RESUMO
Lysophosphatidic acid (LPA) is a phospholipid mediator that plays multiple cellular functions by acting through G protein-coupled LPA receptors. LPAs are known to be key mediators in inflammation, and several lines of evidence suggest a role for LPAs in inflammatory periodontal diseases. A simple and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify LPA species (LPA 18:0, LPA 16:0, LPA 18:1 and LPA 20:4) in human saliva and gingival crevicular fluid (GCF). LPA 17:0 was used as an internal standard and the LPA species were extracted from saliva by liquid-liquid extraction using butanol. Chromatography was performed using a Macherey-Nagel NUCLEODUR® C8 Gravity Column (125 mm × 2.0 mm ID) with a mixture of methanol/water: 75/25 (v/v) containing 0.5% formic acid and 5 mM ammonium formate (mobile phase A) and methanol/water: 99/0.5 (v/v) containing 0.5% formic acid and 5mM ammonium formate (mobile phase B) at a flow rate of 0.5 mL/min. LPAs were detected by a linear ion trap-triple quadrupole mass spectrometer with a total run time of 8.5 min. The limit of quantification (LOQ) in saliva was 1 ng/mL for all LPA species and the method was validated over the range of 1-200 ng/mL. The method was validated in GCF over the ranges of 10-500 ng/mL for LPA 18:0 and LPA 16:0, and 5-500 ng/mL for LPA 18:1 and LPA 20:4. This sensitive LC-MS/MS assay was successfully applied to obtain quantitative data of individual LPA levels from control subjects and patients with various periodontal diseases. All four LPA species were consistently elevated in samples obtained from periodontal diseases, which supports a role of LPAs in the pathogenesis of periodontal diseases.
Assuntos
Cromatografia Líquida , Líquido do Sulco Gengival/química , Lisofosfolipídeos/análise , Periodontite/metabolismo , Saliva/química , Espectrometria de Massas em Tandem , Adulto , Idoso , Biomarcadores/análise , Calibragem , Estudos de Casos e Controles , Cromatografia Líquida/normas , Humanos , Pessoa de Meia-Idade , Nebraska , Periodontite/diagnóstico , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Espectrometria de Massas em Tandem/normasRESUMO
Activation of bovine chromaffin cell neuropeptide Y (NPY) receptors coupled to Gi (Y1) results in the enhancement of ATP-stimulated inositol phosphate formation. NPY alone does not alter inositol phosphate (InsP) formation in these cells, suggesting that some form of receptor cross talk is involved in this process. In some cell types, serial stimulation of Gi-linked and Gs- or Gq-linked receptors results in an increase in intracellular messenger production (cyclic AMP or InsP), a process referred to as heterologous sensitization. NPY preincubation with bovine chromaffin cells followed by the addition of ATP results in a dose-dependent increase in ATP-stimulated InsP formation (EC50 = 2.0 x 10-8 M), which is maximal within 1 min. InsP formation resulting from NPY preincubation persists for more than an hour after NPY removal, declining with time in a linear fashion. [Leu31Pro34]NPY and NPY are equally effective at producing sensitization, whereas NPY13-36 is ineffective, suggesting that NPY acts through the Y1 receptor. Confirmation of the receptor subtype identity was made by including the Y1-selective antagonist HU-404 during the preincubation, which prevented the sensitizing effect of NPY. NPY sensitization was blocked by pertussis toxin pretreatment, demonstrating Gi/Go involvement. ATP-stimulated InsP formation, with and without NPY preincubation, was sensitive to the phospholipase C inhibitor, U73122 [1-(6-([17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino)hexyl)-1H-pyrrole-2,5-dione]. In conclusion, short-term exposure of bovine chromaffin cells to NPY results in a long-lasting increase in the subsequent stimulation of InsP formation by ATP.
