RESUMEN
The action of platelet activating factor (PAF) on subcellular distribution and activity of protein kinase C (PKC) isoforms in rabbit platelets was analyzed. The results showed an increase of PKC alpha in membrane fraction, concomitantly with a decrease in cytosolic fraction after 5 min PAF treatment, indicating that a translocation of PKC alpha occurred. In addition, PKC zeta was redistributed in a "reverse" form, from the membrane to cytosolic fraction after PAF treatment. PAF induced an increase of PKC alpha activity, whereas a decrease rather than increase in PKC zeta was observed by using immunoprecipitation assays. In addition, some results indicated that PI3 kinase activation was not involved in PAF-induced PKC zeta translocation as occur in several cells and with other agonists. These actions were time- and concentration-dependent, and were inhibited by the treatment with a PAF antagonist. No translocation was observed when the platelets were incubated with lysoPAF, a PAF related compound. The redistribution of PKC isoforms take place through the activation of high specificity PAF binding sites. The pretreatment of the rabbit platelets with staurosporine, a putative inhibitor of PKC, completely blocked the PAF-evoked aggregation without affecting to PAF-evoked shape change and serotonin release. All together, these data could suggest that the specific translocation of PKC isoforms play an important role in the activation of rabbit platelets.
Asunto(s)
Plaquetas/enzimología , Isoenzimas/sangre , Factor de Activación Plaquetaria/farmacología , Proteína Quinasa C/sangre , Animales , Plaquetas/citología , Plaquetas/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Factor de Activación Plaquetaria/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Conejos , Serotonina/sangreRESUMEN
In this study we report that protein kinase C zeta (PKC zeta), one of the atypical isoforms of the PKC family located predominantly in cytosol, is redistributed by C2-ceramide treatment in isolated hepatocytes. PKC zeta increased in membrane and nuclear fractions after 30 min of treatment with C2-ceramide in a dose- and time-dependent manner. The action of C2-ceramide was inhibited by wortmannin and LY 294002, indicating that C2-ceramide-induced PKC zeta increase in both nucleus and membrane fractions is mediated by phosphatidylinositol 3-kinase (PI3-kinase) activation. In addition, a significant translocation of PI3-kinase to the nucleus was observed after C2-ceramide treatment.
Asunto(s)
Núcleo Celular/metabolismo , Hepatocitos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Androstadienos/farmacología , Animales , Membrana Celular/metabolismo , Separación Celular , Cromonas/farmacología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Hepatocitos/química , Hepatocitos/efectos de los fármacos , Isoenzimas/metabolismo , Masculino , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Ratas Wistar , Esfingosina/antagonistas & inhibidores , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo , WortmaninaRESUMEN
The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat hippocampal slices. PAF caused an increase in the tyrosine phosphorylation of two phosphoproteins, which we identified by immunoprecipitation assays as the focal adhesion kinase p125FAK and crk-associated substrate p130Ca. The PAF effect was time- and dose-dependent. In addition, the involvement of PAF receptor was demonstrated by using PCA-4248, a specific receptor antagonist. When NO synthase was inhibited by NG-monomethyl-L-arginine (L-NMA), PAF-stimulated protein tyrosine phosphorylation was inhibited. In conclusion, our results indicate that PAF increased the tyrosine phosphorylation of both p125FAK and p130Cas proteins by the production of NO in hippocampus, suggesting that PAF may play a role in the functioning of this cerebral area.
Asunto(s)
Hipocampo/metabolismo , Óxido Nítrico Sintasa/metabolismo , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Factor de Activación Plaquetaria/farmacología , Animales , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Cinética , Masculino , Óxido Nítrico Sintasa/efectos de los fármacos , Fosforilación , Ratas , Ratas Wistar , omega-N-Metilarginina/farmacologíaRESUMEN
Sphingosylphosphorylcholine (SPC) induces a rapid increase of intracellular Ca2+ concentration in isolated synaptosomes. This effect is dose-dependent and is also dependent on extracellular Ca2+. Sphingosine (SPH) has a smaller effect and treatment with psychosine (PSY) is ineffective, which suggests that phosphorylation of the 1-carbon of SPH is required for the SPC to act as a Ca2+ release agonist in synaptosomes. Experiments performed in the presence of heparin or ryanodine indicate that SPC-elicited Ca2+ release is not mediated by IP3 or ryanodine receptors. Finally, our results show that the effect of SPC on Ca2+ concentration is nimodipine-sensitive, suggesting that SPC possibly activates a specific sphingolipid-gated Ca2+ channel in synaptosomes.
