RESUMEN
Ciliary muscle is a smooth muscle characterized by a rapid response to muscarinic receptor stimulation and sustained contraction. Although it is evident that these contractions are Ca(2+)-dependent, detailed molecular mechanisms are still unknown. In order to elucidate the role of Ser/Thr protein phosphatase 2A (PP2A) in ciliary muscle contraction, we examined the effects of okadaic acid and other PP2A inhibitors on contractions induced by carbachol (CCh) and ionomycin in bovine ciliary muscle strips (BCM). Okadaic acid inhibited ionomycin-induced contraction, while it did not cause significant changes in CCh-induced contraction. Fostriecin showed similar inhibitory effects on the contraction of BCM. On the other hand, rubratoxin A inhibited both ionomycin- and CCh-induced contractions. These results indicated that PP2A was involved at least in ionomycin-induced Ca(2+)-dependent contraction, and that BCM had a unique regulatory mechanism in CCh-induced contraction.
Asunto(s)
Cuerpo Ciliar/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/fisiología , Animales , Calcio/fisiología , Carbacol/antagonistas & inhibidores , Carbacol/farmacología , Bovinos , Técnicas In Vitro , Ionomicina/antagonistas & inhibidores , Ionomicina/farmacología , Micotoxinas/farmacología , Polienos/farmacología , Pironas/farmacologíaRESUMEN
Aegeline or N-[2-hydroxy-2(4-methoxyphenyl) ethyl]-3-phenyl-2-propenamide is a main alkaloid isolated from Aegle marmelos Correa collected in Yogyakarta Indonesia. In our study, we investigated the effects of aegeline on the histamine release from mast cell. The study was performed by using (1) rat basophilic leukemia (RBL-2H3) cell line, and (2) rat peritoneal mast cells (RPMCs). DNP(24)-BSA, thapsigargin, ionomycin, compound 48/80 and PMA were used as inducers for histamine release from mast cell. In our study, aegeline inhibited the histamine release from RBL-2H3 cells induced by DNP(24)-BSA. Indeed, aegeline showed strong inhibition when RBL-2H3 cells induced by Ca(2+) stimulants such as thapsigargin and ionomycin. Aegeline is suggested to influence the intracellular Ca(2+) pool only since could not inhibit the (45)Ca(2+) influx into RBL-2H3 cells. Aegeline showed weak inhibitory effects on the histamine release from RPMCs, even though still succeed to inhibit when the histamine release induced by thapsigargin. These findings indicate that aegeline altered the signaling pathway related to the intracellular Ca(2+) pool in which thapsigargin acts. Based on the results, the inhibitory effects of aegeline on the histamine release from mast cells depended on the type of mast cell and also involved some mechanisms related to intracellular Ca(2+) signaling events via the same target of the action of thapsigargin or downstream process of intracellular Ca(2+) signaling in mast cells.
Asunto(s)
Aegle/química , Amidas/farmacología , Interacciones de Hierba-Droga , Liberación de Histamina/efectos de los fármacos , Mastocitos/efectos de los fármacos , Amidas/aislamiento & purificación , Animales , Calcio/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Dinitrofenoles/antagonistas & inhibidores , Dinitrofenoles/farmacología , Ionomicina/antagonistas & inhibidores , Ionomicina/farmacología , Masculino , Mastocitos/metabolismo , Ratas , Ratas Wistar , Albúmina Sérica Bovina/antagonistas & inhibidores , Albúmina Sérica Bovina/farmacología , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/antagonistas & inhibidores , Tapsigargina/farmacología , p-Metoxi-N-metilfenetilamina/antagonistas & inhibidores , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Previously, we showed that in rat cortical neurons, chronic donepezil treatment (10 microM, 4 days) up-regulates nicotinic receptors (nAChR) and makes neurons more sensitive to the neuroprotective effect of donepezil. Here we examined the mechanism of donepezil-induced neuroprotection in neurons chronically treated with donepezil. The mechanism of neuroprotection was examined under different conditions of exposure to glutamate, acute and moderate, that induce cell death associated with necrotic and apoptotic cell death, respectively. Concomitant treatment with antagonists of nAChRs but not muscarinic receptors inhibited donepezil pretreatment-induced neuroprotection against acute glutamate treatment-induced death. Donepezil pretreatment prevented acute glutamate- and ionomycin-induced neurotoxicity, but not S-nitrosocysteine-induced neurotoxicity, suggesting that donepezil protects neurons via nAChR at levels before nitric oxide synthase activation against acute glutamate neurotoxicity. Concomitant treatment with antagonists of nAChR or phosphatidylinositol 3-kinase (PI3K) signaling inhibitors significantly inhibited neuroprotection against moderate glutamate neurotoxicity and decreased the phosphorylation level of Akt. Neuroprotection was also inhibited by treatment with inhibitor of mitogen-activated protein kinase (MAPK) kinase. These results suggest that donepezil protects neurons against moderate glutamate neurotoxicity via nAChR-PI3K-Akt and MAPK signaling pathways. This study provides novel insight into the mechanism of donepezil-induced neuroprotection that involves nAChR up-regulation.