RESUMEN
Eotaxins (CCL11, CCL24, CCL26) originating from airway epithelial cells and leukocytes have been detected in bronchoalveolar lavage of asthmatics. Although the alveolar epithelium is the destination of uncleared allergens and other inflammatory products, scanty information exists on their contribution to the generation and regulation of the eotaxins. We envisioned a state whereby alveolar type II cells, a known source of other inflammatory proteins, could be involved in both the production and regulation of CCL24 and CCL26. Herein, we demonstrated that all three eotaxins are constitutively expressed in A549 cells. IL-4 and IL-13 stimulated a concentration-dependent secretion of CCL24 and CCL26. The cytokines did not act synergistically. Cycloheximide and actinomycin D abrogated IL-4- and IL-13-dependent CCL26 but not CCL24 secretion. Both IL-13 and IL-4 stimulated CCL26 synthesis that was inhibited in a concentration-dependent manner by CCL26 but not CCL24. Only CCL26 reduced expression of CCR3 receptors by 30-40%. On the other hand, anti-CCR3 pretreatment reduced IL-4+IL-13-dependent CCL26 secretion, implying autoregulation. A CCR3-specific antagonist (SB-328437) significantly decreased IL-4-dependent synthesis and release of CCL26. Eosinophils treated with medium from IL-4-stimulated A549 cells preincubated with anti-CCL26 showed a marked decrease of superoxide anion production compared with anti-CCL24 treated. These results suggest that CCL26 is a major eotaxin synthesized and released by alveolar epithelial cells and is involved in autoregulation of CCR3 receptors and other eotaxins. This CCL26-CCR3 ligand-receptor system may be an attractive target for development of therapeutics that limits progress of inflammation in airway disease.
Asunto(s)
Quimiocinas CC/metabolismo , Homeostasis , Neumonía/fisiopatología , Alveolos Pulmonares/fisiopatología , Línea Celular , Quimiocina CCL11 , Quimiocina CCL24 , Quimiocina CCL26 , Quimiocinas CC/biosíntesis , Quimiocinas CC/genética , Eosinófilos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Interleucina-13/farmacología , Interleucina-4/farmacología , Neumonía/metabolismo , Neumonía/patología , Biosíntesis de Proteínas , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Receptores CCR3 , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Transcripción GenéticaRESUMEN
Eosinophilic leukocytes are the cellular hallmark of allergic inflammation. Apart from being potent eosinophils chemoattractants, the eotaxins CCL11, CCL24 and CCL26 are capable of activating eosinophils to generate reactive oxygen species, lipid mediators of inflammation and degranulation of toxic granule proteins. Due to their central role in eosinophil trafficking and activation, understanding the signal transduction mechanism of the eotaxin-induced eosinophil effector functions may provide an innovative therapeutic strategy for eosinophil-associated diseases. Thus, these investigations were conducted to delineate signal transduction mechanisms of CCL11, CCL24 and CCL26-induced eosinophil peroxidase (EPO) degranulation following pretreatment of cells with or without a specific inhibitor of MEK1/MEK2 (U0126), inhibitor of p38 MAP kinase (SB203580) or a specific inhibitor of PI 3-kinase (LY294002). Results have shown that CCR3-mediated eotaxin-induced eosinophilic degranulation was concentration-dependently reduced by specific inhibitors of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. However, the rank order of U0126 with respect to inhibition of chemokine-induced degranulation was CCL11 = CCL24 > CCL26. Potentiation of eotaxin-induced EPO degranulation by IL-5 was also seen. These investigations have not only confirmed the reported co-operativity between IL-5 and the eotaxins but also showed that the eosinophil-degranulating capabilities of the eotaxin CCL11, CCL24 and CCL26 is a consequence of activation of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. Thus, these signaling molecules may provide the biochemical basis for mechanism-based therapy of allergic inflammatory diseases.
Asunto(s)
Degranulación de la Célula/efectos de los fármacos , Factores Quimiotácticos Eosinófilos/farmacología , Inhibidores Enzimáticos/farmacología , Eosinófilos/enzimología , Eosinófilos/fisiología , Butadienos/farmacología , Quimiocina CCL11 , Quimiocina CCL24 , Quimiocina CCL26 , Quimiocinas CC/farmacología , Peroxidasa del Eosinófilo , Eosinófilos/efectos de los fármacos , Células HL-60 , Humanos , Imidazoles/farmacología , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Nitrilos/farmacología , Peroxidasas/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The CNS neurotoxic effects of lead (Pb) are well documented; however, the molecular toxicity targets have not been clearly delineated. Astroglial cells, which are the most abundant cells in the brain and provide critical support to the neurons, are known to accumulate Pb. Although NO generated by inducible NO synthase (iNOS) in glial cells has been associated with many neurotoxic events, it can also serve to protect by modulating blood flow, increase antimicrobial and tumoricidal activities, and promote immune responses following injury or insult. The present investigations were designed to test the hypothesis that Pb exposure may perturb cytokine signal transduction pathways leading to NO production by astroglial cells. Pretreatment with Pb acetate (500 nM-10 microM) attenuated the generation of NO in a concentration-dependent manner up to 90%, and suppressed iNOS protein expression, as well as interfered with the homeostatic functions of calcium in the cytokine-induced NO signal transduction pathway. In addition, pretreatment with staurosporine, a serine-threonine kinase inhibitor, or KT5720, a specific protein kinase A inhibitor (PKA), inhibited cytokine-induced NO production in a concentration-dependent manner with IC(50) values of 26.3 and 346.7 nM, respectively. Therefore, Pb may impede events within the PKA signal transduction pathway; although, based on results from a gel shift assay, Pb does not directly affect PKA enzyme activity. Taken together, these results suggest the possibility that the suppressive effect of Pb acetate on cytokine-induced NO production in glial cells may be implicated in the neurophysiologic changes noted following occupational or environmental exposure to Pb.
