RESUMEN
We have previously reported that transduction of murine colon cancer cells (MC38) with herpes simplex virus thymidine kinase (HSV-tk) gene results in a significant enhancement of tumor growth rate in vivo and overexpression of cyclooxygenase-2 (COX-2). Our current study aimed to investigate the involvement of nuclear factor-kappa B (NF-kappaB), a pivotal transcriptional regulator of COX-2, in the upregulation of COX-2 expression by HSV-tk. It was found that HSV-tk gene transduction of MC38 cells results in significantly enhanced NF-kappaB activity, increased phosphorylation and degradation of inhibitor-kappa Balpha (IkappaBalpha) and enhanced translocation of NF-kappaB to the nucleus. Treatment of HSV-tk-transduced MC38 cells with sulfasalazine, a potent NF-kappaB inhibitor, led to dose-dependent inhibition of NF-kappaB activity, IkappaB phosphorylation and nuclear translocation of NF-kappaB, accompanied by significantly decreased COX-2 expression and reduced release of prostaglandin E2. Transient transfection experiments with COX-2 promoter constructs fused to luciferase reporter gene revealed that mutation in NF-kappaB-responsive element of COX-2 promoter significantly reduced promoter activity in HSV-tk-transduced MC38 and COS-7 cells, whereas it had no effect on promoter activity in the respective wild-type cells. At last, it was found that HSV-tk gene transduction causes significant enhancement of NF-kappaB activity and COX-2 expression in two additional tumor cell lines, 9L and T24. These findings suggest that HSV-tk gene transduction results in NF-kappaB pathway activation, which is essential for COX-2 overexpression by HSV-tk.
Asunto(s)
Neoplasias del Colon/genética , Ciclooxigenasa 2/genética , FN-kappa B/metabolismo , Simplexvirus/genética , Timidina Quinasa/genética , Transporte Activo de Núcleo Celular , Animales , Neoplasias del Colon/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas I-kappa B/efectos de los fármacos , Proteínas I-kappa B/metabolismo , Ratones , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , Fosforilación , Transducción de Señal , Sulfasalazina/farmacología , Transcripción Genética , Transducción Genética , Células Tumorales CultivadasRESUMEN
Ram seminal vesicle microsomes, a rich source of prostaglandin H synthase-1, were incubated with 100 nM of the prostaglandin H synthase-2 inhibitors N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DuP-697) prior to exposure to the prostaglandin H synthase inhibitors aspirin, indomethacin, ibuprofen or naproxen. Activity of the enzyme was measured by following the conversion of arachidonic acid to prostaglandin E(2) and prostaglandin F2alpha. Although prostaglandin H synthase-1 activity was not altered by these concentrations of the prostaglandin H synthase-2 inhibitors, it was found that exposure to these agents prior to aspirin or indomethacin (irreversible prostaglandin H synthase inhibitors) significantly attenuated the inhibition obtained by the latter inhibitors. On the other hand, the same concentrations of the prostaglandin H synthase-2 inhibitors did not interfere with prostaglandin H synthase-1 inhibition that was induced by naproxen or ibuprofen (competitive prostaglandin H synthase inhibitors). Attenuation of the indomethacin inhibition of prostaglandin H synthase-1 by prostaglandin H synthase-2 inhibitors was observed only when the microsomes were pre-exposed to DuP-697 or NS-398 in the absence, but not in the presence, of arachidonic acid. The effect of DuP-697 was found to be irreversible, however, washing away the agent reversed the action of NS-398. Similar phenomena have been reported by us in bovine aortic endothelial cells and in human dermal fibroblasts. Attenuation of the inhibition by aspirin and indomethacin, without altering the enzyme's basal activity or the inhibition induced by ibuprofen or naproxen may suggest the possibility that the prostaglandin H synthase-2 specific inhibitors DuP-697 and NS-398 affect prostaglandin H synthase-1 by interaction with a site different from the enzyme's catalytic site.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Dinoprostona/metabolismo , Isoenzimas/antagonistas & inhibidores , Microsomas/efectos de los fármacos , Animales , Ácido Araquidónico/farmacología , Aspirina/farmacología , Bovinos , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Relación Dosis-Respuesta a Droga , Ibuprofeno/farmacología , Indometacina/farmacología , Isoenzimas/farmacología , Masculino , Microsomas/metabolismo , Nitrobencenos/farmacología , Prostaglandina-Endoperóxido Sintasas , Vesículas Seminales/ultraestructura , Sulfonamidas/farmacología , Tiofenos/farmacologíaRESUMEN
