM). However, incubation in Ca(2+)-free medium did not affect the CCK-8-induced release Ca2+ from internal stores, although it abolished the burst of membrane-associated PKC activity, which showed the importance of Ca2+ influx as opposed to internal release for PKC activation. Thus, glucose, the principal stimulator of insulin secretion, rapidly stimulates Ca2+ influx into islet cells from neonatal rats, and it is probably this influx that stimulates membrane-associated PKC activity.
Asunto(s)
Calcio/metabolismo , Glucosa/farmacología , Islotes Pancreáticos/metabolismo , Potasio/farmacología , Proteína Quinasa C/metabolismo , Sincalida/farmacología , Animales , Animales Recién Nacidos , Membrana Celular/enzimología , Células Cultivadas , Femenino , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Lantano/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
These studies were designed to investigate the role of protein kinase C (PKC) in the regulation of ATP-triggered intracellular Ca2+ ([Ca2+]i) oscillations in chicken granulosa cells. Granulosa cells were obtained from the two largest preovulatory follicles (F1 and F2) of hens and [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. Adenosine triphosphate (100 mumol/l) triggered an immediate, large [Ca2+]i spike that was followed by oscillations that returned to the resting level between spikes. The ATP (100 mumols/l) also stimulated a 1.70 +/- 0.1-fold increase in membrane-associated PKC activity over control levels. The frequency of the ATP-triggered [Ca2+]i oscillations was reduced in a concentration-dependent (1-10 nmol/l) manner by treating the cells for 2 min with a PKC activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA). A higher TPA concentration (100 nmol/l) completely prevented ATP from triggering the initial [Ca2+]i spike and oscillations. Adding TPA during the ATP-triggered [Ca2+]i oscillations immediately stopped the oscillatory activity. Interestingly, PKC inhibitors failed to amplify the ATP-triggered [Ca2+]i oscillations. Instead, adding the PKC inhibitors staurosporine (20 nmol/l), calphostin C (200 nmol/l) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7; 100 mumols/l), either before or during the ATP (100 mumols/l)-triggered [Ca2+]i response, also completely blocked the [Ca2+]i oscillations. Therefore, ATP-triggered [Ca2+]i oscillations in chicken granulosa cells appear to be regulated by a negative feedback loop requiring PKC, because the [Ca2+]i oscillations were prevented by either full activation or inhibition of PKC activity.
Asunto(s)
Adenosina Trifosfato/farmacología , Calcio/metabolismo , Células de la Granulosa/metabolismo , Membranas Intracelulares/metabolismo , Proteína Quinasa C/fisiología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Pollos , Activación Enzimática , Femenino , Naftalenos/farmacología , Concentración Osmolar , Proteína Quinasa C/antagonistas & inhibidores , Estaurosporina/farmacologíaRESUMEN
Low concentrations of the C-terminal parathyroid hormone-related protein (PTHrP) fragments, PTHrP-(107-111) and PTHrP-(107-139), stimulated membrane-associated protein kinase Cs (PKCs), but not adenylyl cyclase or an internal Ca2+ surge, in early passage human skin keratinocytes and BALB/MK-2 murine skin keratinocytes. The fragment maximally stimulated membrane-associated PKCs in BALB/MK-2 cells at 5 x 10(-9) to 10(-8) M. The maximally PKC-stimulating concentrations of PTHrP-(107-111) also stopped or stimulated BALB/MK-2 keratinocyte proliferation depending on whether the cells were, respectively, cycling or quiescent at the time of exposure. Thus, just one brief (30-minute) pulse of 10(-8) M PTHrP-(107-111) stopped the proliferation of BALB/MK-2 keratinocytes for at least 5 days. On the other hand, daily 30-minute pulses of 10(-8) M PTHrP-(107-111) started and then maintained the proliferation of initially quiescent BALB/MK-2 cells. Similarly PTHrP-(107-111) inhibited DNA synthesis by cycling primary adult human keratinocytes, but it stimulated DNA synthesis by quiescent human keratinocytes.