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Citoprotección/efectos de los fármacos , Indanos/farmacología , Degeneración Nerviosa/tratamiento farmacológico , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Piperidinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Inhibidores de la Colinesterasa/farmacología , Citoprotección/fisiología , Donepezilo , Inhibidores Enzimáticos/farmacología , Ácido Glutámico/metabolismo , Ácido Glutámico/toxicidad , Ionomicina/antagonistas & inhibidores , Ionóforos/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Necrosis/tratamiento farmacológico , Necrosis/fisiopatología , Necrosis/prevención & control , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/prevención & control , Neuronas/metabolismo , Antagonistas Nicotínicos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Different inhibitors of the Ca(2+)/calmodulin-stimulated phosphodiesterase 1 family have been described and used for the examination of phosphodiesterase 1 in cellular, organ or animal models. However, the inhibitors described differ in potency and selectivity for the different phosphodiesterase family enzymes, and in part exhibit additional pharmacodynamic actions. In this study, we demonstrate that phosphodiesterase 1C is expressed in the human glioblastoma cell line A172 with regard to mRNA, protein and activity level, and that lower activities of phosphodiesterase 2, phosphodiesterase 3, phosphodiesterase 4 and phosphodiesterase 5 are also present. The identity of the phosphodiesterase 1C activity detected was verified by downregulation of the mRNA and protein through human phosphodiesterase 1C specific small interfering RNA. In addition, the measured K(m) values (cAMP, 1.7 microm; cGMP, 1.3 microm) are characteristic of phosphodiesterase 1C. We demonstrate that treatment with the Ca(2+) ionophore ionomycin increases intracellular Ca(2+) in a concentration-dependent way without affecting cell viability. Under conditions of enhanced intracellular Ca(2+) concentration, a rapid increase in cAMP levels caused by the adenylyl cyclase activator forskolin was abolished, indicating the involvement of Ca(2+)-activated phosphodiesterase 1C. The reduction of forskolin-stimulated cAMP levels was reversed by phosphodiesterase 1 inhibitors in a concentration-dependent way. Using this cellular system, we compared the cellular potency of published phosphodiesterase 1 inhibitors, including 8-methoxymethyl-3-isobutyl-1-methylxanthine, vinpocetine, SCH51866, and two established phosphodiesterase 1 inhibitors developed by Schering-Plough (named compounds 31 and 30). We demonstrate that up to 10 microm 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine had no effect on the reduction of forskolin-stimulated cAMP levels by ionomycin, whereas the more selective and up to 10 000 times more potent phosphodiesterase 1 inhibitors SCH51866, compound 31 and compound 30 inhibited the ionomycin-induced decline of forskolin-induced cAMP at nanomolar concentrations. Thus, our data indicate that SCH51866 and compounds 31 and 30 are effective phosphodiesterase 1 inhibitors in a cellular context, in contrast to the weakly selective and low-potency phosphodiesterase inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine. A172 cells therefore represent a suitable system in which to study the cellular effect of phosphodiesterase 1 inhibitors. 8-Methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine seem not to be suitable for the study of phosphodiesterase 1-mediated functions.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/farmacología , Calcio/metabolismo , Calmodulina/metabolismo , Línea Celular Tumoral , Colforsina/farmacología , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Activación Enzimática/efectos de los fármacos , Glioblastoma/enzimología , Humanos , Ionomicina/antagonistas & inhibidores , Ionomicina/farmacología , Inhibidores de Fosfodiesterasa/clasificaciónRESUMEN
We have previously reported that a novel neuroprotective substance named serofendic acid was purified and isolated from ether extract of fetal calf serum. In the present study, we investigated the effect of serofendic acid on acute neurotoxicity induced by L-glutamate (Glu) using primary cultures of rat cortical neurons. Exposure of cortical cultures to Glu for 1 h caused a marked decrease in cell viability, as determined by trypan blue exclusion. This acute Glu neurotoxicity was prevented by N-methyl-D-aspartate (NMDA) receptor antagonists, extracellular Ca(2+) removal, nitric oxide (NO) synthase inhibitor and NO scavenger. Serofendic acid prevented acute Glu neurotoxicity in a concentration-dependent manner. Acute neurotoxicity was induced by ionomycin, a Ca(2+) ionophore, and S-nitroso-L-cysteine, an NO donor. Serofendic acid also prevented both ionomycin- and S-nitroso-L-cysteine-induced neurotoxicity. Moreover, the protective effect of serofendic acid on acute Glu neurotoxicity was not affected by cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor. These results indicate that serofendic acid protects cultured cortical neurons from acute Glu neurotoxicity by reducing the cytotoxic action of NO and de novo protein synthesis is not required for this neuroprotection.