Bovine aortic endothelial cells produce prostacyclin as their major arachidonic acid metabolite. cAMP, in turn, is the second messenger for prostacyclin. In the present study, we investigated the effects of cAMP-elevating agents on prostacyclin production by bovine aortic endothelial cells. Treatment of resting bovine aortic endothelial cells with cAMP-elevating agents inhibited prostacyclin production and cyclooxygenase activity, without affecting arachidonic acid release. No change was detected in cyclooxygenase-1 protein expression. The specific inhibitor of protein kinase A, Rp-cAMPS (adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, triethylammonium salt), and the phosphatase inhibitor, okadaic acid, both suppressed cAMP-induced inhibition, suggesting that this inhibition is mediated by a phosphorylation-dephosphorylation cascade, which is possibly protein kinase A-dependent. In lipopolysaccharide-treated cyclooxygenase-2 expressing bovine aortic endothelial cells, where cyclooxygenase-1 activity was selectively inhibited, dibutyryl cAMP failed to inhibit cyclooxygenase-2 activity. Cyclooxygenase-2 protein was induced upon treatment with dibutyryl cAMP and further induction of cyclooxygenase-2 protein was effected by IBMX (3-isobutyl-1-methyl-xanthine) and dibutyryl cAMP in bacterial lipopolysaccharide-stimulated cells. These results suggest that increased cellular cAMP selectively inhibits cyclooxygenase-1 activity without altering cyclooxygenase-1 protein expression, and at the same time, up-regulates cyclooxygenase-2 protein. This complex regulation of cyclooxygenase activity and protein expression by cAMP may represent a prostacyclin-induced autoregulatory mechanism in bovine aortic endothelial cells.
Asunto(s)
AMP Cíclico/metabolismo , Endotelio Vascular/efectos de los fármacos , Epoprostenol/metabolismo , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Aorta/efectos de los fármacos , Ácido Araquidónico/metabolismo , Western Blotting , Bucladesina/farmacología , Bovinos , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Lipopolisacáridos/farmacología , Ácido Ocadaico/farmacología , Proteínas/metabolismo , Tionucleótidos/farmacologíaRESUMEN
Since the discovery of the inducible form of prostaglandin (PG) H synthase (PGHS), PGHS-2, considerable effort has been made to design selective inhibitors of this isozyme. N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DuP-697) have been shown to interact reversibly with PGHS-1, while irreversibly inhibiting PGHS-2 in a time-dependent manner. In the present study we have tested the effects of DuP-697 and NS-398 on the activity of PGHS-1 and further explored the interactions between these agents and the inhibition of PGHS-1 by aspirin, indomethacin and ibuprofen. Three independent experimental systems, namely bovine aortic endothelial cells (BAEC), human fibroblasts and ram seminal vesicle microsomes were used to investigate the effects of DuP-697 and NS-398 on PGHS-1. The results show that DuP-697 and NS-398, at concentrations ranges which do not inhibit PGHS-1 activity, significantly attenuated the inhibition of PGHS-1 that was caused by aspirin and indomethacin. The same concentrations of DuP-697 and NS-398 did not affect the inhibition of PGHS-1 that was induced by the competitive reversible inhibitors ibuprofen and naproxen. Similar effects of DuP-697 and NS-393 were obtained with ram seminal vesicle microsomes. These results suggest that PGHS-2 inhibitors DuP-697 and NS-398 possibly interact with PGHS-1 at a site different from the enzyme's catalytic site, thus causing attenuation of PGHS-1 inhibition by aspirin and indomethacin without altering PGHS-1 basal activity or the ibuprofen-induced inhibition.