Asunto(s)
Carbazoles , Neuroglía/efectos de los fármacos , Óxido Nítrico/biosíntesis , Compuestos Organometálicos/toxicidad , Animales , Citocinas/farmacología , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Inhibidores Enzimáticos/farmacología , Glioma , Indoles/farmacología , Neuroglía/enzimología , Inhibidores de Proteínas Quinasas , Pirroles/farmacología , Ratas , Transducción de Señal , Estaurosporina/farmacología , Células Tumorales CultivadasRESUMEN
Eosinophilic leukocytes have been implicated as primary effector cells in inflammatory and allergic diseases. When activated by cytokines, human eosinophils secrete and produce a variety of proinflammatory or tissue damaging substances. Although well known for their chemoattractant effects, little is known about the precise contribution of the eosinophil-selective chemokines, eotaxin, eotaxin-2, and eotaxin-3 to the effector functions of eosinophils. This forms the central focus of these investigations for which clone 15-HL-60 human eosinophilic cells were used as the in vitro model. Investigation results suggest that all three subtypes of eotaxin directly stimulate eosinophil superoxide anion generation that is inhibited by neutralizing eotaxin antibody or pretreatment of cells with the receptor antibody anti-CCR3. Pretreatment or co-treatment with each of the eotaxins augmented phorbol myristate-induced superoxide generation. Concentration-dependent degranulation of eosinophil peroxidase was noted for all three chemokines, and potentiation of calcium ionophore-induced degranulation was observed with eotaxin pretreatments. Results of interleukin-5 pretreatment studies suggest that the eotaxin chemokines may act cooperatively to enhance effector functions of eosinophils. Collectively, the present studies have advanced knowledge of the eotaxin family of chemokines to include eosinophil priming and modulation of eosinophil activation and secretion effector functions.
Asunto(s)
Quimiocinas CC/farmacología , Gránulos Citoplasmáticos/metabolismo , Eosinófilos/efectos de los fármacos , Receptores de Quimiocina/efectos de los fármacos , Superóxidos/metabolismo , Calcimicina/farmacología , Quimiocina CCL11 , Quimiocina CCL24 , Quimiocina CCL26 , Peroxidasa del Eosinófilo , Eosinófilos/metabolismo , Células HL-60/efectos de los fármacos , Células HL-60/metabolismo , Humanos , Interleucina-5/farmacología , Ionóforos/farmacología , Peroxidasas/metabolismo , Receptores CCR3 , Receptores de Quimiocina/fisiología , Proteínas Recombinantes/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
To a series of 21-desoxy-21-chloro-corticosteroids, a metabolically labile methoxycarbonyl group at C-16 has been incorporated. The approach is to synthesize locally active compounds that are hydrolyzed to inactive and readily excretable acid metabolites upon entry into the systemic circulation. Novel antedrugs were evaluated for anti-inflammatory activity and their adverse effects in an acute and semichronic croton oil-induced ear edema bioassay. Binding affinity to glucocorticoid receptors and induction of L-tyrosine-2-oxoglutarate aminotransferase were studied in hepatoma tissue culture cells. After a single topical application in the croton oil-induced ear edema bioassay, treatment with all the compounds resulted in dose-dependent inhibition of edema. From these dose-response profiles, the following ID(50) values (nmol/ear resulting in a 50% reduction of edema) were calculated: 540, 618, 454, and 346 nmol for prednisolone (P), methyl 21-desoxy-21-chloro-11beta,17alpha-dihydroxy-3,20-dioxo-1, 4-pregnadien-16alpha-carboxylate (PClCM), methyl 21-desoxy-21-chloro-11beta,17alpha-dihydroxy-9alpha-fl uoro-3, 20-dioxo-1,4-pregnadien-16alpha-carboxylate (FPClCM), and methyl 21-desoxy-21-chloro-9alpha-fluoro-11beta-hydroxy-3,20-dioxo- 1, 4-pregnadien-16alpha-carboxylate (FDPClCM), respectively. Results of the 5-day rat croton oil ear edema bioassay indicated that, in contrast with the parent compound P, the novel steroidal antedrugs did not significantly alter body weight gain, thymus weights, or plasma corticosterone levels. The binding affinities for cytosolic hepatoma tissue culture glucocorticoid receptors were 33, 201, 471, 5304, and 3765 nM for P, PClCM, FPClCM, methyl 21-desoxy-21-chloro-11beta-hydroxy-3,20-dioxo-1, 4-pregnadien-16alpha-carboxylate (DPClCM), and FDPClCM, respectively. Collectively, results of these investigations suggest that modifications of P, which included replacement of 21-hydroxyl group with chlorine and addition of 16-methoxycarbonyl group with or without 17-hydroxyl moiety, retained the topical anti-inflammatory activity of the parent compound P without significant adverse systemic effects.
Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/farmacología , Pregnadienos/farmacología , Animales , Antiinflamatorios/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Aceite de Crotón , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Enfermedades del Oído/inducido químicamente , Enfermedades del Oído/tratamiento farmacológico , Edema/inducido químicamente , Edema/tratamiento farmacológico , Masculino , Prednisolona/metabolismo , Prednisolona/farmacología , Pregnadienos/química , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Células Tumorales Cultivadas , Tirosina Transaminasa/efectos de los fármacos , Tirosina Transaminasa/metabolismoRESUMEN
The eosinophilic (EOS) leukocyte has been implicated as a primary effector cell in inflammatory and allergic diseases. Cytokines are among the mediators of inflammatory and allergic diseases which modulate the effector functions of EOS. Certain cytokines, elevated in patients with various allergies, are thought to modulate EOS reactive oxygen species superoxide anion and nitric oxide (NO) responses. Though EOS transcribe and translate mRNA for inducible NO synthase, the effects of cytokines on NO generation remain largely unknown. Thus, we have investigated effects of IL-3, IL-5, GM-CSF, IL-8, RANTES and the proinflammatory cytokines TNF-alpha and IFN-gamma, on superoxide anion and NO generation by clone 15 HL-60 human eosinophilic cells. Cytokine treatments (3 and 18 h) resulted in production of small amounts of superoxide anion which were enhanced by the NO inhibitor L-NAME. In the presence of L-NAME, PMA (1 nM) stimulation significantly increased superoxide anion generation following 3 h treatments with IL-3, TNF-alpha or IFN-gamma. Eighteen hour cytokine treatments with GM-CSF, IL-8, RANTES, IFN-gamma or TNF-alpha primed the cells for enhanced reactive oxygen species following exposure to an EOS stimulant. Inhibition of NO synthesis resulted in increased levels of superoxide anion. Collectively, these results suggest that an environment of proinflammatory cytokines may potentiate the generation of reactive oxygen species by EOS. These results further suggest that at an inflammatory site or during an allergic response, EOS may concomitantly synthesize NO and generate superoxide anion, fractions of which may rapidly react to form the potent oxidant peroxynitrite.
Asunto(s)
Citocinas/farmacología , Inhibidores Enzimáticos/farmacología , Eosinófilos/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Superóxidos/metabolismo , Arginina/farmacología , Células Cultivadas , Sinergismo Farmacológico , Eosinófilos/metabolismo , Humanos , Interferón gamma/farmacología , Óxido Nítrico/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
In a continuing effort to discover potent anti-inflammatory steroids without systemic side effects, diethyl oxalate was condensed with the 17beta-ketol side chain of prednisolone derivatives. Prednisolone gave the most interesting result in forming a novel spiro enone with alpha-hydroxy and beta-carboxylic ester substitutions, and a facile one-pot procedure has been established for the synthesis of this highly functionalized spiro enone structure. The spiro products were tested for their binding potency to the RAW 264.7 macrophage glucocorticoid receptor, for their effect on LPS-induced nitric oxide generation in RAW 264.7 cells, and for their inhibition of rats ear edema induced by croton oil. The new compounds showed weak activities in all of the bioassays. Because the corresponding acid metabolites of two representative spiro enone esters gave no activity in either the binding assay or the nitric oxide generation test, the novel steroids are probably antedrugs. The reduced potency as compared to their parent compounds suggests that the rigid spiro structure is unfavorable to anti-inflammatory activities. Molecular modeling studies confirm that the spiro enones adopt a rigid planar geometry with the ester group in the plane.
Asunto(s)
Prednisolona/análogos & derivados , Compuestos de Espiro/síntesis química , Compuestos de Espiro/uso terapéutico , Animales , Línea Celular , Aceite de Semillas de Algodón/efectos adversos , Oído , Edema/inducido químicamente , Edema/tratamiento farmacológico , Macrófagos/química , Masculino , Ratones , Modelos Moleculares , Estructura Molecular , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/farmacología , Óxido Nítrico Sintasa de Tipo II , Oxalatos/química , Prednisolona/síntesis química , Prednisolona/uso terapéutico , Isoformas de Proteínas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismoRESUMEN
In continuing efforts to synthesize potent, anti-inflammatory steroids devoid of systemic side effects, methyl 9 alpha-fluoro-11 beta,17 alpha,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16 alpha-carboxylate (FP16CM) and its 21-acetate derivative (FP16CMAc) were recently synthesized and screened in animal models of inflammation. The compounds have now been assessed for high-affinity glucocorticoid receptor binding and glucocorticoid-mediated inhibition of nitric oxide (NO) generation in an in vitro RAW 264.7 macrophage cell culture system. Relative potencies for glucocorticoid receptor binding were 1, 1.7, and 2.4 for prednisone (P) (IC50 = 287 nM), FP16CM, and FP16CMAc, respectively. Concomitant relative potencies for inhibition of NO generation by macrophages stimulated with lipopolysaccharide were 1, 0.92 and 1.9 for P (IC50 = 126 nM), FP16CM, and FP16CMAc, respectively. Collectively, results suggest that the novel antedrugs are active anti-inflammatory agents. The 9 alpha-fluoro and 21-acetate substituent may contribute to enhanced topical potency, increased receptor binding affinity and inhibitory effects on NO generation. Inhibition of vasoactive NO may be one anti-inflammatory action of the steroidal antedrugs in vivo. Collectively, results suggest that these agents may be useful for topical application in allergic/inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Macrófagos/efectos de los fármacos , Óxido Nítrico/antagonistas & inhibidores , Receptores de Glucocorticoides/efectos de los fármacos , Animales , Antiinflamatorios/metabolismo , Línea Celular , Macrófagos/metabolismo , Ratones , Unión Proteica , EsteroidesRESUMEN