Asunto(s)
Calcio/metabolismo , Fosforilcolina/farmacología , Esfingolípidos/farmacología , Esfingosina/farmacología , Sinaptosomas/metabolismo , Animales , Anticoagulantes/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Heparina/farmacología , Nimodipina/farmacología , Fosforilcolina/análogos & derivados , Psicosina/farmacología , Ratas , Rianodina/farmacología , Esfingosina/análogos & derivados , Sinaptosomas/efectos de los fármacosRESUMEN
When isolated rat liver nuclei were treated with platelet-activating factor (PAF), a rapid increase in the mass of diacylglycerol (DAG) occurred. This effect was dose- and time-dependent. The maximum effect was observed after 1 min of 10(-7) M PAF treatment. A concomitant decrease of polyphosphoinositides and phosphatidic acid (PA) levels was observed. PAF-induced DAG accumulation was inhibited by the treatment with WEB 2086 or PCA-4248, specific PAF-receptor antagonists. This result may suggest that PAF exerts its action in the nucleus through specific nuclear PAF binding sites. The findings described herein are due to the activation of phospholipase C, as the results from experiments using U73122, a phospholipase C inhibitor, indicate. These are the first data on the action of
Asunto(s)
Núcleo Celular/efectos de los fármacos , Diglicéridos/biosíntesis , Hepatocitos/metabolismo , Fosfatidilinositoles/metabolismo , Factor de Activación Plaquetaria/farmacología , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Adenosina Trifosfato/metabolismo , Animales , Núcleo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Hidrólisis , Masculino , Glicoproteínas de Membrana Plaquetaria/metabolismo , Ratas , Ratas Wistar , Factores de Tiempo , Fosfolipasas de Tipo C/metabolismoAsunto(s)
Plaquetas/metabolismo , Endotelina-1/farmacología , Serotonina/farmacología , Animales , Antihipertensivos/farmacología , Plaquetas/ultraestructura , Western Blotting , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Endotelina-1/agonistas , Endotelina-1/metabolismo , Depuradores de Radicales Libres/farmacología , Péptidos Cíclicos/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica , Conejos , Serotonina/metabolismo , Serotonina/farmacocinética , Tirosina/metabolismo , Vasoconstrictores/metabolismo , Vasoconstrictores/farmacología , Venenos de Víboras/farmacologíaRESUMEN
In this study we have analyzed the distribution of protein kinase C isoforms in cytosol, membrane, and nucleus in HL60 cells. Furthermore, we have studied the redistribution of these isoforms after cyclic AMP treatment. Protein kinase C localization and cyclic AMP-induced translocation was demonstrated by Western blot analysis. Cytosol, membrane and nucleus in HL60 cells expressed the abundance of protein kinase C alpha, betaI, betaII, delta, lambda, and zeta isoforms. After cyclic AMP treatment, the amount of protein kinase C betaI and zeta increased only in the nucleus, while protein kinase C delta increased in the three fractions tested. These effects were dependent on the cyclic AMP concentration and duration of action. Our results suggest the existence of cross-talk between the cyclic AMP system and protein kinase C in HL60 cells. Taking into account the processes regulated by protein kinase C, these findings also suggest that cyclic AMP plays a regulatory role in various cellular responses in HL60 cells, such as differentiation and gene expression. The increase observed in PKC delta was due to cyclic AMP-dependent protein kinase C activation, and the synthesis of enzyme was probably activated by the nucleotide.