Assuntos
Trifosfato de Adenosina/farmacologia , Células Cromafins/efeitos dos fármacos , Fosfatos de Inositol/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Análise de Variância , Animais , Bovinos , Células Cultivadas , Células Cromafins/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neuropeptídeo Y/farmacologiaRESUMO
Simultaneous treatment of human airway smooth muscle (HASM) cells with lysophosphatidic acid (LPA) and epidermal growth factor (EGF) leads to strikingly synergistic stimulation of mitogenesis. The purpose of this study was to explore potential sites for signal integration mediating synergism, focusing on extracellular signal-regulated kinase (ERK) and transcription factors involved in proliferation and inflammation as likely candidates. Activation of ERK was analysed by immunoblotting. Transcription factor activation was assessed using HASM cells transduced with luciferase reporter gene constructs. LPA and EGF both activated ERK but had no synergistic effect when combined. LPA and EGF both activated activator protein (AP)-1, cyclic adenosine monophosphate response element-binding protein, nuclear factor of activated T-cells and the serum response element; however, only AP-1 activation exhibited synergism. Activation of the inhibitory guanine nucleotide-binding protein and of ERK signalling pathways were required for most transcription factor responses to LPA. In contrast, nuclear factor (NF)-kappaB was activated by LPA but not EGF and NF-kappaB activation was completely blocked only when Rho was inhibited. Rapid activation of Rho was observed in response to LPA but not to EGF. Importantly, inhibition of Rho selectively blocked synergism in both AP-1 activation and mitogenesis. In summary, extracellular signal-regulated kinase activation is required for many transcription factor responses to lysophosphatidic acid and epidermal growth factor, however it is not synergistic. Activation of activator protein-1 is synergistic, and Rho activation by lysophosphatidic acid is required for synergism in both activator protein-1 activation and mitogenesis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Mitógenos/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Fase Aguda/metabolismo , Toxinas Botulínicas/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Humanos , Lisofosfolipídeos/farmacologia , Miócitos de Músculo Liso/patologia , Traqueia , Fator de Transcrição AP-1/metabolismoRESUMO
Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10(-9) and 10(-8) M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+ mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.
Assuntos
Bradicinina/farmacologia , Colágeno/efeitos dos fármacos , Colágeno/fisiologia , Fibroblastos/fisiologia , Animais , Bradicinina/antagonistas & inibidores , Células Cultivadas , Géis , Humanos , Concentração Osmolar , Inibidores de Proteases/farmacologia , Ratos , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
Lysophosphatidic acid (LPA) is a phospholipid growth mediator found in serum at 2-20 microM. In many cell types, including human airway smooth muscle (HASM) cells, LPA-induced proliferation occurs at 10-100 microM LPA. At these concentrations LPA forms Ca2+ precipitates. The potential involvement of Ca2+ and Ca2+ LPA precipitates in LPA-induced HASM cell mitogenesis was investigated. In the absence of extracellular Ca2+, 10 and 30 microM LPA stimulated HASM cell mitogenesis. However, with 100 microM LPA in the absence of extracellular Ca2+, HASM cells exhibited a profound shape change and loss of viability, determined to be apoptosis by both DNA staining and assessment of cytosolic nucleosomal reactivity. A bioassay based on the adenosine 3':5'-cyclic monophosphate response of C62B rat glioma cells was used to measure the bioactivity of LPA solutions prepared in Ca2+ free and Ca2+ containing medium. After 24 h, a 100 microM LPA solution in Ca2+ free medium contained markedly greater bioactivity than a 100 microM LPA solution made in Ca2+ containing medium. In summary, formation of Ca2+ LPA precipitates decreases the amount of biologically active LPA in solution, and high concentrations of bioactive LPA achieved in Ca2+ free but not in Ca2+ containing medium induce apoptosis of HASM cells.