Mast cells are effectors of inflammatory responses. When triggered by immunological or nonimmunological mechanisms, mast cells release potent biological mediators from preformed stores and synthesize others de novo. In previous investigations from this laboratory, the signal transduction pathways of cloned 10P2 cytokine-independent mast cells were explored. Results suggested that 10P2 cells undergo activation-secretion coupling assessed as release of stored [14C]serotonin (5-HT) when challenged with IgE-specific antigen, influx of extracellular calcium, release of intracellular calcium stores, or by direct activation of protein kinase C isozymes. In the present investigations, cytokine proliferative effects and modulatory roles on release of stored [14C]5-HT have been explored. Following passive sensitization with anti-dinitrophenol (anti-DNP) IgE and challenge with DNP, mast cells released up to 32% of the stored [14C]5-HT. Pretreatment of cells with 10, 30, or 50 ng/ml stem cell factor (SCF) did not alter the response. SCF did not directly induce [14C]5-HT release. Pretreatment with 25 ng/ml interleukin-9 (IL-9) significantly potentiated the IgE-antigen release by 51.1%, 35.7%, or 31.6% when challenged with 3, 10 or 30 ng/ml DNP-HSA. Treatment of cells with 1-100 ng/ml SCF for 72 hr resulted in significantly enhanced proliferation whereas this did not occur when cells were treated with 1-100 ng/ml IL-9. Collectively, these results suggest that SCF alone has a proliferative effect, does not alter the IgE-specific antigen signal transduction pathway, and does not directly stimulate mast cell degranulation. In contrast, IL-9 potentiates the IgE-antigen signal transduction response but exerts no proliferative response. Reports of effects of orally administered cytokines are now beginning to emerge. This raises the possibility that cytokines may be a future therapeutic approach to treatment of allergic and nonallergic inflammatory diseases. The 10P2 cytokine-independent mast cell line may be a valuable adjunct to existing mast cell models as this avenue of drug discovery is explored.
Asunto(s)
Interleucina-9/farmacología , Mastocitos/citología , Mastocitos/metabolismo , Serotonina/metabolismo , Factor de Células Madre/farmacología , Animales , Antígenos/inmunología , Degranulación de la Célula , División Celular , Línea Celular , Dinitrofenoles/inmunología , Haptenos/inmunología , Inmunización Pasiva , Inmunoglobulina E/inmunología , Mastocitos/inmunología , Ratones , Albúmina Sérica/inmunología , Transducción de SeñalRESUMEN
Focused efforts have been made to increase local-to-systemic activity ratios of potent anti-inflammatory steroids for local and/or topical applications. The approach taken in the present investigation is based upon the concept of "antedrug," defined as a locally active compound that exerts its action at the application site but rapidly undergoes a predictable biotransformation to an inactive metabolite that is readily excreted upon entry into the systemic circulation. In continuing efforts to synthesize potent, anti-inflammatory steroids without systemic glucocorticoid activities, 9 alpha-fluoro-methyl 11 beta, 17 alpha, 21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16 alpha-carboxylate (FP16CM) and its 21-acetate derivative (FP16CMAc) have been synthesized and screened. Novel antedrugs were evaluated for antiinflammatory activity in the acute croton oil-induced ear edema bioassay, adverse systemic effects in the 5-day croton oil model, receptor binding, and concomitant L-tyrosine-2-oxoglutarate aminotransferase (EC 2.6.1.5) (TAT) enzyme induction in HTC cells in culture. Following a single topical application in the croton oil-induced ear edema bioassay, treatment with all compounds resulted in dose-dependent inhibition of edema. From these dose-response profiles, the following ID50 values (nmol resulting in a 50% reduction of edema) were calculated: 817, 540, 266, and 67 for hydrocortisone (HC), prednisolone (P), FP16CM, and FP16CMAc, respectively. Calculated relative potencies, setting HC = 1.0, were P, 1.5; FP16CM, 3.1, and FP16CMAc, 12.2. Results of the 5-day rat croton oil ear edema bioassay indicated that, in contrast to the parent compound P, the novel steroidal antedrugs did not significantly alter body weight gain, thymus weights, or plasma corticosterone levels. Relative binding potencies for cytosolic HTC glucocorticoid receptors were 1.0, 20.1, 5.4, and 2.5 for HC, P, FP16CM, and FP16CMAc, respectively. As predicted by the antedrug concept, FP16CM and FP16CMAc were very weak agonists for induction of TAT in HTC cells. Collectively, results of these investigations suggest that modification of P, which included addition of the 9-fluoro and 16-methoxycarbonyl group alone or in conjunction with a 21-acetoxy moiety, increase topical anti-inflammatory activity without significant adverse systemic effects. These new antedrugs may be useful as anti-inflammatory steroids for local applications.