Asunto(s)
Aspirina/antagonistas & inhibidores , Inhibidores de la Ciclooxigenasa/farmacología , Indometacina/antagonistas & inhibidores , Nitrobencenos/farmacología , Sulfonamidas/farmacología , Tiofenos/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Bovinos , Células Cultivadas , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Indometacina/farmacología , Isoenzimas/efectos de los fármacos , Masculino , Proteínas de la Membrana , Microsomas/efectos de los fármacos , Microsomas/enzimología , Prostaglandina-Endoperóxido Sintasas/efectos de los fármacos , Vesículas Seminales/efectos de los fármacos , Vesículas Seminales/enzimología , OvinosRESUMEN
PGE2 and prostacyclin each enhance cAMP synthesis in the osteoblast-like cell line UMR-106. The amount of cAMP induced by PGE2 was 5-7-fold greater than the amount induced by cicaprost or iloprost, stable prostacyclin analogues. Both PGE2 and the two prostacyclin analogues enhanced cAMP synthesis with similar time dependence. The EC50 values of PGE2 and cicaprost were 3 X 10(-6) and 5 x 10(-8) M, respectively. Short-term incubation of the cells with 12-o-tetradecanoylphorbol 13-acetate (TPA) markedly reduced the PGE2-induced cAMP synthesis. In contrast, cells that were incubated with the same concentrations of TPA in the presence of cicaprost or iloprost showed a 1.6-fold increase in cAMP formation. The marked disparity between the cAMP response to cicaprost and PGE2 in the presence of TPA suggests that the two prostanoids induce cAMP synthesis in the UMR-106 cells by interaction with different receptors. These observations support the idea that the osteoblastic UMR-106 cells may express specific prostacyclin receptors and suggest that prostacyclin may have a unique role in osteoblasts.
Asunto(s)
AMP Cíclico/biosíntesis , AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Epoprostenol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Animales , Línea Celular , Colforsina/farmacología , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Epoprostenol/farmacología , Fluoruro de Sodio/farmacología , Factores de TiempoRESUMEN
Corticotropin releasing factor (CRF) is a predominant regulator of the neuroendocrine, autonomic and behavioral responses to stress. In addition, numerous studies support autocrine/paracrine roles for this peptide at peripheral sites. CRF and CRF binding sites have been identified in different regions of the central nervous system as well as in the heart, spleen, adrenal and testis, and high levels of CRF were detected in inflamed fibroblasts. However, the precise physiological or pathophysiological role of peripheral CRF cannot yet be discerned. Here we show that CRF, through interaction with specific membrane receptors, blocks the interleukin-1alpha (IL-1alpha)-stimulated prostaglandin (PG) synthesis in fibroblasts. Binding of [125I]-labeled CRF in fibroblasts was saturable and fitted a two sites model. K(D) for the higher-affinity class of receptors was 20+/-2.2 pM, and Bmax 1.95+/-0.22 fmol/mg protein. For the lower-affinity class of receptors K(D) was 160+/-17 nM, and Bmax 2.38+/-0.27 fmol/mg protein. CRF blocked the effect of IL-1alpha on PGE2 synthesis, and this was antagonised by D-PheCRF12-41. In addition, the CRF receptor antagonists alpha helical CRF9-41 and D-PheCRF12-41 at high concentrations inhibited the IL-1alpha-induced PG synthesis similarly to CRF, suggesting partial agonistic action. Taken together, these results suggest a modulatory role of CRF in inflammation.