Assuntos
Lisofosfolipídeos/farmacologia , Mitógenos/farmacologia , Músculo Liso/efeitos dos fármacos , Apoptose , Cálcio/análise , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Meios de Cultura/química , Humanos , Traqueia/efeitos dos fármacosRESUMO
Previous studies showed that human airway smooth muscle (HASM) cells treated with lysophosphatidic acid (LPA), a pertussis toxin (PTX)-sensitive G protein-coupled (GPC) mitogen, simultaneously with epidermal growth factor (EGF), a receptor tyrosine kinase (RTK) mitogen, exhibit markedly synergistic stimulation of mitogenesis. We now show that the RTK mitogens basic fibroblast growth factor, insulin-like growth factor-1, insulin, platelet-derived growth factor-AA, and platelet-derived growth factor-BB, as well as transforming growth factor-beta, all induced synergistic stimulation of mitogenesis in the presence of LPA. The PTX-sensitive GPC mitogens carbachol and endothelin-1 and the PTX-insensitive GPC mitogens sphingosine-1-phosphate and thrombin exhibited synergistic stimulation together with EGF. Several RTK-RTK growth factor pairs and GPC-GPC mitogen pairs were also synergistic. HASM cells showed synergistic responses to serum plus EGF but not to serum plus LPA. Testing various other cell types showed that synergism between LPA and EGF occurred in other smooth muscle cells because both vascular smooth muscle cells and mesangial cells exhibited synergism. Additionally, human fetal lung fibroblasts also showed striking synergism. These results indicate that HASM cells can respond synergistically to a wide variety of mitogen combinations and that this synergism is a feature shared with other contractile cell types.
Assuntos
Substâncias de Crescimento/fisiologia , Pulmão/citologia , Músculo Liso/citologia , Traqueia/citologia , Divisão Celular , Células Cultivadas , DNA/biossíntese , Sinergismo Farmacológico , Fator de Crescimento Epidérmico/farmacologia , Fibroblastos/citologia , Proteínas de Ligação ao GTP/metabolismo , Mesângio Glomerular/citologia , Substâncias de Crescimento/farmacologia , Humanos , Lisofosfolipídeos/farmacologia , Mitógenos/farmacologia , Músculo Liso Vascular/citologia , Receptores Proteína Tirosina Quinases/farmacologia , Fase S , Pele/citologiaRESUMO
The roles of the carboxyl-terminal tail of the alpha(1B)-adrenergic receptor in its expression, function, and regulation were investigated by site-directed mutagenesis. The receptor construct truncated after residue 363 seemed not to be properly expressed. In contrast, the receptor truncated after residue 366 and all of the longer receptor constructs were properly expressed and exhibited agonist and antagonist binding and activation of phosphoinositide hydrolysis similar to the wild-type receptor. Agonist-induced sequestration of receptors within the plasma membrane, endocytosis into intracellular vesicles, and eventual down-regulation were all absent in the receptor truncated after residue 366. A series of sequential truncations and a deletion mutation identified a critical role for residues 403 to 425, which include the previously identified sites for G protein-coupled receptor kinase phosphorylation, in agonist-induced internalization of the receptor. Similar studies identified a critical role for residues 367 to 380 in agonist-induced down-regulation. Individual point mutations converting either cysteine 367 or serine 369 to alanine selectively eliminated down-regulation, thus identifying two specific amino acid residues required for down-regulation. Importantly, several of the mutated receptors that failed to show rapid agonist-induced internalization nonetheless exhibited normal agonist-induced down-regulation. In addition to identifying specific regions and individual residues of the alpha(1B)-adrenergic receptor involved in internalization and down-regulation, these studies provide mutated receptors that internalize but do not down-regulate, that down-regulate without internalization, and that are defective in both internalization and down-regulation, all of which should be useful tools for further studies of the specific cellular compartments and molecular mechanisms involved in receptor internalization and down-regulation.
Assuntos
Receptores Adrenérgicos alfa 1/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Cricetinae , Regulação para Baixo , Deleção de Genes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Receptores Adrenérgicos alfa 1/química , Receptores Adrenérgicos alfa 1/genéticaRESUMO
The effects of the simple phospholipid mediator lysophosphatidic acid (LPA) on the contractile responsiveness of isolated tracheal rings from rabbits and cats were assessed. In both species, LPA increased the contractile response to the muscarinic agonist methacholine, but LPA did not induce contraction on its own. Conversely, LPA decreased the relaxation response to the beta-adrenergic-agonist isoproterenol in both species. Concentrations of LPA as low as 10(-8) M were effective, and the effects of LPA were rapidly reversed on washing. Phosphatidic acid was much less effective, requiring higher concentrations and producing only a minimal effect. Contractions induced by serotonin and by substance P also were enhanced by LPA, but KCl-induced contractions were unaffected. LPA inhibited the isoproterenol-induced relaxation of KCl-precontracted rings, similar to its effects on methacholine-precontracted rings, and relaxation induced by the direct adenylyl cyclase activator forskolin was inhibited in a manner similar to that induced by isoproterenol. Epithelium removal did not alter the contraction-enhancing effect of LPA. The ability of LPA to both enhance contraction and inhibit relaxation of airway smooth muscle suggests that LPA could contribute to airway hypercontractility in asthma, airway inflammation, or other types of lung injury.