Asunto(s)
Antiinflamatorios/síntesis química , Pregnadienodioles/síntesis química , Pregnadienotrioles/síntesis química , Administración Tópica , Animales , Antiinflamatorios/farmacología , Aceite de Crotón , Relación Dosis-Respuesta a Droga , Edema/tratamiento farmacológico , Inducción Enzimática , Pregnadienodioles/farmacología , Pregnadienotrioles/farmacología , Ratas , Receptores de Glucocorticoides/metabolismo , Células Tumorales Cultivadas , Tirosina Transaminasa/biosíntesis , Tirosina Transaminasa/metabolismoRESUMEN
Specific cellular sites of lead action have not been completely defined. To elucidate the effects of lead exposure on glucocorticoid-mediated signal transduction in hepatic hormonal target tissues, the induction of tyrosine aminotransferase (TAT) specific activity in the H4-IIE-C3 hepatoma cell culture model system was employed. It had been found that lead acetate (3-10 microM) exposure of HTC cells significantly reduced TAT specific activity in a concentration- and time-dependent manner. Two possible molecular targets of the lead-induced effect were investigated: interference with calcium-mediated cellular processes and calcium- and phospholipid-dependent protein kinase C (PKC) activity and isoform-type interactions. Lead acetate treatment (5 microM) reduced TAT specific activity below sodium acetate treated controls by 31%. One-half of the TAT specific activity was recovered by co-treatment with 5 microM lead acetate and 10 mM calcium chloride. As the concentration of lead acetate was increased to 10 microM, interference in calcium-mediated events also increased. Potentiation of glucocorticoid induction by phorbol myristate acetate (PMA) (300 nM) in control cells was 34%, but was abolished by exposure of cells to 10 microM lead acetate (48 h). Treatment with the kinase inhibitor genistein decreased TAT specific activity by 55% and 45% in control and lead acetate exposed cells, respectively. Following treatment with dexamethasone (100 nM), significant increases in both cytosolic and particulate PKC were noted in control cells but not lead acetate exposed cells. Western blot results indicated that lead exposure may increase PKC beta and decrease PKC alpha translocation from cytosolic to particulate fractions, respectively. Taken together, these results suggest that glucocorticoid signal transduction pathways in HTC cells involve calcium-mediated cellular events and PKC isoforms. Exposure of cells to lead results in interference with calcium-mediated events and aberrant modulation of PKC activities. Within hormonal target cells, these may be toxic molecular sites of action of the heavy metal lead and may contribute to the overall toxicity of lead exposure.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Dexametasona/farmacología , Glucocorticoides/farmacología , Plomo/toxicidad , Neoplasias Hepáticas/enzimología , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Western Blotting , Cloruro de Calcio/toxicidad , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genisteína , Isoenzimas/efectos de los fármacos , Isoflavonas/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Compuestos Organometálicos/toxicidad , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Tirosina Transaminasa/efectos de los fármacos , Tirosina Transaminasa/metabolismoRESUMEN
Mast cell activation-secretion by several signal transduction pathways results in the release of proinflammatory mediators including histamine, proteases, arachidonic acid metabolites and multifunctional cytokines. In the present investigations the activation-secretion responses of the cytokine-independent, cloned 10P2 cell line have been explored. [14C]Serotonin (5-HT) preloaded cells were stimulated with antigen, with and without IL-4, ionophore A23187, thapsigargin or phorbol myristate acetate (PMA). Following passive sensitization with anti-dinitrophenol (anti-DNP) IgE, mast cells released up to 31% of incorporated [14C]5-HT when stimulated with specific antigen (DNP-human serum albumin). This response was potentiated by pretreatment with IL-4. Significant degranulation (50%) was noted following treatment with calcium ionophore A23187, thapsigargin and ionophore A23187/PMA. Collectively, these results suggest that 10P2 cells undergo activation-secretion responses, assessed as degranulation of preloaded [14C]5-HT when challenged with IgE antigen, by influx of extracellular calcium or release of intracellular calcium stores, or by direct activation of protein kinase isozymes. As a growth factor-independent cell line, 10P2 cells may be a valuable adjunct to existing mast cell model systems currently used for pharmacologic investigations.