Asunto(s)
Núcleo Celular/enzimología , AMP Cíclico/farmacología , Citosol/enzimología , Membranas Intracelulares/enzimología , Proteína Quinasa C/metabolismo , Transporte Biológico , Compartimento Celular , Células HL-60 , Humanos , Isoenzimas/metabolismo , Transducción de SeñalRESUMEN
We have studied whether endothelin isopeptides have any effects on histamine-induced cyclic AMP in [(3)H]adenine-prelabeled brain vessels isolated from bovine brain. Basal levels of [(3)H]cyclic AMP were enhanced by histamine in a concentration-dependent manner (EC(50) = 1.1 +/- 0.3 microM). Endothelin-1 inhibited histamine-elicited [(3)H]cyclic AMP generation with an IC(50) value of 3 +/- 2.5 nM. Sarafotoxin 6c, an ET-B receptor agonist, had no effect. ET-1 inhibition of histamine-induced [(3)H]cyclic AMP was reversed by the ET-A receptor antagonist BQ-123 while the ET-B receptor antagonist BQ-788 had no effect. The levels of [(3)H]cyclic AMP induced by isoprenaline were not altered by endothelin-1. Taken together, these results show that endothelins modulate the actions of histamine on the blood-brain barrier, probably by type A endothelin receptors.
Asunto(s)
Encéfalo/irrigación sanguínea , AMP Cíclico/metabolismo , Endotelina-1/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Histamina/farmacología , Sistemas de Mensajero Secundario/efectos de los fármacos , 1-Metil-3-Isobutilxantina/farmacología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Capilares/efectos de los fármacos , Capilares/metabolismo , Bovinos , Antagonistas de los Receptores de Endotelina , Isoproterenol/farmacología , Microcirculación/efectos de los fármacos , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Piperidinas/farmacología , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/efectos de los fármacos , Receptores de Endotelina/fisiología , Venenos de Víboras/farmacologíaRESUMEN
We examined the effect of ET-1 on cyclic AMP levels in rat cerebral cortex. The peptide caused a concentration-dependent increase of [(3)H]cyclic AMP accumulation after 10 min of treatment. This effect was due to adenosine accumulation since it was inhibited by the treatment with adenosine deaminase. ET-1, apart from being able to increase cyclic AMP, also potentiated the cyclic AMP generated by isoprenaline in the presence of adenosine deaminase. Experiments performed in the presence of BQ-123 or BQ-788, specific ET(A) or ET(B) receptor antagonists respectively indicated that ET(B) was the receptor involved. This effect was dependent on extracellular and intracellular calcium concentration. These findings suggest that ET-1 plays a modulatory role in cyclic AMP generation systems in cerebral cortex.
Asunto(s)
Agonistas Adrenérgicos beta/metabolismo , Corteza Cerebral/metabolismo , AMP Cíclico/metabolismo , Endotelina-1/metabolismo , Isoproterenol/metabolismo , Adenosina Desaminasa/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Calcio/metabolismo , Corteza Cerebral/efectos de los fármacos , Antagonistas de los Receptores de Endotelina , Endotelina-1/farmacología , Isoproterenol/farmacología , Masculino , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Piperidinas/farmacología , Ratas , Ratas WistarRESUMEN
This study examined the temporal relationships of endothelin-1-stimulated rabbit platelets tyrosine phosphorylated proteins. The effect of endothelin-1 on tyrosine phosphorylation was dose- and time-dependent and caused a rapid tyrosine phosphorylation of three groups of proteins in the molecular mass range 70-100 kDa, 100-150 kDa and 150-200 kDa. Significant protein tyrosine phosphatase activity and amount were found to be associated with the cytoskeleton of endothelin-1-stimulated rabbit platelets. Under our experimental conditions, translocation from the cytosolic fraction to the cytoskeleton reached its highest levels within 10-20 sec of endothelin-1 stimulation. Endothelin-1-induced translocation of protein tyrosine phosphatase, associated with the increase in its activity was demonstrated by immunoblotting and immunoelectron microscopy.