Assuntos
Lisofosfolipídeos/farmacologia , Músculo Liso/efeitos dos fármacos , Agonistas Adrenérgicos beta/farmacologia , Animais , Gatos , Relação Dose-Resposta a Droga , Endotélio/fisiologia , Técnicas In Vitro , Contração Isométrica/efeitos dos fármacos , Isoproterenol/antagonistas & inibidores , Isoproterenol/farmacologia , Cloreto de Metacolina/farmacologia , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Parassimpatomiméticos/farmacologia , Cloreto de Potássio/farmacologia , CoelhosRESUMO
An NP(X)nY motif is highly conserved among G protein-coupled receptors and is similar to an NPXY motif involved in receptor-mediated endocytosis for several non-G protein-coupled receptors. We investigated the role of this motif in alpha1B-adrenergic receptor function and regulation. Y348A alpha1B-adrenergic receptors in which this sequence was mutated from NPIIY to NPIIA were prepared by site-directed mutagenesis and transfected into Chinese hamster ovary cells. Binding of the antagonist prazosin to Y348A receptors was similar to that of wild-type receptors, but affinity of the Y348A receptors for the agonist epinephrine was increased by approximately 10-fold. Despite this increase in agonist binding affinity, the Y348A mutation completely uncoupled the receptors from stimulation of phosphoinositide hydrolysis and mobilization of intracellular Ca2+. Exposure of cells expressing Y348A receptors to the agonist epinephrine resulted in receptor "sequestration," defined as a loss of cell surface receptors accessible to radioligand in binding assays with intact cells on ice, similar to that for the wild-type receptor. In contrast, Y348A receptors did not undergo "endocytosis" into the light vesicle fraction in sucrose density gradient centrifugation assays, as did the wild-type receptor. These results (i) indicate an important role for Tyr348 in coupling the alpha1B-adrenergic receptor to G protein and subsequent effector activation, (ii) provide further evidence that alpha1B-adrenergic receptor internalization can be separated into a sequestration step and an endocytosis step, (iii) indicate that effector activation and second messenger formation are not required for the sequestration of these receptors but may be involved in endocytosis, and (iv) provide a useful new tool for further investigation of the nature of the subcellular compartments and the molecular modifications involved in the multiple steps involved in internalization of G protein-coupled receptors.
Assuntos
Receptores Adrenérgicos alfa 1/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Compartimento Celular , Temperatura Baixa , Cricetinae , Regulação para Baixo , Endocitose , Fosfatidilinositóis/metabolismo , Mutação Puntual , Ensaio Radioligante , Receptores Adrenérgicos alfa 1/genética , Proteínas Recombinantes , Transdução de Sinais , Relação Estrutura-Atividade , Frações Subcelulares/química , Transfecção , Tirosina/químicaRESUMO
Enhanced proliferation of airway smooth muscle is thought to contribute to the pathogenesis of asthma and other obstructive airway diseases. Lysophosphatidic acid (LPA) is a simple bioactive lipid mediator that stimulates mitogenesis in fibroblasts and some other cell types. The effects of LPA on mitogenesis of cultured human airway smooth muscle cells were determined by measuring [3H]thymidine incorporation into cellular DNA. LPA induced a concentration-dependent stimulation of [3H]thymidine incorporation of a similar magnitude to that induced by serum, with the effects of 50 microM LPA being similar to those of 5% serum. Stimulation by LPA and by serum was almost completely eliminated in cells exposed to pertussis toxin, indicating involvement of a pertussis toxin-sensitive G protein in mitogenic signaling by these agents. Epidermal growth factor (EGF) induced stimulation of a similar magnitude as that with LPA, but the stimulation by EGF was insensitive to pertussis toxin. LPA and EGF, when added together, exhibited a markedly synergistic stimulation of [3H]thymidine incorporation that was typically 10-fold greater than the stimulation with either agent alone. LPA and EGF also stimulated mitogenesis assessed by cell growth, and again LPA and EGF together exhibited synergism. These results suggest the possibility that stimulation of airway smooth muscle cell proliferation by LPA, either alone or by enhancing effects of other growth factors, could play a role in normal airway remodeling or in the pathological proliferation of smooth muscle in various airway diseases.