Asunto(s)
Antígenos/toxicidad , Calcimicina/toxicidad , Carcinógenos/toxicidad , Interleucina-4/toxicidad , Ionóforos/toxicidad , Mastocitos/efectos de los fármacos , Acetato de Tetradecanoilforbol/toxicidad , Tapsigargina/toxicidad , 2,4-Dinitrofenol/metabolismo , Análisis de Varianza , Animales , Línea Celular , Inmunización Pasiva , Inmunoglobulina E/inmunología , Mastocitos/citología , Mastocitos/metabolismo , Ratones , Serotonina/metabolismo , Serotonina/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Lead exerts significant toxic effects on the nervous system, the hematopoietic system and the kidney. Specific cellular sites of action of this environmental pollutant have not been elucidated in the central nervous system. The present investigations were conducted to test the hypothesis that lead exposure perturbs glucocorticoid-mediated events in central nervous system hormonal target tissues. Utilizing the C6 glioma cell culture model in these studies, glucocorticoid receptor binding to its cytosolic receptor was investigated. Receptor binding studies yielded a Kd= 10.5 +/- 0.5 nM and a Bmax = 486 +/- 27 fmol/mg protein in untreated cells versus a Kd = 23.1 +/- 2.6 nM and Bmax = 472 +/- 35 fmol/mg protein in cells exposed to 10 microM lead acetate for 24 h. Presence of lead in these glial cells may decrease affinity of the glucocorticoid for its receptor without affecting receptor number. Treatment with 10 microM lead acetate for 48 h, resulted in a significant reduction in glucocorticoid-regulated glycerol phosphate dehydrogenase (GPDH) specific activity. These effects were not due to cell cytotoxicity assessed as cell number growth curves, [3H]thymidine incorporation or trypan blue exclusion. In protein kinase C (PKC) activity assays, treatment of cells with sodium or lead acetate and dexamethasone indicated that both lead and dexamethasone affect the distribution of PKC. In lead-treated cells cytosolic PKC activity was reduced 48% when compared to sodium acetate treated controls. Taken together, these results suggest that acute exposure of C6 cells to lead may inhibit processes involved in glucocorticoid-mediated signal transduction events within central nervous system hormonal target cells. Lead may perturb initial glucocorticoid binding events possibly by affecting PKC-mediated phosphorylations in the glucocorticoid signal transduction system.
Asunto(s)
Glioma/ultraestructura , Compuestos Organometálicos/toxicidad , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Animales , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dexametasona/metabolismo , Activación Enzimática , Inducción Enzimática/efectos de los fármacos , Glioma/enzimología , Glioma/metabolismo , Glicerolfosfato Deshidrogenasa/biosíntesis , Glicerolfosfato Deshidrogenasa/metabolismo , Cinética , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/metabolismo , Ratas , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Specific cellular sites of action of the environmental pollutant, lead, have not been completely defined. The present investigations were conducted to test the hypothesis that lead exposure perturbs glucocorticoid-mediated effects in hormonal target tissues. The cell culture model chosen for these investigations was the effects of lead on glucocorticoid-regulated tyrosine aminotransferase (TAT) specific activity in the H4-II-C3 hepatoma cells. Cells were treated with 300 nM-10 microM lead acetate for 24 or 48 h in absence or presence of the inducing agent, dexamethasone. Lead dose-dependently inhibited TAT specific activity up to 52% and 61% following 24 and 48 h lead treatments, respectively. These treatment times and concentrations of lead acetate did not significantly alter total cell numbers, [3H]thymidine incorporation or trypan blue exclusion. Glucocorticoid receptor-binding studies yielded a Kd = 8.3 nM and a Bmax = 290 fmol/mg protein in untreated cells versus a Kd = 9.2 nM and Bmax = 262 fmol/mg protein in cells exposed to 10 microM lead acetate for 48 h. Treatment with lead did not significantly perturb uptake of the inducing glucocorticoids or initial cytosolic receptor-binding events. To sustain induced levels of TAT, glucocorticoid must be continuously present. Following steroid withdrawal, enzyme de-induction was significantly altered in lead-treated cells. At 6 h following dexamethasone withdrawal, TAT levels had decreased to 51% of maximum in sodium acetate-treated cells. This was significantly reduced to 33% of maximum in lead acetate-treated cells. Lead treatment of HTC cells was also shown to ameliorate PMA amplification of dexamethasone-induced TAT activity. Taken together, these results suggest that acute exposure of cells to lead may inhibit processes involved in glucocorticoid-mediated enzyme induction within the hormonal target cell. Results suggest that lead may be acting to increase the turnover of TAT by actions at the transcription, translation and/or posttranslational level. Lead may also be affecting PKC-mediated phosphorylations in the glucocorticoid-TAT signal transduction system.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Dexametasona/farmacología , Plomo/toxicidad , Neoplasias Hepáticas/enzimología , Compuestos Organometálicos/toxicidad , Tirosina Transaminasa/metabolismo , Animales , Carcinoma Hepatocelular/patología , División Celular/efectos de los fármacos , Dexametasona/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Contaminantes Ambientales/toxicidad , Inducción Enzimática/efectos de los fármacos , Neoplasias Hepáticas/patología , Fosforilación , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Proteína Quinasa C/metabolismo , Receptores de Glucocorticoides/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Timidina/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Tritio/metabolismo , Azul de Tripano/metabolismo , Células Tumorales Cultivadas , Tirosina Transaminasa/genéticaRESUMEN
Release of inflammatory mediators from mast cells following immunoglobulin bridging by specific allergens triggers episodes of asthma and bronchial hyperreactivity. Recent evidence has shown that neuropeptides, such as substance P (SP), may modulate the pulmonary inflammatory response in these airway diseases. This suggests that SP may affect secretory events of mast cells. To investigate these effects, resident peritoneal mast cells were collected from Sprague-Dawley male rats and stimulated with Con A (utilizes surface-bound immunoglobulin), compound 48/80 (acts in a peptide-like manner) and SP. Secretion was assessed as the release of preloaded [14C]serotonin. All secretagogues induced dose-dependent release. Pharmacologic modulation of release was then studied with two drugs employed for treatment of airway disease, hydrocortisone, a classical anti-inflammatory steroid, and disodium cromoglycate (DSCG). Following pretreatment with 5 mumol/l hydrocortisone, serotonin release induced by Con A was inhibited by 59%. No inhibition was noted with compound 48/80 or SP release. Similarly, following DSCG (300 mumol/l) pretreatment, 40% inhibition of release was noted with Con A, but no inhibition occurred following compound 48/80- or SP-stimulated release. Collectively, these results suggest that mast cells possess multiple activation-secretion coupling pathways which respond differently to clinically used pharmacologic agents. Diseases involving SP modulation of mast cell mediator release may not be successfully treated with anti-inflammatory steroids or DSCG.