Asunto(s)
Hormona Liberadora de Corticotropina/farmacología , Dinoprostona/biosíntesis , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Sitios de Unión/fisiología , Unión Competitiva/fisiología , Hormona Liberadora de Corticotropina/análogos & derivados , Fibroblastos , Humanos , Interleucina-1/farmacología , Radioisótopos de Yodo/metabolismo , Masculino , Fragmentos de Péptidos/farmacología , Unión Proteica/fisiología , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidoresRESUMEN
Corticotropin releasing factor (CRF) is a hypothalamic hormone that also displays autocrine/paracrine roles at peripheral sites. High concentrations of CRF have been identified in endothelial cells and other inflammatory tissues. We investigated the effects of CRF and antagonists in the regulation of prostaglandin synthesis in bovine aortic endothelial cells, and also characterized the binding of CRF in these cells. Interleukin-1alpha increased prostacyclin (prostaglandin I2) synthesis in endothelial cells and this response to interleukin-1alpha was abolished by simultaneous exposure to CRF. The effect of CRF on interleukin-1alpha-induced prostaglandin synthesis was antagonised by the CRF receptor antagonist alpha-helical CRF-(9-41). In addition, this as well as another CRF receptor antagonist, namely [D-Phe12]CRF-(12-41), when applied alone at low concentrations inhibited the interleukin-1alpha-induced prostaglandin synthesis similarly to CRF, suggesting partial agonistic action. Binding of [125I]-labeled CRF in endothelial cells was saturable and fitted a two sites model. Kd for the higher-affinity class of receptors was 0.2 +/- 0.02 nM, and Bmax 0.79 +/- 0.095 fmol/mg protein. The lower-affinity class of receptors had a Kd of 1.77 +/- 0.14 microM and Bmax 0.97 +/- 0.12 fmol/mg protein. These findings suggest a direct role for CRF in the local regulation of inflammation.
Asunto(s)
Hormona Liberadora de Corticotropina/farmacología , Endotelio Vascular/efectos de los fármacos , Epoprostenol/biosíntesis , Animales , Bovinos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Interleucina-1/farmacología , Fragmentos de Péptidos/farmacología , Receptores de Hormona Liberadora de Corticotropina/antagonistas & inhibidoresRESUMEN
Bovine aortic endothelial cells contain a prostaglandin site which binds with similar low-affinity PGE2, PGF2alpha and the thromboxane agonist U-46619. Treatment of the cells with agents that increase the level of cellular cAMP such as forskolin, a direct activator of adenylate cyclase or IBMX, a phosphodiesterase inhibitor, decreased the binding of PGE2 to the cells. Addition of dibutyryl cAMP to intact cells caused a quick reduction in PGE2 binding with a half time of less than 2 min. The reduction in PGE2 binding was completely reversible after removing the dibutyryl cAMP. The reduction in PGE2 binding after addition of dibutyryl cAMP to the intact cells was also observed after a mechanical disruption of the cells or after permeabilization with digitonin. Incubation of the cells with myristoylated PKI(14-22) amide, a specific protein kinase A inhibitor, resulted in partial suppression of the reduction of PGE2 binding by dibutyryl cAMP. Pretreatment of intact cells for 24 h with 10(-6) M PGE2 or a PKC activator did not reduce the specific binding of [3H]-PGE2. These results suggest that PKA, but not PKC, is involved in a fast reversible regulation of the common prostanoid receptor on bovine endothelial cells.
Asunto(s)
Endotelio Vascular/metabolismo , Prostaglandinas/metabolismo , Animales , Aorta Torácica , Sitios de Unión/fisiología , Bucladesina/farmacología , Carcinógenos/farmacología , Bovinos , Células Cultivadas , AMP Cíclico/metabolismo , Citosol/química , Citosol/metabolismo , Digitonina/farmacología , Dinoprost/metabolismo , Dinoprostona/metabolismo , Dinoprostona/farmacología , Endotelio Vascular/citología , Indicadores y Reactivos/farmacología , Oxitócicos/farmacología , Unión Proteica/efectos de los fármacos , Cloruro de Sodio , Soluciones/farmacología , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , TritioRESUMEN
The objective of the present study was to examine whether prostaglandin H synthase (PGHS) can be regulated by pathways independent of de novo synthesis of PGHS. Incubation of bovine aortic endothelial cells (BAEC) for as short as 5 min with NaF (40 mM) resulted in a 60% increase in PGHS activity. PGHS activity induced by NaF was unaffected by either 10 microM cycloheximide or 1 microM actinomycin D. Aspirin (25 microM) completely inhibited resting PGHS activity, and NaF did not induce further stimulation. NS-398 (500 nM), a specific PGHS-2 inhibitor, was ineffective. Basic fibroblast growth factor (bFGF) induced a significant increase in PGHS activity within 30 min and was insensitive to cycloheximide. The levels of PGHS-1 and PGHS-2 proteins, as measured by Western blots, were not affected by NaF or bFGF. The tyrosine kinase inhibitor genistein attenuated PGHS activity that was induced by NaF and bFGF, whereas the tyrosine phosphatase inhibitor, sodium orthovanadate, augmented these responses. The G protein activators 5'-guanylyl imidodiphosphate and guanosine 5'-O-(3-thiotriphosphate) inhibited both resting and NaF-induced PGHS activities. These results suggest-that, in BAEC, PGHS-1 activity can be regulated by tyrosine kinase and/or G proteins, independently of de novo protein synthesis.