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Lisofosfolipídeos/farmacologia , Músculo Liso/citologia , Traqueia/citologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , DNA/biossíntese , Sinergismo Farmacológico , Humanos , Cinética , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Toxina Pertussis , Timidina/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The agonist-induced up-regulation of alpha1B-adrenoceptors in clone H99 of transfected Chinese hamster ovary cells that we reported previously (Zhu et al., 1996) was further investigated. Studies with a larger number of clones revealed that the up-regulation observed in H99 cells is atypical and that most other clones exhibit down-regulation under the same conditions. The role of protein kinases in the up-regulation of alpha1B-adrenoceptors in clone H99 was further investigated. Surprisingly, the protein kinase inhibitor staurosporine induced a similar up-regulation. Neither the selective protein kinase C inhibitor GF109203X nor the activator phorbol 12-myristate, 13-acetate altered receptor expression. The tyrosine kinase inhibitors genistein and its weaker analog daidzein did not induce up-regulation but blocked the up-regulation induced by epinephrine and by staurosporine. Up-regulation was blocked by the protein synthesis inhibitor cycloheximide. These studies suggest multiple mechanisms by which different protein kinases can modulate the expression of transfected alpha1B-adrenoceptors.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Proteínas Quinases , Receptores Adrenérgicos alfa 1/biossíntese , Animais , Células CHO , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Epinefrina/farmacologia , Genisteína/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Chinese hamster ovary (CHO) cells were stably transfected to express the hamster alpha 1B-adrenoeceptor, and the function and agonist-induced regulation of the binding properties of these receptors were characterized. The cells expressed approximately 230,000 receptors per cell, with a KD for [3H]prazosin of 140 pM. In assays of competition by epinephrine for [3H]prazosin binding to receptors on intact cells, 88% of the receptors were in a low affinity form. The protein kinase C activator phorbol 12-myristate, 13-acetate (PMA) did not further increase the fraction in the low affinity form, but the protein kinase C inhibitor staurosporine reduced the low affinity fraction to 51%. In sucrose density gradient centrifugation assays of receptor internalization, the percentage of receptors in the light vesicle fraction was 25% for control cells, 53% for epinephrine-pretreated cells, 44% for PMA-pretreated cells, and 53% for cells pretreated with epinephrine plus PMA. Staurosporine completely blocked PMA-induced internalization, but only partially inhibited epinephrine-induced internalization. These results suggest a relationship between low affinity binding and internalization for alpha 1B-adrenoceptors and the involvement of protein kinase C in both processes. Longer-term (24 h) exposure of cells to epinephrine induced an unexpected up-regulation of receptor density of approximately 2-fold that was accompanied by an increase in maximal agonist-stimulated phosphoinositide turnover. These studies document several regulatory differences between alpha 1B-adrenoceptors expressed in transfected CHO cells and those natively expressed in DDT1 MF-2 hamster smooth muscle cells, and they provide additional information on the molecular mechanisms involved in agonist-induced regulation of alpha 1B-adrenoceptors.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Epinefrina/farmacologia , Fosfatidilinositóis/biossíntese , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas alfa-Adrenérgicos/metabolismo , Animais , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Cricetinae , Prazosina/metabolismo , Proteína Quinase C/metabolismo , Receptores Adrenérgicos alfa 1/genética , Proteínas Recombinantes/metabolismo , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
The effects of hypertonic sucrose and of intracellular potassium depletion on the intact-cell-binding properties of beta (beta AR) and alpha 1 (alpha 1AR) adrenergic receptors of DDT1 MF-2 hamster smooth muscle cells were investigated. These treatments are known to block clathrin assembly into coated pits, an early step in the pathway of receptor endocytosis. Conducting intact-cell-binding assays in the presence of 0.4 M sucrose markedly decreased the fraction of both beta ARs and alpha 1ARs converted during the assay to a form exhibiting low apparent affinity for agonists. Intracellular potassium depletion also decreased the fraction of both beta ARs and alpha 1ARs converted to this low-affinity form. In contrast the intact-cell-binding properties of antagonists were unaltered by these treatments. These results suggest a role for receptor internalization in conversion of both beta ARs and alpha 1ARs to their low-affinity forms. A model is proposed in which either an initial agonist-induced receptor sequestration within the plasma membrane or the subsequent endocytosis of receptors into intracellular vesicles allows conversion of these receptors to the form exhibiting low affinity for agonists in binding assays with intact cells.