Asunto(s)
Queratinocitos/enzimología , Proteína Relacionada con la Hormona Paratiroidea , Hormona Paratiroidea/fisiología , Fragmentos de Péptidos/fisiología , Proteína Quinasa C/metabolismo , Proteínas/fisiología , Adenilil Ciclasas/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Ciclo Celular/fisiología , División Celular/fisiología , Células Cultivadas/citología , Células Cultivadas/enzimología , Medios de Cultivo , Células Epidérmicas , Humanos , Queratinocitos/citología , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fosforilación , Temperatura , Timidina/metabolismo , Factores de Tiempo , TritioRESUMEN
Murine neuroblastoma cells, N1E-115, were induced to differentiate into neuron-like cells by serum deprivation for 18 h. As previous studies have shown that the suppression of protein kinase C (PKC) activity by selective inhibitors or neutralizing antibodies induces neuroblastoma cells to differentiate, we tested the hypothesis that serum deprivation may cause a rapid loss in membrane PKC activity that occurs well before the morphological changes that are characteristic of cell differentiation. A significant reduction in particulate (membrane) PKC activity was indeed observed within 3 h of serum withdrawal when enzyme activity was measured in intact native membranes by the recently described in vitro "direct" assay. This rapid reduction in enzyme activity was confirmed by the decreased phosphorylation of the MARCKS protein, an endogenous PKC-selective substrate, in intact cells. The decrease in membrane PKC activity occurred without any loss in the amount of membrane-associated enzyme, suggesting that some factor(s) resident in neuroblastoma membranes was suppressing PKC activity. Indeed, results indicate the presence of an endogenous inhibitor of PKC tightly associated with neuroblastoma membranes. This inhibitory activity increased in the membranes of cells subjected to serum deprivation, raising the possibility that it was likely responsible for the decline in membrane PKC activity in differentiating N1E-115 cells. Preliminary characterization indicated that the inhibitory activity is a protein and is localized mainly in the membrane fraction. Thus, these results demonstrate directly that endogenous inhibitor can regulate membrane-associated PKC activity in cells and thereby modulate PKC-related neuronal functions.
Asunto(s)
Neuroblastoma/patología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Animales , Diferenciación Celular , Membrana Celular/enzimología , Ratones , Ratas , Células Tumorales CultivadasRESUMEN
Ionomycin stimulated membrane-associated protein kinase Cs (PKCs) activity in C6 rat glioma cells as much as the potent PKCs stimulator 12-O-tetradecanoyl phorbol 13-acetate (TPA). However, while TPA, as expected, powerfully stimulated the phosphorylation of the PKCs' 85-kDa myristoylated alanine-rich protein kinase C substrate (MARCKS) protein, ionomycin unexpectedly did not. Instead, ionomycin reduced the basal MARCKS phosphorylation. Pretreating the glioma cells with ionomycin prevented TPA-stimulated PKCs from phosphorylating the MARCKS protein. The stimulation of membrane PKCs activity and the prevention of MARCKS phosphorylation by ionomycin required external Ca2+ because they were both abolished by adding 5 mM EGTA to the culture medium. Recently (Chakravarthy, B. R., Isaacs, R. J., Morley, P., Durkin, J. P., and Whitfield, J. F. (1995) J. Biol. Chem. 270, 1362-1368), we proposed that Ca2+ x calmodulin complexes block MARCKS phosphorylation by the activated PKCs in keratinocytes stimulated by raising the external Ca2+ concentration. In the present experiments calmodulin prevented MARCKS phosphorylation by TPA-stimulated PKCs in glioma cell lysates, and this blockade was lifted by a calmodulin antagonist, the calmodulin-binding domain peptide. But, physiologically more significant, pretreating intact glioma cells with a cell-permeable calmodulin antagonist, calmidazolium, prevented ionomycin from blocking MARCKS phosphorylation by PKCs in unstimulated and TPA-stimulated cells. The effect of ionomycin on MARCKS phosphorylation was not due to the stimulation of Ca2+ x calmodulin-dependent phosphoprotein phosphatase, calcineurin, because cyclosporin A, a potent inhibitor of this phosphatase, did not stop ionomycin from preventing MARCKS phosphorylation. The ability of ionomycin to prevent TPA-stimulated PKCs from phosphorylating MARCKS depended on whether ionomycin was added before, with, or after TPA. Maximum blockade occurred when ionomycin was added before TPA but was less effective when added with or after TPA. These results indicate that Ca2+ x calmodulin can profoundly affect PKCs' signaling at the substrate level.