Asunto(s)
Plaquetas/fisiología , Endotelina-1/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Conejos , Factores de Tiempo , Tirosina/metabolismoRESUMEN
In this study, we present evidence showing that endothelin (ET) potentiates the responses to adenosine, to 5'-N-ethylcarboxamidoad, a nonhydrolyzable adenosine agonist, and to isoprenaline. These responses seem to occur through ET-B receptors, as all three endothelin isopeptides have the same potency, sarafotoxin 6c has the same effect as ET-1, BQ-123, an ET-A receptor antagonist has no effect, and BQ-788, an ET-B receptor antagonist that totally suppresses the responses analyzed.
Asunto(s)
Adenosina/farmacología , Cerebelo/efectos de los fármacos , AMP Cíclico/metabolismo , Endotelina-1/farmacología , Isoproterenol/farmacología , Adenosina/metabolismo , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Cerebelo/metabolismo , Colforsina/farmacología , Antagonistas de los Receptores de Endotelina , Técnicas In Vitro , Masculino , Ratas , Ratas Wistar , Receptores de Endotelina/agonistas , Receptores de Endotelina/metabolismo , TritioRESUMEN
The effect of platelet activating factor (PAF) on subcellular distribution of protein kinase C isoforms in rat cerebral cortex was investigated. PAF induced an increase in levels of protein kinase C epsilon and gamma in membrane fraction. Results also indicate that PAF induced an increase in protein kinase C delta levels in both cytosolic and membrane fraction. This effect is possibly due to an increase in enzyme synthesis, as indicated by the results obtained from the experiments performed in the presence of cycloheximide and actinomycin. All the effects induced by PAF were time- and dose-dependent, and were mediated through the activation of PAF receptor. These findings indicate that the three isoforms may be involved in signal transduction of PAF in the brain.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Isoenzimas/metabolismo , Factor de Activación Plaquetaria/farmacología , Proteína Quinasa C/metabolismo , Animales , Corteza Cerebral/enzimología , Masculino , Ratas , Ratas Wistar , Transducción de Señal , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimologíaRESUMEN
Stimulation of rat cerebral cortex with endothelin-1 (ET-1) caused an increase in the tyrosine phosphorylation of several proteins. Two of these phosphoproteins were identified by the immunoprecipitation assays as being the focal adhesion kinase p125FAK and crk-associated substrate p130Cas. This effect was time- and dose-dependent, with an EC50 value of 3.9 x 10(-8) M. In addition, the cerebral cortex ET receptor subtype involved in this action was determined by using BQ-123 and BQ-788, which are ET(A) and ET(B) receptor antagonists respectively. Our results indicate that the ET-1 effect on protein tyrosine phosphorylation occurred through ET(B) receptors. The requirement for extracellular Ca2+ on ET-1 action was also studied. ET-1-stimulated tyrosine phosphorylation of both p125FAK and p130Cas was abolished in the absence of external Ca2+ or in the presence of nimodipine, a Ca2+ channel-blocker. These results suggest that the ET-1-stimulated protein tyrosine phosphorylation was secondary to Ca2+ influx through the dihydropyridine Ca2+-channel. In slices where protein kinase C was inhibited, ET-1-stimulated tyrosine phosphorylation of both proteins was reduced. These results indicate that ET-1 modulates the tyrosine phosphorylation of specific proteins, which may be involved in adhesion processes in the brain.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Corteza Cerebral/metabolismo , Endotelina-1/farmacología , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas , Tirosina/metabolismo , Animales , Calcio/metabolismo , Corteza Cerebral/efectos de los fármacos , Proteína Sustrato Asociada a CrK , Antagonistas de los Receptores de Endotelina , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Masculino , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Fosforilación , Piperidinas/farmacología , Ratas , Ratas Wistar , Proteína p130 Similar a la del RetinoblastomaRESUMEN
The effect of platelet-activating factor (PAF) on protein tyrosine phosphorylation was studied in rat brain slices. PAF induced a time- and concentration-dependent increase in tyrosine phosphorylation of a doublet of approximately 125 kDa. These proteins were identified by immunoprecipitation as p125(FAK) and p130(Cas), using monoclonal antibodies. This effect was mediated by PAF receptors, as shown by its inhibition by the action of a PAF antagonist. The tyrosine phosphorylation evoked by PAF was dependent, at least in part, on external calcium. The involvement of protein kinase C was demonstrated by the synergistic effect of TPA on PAF-stimulated tyrosine phosphorylation. The finding that PAF stimulates tyrosine phosphorylation of both focal adhesion protein p125(FAK) and p130(Cas) suggests that PAF might modulate the integrin mediated signal transduction in the brain.