Asunto(s)
Cromolin Sódico/farmacología , Hidrocortisona/farmacología , Mastocitos/inmunología , Serotonina/metabolismo , Alcaloides/farmacología , Análisis de Varianza , Animales , Concanavalina A/inmunología , Técnicas In Vitro , Masculino , Mastocitos/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Estaurosporina , Sustancia P/inmunología , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Novel steroidal antiinflammatory antedrugs, 11 beta,20-dihydroxy-3,20-dioxo-3'-(ethoxycarbonyl)-isoxazolino[16,17 - d]pregna-1,4-diene (2a) and 9-fluoro-11 beta,20-dihydroxy-3,20-dioxo-3'-ethoxy- carbonylisoxazolino[16,17-d]pregna-1,4-diene (2b) were prepared in 97% yield via 1,3-dipolar cycloaddition of carbethoxyformonitrile (CEFNO) to 11 beta,21-dihydroxy-3,20-dioxopregna-1,4,-16-triene (1a) and 11 beta,21-dihydroxy-3,20-dioxo-9-fluoropregna-1,4,16-triene (1b), respectively, which were prepared via five steps from prednisolone and 9-fluoroprednisolone, respectively. The treatment of steroids 2a and 2b with acetic anhydride in pyridine led to the corresponding 21-acetates 3a and 3b, respectively, in 95% yield. Dose-response profiles of the croton oil-induced ear edema bioassay in rats were used to calculate the following ID50 values (nmol/ear resulting in a 50% reduction of edema): prednisolone (P), 540 nmol; 2b, 135 nmol; and 3b, 101 nmol. Inhibition of edema did not exceed 50% following application of either 2a or 3a. Relative potency calculations indicated that 2b was 4-fold and 3b 5.3-fold more potent than the parent compound P when applied topically. No significant adverse systemic effects were seen following treatments with 3b. These results suggest that C-9-fluorination, side-chain hydroxy group esterification, and [16 alpha,17 alpha-d]-3'-carbethoxyisoxazoline additions to the conventional steroid P improve topical antiinflammatory activity without concomitant increases in adverse systemic activity.
Asunto(s)
Antiinflamatorios/síntesis química , Antiinflamatorios/farmacología , Isoxazoles/síntesis química , Isoxazoles/farmacología , Animales , Aceite de Crotón , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Oído Externo/efectos de los fármacos , Edema/inducido químicamente , Edema/tratamiento farmacológico , Masculino , Ratas , Ratas Sprague-Dawley , EsteroidesRESUMEN
Systemic side effects of antiinflammatory steroids may be minimized by incorporation of a metabolically labile group which is metabolized to make the steroid inactive upon entry into the systemic circulation (antedrug concept). In continuing efforts to minimize systemic adverse effects of potent antiinflammatory steroids, we have recently synthesized methyl 11 beta, 17 alpha, 21-trihydroxy-3,20-dioxopregna-1,4-diene-6-carboxylate (P6CM), its 21-acetoxys (P6CMa, P6CMb) and 17,21-acetonide (P6CMacet) derivatives. Structure-activity relationships have now been assessed and compared with prednisolone (P) for glucocorticoid receptor affinity (P IC50 = 28 nM), gluconeogenic activity as induction of tyrosine aminotransferase (EC50 = 4.4 nM) in H4-II-C3 HTC cells and antiproliferative effects (P = 48% inhibition of [3H]thymidine incorporation at 1 microM). Relative potencies for receptor binding (P = 1) were 0.12, 0.03, 0.004, and 0.0008 for P6CM, P6CMa, P6CMb, and P6CMacet, respectively, and enzyme induction relative potencies were 0.13, 0.05, 0.01, and 0.008, respectively. Antiproliferative effects of all derivatives were also less than that of P. These decreases suggest that addition of the 6-carboxymethyl group to prednisolone results in the general reduction of glucocorticoid activities. Taken together with previously reported results demonstrating retention of topical antiinflammatory activity of these novel steroids, P6CM and its derivatives may represent new locally active antiinflammatory steroids with reduced propensity to cause gluconeogenic and antiproliferative adverse effects.