Asunto(s)
Células Cultivadas , Corteza Cerebral/citología , Cisteína/análogos & derivados , Diterpenos/uso terapéutico , Neuronas/patología , Síndromes de Neurotoxicidad/prevención & control , Glutamato de Sodio/efectos adversos , Valina/análogos & derivados , Animales , Calcio/antagonistas & inhibidores , Calcio/farmacología , Bovinos , Supervivencia Celular/efectos de los fármacos , Cisteína/efectos adversos , Cisteína/antagonistas & inhibidores , Cisteína/metabolismo , Diterpenos/aislamiento & purificación , Diterpenos/farmacología , Maleato de Dizocilpina/farmacología , Maleato de Dizocilpina/uso terapéutico , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Sangre Fetal/química , Feto/anatomía & histología , Ionomicina/efectos adversos , Ionomicina/antagonistas & inhibidores , Neuronas/citología , Neuronas/efectos de los fármacos , Óxido Nítrico/efectos adversos , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Quinoxalinas/farmacología , Quinoxalinas/uso terapéutico , Ratas , S-Nitrosotioles/efectos adversos , S-Nitrosotioles/antagonistas & inhibidores , S-Nitrosotioles/metabolismo , Glutamato de Sodio/antagonistas & inhibidores , Factores de Tiempo , Valina/farmacología , Valina/uso terapéuticoRESUMEN
The tumour necrosis factor (TNF) ligands CD154, CD70 and TNF receptors CD134 and CD137 are all involved in allograft rejection. Because these molecules are not present on resting T cells, we investigated whether immunosuppressive drugs could inhibit their induction. Expression was induced in vitro on T cells by phorbol 12-myristate 13-acetate and ionomycin or by allogeneic dendritic cells in the presence or absence of cyclosporin A (CsA), tacrolimus (TAC), rapamycin derivative (SDZ RAD), or mycophenolic acid (MPA), and determined by flow cytometry. To study the effect of in vivo exposure to immunosuppressive drugs on these molecules, immunohistochemistry was performed on human lymph nodes from patients treated with TAC or TAC and MMF. The calcineurin inhibitors (CI) CsA and TAC strongly suppressed the induction of CD70, CD137 and CD154, but not of CD134, upon pharmacological stimulation of T cells in vitro. In allogeneic stimulations only CD137 and CD154 were inhibited by CI. SDZ RAD did not inhibit pharmacological induction, but in allogeneic stimulations all the investigated molecules were partially suppressed. Both in pharmacological and in allogeneic stimulations, MPA inhibited induction of all tested molecules on T cells nearly completely at 4 micro g/ml. However, in lymph nodes obtained from patients chronically treated with MMF and TAC no reduction of the expression of these molecules was observed. This was possibly caused by trough levels which were in vivo lower (mean: 2.3 micro g/ml) than the concentration giving complete inhibition in vitro. In conclusion, in contrast to CsA, TAC and SDZ RAD, MPA is a potent inhibitor of the induction of TNF- and TNF-receptor superfamily molecules on T cells. To obtain long-term suppression of these molecules in vivo, a plasma trough level of 4 micro g/ml is indicated.
Asunto(s)
Antígenos CD/metabolismo , Inmunosupresores/farmacología , Ácido Micofenólico/farmacología , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Ligando CD27 , Ligando de CD40/metabolismo , Células Cultivadas , Células Dendríticas/inmunología , Humanos , Ionomicina/antagonistas & inhibidores , Ionomicina/inmunología , Ganglios Linfáticos , Activación de Linfocitos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores OX40 , Receptores del Factor de Necrosis Tumoral/metabolismo , Linfocitos T/inmunología , Tacrolimus/farmacología , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Vascular smooth muscle cell (VSMC) hyperproliferation is a characteristic feature of both atherosclerosis and restenosis seen after vascular surgery. A number of studies have shown that heparin inhibits VSMC proliferation in vivo and in culture. To test our hypothesis that heparin mediates its antiproliferative effect by altering Ca(2+) regulated pathways involved in mitogenic signaling in VSMC, we analyzed the effect of heparin on multifunctional Ca(2+)/calmodulin dependent protein kinase II (CaM kinase II) which is abundantly expressed in VSMC. Using activity assays, radioactive labeling, and immunoprecipitation it was found that heparin inhibits the overall phosphorylation of the delta-subunit of CaM kinase II which is consistent with inhibition of autophosphorylation-dependent, Ca(2+)/calmodulin-independent CaM kinase II activity. This effect was less evident in heparin-resistant cells, consistent with a role for CaM kinase II in mediating the antiproliferative effect of heparin. Finally, the effects of pharmacological inhibitors of phosphatases like okadaic acid, calyculin, and tautomycin suggest that heparin inhibits CaM kinase II phosphorylation by activating protein phosphatases 1 and 2A. These findings support the hypothesis that alterations in calcium-mediated mitogenic signaling pathways may be involved in the antiproliferative mechanism of action of heparin.