Asunto(s)
Calcio/farmacología , Calmodulina/farmacología , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Glioma/metabolismo , Ionomicina/farmacología , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Fosforilación , Ratas , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Raising the external Ca2+ concentration from 0.05 to 1.8 mM stimulated membrane-associated protein kinase Cs (PKCs) activity as strongly as the specific PKCs activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) in BALB/MK mouse keratinocytes. This was indicated by the increased phosphorylation of a PKC-selective peptide substrate, Ac-FKKSFKL-NH2, by membranes isolated from the Ca(2+)- or TPA-stimulated keratinocytes. Raising the external Ca2+ concentration to 1.8 mM also triggered a 4-fold rise in the intracellular free Ca2+ concentration. As reported elsewhere (Moscat, J. Fleming, T. P., Molloy, C. J. Lopez-Barahona, M., and Aaronson, S. A. (1989) J. Biol. Chem. 264, 11228-11235), TPA stimulated the phosphorylation of the PKCs substrate, the 85-kDa myristoylated alanine-rich kinase C substrate (MARCKS) protein, in intact keratinocytes, but Ca2+ did not. Furthermore, Ca(2+)-pretreatment reduced the TPA-induced phosphorylation of the 85-kDa protein in intact cells. There was no significant increase in MARCKS phosphorylation when keratinocytes were treated with a Ca2+.CaM-dependent phosphatase inhibitor, cyclosporin A, before stimulation with 1.8 mM Ca2+.Ca2+.calmodulin suppressed the ability of isolated membranes to phosphorylate the 85-kDa MARCKS holoprotein in vitro in the presence of phosphatase inhibitors such as fluoride, pyrophosphate, and vanadate, and this inhibition was overcome by a calmodulin antagonist, the calmodulin-binding domain peptide. Thus, the ability of 1.8 mM Ca2+ to strongly stimulate the membrane PKCs activity without stimulating the phosphorylation of the MARCKS protein in keratinocytes is consistent with the possibility of Ca2+.calmodulin complexes, formed by the internal Ca2+ surge, binding to, and blocking the phosphorylation of, this PKC protein substrate.
Asunto(s)
Calcio/farmacología , Calmodulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Queratinocitos/efectos de los fármacos , Proteínas de la Membrana , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular/efectos de los fármacos , Activación Enzimática , Queratinocitos/citología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Fosforilación , Especificidad por Sustrato , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Ca2+ and Ca(2+)-binding proteins are involved in running the cell cycle. Ca2+ spikes and signals from integrin-activated focal adhesion complexes and Ca2+ receptors on the cell surface along with cyclic AMP begin the cycle of cyclin-dependent protein kinases (PKs). These transiently expressed PKs stimulate the coordinate expression of DNA-replicating enzymes, activate replication enzymes, inactivate replication suppressors (e.g., retinoblastoma susceptibility protein), activate the replicator complexes at the end of the G1 build-up, and when replication is complete they and a Ca2+ spike trigger mitotic prophase. Another Ca2+ surge at the end of metaphase triggers the destruction of the prophase-stimulating PKs and starts anaphase. Ca2+ finally stimulates cytoplasmic division (cytokinesis). However, Ca2+ does more than this in epithelial cells, such as those lining the colon, and skin keratinocytes. These cells also need Ca2+, integrin signals, and only a small amount (e.g., 0.05-0.1 mM) of external Ca2+ to start DNA replication. Signals from their surface Ca2+ receptors trigger a combination of differentiation and apoptosis ("diffpoptosis") when external Ca2+ concentration reaches their setpoints. The skin's steep, upwardly directed, Ca2+ gradient has a low concentration in the basal layer to allow stem and precursor keratinocytes to proliferate, and higher concentrations in the suprabasal layers to trigger the differentiation-apoptosis ("diffpoptosis") mechanism that converts granular cells into protective, hard-shelled, dead corneocytes. A similar Ca2+ gradient may exist in the colon crypt allowing the stem cell and its amplifying transit or precursor offspring to cycle in the lower parts of the crypt, while stopping proliferation and stimulating terminal differentiation in the upper crypt and flat mucosa. Raising the amount of Ca2+ in fecal water above a critical level reduces proliferation and thus colorectal carcinogenesis in normal rats and some high-risk humans. But during carcinogenesis the Ca2+ sensors malfunction or their signals become ineffective: high Ca2+ does not stop, and may even stimulate, the proliferation of initiated mutants. Therefore, Ca2+ may either not affect, or even promote, the growth of epithelial cells in carcinogen-initiated rat colon and human adenoma patients. Clearly, a much greater understanding of how Ca2+ controls the proliferation and differentiation of epithelial cells and why initiated cells lose their responsiveness to Ca2+ are needed to assess the drawbacks and advantages of using Ca2+ as a chemopreventor.