Assuntos
Potássio/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos beta/metabolismo , Sacarose/metabolismo , Sacarose/farmacologia , Animais , Linhagem Celular , Cricetinae , Epinefrina/farmacologia , Soluções Hipertônicas , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/fisiologia , Receptores Adrenérgicos beta/fisiologiaRESUMO
The effects of the simple bioactive lipid mediator lysophosphatidic acid (LPA) on cAMP accumulation were investigated in cultured human airway smooth muscle cells (ASMC). Pretreatment of cells with LPA induced an increase in subsequent stimulation of cAMP accumulation by forskolin and by isoproterenol. When included during the assay of cAMP accumulation rather than as a pretreatment, LPA inhibited forskolin stimulation but enhanced isoproterenol stimulation. Both effects of LPA on forskolin stimulation were completely blocked by pertussis toxin treatment, whereas the effects on isoproterenol stimulation appeared relatively insensitive to pertussis toxin. The protein kinase C activator phorbol-12-myristate-13-acetate (PMA) sensitized forskolin stimulation to a similar extent as did LPA, and the combination of LPA plus PMA caused markedly more sensitization than either agent alone. In contrast, PMA inhibited isoproterenol stimulation and markedly decreased the sensitization induced by LPA. Serum also induced sensitization, and sensitization by LPA plus serum was no greater than that with LPA alone. LPA-induced sensitization appeared to be independent of protein kinase C activation because it was unchanged in cells treated to down-regulate protein kinase C. LPA also stimulated polyphosphoinositide hydrolysis, and this stimulation was partially inhibited by pertussis toxin treatment. These results suggest that LPA activates receptors coupled to both the pertussis toxin-sensitive G protein Gi and the pertussis toxin-insensitive G protein Gq. The complex effects of LPA, PMA, and pertussis toxin on cAMP accumulation in these cells are consistent with the expression of the type 2 isozyme of adenylyl cyclase in these cells.
Assuntos
AMP Cíclico/metabolismo , Lisofosfolipídeos/fisiologia , Músculo Liso/metabolismo , Traqueia/metabolismo , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática , Humanos , Hidrólise , Lisofosfolipídeos/farmacologia , Músculo Liso/citologia , Músculo Liso/fisiologia , Fosfatidilinositóis/metabolismo , Proteína Quinase C/metabolismo , Sensibilidade e Especificidade , Estimulação Química , Acetato de Tetradecanoilforbol/farmacologia , Traqueia/citologia , Traqueia/fisiologiaRESUMO
Tyrosine kinases are ubiquitous enzymes that have been shown to be involved in many cellular functions, including growth and differentiation. Recent studies have shown that they are also involved in integrin signal transduction pathways. Since integrins are known to be involved in cellular adhesion and thus in invasion and metastasis, the possible involvement of tyrosine kinases in invasion was tested. Tumor cell invasion was measured using filter inserts coated with Matrigel, a substance that closely resembles the natural basement membrane. A highly metastatic subline of BALB/c mammary carcinoma (410.4) cells was shown to invade nearly three times as much as a low metastatic subline (168.1). Genistein, an inhibitor of tyrosine kinases, was found to inhibit invasion of 410.4 cells with an EC50 of approximately 1 microM. At a concentration of 37 microM, there was almost complete inhibition of invasion by genistein, whereas the structural analog, daidzein, which does not inhibit tyrosine kinases, had only a small effect. At higher concentrations (370 microM), daidzein also caused marked inhibition. Genistein was able to inhibit invasion at concentrations having little effect on cell growth. However, for daidzein, most of the effect on invasion was apparently due to its effect on growth inhibition. The relatively specific effect of genistein to inhibit tumor invasion suggests a role for tyrosine phosphorylation in this process. Genistein or other tyrosine kinase inhibitors may be effective inhibitors of tumor invasion and metastasis.