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores de Crecimiento/farmacología , Heparina/farmacología , Músculo Liso Vascular/enzimología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Células Cultivadas , Ionomicina/antagonistas & inhibidores , Músculo Liso Vascular/efectos de los fármacos , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Pulmonary surfactant is a lipoprotein complex that lowers surface tension at the air-liquid interface of the lung and participates in pulmonary host defense. Surfactant proteins (SP), SP-A and SP-D, modulate a variety of immune cell functions, including the production of cytokines and free radicals. Previous studies showed that SP-A and SP-D inhibit lymphocyte proliferation in the presence of accessory cells. The goal of this study was to determine whether SP-A and SP-D directly suppress Th cell function. Both proteins inhibited CD3(+)/CD4(+) lymphocyte proliferation induced by PMA and ionomycin in an IL-2-independent manner. Both proteins decreased the number of cells entering the S and mitotic phases of the cell cycle. Neither SP-A nor SP-D altered cell viability, apoptosis, or secretion of IL-2, IL-4, or IFN-gamma when Th cells were treated with PMA and ionomycin. However, both proteins attenuated ionomycin-induced cytosolic free calcium ([Ca(2+) ](i)), but not thapsigargin-induced changes in [Ca(2+)](i). In summary, inhibition of T cell proliferation by SP-A and SP-D occurs via two mechanisms, an IL-2-dependent mechanism observed with accessory cell-dependent T cell mitogens and specific Ag, as well as an IL-2-independent mechanism of suppression that potentially involves attenuation of [Ca(2+)](i).
Asunto(s)
Complejo CD3/biosíntesis , Linfocitos T CD4-Positivos/inmunología , Regulación hacia Abajo/inmunología , Inmunosupresores/farmacología , Proteína A Asociada a Surfactante Pulmonar/farmacología , Proteína D Asociada a Surfactante Pulmonar/farmacología , Subgrupos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Calcio/metabolismo , Bovinos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/inmunología , Técnicas de Cocultivo , Citosol/efectos de los fármacos , Citosol/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Epítopos de Linfocito T/fisiología , Inhibidores de Crecimiento/farmacología , Humanos , Hibridomas/inmunología , Hibridomas/metabolismo , Interleucina-2/antagonistas & inhibidores , Interleucina-2/biosíntesis , Interleucina-2/fisiología , Ionomicina/antagonistas & inhibidores , Ionomicina/farmacología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Ratas , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/farmacologíaRESUMEN
Cross-linking the high affinity IgE receptor on the rat basophil leukemia clone 2H3 (RBL-2H3) cell line, an vitro model for mast cell signaling, results in granule release. A great deal of research has focused on the earliest steps in this signaling cascade resulting in models which include the participation of lyn, syk, phospholipase C (PLC), protein kinase C (PKC) and intracellular calcium mobilization. In an effort to look at pathways downstream of calcium mobilization, ionomycin-mediated granule release was studied. The kinase inhibitors PP1 (src family), GF109203X (PKC), PD98059 (MEK1/2), and U0126 (MEK1/2) substantially inhibited ionomycin-mediated granule release, while the p38 kinase inhibitor SB203580 did not. Both p38 and erk were phosphorylated upon ionomycin treatment, but only extracellular regulated kinase (erk) activation was completely inhibited by PP1 treatment and partially inhibited by the MEK inhibitors, thus, correlating with the granule release data. Interestingly, while GF109203X alone had no affect on erk activation, combining it with U0126 completely blocked this response. This suggests the existence an alternate pathway for erk activation that is MEK independent and PKC dependent. Experiments in which ionomycin and PP1 were titrated (independently) demonstrated a correlation between erk phosphorylation and granule release, implicating erk in a PP1-inhibitable pathway operating downstream of calcium and controlling mast cell degranulation.