Asunto(s)
Anticarcinógenos/uso terapéutico , Apoptosis/efectos de los fármacos , Calcio/uso terapéutico , Carcinógenos/toxicidad , Ciclo Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Anticarcinógenos/toxicidad , Calcio/toxicidad , Diferenciación Celular/efectos de los fármacos , Humanos , RatasRESUMEN
The activation of the multifunctional cell signalling enzymes, the protein kinase Cs (PKCs), is generally thought to result from the translocation of inactive cytosolic enzymes to activation sites in cell membranes. However, recent studies suggest that PKCs may also be stimulated in cells by processes independent of translocation. One possible mechanism is the modulation of the activity of PKCs already resident in membranes. A PKC assay that measures enzyme activity directly in isolated native membranes has revealed the presence of an activatable pool of PKCs resident in native membranes of various cells and tissues. In 3T3-L1 cells, some or all of this pool of membrane PKCs was stimulated within 10 min of exposing the cells to 10 ng/ml epidermal growth factor or 100 ng/ml fibroblast growth factor. Similar increases in PKC activity were observed in native membranes isolated from CTLL-2, WEHI-231 and S49 lymphoma cells that had been exposed to interleukin-2. These growth factors all stimulated membrane PKC activity without detectably translocating cytosolic enzymes to the membranes. In intact WEHI cells, low concentrations (5-10 microM) of a diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), or low concentrations (2-10 nM) of phorbol 12-myristate 13-acetate sufficed to activate PKCs already resident in membranes, but much higher concentrations (50-100 microM and 50-100 nM respectively) were needed to detectably stimulate the translocation of cytosolic PKCs. A phosphatidylcholine-specific phospholipase C also selectively stimulated membrane PKCs in WEHI cells at concentrations that were much less than those needed to induce the translocation of cytosolic enzymes. Furthermore, interleukin-2 and low concentrations of OAG both stimulated the phosphorylation of the 85 kDa PKC-selective substrate protein in intact WEHI cells in which translocation of PKCs was not evident. These results suggest that the membranes of some cells maintain a pool of activatable PKCs that respond to lower levels of extracellular stimuli than cytosolic PKCs, and that can be stimulated by signals which produce diacylglycerols through the hydrolysis of phospholipids other than polyphosphoinositides.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Membrana Celular/enzimología , Diglicéridos/farmacología , Activación Enzimática , Sustancias de Crecimiento/farmacología , Sustancias de Crecimiento/fisiología , Humanos , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fosforilación , Proteína Quinasa C/fisiología , Ratas , Estimulación Química , Acetato de Tetradecanoilforbol/farmacología , Translocación GenéticaRESUMEN
Membrane-associated protein kinase C (PKC) activity in lymphocytes freshly isolated from rat spleen was stimulated by the C-terminal parathyroid hormone-related protein fragments, PTHrP-(107-111) and PTHrP-(107-139), at concentrations from 10(-3) to 10(4) pM. By contrast, the same concentrations of PTHrP-(120-139), without the 107-111 TRSAW (-Thr-Arg-Ser-Ala-Trp-) sequence of the other C terminal fragments, did not stimulate spleen lymphocyte PKC. Low concentrations of the N-terminal PTHrP-(1-40) fragment also stimulated membrane-associated PKC activity in the spleen lymphocytes. These results suggest that PTHrP might be an important physiological regulator of the immune response.