Asunto(s)
Encéfalo/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Fosfoproteínas/metabolismo , Factor de Activación Plaquetaria/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas , Animales , Encéfalo/metabolismo , Proteína Sustrato Asociada a CrK , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Técnicas In Vitro , Fosforilación , Pruebas de Precipitina , Ratas , Proteína p130 Similar a la del Retinoblastoma , Estimulación QuímicaRESUMEN
In the present study the modulatory action of platelet-activating factor (PAF) on sphingolipid metabolism in cerebral cortical slices was studied. PAF did not alter the basal levels of either sphingomyelin (SM) or ceramide. However, the SMase-elicited reciprocal alterations in SM and ceramide levels were partially prevented by the PAF treatment. The PAF effect was dose-dependent, with 10-8 m being the lowest effective concentration, and receptor-mediated as it was abolished by WEB 2086, a PAF receptor antagonist. Neither N-oleoylethanolamine (OE, ceramidase inhibitor) or d,l-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP, an inhibitor of glucosylceramide synthase and the formation of 1-O-acyl ceramides) prevented the action of PAF. Therefore, the effect of PAF was unlikely to be dependent upon transformation of ceramides into glycosphingolipids, 1-O-acyl ceramides or sphingosine. Experiments with different labeled compounds ([14C]serine, [14C]arachidonate and phosphatidyl [N-methyl-3H]choline) were also performed to test whether PAF could affect the resynthesis of SM. Data obtained agree with the idea that selective pools of both choline and ethanolamine phospholipids were used as precursors for the resynthesis of SM elicited by SMase treatment. PAF itself did not evoke any variation in the lipids analyzed but always prevented the SMase-evoked alterations. Together the data suggest the interesting possibility that PAF increases the overall turnover of SM. In summary, the present data demonstrate that PAF is able to regulate the cellular ceramide levels in brain by accelerating the SM cycle.
Asunto(s)
Encéfalo/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Esfingomielinas/metabolismo , Animales , Encéfalo/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
The involvement of glutamate in PAF-increased cyclic GMP levels was studied. Glutamate treatment caused a dose-response increase of cyclic GMP levels in hippocampal slices. The presence of 1 mM glutamate did not modify the effect caused by 10(-7)M PAF. To elucidate the involvement of glutamate in this action, slices were treated with PAF in the presence of MK-801, a NMDA receptor antagonist. Results indicate that PAF-increased cyclic GMP levels were obtained by NMDA receptors activation. Finally, results obtained from the experiments performed with PAF in the presence of riluzole, to inhibit the glutamate release, demonstrated that glutamate release is a stage in the PAF-induced increase of cyclic GMP levels in hippocampus.
Asunto(s)
GMP Cíclico/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Animales , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/farmacología , Masculino , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Riluzol/farmacologíaRESUMEN
Sphingosylphosphorylcholine (SPC) caused a rapid increase of Ca2+ concentration in isolated brain nuclei. This effect was prevented by nimodipine, an inhibitor of L-type Ca2+ channels, and by thapsigargin, an inhibitor of Ca(2+)-ATPase. Neither heparin nor U73122 modified this effect, suggesting that phospholipase C activation and inositol 1,4,5-trisphosphate (IP3) production are not involved. Results also indicated that SPC-induced increase in Ca2+ concentration is not protein kinase C-dependent.