Asunto(s)
Dactinomicina/farmacología , Endotelio Vascular/enzimología , Isoenzimas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Inhibidores de la Síntesis de la Proteína/farmacología , Fluoruro de Sodio/farmacología , Compuestos de Aluminio/farmacología , Animales , Aorta , Aspirina/farmacología , Bovinos , Células Cultivadas , Cicloheximida/farmacología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fluoruros/farmacología , Proteínas de Unión al GTP/metabolismo , Genisteína/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanilil Imidodifosfato/farmacología , Cinética , Nitrobencenos/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Sulfonamidas/farmacología , Factores de Tiempo , Vanadatos/farmacologíaRESUMEN
Neuronal apoptosis is the subject of intense investigation and is beginning to be understood in some molecular detail. In the present study, we show that PC12 cells, like certain other cell types, redistribute phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the plasma membrane early in the process of apoptosis. The externalised PS can be readily visualised by incubating intact cells with a fluorescent derivative of the protein annexin V. When apoptosis is blocked with an inhibitor of interleukin-1beta-converting-enzyme-like proteases, the increased annexin binding is also blocked. Fluorescent annexin V binding provides a rapid and convenient way to identify apoptotic neurones.
Asunto(s)
Apoptosis , Membrana Celular/metabolismo , Células PC12/metabolismo , Fosfatidilserinas/metabolismo , Animales , Anexina A5/metabolismo , Biomarcadores , Caspasa 1 , Diferenciación Celular/efectos de los fármacos , Cisteína Endopeptidasas/fisiología , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/fisiología , Células PC12/citología , Células PC12/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Unión Proteica , Ratas , Estaurosporina/farmacologíaRESUMEN
[3H]PGE2 and [3H]PGF2 alpha were shown to bind with similar binding capacity and dissociation constants to bovine aorta endothelial cells. The similarity in the binding parameters suggests that both agonists may bind to the same binding site. Displacement of [3H]PGE2 performed with PGE2, PGF2 alpha or U-46619, a thromboxane agonist, shows that all three prostanoids displaced the bound [3H]PGE2 with comparable potency (IC50 = 10(-7) M). These results indicated that the three different prostanoids, which serve as specific agonists to different prostanoid receptors, also compete for the same binding site in bovine endothelial cells with similar affinity. Comparison of the displacement of [3H]PGE2 or [3H]PGF2 alpha by a number of prostaglandin agonists and antagonists further supports the notion that the natural prostanoids bind with similar affinities to the same binding site. Thus, sulprostone, an EP1/EP3 agonist, displaced bound [3H]PGE2 and [3H]PGF2 alpha with IC50 of about 10(-7) M. On the other hand, thromboxane antagonists (BAY u-3405 and GR-32191B), EP1 specific antagonist (SC-19220) EP1/DP antagonist (AH-6809) and iloprost, a stable prostacyclin agonist, failed to displace bound [3H]PGE2 or [3H]PGF2 alpha at a concentration range of 10(-9)-10(-6) M. Gradual increase of sodium fluoride (NaF), a general activator of G binding proteins, or incubation of permeabilized cells with GTP gamma S resulted in a decrease in [3H]PGE2 binding, suggesting that the binding site represents a low-affinity common prostanoid receptor which, similar to other prostanoid receptors, is probably coupled with G binding proteins.