Asunto(s)
Linfocitos/citología , Linfocitos/enzimología , Fragmentos de Péptidos/farmacología , Proteína Quinasa C/metabolismo , Proteínas/farmacología , Bazo/citología , Bazo/enzimología , Animales , División Celular , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Células Cultivadas , Cromatografía Líquida de Alta Presión , Activación Enzimática , Linfocitos/ultraestructura , Masculino , Proteína Relacionada con la Hormona Paratiroidea , Proteína Quinasa C/fisiología , Ratas , Ratas Sprague-Dawley , Bazo/ultraestructuraRESUMEN
The rapid redistribution of cytosolic protein kinase C (PKC) to membranes and its subsequent proteolytic activation to PKM have been implicated in the DMSO/HMBA-induced differentiation of murine erythroleukemia (MEL) cells. However, DMSO was found not to induce detectable changes in PKC distribution in a MEL cell subline (MEL1) which differentiated normally in response to the agent. Nevertheless, the differentiation of MEL1 cells appeared dependent on an early PKC-related event because hemoglobinization was partially blocked by the PKC inhibitor H-7 added to cells within the first 2 h after DMSO induction. Indeed, a rapid (15-60 min) increase in membrane PKC activity was detected in DMSO-treated MEL1 cells using a novel method which quantitates the amount of 'active' PKC in intact membranes. This transient PKC increase resulted from the activation of 'inactive' enzyme already associated with membranes, and not from the translocation of cytosolic PKC. Conventional PKC assays cannot distinguish between active and inactive membrane PKC pools. DMSO also activated inactive membrane PKC in HL-60 cells, but not in S49T-lymphoma and WEHI-231 B-lymphoma cells which do not differentiate in response to DMSO. The results suggest that a rapid and transient increase in membrane PKC activity may be an important early step in DMSO-induced differentiation of erythroleukemia cells.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Proteína Quinasa C/metabolismo , Animales , Membrana Celular/enzimología , Activación Enzimática , Cinética , Leucemia Eritroblástica Aguda , Ratones , Células Tumorales CultivadasRESUMEN
5'-Nucleotidase (EC 3.1.3.5) is widely distributed in nature. However, it could not be detected in rat liver, because of the presence of specific inhibitors. Such inhibitors were also found in other tissues of rat, but at lower concentrations than that in the liver. The inhibitor activity was enriched in the membrane fraction and was also present in the cytosol fraction. It was sensitive to treatment with 6M urea and trypsin, while heating in a boiling water bath for 10 min or dialysis reduced the activity only slightly. Gel filtration or Sephadex G-50 yielded two types of inhibitors. Inhibitor I inhibited brain 5'-nucleotidase while inhibitor II inhibited both the brain and liver enzymes. Inhibitor II on further purification on CM Sephadex C-25 yielded five fractions with inhibitor activity of which inhibitor IIC was electrophoretically homogeneous. It had a molecular weight of 8500 by SDS gel electrophoresis, was rich in basic amino acids and had a high proportion of beta structure. Interaction of the inhibitor with 5'-nucleotidase brought about modifications in the secondary structure of the inhibitor as seen from the circular dichroism spectrum.
Asunto(s)
5'-Nucleotidasa/antagonistas & inhibidores , Encéfalo/enzimología , Inhibidores Enzimáticos/aislamiento & purificación , Hígado/enzimología , Proteínas/aislamiento & purificación , 5'-Nucleotidasa/metabolismo , Animales , Cromatografía en Gel , Masculino , Especificidad de Órganos , RatasRESUMEN
Intact human parathyroid hormone, hPTH [1-84], and the hPTH [1-34] fragment stimulated membrane-associated protein kinase C (PKC) activity in immortalized (but still differentiation-competent) murine BALB/MK-2 skin keratinocytes. Unexpectedly, the hormone and its fragment did not stimulate adenylate cyclase. The failure of PTH to stimulate adenylate cyclase activity was not due to the lack of a functioning receptor-cyclase coupling mechanism because the cells were stimulated to synthesize cyclic adenosine monophosphate (cyclic AMP) by the beta-adrenergic drug isoproterenol. Thus, skin keratinocytes seem to have an unconventional PTH receptor that is coupled to a PKC-activating mechanism but not to adenylate cyclase. These observations suggest that normal and neoplastic skin keratinocytes respond to the PTH-related peptide that they make and secrete.
Asunto(s)
Adenilil Ciclasas/metabolismo , Queratinocitos/enzimología , Hormona Paratiroidea/farmacología , Proteína Quinasa C/metabolismo , Secuencia de Aminoácidos , Animales , AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Células Epidérmicas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Osteosarcoma/metabolismo , Hormona Paratiroidea/química , Péptidos/química , Péptidos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
A protein kinase C (PKC)-selective peptide substrate was used to develop a method for measuring PKC activity directly and quantitatively in isolated cell membranes without prior detergent extraction and reconstitution of the enzyme with phosphatidylserine and TPA in the presence of excess Ca2+. This simple and rapid method can reliably measure changes in membrane-associated PKC activity induced by various bioactive compounds such as hormones and growth factors. Also, this method, which measures PKC activity in its native membrane-associated state, has the advantage of being able to distinguish between active and inactive PKC associated with cell membranes.