Asunto(s)
Antiinflamatorios , División Celular/efectos de los fármacos , Prednisolona/análogos & derivados , Receptores de Glucocorticoides/metabolismo , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Unión Competitiva , ADN/biosíntesis , Dexametasona/metabolismo , Inducción Enzimática/efectos de los fármacos , Ratones , Prednisolona/química , Prednisolona/metabolismo , Prednisolona/farmacología , Linfocitos T/citología , Linfocitos T/metabolismo , Tritio , Células Tumorales Cultivadas , Tirosina Transaminasa/biosíntesisRESUMEN
In an effort to test the hypothesis that 9 alpha-fluorination of a steroidal antedrug would enhance receptor binding affinity and local antiinflammatory activity, without concomitantly increasing adverse systemic effects, a fluorinated analog, 10, of methyl 11 beta, 21-dihydroxy- 3,20-dioxo-1,4-pregnadiene-16 alpha-carboxylate (DP16CM, 1) was synthesized and evaluated. In the acute rat croton oil-induced ear edema bioassay, 10 was found to be twice as potent as 1. This increase in topical potency was consistent with enhanced binding affinity of 10, relative to 1. The IC50 values for displacement of [3H]dexamethasone from glucocorticoid receptors of rat hepatoma tissue culture cells were 0.16, 1.2, and 0.03 microM for 10, 1, and prednisolone, respectively. Following multiple topical ID50 applications of predniosolone, 1, and its new fluorinated analog, 10, in the rat subacute croton oil-induced ear edema bioassay, only prednisolone exhibited significant untoward effects, such as reduction in relative thymus and adrenal weights, plasma corticosterone levels, and normal body weight gain. Thus, while fluroination of 1 enhanced its topical potency, there was not a concomitant increase in untoward systemic effects. This lack of adverse systemic effects is ostensibly due to the presence of the metabolically labile 16-carboxylate ester moiety.
Asunto(s)
Antiinflamatorios/síntesis química , Fluprednisolona/análogos & derivados , Administración Tópica , Animales , Antiinflamatorios/efectos adversos , Antiinflamatorios/farmacocinética , Antiinflamatorios/farmacología , Unión Competitiva/efectos de los fármacos , Fenómenos Químicos , Química Física , Aceite de Crotón , Dexametasona/farmacocinética , Oído Externo/patología , Edema/inducido químicamente , Edema/prevención & control , Fluprednisolona/efectos adversos , Fluprednisolona/síntesis química , Fluprednisolona/farmacología , Neoplasias Hepáticas Experimentales/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Tirosina Transaminasa/metabolismoRESUMEN
The synthesis and pharmacological evaluation of 6-(methoxycarbonyl)prednisolone (11) (a 3:1 mixture of 6 alpha-isomer 11a and 6 beta-isomer 11b), its 21-ol acetates 13a (6 alpha-isomer) and 13b (6 beta-isomer), and 17,21-diol acetonide 14 (a 6:1 mixture of 6 alpha-isomer 14a and 6 beta-isomer 14b) as local antiinflammatory steroidal antedrugs are described. The lead compound 11 was prepared via 12 steps from hydrocortisone (1). In the croton oil-induced ear edema assay, the topical antiinflammatory activity of 13a was higher than that of its epimer 13b. Except for 13a, the compounds (11, 13b, and 14) showed less activity than prednisolone. The systemic activities were assessed after 5 days of consecutive administration of these compounds at equiactive doses. Neither 11 nor 14 depressed plasma corticosteroid levels or significantly altered adrenal weights. Thymic involution was absent for 14, 15% for 11, and 47% for prednisolone at the equiactive doses. Both 13a and 13b showed significant reduction of adverse systemic effects assessed as the increase of body weight and the decreases of adrenal and thymus weights. The putative metabolite, carboxylic acid 12, showed 26 times less topical antiinflammatory activity than prednisolone. These results suggest that introduction of a labile methoxycarbonyl group at the C-6 position of prednisolone results in retention of antiinflammatory activity while reducing systemic effects noted following topical application of the parent compound prednisolone.
Asunto(s)
Antiinflamatorios/síntesis química , Prednisolona/análogos & derivados , Profármacos/síntesis química , Administración Tópica , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacología , Corticosterona/sangre , Aceite de Crotón , Relación Dosis-Respuesta a Droga , Oído Externo/patología , Edema/inducido químicamente , Edema/prevención & control , Masculino , Tamaño de los Órganos/efectos de los fármacos , Prednisolona/administración & dosificación , Prednisolona/síntesis química , Prednisolona/farmacología , Profármacos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/efectos de los fármacos , Aumento de Peso/efectos de los fármacosRESUMEN
The steroidal 20-carboxamides [(20R)- and (20S)-21-(N-substituted amino)-11 beta,17,20-trihydroxy-3,21-dioxo-1,4-pregnadiene] recently have been shown to possess anti-inflammatory activity in animal models of inflammation. These N-substituted methyl, ethyl, n-propyl, and benzyl derivatives also exhibited suppressive effects on plasma corticosterone and thymus function. Generally, the (20R)-hydroxy-20-carboxamides were more potent than the corresponding (20S)-epimers. In continuing investigations on the glucocorticoid effects of these compounds, we have studied their ability to induce tyrosine aminotransferase (TAT), inhibit uptake of [3H]thymidine into DNA, and complete with [3H] dexamethasone for binding to the hepatoma tissue culture glucocorticoid receptor. Results indicated that the N-substituted methyl, ethyl, and n-propyl derivatives were full glucocorticoid agonists in the three measurements. Receptor binding affinities of the N-substituted carboxamides correlated well with their ability to induce TAT activity and to inhibit thymocyte proliferation. Structure-activity relationships indicated that the larger the N-substituent, the weaker the agonist activity in this system, and 20R isomers exhibited higher glucocorticoid agonist activity than the corresponding 20S isomers. This investigation is part of our effort to elucidate structure-activity relationships of steroidal carboxamides synthesized on the basis of the antedrug concept.