Asunto(s)
Encéfalo/metabolismo , Calcio/metabolismo , Núcleo Celular/metabolismo , Fosforilcolina/análogos & derivados , Esfingosina/análogos & derivados , Animales , Encéfalo/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Núcleo Celular/efectos de los fármacos , Estrenos/farmacología , Heparina/farmacología , Técnicas In Vitro , Cinética , Nimodipina/farmacología , Fosforilcolina/farmacología , Pirrolidinonas/farmacología , Ratas , Esfingosina/farmacología , Tapsigargina/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
The effect of PCA-4230, a new dihydropyridine derivative with a potent antithrombotic activity, on cyclic nucleotide phosphodiesterase in platelets was studied. PCA-4230 inhibited (54%) cyclic GMP hydrolytic activity of a platelet cytosolic fraction, whereas it did not affect that of cyclic AMP. Results suggested that PCA-4230 inhibited a cyclic GMP-dependent phosphodiesterase, known as cGB PDE or type V, on a purified enzyme from rabbit platelets by a non-competitive-uncompetitive type inhibition. In addition, PCA-4230 potentiated the increase in both cyclic GMP and cyclic AMP levels evoked by sodium nitroprusside. Furthermore, PCA-4230 and forskolin caused a synergistic effect in cyclic AMP, and also potentiated the phosphorylation of 50 kDa and 22 kDa proteins, reported as substrates of cyclic GMP- and cyclic AMP-dependent protein kinases that are related to the inhibition of platelet functions. Finally, PCA-4230 also potentiated the forskolin- and sodium nitroprusside-inhibited serotonin release evoked by thrombin, probably related to the increased cyclic nucleotide level.
Asunto(s)
Plaquetas/enzimología , GMP Cíclico/farmacología , Dihidropiridinas/farmacología , Fibrinolíticos/farmacología , 1-Metil-3-Isobutilxantina/farmacología , 3',5'-GMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Animales , Plaquetas/efectos de los fármacos , Colforsina/farmacología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Sinergismo Farmacológico , Cinética , Nitroprusiato/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación/efectos de los fármacos , Conejos , Serotonina/metabolismo , Trombina/farmacología , Vasodilatadores/farmacologíaRESUMEN
The sphingolipids, sphingosine (SPH), sphingosylphosphorylcholine (SPC) and psycosine induce a rapid and transient rise in nuclear free Ca2+ concentration in a dose dependent manner. To determine whether these sphingolipids act by a IP3-dependent pathway, we tested the increase of Ca2+ in the presence of heparin, an antagonist of IP3 receptor or U70122, an inhibitor of phospholipase C. Results indicate that the effect of both SPH and SPC, but not that of psychosine, is partially mediated by IP3 production. The sphingolipid-induced Ca2+ mobilization was unaffected by the inhibition of protein kinase C, but was totally abolished in the presence of nimodipine, a L-type Ca2+ channel inhibitor. The results could indicate the existence of a sphingosine-gated Ca2+-permeable channel in liver nuclei.
Asunto(s)
Calcio/metabolismo , Núcleo Celular/metabolismo , Hígado/metabolismo , Esfingolípidos/farmacología , Animales , Colorantes Fluorescentes , Fura-2 , Hígado/ultraestructura , Masculino , Ratas , Ratas WistarRESUMEN
Protein tyrosine phosphorylation, modulated by the rate of both protein tyrosine kinase and protein tyrosine phosphatase activities, is critical for cellular signal transduction cascades. We report that endothelin-1 stimulation of rabbit platelets resulted in a dose- and time-dependent tyrosine phosphorylation of four groups of proteins in the molecular mass ranges of 50, 60, 70-100 and 100-200 kDa and that one of these corresponds to focal adhesion kinase. This effect is also related to the approximately 60% decrease in protein tyrosine phosphatase activity. Moreover, this inhibited activity was less sensitive to orthovanadate. In the presence of forskolin that increases the cAMP level a dose-dependent inhibition of the endothelin-stimulated tyrosine phosphorylation of different protein substrates and a correlation with an increase in the protein tyrosine phosphatase activity (11.6-fold compared to control) have been found. Further studies by immunoblotting of immunoprecipitated soluble fraction with anti-protein tyrosine phosphatase-1C from endothelin-stimulated platelets have demonstrated that the tyrosine phosphorylation of platelet protein tyrosine phosphatase-1C is correlated with the decrease in its phosphatase activity. As a consequence, modulation and regulation by endothelin-1 in rabbit platelets can be proposed through a cAMP-dependent pathway and a tyrosine phosphorylation process that may affect some relevant proteins such as focal adhesion kinase.