Asunto(s)
Basófilos/inmunología , Degranulación de la Célula , Ionomicina/farmacología , Ionóforos/farmacología , Animales , Basófilos/efectos de los fármacos , Basófilos/enzimología , Butadienos/farmacología , Degranulación de la Célula/efectos de los fármacos , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Indoles/farmacología , Ionomicina/antagonistas & inhibidores , Ionóforos/antagonistas & inhibidores , Leucemia Mieloide , Maleimidas/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitrilos/farmacología , Fosforilación , Inhibidores de Proteínas Quinasas , Pirazoles/farmacología , Pirimidinas/farmacología , Ratas , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
AIM: To compare the suppressions of kanglemycin C (Kan) with that of cyclosporine (Cyc) on lymphocyte proliferations induced by tetradecanoylphorbol acetate (TPA) with ionomycin (IM), and concanavalin A (Con A). METHODS: Cell proliferation was quantified with 3-(4, 5-dimetyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) or [3H]thymidine ([3H]TdR) incorporating method. Calcineurin (CN) activity was measured with molybdate dye colorimetry. RESULTS: Kan (8, 40, 80, and 400 nmol.L-1) strongly suppressed splenocyte proliferation induced by TPA and IM, and the suppressive effect of Kan gradually decreased along with the increasing concentrations of TPA (1-100 micrograms.L-1) and IM (125-500 micrograms.L-1). But, the suppression of Cyc on the splenocyte proliferation induced by TPA was mild. Cyc suppressed CN activity of mouse splenocytes stimulated by Con A stronger than Kan. Moreover, Kan and Cyc strongly suppressed spleen enriched T-cell proliferation induced by Con A and TPA + IM, and the suppression of Kan on proliferation was partly attenuated by exogenous IL-2. CONCLUSION: Kan competitively suppressed the proliferation induced by TPA and IM, and Cyc mainly suppressed IM part of the proliferation induced by TPA and IM.
Asunto(s)
Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Rifamicinas/farmacología , Animales , Calcineurina/metabolismo , División Celular/efectos de los fármacos , Ciclosporina/farmacología , Femenino , Ionomicina/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/metabolismo , Acetato de Tetradecanoilforbol/antagonistas & inhibidoresRESUMEN
Hepatic Kupffer cells (KC), the major tissue macrophage population, produce the septic response mediators, tumor necrosis factor alpha (TNF-alpha) and prostaglandin E2 (PGE2), and have been shown to internalize gadolinium chloride (GD), a rare earth metal of the lanthanide series. Because GD pretreatment of rats has been shown to inhibit the mortality of sepsis, we studied the secretory response to lipopolysaccharide (LPS) by KC isolated from rats injected with either saline or GD (7 mg/kg, intravenously) on the 2 days before KC isolation. Using culture conditions modified to reflect the intrasinusoidal milieu of arginine (RPMI-1640 media with 10 or 100 micromol/L arginine), KC from GD-treated rats responded to LPS (0. 0025 microg/mL) with significantly (P <.01) reduced PGE2 release. In contrast, TNF-alpha release by treated KC was significantly (P <.05) enhanced, consistent with the loss of PGE2 autocoid inhibition of TNF-alpha. Calcium flux is an early signaling event in eicosanoid synthesis, and GD is known to block calcium channels. Therefore, KC were loaded with fura-2-AM to study the effect of GD on KC calcium flux. GD prevented ionomycin and platelet-activating factor (PAF)-mediated [Ca++]i increase and calcium-dependent PGE2 synthesis, while GD did not affect PGE2 synthesis when protein kinase C (PKC) was directly activated with tetradecanoylphorbolacetate (TPA). The inhibition of calcium flux and calcium-dependent PGE2 synthesis in the major cell of the monocytic phagocytic system by GD may explain the previously reported ability of this lanthanide to prevent the mortality of endotoxemia.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Gadolinio/farmacología , Macrófagos del Hígado/metabolismo , Animales , Calcio/metabolismo , Calcio/fisiología , Dinoprostona/antagonistas & inhibidores , Dinoprostona/biosíntesis , Infecciones/mortalidad , Ionomicina/antagonistas & inhibidores , Ionomicina/farmacología , Ionóforos/antagonistas & inhibidores , Ionóforos/farmacología , Lipopolisacáridos/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The marine toxin zooxanthellatoxin-A (ZT-A) and the Ca2+ ionophore ionomycin caused aggregation in rabbit platelets. While ZT-A-induced platelet aggregation was inhibited by indomethacin, ionomycin-induced aggregation was potentiated by the drug. In contrast, both ZT-A- and ionomycin-induced accumulations of thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2), were completely inhibited by indomethacin. 12S-Hydroxyeicosatetraenoic acid (12-HETE), which could be accumulated in the presence of indomethacin, inhibited ZT-A-induced aggregation, but it potentiated the ionomycin-induced one. These results suggest that the different effects of indomethacin may be attributed to the distinct effects of 12-HETE on ZT-A- and ionomycin-induced platelet aggregations.