Assuntos
Isoflavonas/farmacologia , Neoplasias Mamárias Experimentais/patologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Genisteína , Inibidores do Crescimento/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Metástase Neoplásica , Células Tumorais CultivadasRESUMO
Incubation of bovine hippocampal membranes with [alpha-32P]GTP and exposure to ultraviolet light resulted in the labelling of seven species with apparent molecular masses of 200, 74, 55, 53, 50, 43 and 40 kDa. Labelling of the 55 kDa species was greatly enhanced in the presence of carboxyl terminal fragments [neuropeptide Y-(18-36)] of neuropeptide Y. Labelling occurred with [alpha-32P]GTP but not [alpha-32P]ATP. A group of putative direct G protein activating peptides including mastoparan, melittin, substance P and adrenocorticotropic hormone (ACTH)-(1-24), were also able to stimulate the labelling of this protein. Labelling of the 55 kDa protein could be demonstrated in bovine brain but not peripheral tissues. Western blot analysis using an antibody against the common alpha subunit of G proteins recognized a protein co-migrating with the 55 kDa GTP-binding protein. These findings demonstrate the existence of a previously uncharacterized neuronal protein, with an apparent molecular mass of 55 kDa, that binds GTP in response to neuropeptide Y and other peptides.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Neuropeptídeo Y/farmacologia , Fragmentos de Peptídeos/farmacologia , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Anuros , Western Blotting , Bovinos , Embrião de Galinha , Peixes , Hipocampo/metabolismo , Hipocampo/efeitos da radiação , Técnicas In Vitro , Membranas/metabolismo , Membranas/efeitos da radiação , Dados de Sequência Molecular , Peso Molecular , Terminações Nervosas/metabolismo , Fotoquímica , Ligação Proteica , Especificidade da Espécie , Raios UltravioletaRESUMO
Pretreatment of cultured human tracheal smooth muscle cells with transforming growth factor-beta 1 (TGF-beta 1) decreased adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by intact cells stimulated with the beta-adrenergic agonist isoproterenol. The maximal inhibition of isoproterenol-stimulated cAMP accumulation by TGF-beta 1 was 31 +/- 3%, and the mean effective concentration (EC50) of TGF-beta 1 was approximately 1.5 pM. TGF-beta 1 decreased the maximal response to isoproterenol but did not change the EC50 value of isoproterenol. TGF-beta 1 did not change cAMP accumulation stimulated by forskolin. TGF-beta 1 pretreatment decreased isoproterenol-stimulated adenylyl cyclase activity measured in broken cell preparations, but did not change the fluoride-stimulated adenylyl cyclase activity. Together these results suggest that the TGF-beta 1 effect is not by direct inhibition of adenylyl cyclase or by decreased activity of the stimulatory GTP-binding protein. Saturation binding experiments with the beta-adrenergic receptor radioligand [125I]iodopindolol showed that TGF-beta 1 pretreatment decreased the beta-adrenergic receptor number. The protein synthesis inhibitor cycloheximide abolished the effect of TGF-beta 1 on both cAMP accumulation and on beta-adrenergic receptor number, indicating that protein synthesis is involved. These results suggest that TGF-beta 1 in the lung could play a role in changing the responsiveness of airway smooth muscle cells to endogenous catecholamines and to beta-adrenergic agonists used in therapy.