Asunto(s)
Endotelio Vascular/metabolismo , Prostaglandinas/metabolismo , Xantonas , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Animales , Aorta , Sitios de Unión , Compuestos de Bifenilo/farmacología , Carbazoles/farmacología , Bovinos , Células Cultivadas , Ácido Dibenzo(b,f)(1,4)oxazepina-10(11H)-carboxílico, 8-cloro-, 2-acetilhidrazida/farmacología , Dinoprostona/análogos & derivados , Dinoprostona/metabolismo , Dinoprostona/farmacología , Endotelio Vascular/citología , Epoprostenol/agonistas , Epoprostenol/análogos & derivados , Epoprostenol/farmacología , Ácidos Heptanoicos/farmacología , Iloprost/farmacología , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Sulfonamidas/farmacología , Tromboxano A2/análogos & derivados , Tromboxano A2/farmacología , Tromboxanos/agonistas , Xantenos/farmacologíaRESUMEN
NaF, a nonselective activator of heterotrimeric guanine nucleotide-binding proteins (G proteins), increased the release of arachidonic acid (AA) and prostacyclin (PGI2) production in bovine aortic endothelial cells (BAEC) at low concentrations (40-60 mM). On the other hand, higher concentrations (100 mM) inhibited phospholipase A2 (PLA2) compared with the basal activity. Intracellular Ca2+ levels did not rise after treatment with stimulatory concentrations of NaF, and, moreover, neither neomycin nor Ca(2+)-free medium affected the biphasic pattern of PGI2 synthesis in response to NaF. CGP-43187, an inhibitor of the 14-kDa secretory PLA2, did not affect NaF-induced AA release. However, AACOCF3, a specific inhibitor of the cytosolic 85-kDa PLA2 (cPLA2), abrogated AA release and PGI2 production in response to 60 mM NaF. A biphasic pattern of PGI2 production was also obtained with the guanosine 5'-triphosphate analogues guanosine 5'-O-(3-thiotriphosphate) and guanylylimidodiphosphate in permeabilized BAEC. Pretreatment of the cells with guanosine 5'-O-(2-thiodiphosphate) suppressed the inhibition and the stimulation of AA release induced by guanylylimidodiphosphate. In addition, phenylisopropyl adenosine inhibited the release of AA and PGI2, whereas ATP and bradykinin increased PGI2. Pertussis toxin not only inhibited ATP- and bradykinin-stimulated PGI2 release, it also reversed the inhibitory effect of phenylisopropyl adenosine, resulting in a significant stimulation. These findings strongly suggest that, in BAEC, cPLA2 is coupled with more than one G protein that are involved in inhibition and stimulation of cPLA2 activity.
Asunto(s)
Aorta/metabolismo , Endotelio Vascular/metabolismo , Epoprostenol/biosíntesis , Proteínas de Unión al GTP/fisiología , Fosfolipasas A/biosíntesis , Animales , Aorta/citología , Ácido Araquidónico/metabolismo , Calcio/metabolismo , Bovinos , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Guanilil Imidodifosfato/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Prostaglandina-Endoperóxido Sintasas/metabolismo , Fluoruro de Sodio/farmacologíaRESUMEN
We used mice transgenic for a major histocompatibility complex class I-restricted T cell receptor to study the changes of phenotype in vivo which follow priming by antigen of CD8 T cells. We show that following priming with peptide, CD44 on CD8 T cells is up-regulated. The change of phenotype was relatively stable, as primed CD8 cells isolated from thymectomized mice 6 weeks after priming still expressed increased levels of CD44. CD8 T cells in these mice are still responsive to peptide and could represent long-lived primed cells. No down-regulation in vivo of the CD45RA or CD45RB isoforms was found, indicating that there is a differential regulation of the expression of CD44 and CD45RB by activated CD8 transgenic T cells. These results contradict earlier studies in vitro which showed that CD8 T cells which have been primed earlier belong to the CD45RA- or CD45RB- subset.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Proteínas Portadoras/inmunología , Antígenos Comunes de Leucocito/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Superficie Celular/inmunología , Receptores Mensajeros de Linfocitos/inmunología , Animales , Proteínas Portadoras/biosíntesis , Receptores de Hialuranos , Inmunización , Antígenos Comunes de Leucocito/biosíntesis , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Superficie Celular/biosíntesis , Receptores Mensajeros de Linfocitos/biosíntesisRESUMEN
During the past year, apoptosis has been recognized as a process that is perpetually poised to be initiated--often from the cytoplasm rather than from the nucleus--unless it is suppressed by survival factors. Suspected mediators of apoptosis that have recently been investigated include the cysteine protease interleukin-1 beta-converting enzyme, free radicals and cell cycle kinases. Known inhibitors of programmed cell death, such as Bcl-2 and its homologues, have been further studied, and the results suggest that cell death may be regulated by multiple pathways. With the recent identification of the Drosophila gene reaper, which appears to play a role in the initiation of apoptosis, another genetic system for studying cell death has become available.