Asunto(s)
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Antiinflamatorios no Esteroideos/farmacología , Indometacina/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Animales , Interacciones Farmacológicas , Ionomicina/antagonistas & inhibidores , Ionóforos/antagonistas & inhibidores , Toxinas Marinas/antagonistas & inhibidores , Polienos/antagonistas & inhibidores , Conejos , Tromboxano A2/sangre , Tromboxano B2/sangreRESUMEN
We examined the effects of nicotine on glutamate-induced cytotoxicity using primary cultures of rat cortical neurons. The cell viability decreased significantly when cultures were exposed to glutamate for 10 min and then incubated with glutamate-free medium for 1 h. The exposure of cultures to nicotine (10 microM) for 8-24 h prior to glutamate application ameliorated the glutamate-induced cytotoxicity, with no significant effect of nicotine alone on the cell viability. Neuroprotection by nicotine was dependent on the incubation period. alpha-bungarotoxin (alpha-BTX) and methyllycaconitine (MLA), both of which are alpha7-neuronal receptor antagonists, and dihydro-beta-erythroidine (DHbetaE), a neuronal central nervous system (CNS) receptor antagonist, each significantly antagonized the protection by nicotine against glutamate-induced cytotoxicity. Ionomycin, a calcium ionophore, and S-nitrosocysteine (SNOC), a nitric oxide (NO) donor, also induced cytotoxicity in a manner similar to glutamate. Nicotine protected cultures against ionomycin-induced cytotoxicity, but not against SNOC-induced cytotoxicity. These results suggest that nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via alpha7-neuronal receptors and neuronal CNS receptors by reducing NO-formation triggered by Ca2+ influx.
Asunto(s)
Corteza Cerebral/citología , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/toxicidad , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Nicotina/farmacología , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/efectos de los fármacos , S-Nitrosotioles , Aconitina/análogos & derivados , Aconitina/farmacología , Animales , Bungarotoxinas/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Cisteína/análogos & derivados , Cisteína/antagonistas & inhibidores , Cisteína/toxicidad , Dihidro-beta-Eritroidina/farmacología , Ionomicina/antagonistas & inhibidores , Ionomicina/toxicidad , Ionóforos/antagonistas & inhibidores , Ionóforos/toxicidad , Ratas , Toxinas Biológicas/farmacologíaRESUMEN
The effect on human neutrophil NADPH-oxidase activity of the serine protease inhibitor diisopropylfluorophosphate (DFP) was investigated. Pretreatment of neutrophils with the protease inhibitor did not affect the release of reactive oxygen species induced by fMLP. However, the intracellular production of reactive oxygen species induced by ionomycin and yeast particles was largely inhibited in DFP treated cells. Production of reactive oxygen species in subcellular fractions was not affected by the protease inhibitor, neither when the plasma membrane nor when the specific granules were used as source for the b cytochrome subunit of the oxidase. This shows that DFP does not affect the assembly of the oxidase or the activity of the assembled complex. We suggest that serine protease activity is of importance for the signal(s) induced by the Ca2+ ionophore and the yeast particles to reach the dormant NADPH oxidase present in the specific granules and phagolysosomes.
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Ionomicina/antagonistas & inhibidores , Ionóforos/antagonistas & inhibidores , Isoflurofato/farmacología , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Neutrófilos/enzimología , Inhibidores de Serina Proteinasa/farmacología , Levadura Seca/farmacología , Adulto , Biomarcadores , Humanos , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , Ionomicina/farmacología , Ionóforos/farmacología , Mediciones Luminiscentes , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Proteínas Opsoninas/farmacología , Fagocitosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Superóxidos/metabolismoRESUMEN
The regulation of phosphatidylcholine (PtdCho) hydrolysis by Ca2+ and protein kinase C (PKC) was measured in [3H]palmitate-labelled cultured guinea-pig airway smooth-muscle cells as phosphatidylbutanol ([3H]PtdBut) and phosphatidate ([3H]PtdOH) formation in the presence of butanol. The former is a direct measure of phospholipase D (PLD) activity, whereas the latter, in airway smooth muscle, is indicative of net PtdCho-specific phospholipase C (PLC)-like/diacylglycerol (DG) kinase activity. Bradykinin-stimulated responses exhibited a requirement for extracellular Ca2+ influx, since they were inhibited in the presence of EGTA. This influx was independent of voltage-operated channels, since the L-type channel blocker nifedipine (up to 10 microM) was without effect on bradykinin-stimulated responses. In support of this, membrane depolarization with KCl (30 mM) failed to elicit either response. However, bradykinin-stimulated formation of both [3H]PtdBut and [3H]PtdOH was partially inhibited by 100 microM SKF96365. Ionomycin, a Ca2+ ionophore, induced PtdCho hydrolysis to a greater extent than bradykinin, also in an extracellular-Ca(2+)-dependent manner. Thapsigargin-induced emptying of intracellular Ca2+ pools elicited the formation of both [3H]PtdBut and [3H]PtdOH and displayed a requirement for extracellular Ca2+. Bradykinin-stimulated PtdCho-specific PLC-like/DG kinase pathway and PLD responses were unaffected by thapsigargin pretreatment, thereby questioning the role of Ins(1,4,5)P3/Ins(1,3,4,5)P4-dependent Ca2+ stores in the receptor stimulation of these activities in airway smooth-muscle cells. In this regard, we have previously demonstrated that the bradykinin-stimulated PtdCho-specific PLD and PLC-like activities can occur under conditions of apparent complete blockade of bradykinin-stimulated Ins(1,4,5)P3 formation by receptor antagonist in guinea-pig airway smooth muscle. The PKC inhibitor, Ro31-8220, selectively blocked both bradykinin- and ionomycin-stimulated PLD activity in a concentration-dependent manner (IC50 approx. 1 microM), but was without effect on bradykinin-stimulated PtdCho-PLC-like/DG kinase-derived PtdOH formation. In contrast, an inhibitor of PtdCho-PLC, D609, selectively blocked the formation of [3H]PtdOH in the presence of butanol (PtdCho-PLC-like/DG kinase activity), but not [3H]PtdBut formation. In conclusion, PtdCho hydrolysis appears to occur via two distinguishable routes which both require extracellular Ca2+, whereas only the PLD route is regulated by PKC.