Asunto(s)
Membrana Celular/enzimología , Proteína Quinasa C/análisis , Secuencia de Aminoácidos , Calcio/farmacología , Línea Celular , Cinética , Linfoma , Métodos , Datos de Secuencia Molecular , Péptidos/metabolismo , Fosforilación , Especificidad por Sustrato , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
The parathyroid hormone (PTH) fragment [1-34] strongly stimulated both adenylate cyclase and membrane-associated PKC activities in rat 17/2 osteosarcoma cells. By contrast, the PTH [3-34] fragment, which was unable to stimulate adenylate cyclase, remained a potent stimulator of membrane-associated PKC activity in these cells. Both PTH fragments also strongly stimulated membrane-PKC activity in cyc-S49T-lymphoma cells possessing a defective adenylate cyclase system. This ability of PTH [3-34] to stimulate membrane-associated PKC activity could explain the residual bioactivity of this fragment.
Asunto(s)
Hormona Paratiroidea/farmacología , Fragmentos de Péptidos/farmacología , Proteína Quinasa C/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Membrana Celular/enzimología , AMP Cíclico/metabolismo , Cinética , Linfoma , Ratones , Osteosarcoma , RatasRESUMEN
Physiological activation of protein kinase C (PKC) is believed to occur by redistributing soluble enzyme to the phospholipid environment of membranes. Currently available in vitro methods of measuring PKC activation all involve prior extraction of membrane-associated enzyme and its reconstitution in an artificial phospholipid environment or modification (such as partial trypsinization) of the enzyme itself. Here we report a novel method which, for the first time, allows measurement of active PKC still in its native, membrane-associated state using a specific, physiological substrate. Thus, with this new method PKC activity can be measured while still in an environment that approximates the in vivo situation.
Asunto(s)
Membrana Celular/enzimología , Proteína Quinasa C/metabolismo , Animales , Bombesina/farmacología , Calcio/farmacología , Citosol/enzimología , Ácido Egtácico/farmacología , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Humanos , Linfoma/metabolismo , Ratones , Peso Molecular , Fosfoproteínas/metabolismo , Ratas , Especificidad por Sustrato , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.
Asunto(s)
Ácidos Grasos/metabolismo , Lípidos de la Membrana/metabolismo , Neuroblastoma/metabolismo , Fosfolípidos/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Acilación , Animales , Fraccionamiento Celular , Línea Celular , Membrana Celular/metabolismo , Cinética , Ratones , Fracciones Subcelulares/metabolismo , TritioRESUMEN
A purified plasma membrane fraction was isolated from cultured neuroblastoma (N1E-115) cells on a discontinuous gradient of 5, 25 and 35% Percoll within 1 h of cell disruption by nitrogen cavitation. Yield of plasma membrane, banding in the 25% Percoll (d = 1.051), was high as judged by the recoveries of the marker enzymes, 5'-nucleotidase (58.0 +/- 5.4%, n = 5), alkaline phosphatase (46.0 +/- 3.0%, n = 4) and Mg2+-stimulated neutral sphingomyelinase (48.0 +/- 4.2%, n = 3); enrichment of specific activities of these enzymes relative to total cell homogenate (lysate) were 10.9 +/- 1.0-, 9.1 +/- 1.0- and 9.6 +/- 0.4-fold, respectively. Levels of marker enzymes for other organelles were less than 3% of total activity, except for microsomes (less than 9%). The plasma membrane fraction was further characterized by 2-, 5- and 6-fold higher content (nmol/mg protein) of total phospholipids, free cholesterol and sphingomyelin, respectively, compared to lysate. Ratios of free cholesterol to phospholipids and of sphingomyelin to phosphatidylcholine in the plasma membrane fraction were about 2-fold greater than that of lysate. The cholesterol ester content of plasma membrane (36 +/- 8 nmol/mg protein) was 2-3-fold higher than that of lysate. Sphingomyelin of the plasma membrane fraction had a higher concentration of long-chain fatty acids (more than 18 carbon atoms) relative to lysate or microsomes. Significant differences also were observed in the fatty acyl composition of diphosphatidylglycerol, cholesterol esters and triacylglycerol of plasma membrane. Thus, we have devised a rapid and reliable method for isolation of highly purified plasma membranes of cultured neuroblastoma cells that is suitable for comparison of metabolic relationships between the plasma membrane and other cellular organelles.