Asunto(s)
Apoptosis , Neuronas/fisiología , Animales , Caspasa 1 , Ciclo Celular , Muerte Celular , Cisteína Endopeptidasas/fisiología , Drosophila/genética , Humanos , Degeneración Nerviosa , Enfermedades del Sistema Nervioso/patología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
In a previous work, we showed that picomolar concentrations of prostaglandin E2 (PGE2) inhibit Na-K-ATPase activity and ouabain binding in a clone of Madin-Darby canine kidney (MDCK) cells. In the present study, we demonstrate that the inhibitory effects of PGE2 on Na-K-ATPase activity, ouabain-sensitive Rb+ uptake, and ouabain binding in MDCK cells were diminished by treatment of the cells with nonsteroidal anti-inflammatory drugs. These results suggested that products of arachidonic acid synthesized through the cyclooxygenase pathway are involved in the inhibitory mechanism of PGE2. Treatment of the cells with arachidonic acid resulted in inhibition of ouabain binding, and the inhibition was eliminated by cyclooxygenase inhibitors. These observations further support the involvement of cyclooxygenase products in the PGE2-induced inhibitory process. Finally, we demonstrated that dopamine inhibits Rb+ influx and ouabain binding in MDCK cells similarly to PGE2. Cyclooxygenase inhibitors suppressed the inhibition of ouabain binding by dopamine, thus also suggesting the involvement of cyclooxygenase products in the inhibitory effect of dopamine.
Asunto(s)
Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/farmacología , Riñón/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Línea Celular , Perros , Riñón/citologíaRESUMEN
We present evidence that the neurite out-growth stimulated by the binding of Thy-1 antibodies to PC12 cells is mediated by calcium influx through both N- and L-type calcium channels. PC12 cells cultured on a noncellular substratum in the presence of NGF, or on a cellular substratum in the absence of NGF, responded to soluble Thy-1 antibody by extending longer neurites. The response required bivalent antibody and could be blocked by removing Thy-1 from the surface of PC12 cells with phosphatidylinositol specific phospholipase C. The response could also be blocked by reducing extracellular calcium to 0.25 mM, or by antagonists of L- and N-type calcium channels. Additionally, the response could be fully inhibited by preloading PC12 cells with BAPTA/AM which buffers changes in intracellular calcium. A heterotrimeric G-protein is also implicated in the pathway as the response could be fully inhibited by pertussis toxin. These data suggest that antibody-induced clustering of Thy-1 stimulates neurite outgrowth by activating a second messenger pathway that has previously been shown to underlie cell adhesion molecule (NCAM, N-cadherin, and L1), but not integrin or NGF-dependent neurite outgrowth.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos de Superficie/metabolismo , Canales de Calcio/fisiología , Calcio/metabolismo , Isoanticuerpos/farmacología , Glicoproteínas de Membrana/metabolismo , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/metabolismo , Neuritas/fisiología , Neuronas/fisiología , Células 3T3 , Animales , Canales de Calcio/efectos de los fármacos , Diltiazem/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Ratones , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células PC12 , Toxina del Pertussis , Antígenos Thy-1 , Factores de Virulencia de Bordetella/farmacologíaAsunto(s)
Tejido Adiposo/metabolismo , Citosol/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Proteínas/metabolismo , Receptores de Prostaglandina/metabolismo , Albúmina Sérica/farmacología , Animales , Proteínas Portadoras/farmacología , Epidídimo/metabolismo , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Proteínas de Unión al GTP/metabolismo , Masculino , Ratas , Receptores de Prostaglandina/efectos de los fármacos , Receptores de Prostaglandina ERESUMEN