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Bradiquinina/farmacología , Calcio/fisiología , Músculo Liso/metabolismo , Fosfatidilcolinas/metabolismo , Proteína Quinasa C/fisiología , Animales , Bradiquinina/antagonistas & inhibidores , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Cobayas , Hidrólisis , Imidazoles/farmacología , Indoles/farmacología , Ionomicina/antagonistas & inhibidores , Ionomicina/farmacología , Ionóforos/antagonistas & inhibidores , Ionóforos/farmacología , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Fosfolipasa D/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Sistema Respiratorio/citología , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Chimeric polypeptides composed of the homeodomain of Antennapedia and of the C-terminus of several low molecular weight GTP-binding proteins of the rab family have been found to translocate through the membrane of cells in culture and to accumulate in the cytoplasm and nucleus. We have used these chimeric peptides to study, in intact endocrine cells, a putative role for the C-terminal domain of rab proteins in the exocytotic process. We show that the internalization of 33- and 32-amino acid polypeptides corresponding to the C-terminal domains of rab3A and rab3B blocks calcium-triggered PRL release from adult rat anterior pituitary cells maintained in primary culture. This effect is specific to rab3 since it is not observed after internalization of either rab1 or rab2 C-terminal peptides. In addition, we demonstrate that this inhibition does not require the geranylgeranylation of the internalized C-termini. As rab3B normally shows a permissive effect on exocytosis in PRL-secreting cells, we suggest that the C-terminal domains of rab3A and rab3B contain structural elements that compete with endogenous rab3 necessary for calcium-induced exocytosis.
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Proteínas de Unión al GTP/fisiología , Proteínas de Homeodominio , Proteínas Nucleares , Fragmentos de Péptidos/farmacología , Adenohipófisis/efectos de los fármacos , Prolactina/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Proteína con Homeodominio Antennapedia , Secuencia de Bases , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Exocitosis/efectos de los fármacos , Femenino , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Ionomicina/antagonistas & inhibidores , Datos de Secuencia Molecular , Adenohipófisis/metabolismo , Prenilación de Proteína , Procesamiento Proteico-Postraduccional , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Tasa de Secreción/efectos de los fármacos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rab3RESUMEN
Prostate tissue is composed of both androgen-dependent and -independent cells. To identify the gene program induced by effectors of apoptosis in both of these cell types, we performed differential hybridization on a complementary DNA library prepared from an androgen-independent prostate cancer cell line, AT-3, exposed to ionomycin. Five distinct complementary DNAs representing ionomycin-inducible genes, designated prostate apoptosis response (par) -1, -2, -3, -4, and -5, were identified. Nucleotide sequencing identified par-1 as the rat homologue of a serum- and oxidative stress-inducible gene, 3CH134/erp/CL100; par-2 as the injury-inducible gene HB-EGF encoding a heparin-binding epidermal growth factor-like growth factor; par-3 as the serum-inducible gene cyr-61; whereas par-4 and par-5 were novel, as judged by a GenBank search. par-1, -3, -4, and -5 were also induced in rat ventral prostate following castration, which causes androgen ablation, leading to apoptosis of androgen-dependent prostate cells. Pretreatment of rats with nifedipine prior to castration abrogated inducible expression of the par genes, indicating that their expression was downstream to Ca2+ elevation. Further characterization of these genes revealed that induction of par-4 is apoptosis specific: it is not induced by effectors of growth stimulation, oxidative stress and necrosis, or growth arrest in prostate cells. Together, par-1, -3, -4, and -5 represent an apoptosis response gene program common to both androgen-dependent and -independent prostate cells. Thus, cell death programs in prostate cells are comprised of genes specifically associated with apoptosis as well as those with multifunctional roles in growth regulation. Since elevation of intracellular Ca2+ is central to apoptosis in many cell types, we predict that par genes will be important components of diverse effector-driven apoptotic pathways.
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Andrógenos/fisiología , Apoptosis/genética , Regulación de la Expresión Génica/efectos de los fármacos , Próstata/fisiología , Animales , División Celular/efectos de los fármacos , División Celular/genética , Línea Celular , Cicloheximida/farmacología , Dactinomicina/farmacología , Ionomicina/antagonistas & inhibidores , Cinética , Masculino , Nifedipino/farmacología , Orquiectomía , Próstata/citología , Ratas , Ratas Sprague-Dawley , Receptores Androgénicos/biosíntesisRESUMEN
The effect of methylene blue on the metabolism of L-arginine to L-citrulline coupled with nitric oxide synthesis in cultured endothelial cells was investigated. The addition of the calcium ionophore ionomycin (10(-8)-10(-5) M) to endothelial cells stimulated L-citrulline formation from L-arginine. Ionomycin-stimulated L-citrulline formation was inhibited by NG-nitro-L-arginine (10(-6)-10(-4) M), a potent inhibitor of nitric oxide synthase. These results indicate that ionomycin-stimulated L-citrulline formation was coupled to nitric oxide synthesis. Therefore, L-citrulline formation from L-arginine was used as a marker for nitric oxide synthesis. Methylene blue (10(-4)-10(-3) M) inhibited ionomycin-stimulated L-citrulline formation in bovine aortic endothelial cells. Moreover, the formation of L-citrulline by the particulate fraction obtained from bovine cultured endothelial cells was also inhibited by methylene blue concentrations in excess of 10(-5) M. Although the addition of methylene blue to various tissues has been reported to release superoxide anions (Wolin et al., 1990; Marczin et al., 1992), inhibition of L-citrulline formation by methylene blue was not affected by the presence of superoxide dismutase (100 micrograms/mL) in either cultured endothelial cells or the particulate fraction. These findings suggest that methylene blue is a direct inhibitor of nitric oxide synthase in bovine aortic endothelial cells.
Asunto(s)
Arginina/antagonistas & inhibidores , Citrulina/biosíntesis , Endotelio Vascular/metabolismo , Azul de Metileno/farmacología , Óxido Nítrico/biosíntesis , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Animales , Aorta Torácica , Arginina/análogos & derivados , Arginina/metabolismo , Arginina/farmacología , Bovinos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Ionomicina/antagonistas & inhibidores , Ionomicina/farmacología , Óxido Nítrico Sintasa , Nitroarginina , Superóxido Dismutasa/farmacologíaRESUMEN
1. Acetylcholine (ACh) evoked secretion by the calcium ionophore, ionomycin, was studied at frog motor nerve endings. 2. Bath application of ionomycin stimulated an irreversible increase in the rate of spontaneous, quantal ACh release in the presence of extracellular Ca2+. In contrast, local application of ionomycin stimulated a rapid, reversible acceleration of spontaneous ACh release. 3. The magnitude of the secretory response to ionomycin was dependent both upon the concentration of ionophore and the concentration of extracellular Ca2+. 4. Adenosine or 2-chloroadenosine inhibited ionomycin-stimulated ACh release with the same potency and efficacy observed previously for these adenosine analogues as inhibitors of ACh secretion evoked by nerve impulses. 5. These results support the conclusion that adenosine receptor activation inhibits quantal ACh secretion at a site distal to that of Ca2+ entry at frog motor nerve endings.
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Acetilcolina/metabolismo , Adenosina/farmacología , Ionomicina/farmacología , Neuronas Motoras/metabolismo , Unión Neuromuscular/metabolismo , 2-Cloroadenosina/farmacología , Animales , Calcio/metabolismo , Técnicas In Vitro , Ionomicina/antagonistas & inhibidores , Potenciales de la Membrana/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Rana pipiensRESUMEN
PROBLEM: This study was undertaken to evaluate the effects of cyclosporin A on prostanoid (prostaglandin and thromboxane) production by human decidua. METHOD: Decidual cells were isolated from term placentae obtained at elective cesarean section before the onset of labor. Cells were grown to confluence and then incubated for 16 h with cyclosporin A (0.1-100 ng/ml) in the presence and absence of interleukin 1 beta (IL-1 beta, 10 ng/ml), phorbol 12-myristate 13-acetate (PMA, 10(-7) M) and ionomycin (0.5 micron). Prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) were measured by radioimmunoassay, and cellular protein was determined. RESULTS: IL-1 beta, PMA, and ionomycin all stimulated decidual PGE2 and TXB2 production as expected. However, these stimulatory actions were attenuated by 20% when cells were coincubated with cyclosporin A (100 ng/ml). All concentrations of cyclosporin A tested were within the therapeutic range. CONCLUSIONS: Our results indicate that cyclosporin A does not stimulate decidual prostanoid production and is probably unrelated to preterm labor and delivery in allograft recipients.