In the present study we report on a direct effect of prostaglandin E2 (PGE2) on ouabain binding and Na-K-adenosinetriphosphatase (Na-K-ATPase) activity in a clone of Madin-Darby canine kidney cells, a renal cell line with collecting duct properties. Incubation of the cells with low concentrations (pM) of PGE2 produced a concomitant reduction of approximately 50% in the activity of Na-K-ATPase in the cell homogenate and in ouabain binding to the intact cells (half-maximal inhibition of approximately 0.1 pM). The inhibition was apparent within 10 min of preincubation of the cells with PGE2. Scatchard analysis of the binding demonstrated that the treatment with PGE2 reduced the number of ouabain binding sites without a change in the dissociation constant. PGE1 and PGF2 alpha (10 nM) did not affect ouabain binding or Na-K-ATPase activity. The fast, potent, and specific effect of PGE2 suggests that the diuretic/natriuretic effect of prostaglandins of the E series in the collecting tubule, in addition to the interference with the activity of arginine vasopressin, may result from a direct reduction in the number of the Na-K-ATPase active units, via a prostaglandin receptor.
Asunto(s)
Dinoprostona/farmacología , Riñón/metabolismo , Ouabaína/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Sitios de Unión/efectos de los fármacos , Línea Celular , Perros , Riñón/citología , Concentración Osmolar , Prostaglandinas/farmacologíaRESUMEN
A full-length cDNA encoding 180-kDa neural cell adhesion molecule (NCAM 180) has been transfected into mouse NIH-3T3 fibroblasts, and stable clones expressing the transgene have been isolated and characterised. Transfection was associated with the expression of a major protein band of 180 kDa and a minor related band of 140 kDa. Antibodies reactive exclusively with human NCAM immunoprecipitated both proteins but failed to coprecipitate any other proteins. The ability of transfected NCAM to stimulate neurite outgrowth was determined by culturing rat cerebellar neurons on top of confluent monolayers of parental 3T3 cells or clones of transfected 3T3 cells expressing either NCAM 140 or NCAM 180. The results show that NCAM 180 is less able to act as a substrate for neurite outgrowth than NCAM 140.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/fisiología , ADN Recombinante/genética , Neuritas/fisiología , Empalme del ARN , Animales , Western Blotting , División Celular/fisiología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Citoplasma/fisiología , Exones , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/fisiología , Isomerismo , Ratones , Neuronas/citología , Neuronas/fisiología , Pruebas de Precipitina , Ratas , Factores de Tiempo , TransfecciónRESUMEN
Measurements of prostaglandin E2 (PGE2)-induced adenylyl cyclase activity in membranes isolated from epididymal rat adipocytes revealed inhibition of cAMP production at low concentrations of PGE2 (less than 10 mM) and stimulation at higher concentrations. This biphasic effect of PGE2 was obtained when adenylyl cyclase was stimulated with GTP or NaF. In the presence of forskolin only the inhibitory phase by PGE2 was observed. Sulprostone, a PGE2 analogue, did not affect cAMP synthesis in the presence of either GTP or NaF; however, in the presence of forskolin, it inhibited cAMP production similarly to PGE2. Treatment of the membranes with cholera or pertussis toxin did not alter the biphasic effect of PGE2 on cAMP production. These findings raise the possibility that PGE2 acts through several receptor subtypes which are coupled to GTP binding proteins different from the classical Gi or Gs proteins.