Assuntos
Músculo Liso/metabolismo , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/metabolismo , Traqueia/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Adenilil Ciclases/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Cicloeximida/farmacologia , Fluoretos/farmacologia , Humanos , Membranas Intracelulares/metabolismo , Isoproterenol/farmacologia , Músculo Liso/citologia , Traqueia/citologiaRESUMO
During the course of equilibrium competition binding assays with intact cells, agonists induce conversion of beta-adrenergic receptors (BARs) from a native form with high affinity for agonists to a form with a markedly lower apparent affinity. The roles of receptor internalization, receptor-Gs coupling, and receptor phosphorylation in this agonist-induced conversion to the low affinity form were investigated. Agonist and antagonist competition for [125I]iodopindolol binding to intact cells was measured in mouse L cells expressing wild-type BARs (C+I+), mutated BARs that do not couple to Gs but do internalize (C-I+), and mutated BARs that do not couple to Gs and do not internalize (C-I-). For C+I+ and C-I+ cells, most of the receptors exhibited apparent affinities for the agonist isoproterenol that were 500-900-fold lower in equilibrium assays with intact cells than in short-time assays with intact cells or in equilibrium assays with isolated membranes, similar to previous results with cells expressing native BARs. The extent of conversion to this lower affinity form for C-I- cells was markedly decreased. Binding properties for the antagonist metoprolol were similar for all three BARs in both short-time and equilibrium assays. Isoproterenol competition in short-time and equilibrium assays also was compared in Chinese hamster fibroblasts expressing wild-type BARs, mutated BARs that lack BAR kinase sites, mutated BARs that lack cAMP-dependent protein kinase sites, and mutated BARs that lack both types of phosphorylation sites. All three BAR phosphorylation mutants showed only small but significant decreases, relative to the wild-type BAR, in the extent of conversion to the low affinity form. These results provide additional evidence that receptor internalization is the major determinant for the conversion of intact cell BARs to the low affinity form. Receptor phosphorylation may play a minor role in conversion to the low affinity form, whereas receptor coupling to Gs is apparently not required.
Assuntos
Agonistas Adrenérgicos beta/metabolismo , Antagonistas Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Ligação Competitiva , Linhagem Celular , Cricetinae , Cricetulus , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Isoproterenol/metabolismo , Células L , Metoprolol/metabolismo , Metoprolol/farmacologia , Camundongos , Mutação , Fosforilação , Pindolol/análogos & derivados , Pindolol/metabolismo , Receptores Adrenérgicos beta/genética , Transfecção , Quinases de Receptores Adrenérgicos betaRESUMO
Radioligand binding assays are a relatively simple but extremely powerful tool for studying receptors. They allow an analysis of the interactions of hormones, neurotransmitters, growth factors, and related drugs with the receptors, studies of receptor interactions with second messenger systems, and characterization of regulatory changes in receptor number, subcellular distribution, and physiological function. As a result, these assays are widely used (and often misused) by investigators in a variety of disciplines, including pharmacology, physiology, biochemistry, immunology, and cell biology. This article presents a broad overview of the radioligand binding assay technique, primarily for the investigator who has limited experience with this technique. Practical guidelines for setting up a new assay are presented, including the receptor preparation to be used, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. Tips for avoiding some of the common pitfalls in application of these assays are also included. The primary focus is on radioligand binding assays of membrane-bound receptors studied in membrane preparations. However, similar assay techniques can be used to study receptors on intact cells. The unique advantages and disadvantages of these intact cell binding assays are also discussed. In particular, the occurrence of regulatory changes in receptors during the course of intact cell binding assays is considered, with approaches for circumventing these complications and for using intact cell assays to advantage in studying these regulatory changes.
Assuntos
Ensaio Radioligante/métodos , Receptores de Superfície Celular/análise , Animais , Ligação Competitiva , Células/metabolismo , Cinética , Fosforilação , Receptores de Superfície Celular/metabolismoRESUMO
Pretreatment of 1321N1 human astrocytoma cells with serum induces a pronounced increase in subsequent stimulation by forskolin and other agents of intracellular cyclic AMP accumulation, a phenomenon referred to as sensitization (Mol. Pharmacol. 39, 399-406, 1991). Pretreatment of these cells with lysophosphatidic acid induced sensitization to a similar extent as that with serum (approximately fivefold for forskolin stimulation and twofold for isoproterenol and prostaglandin E1 stimulation), with half-maximal effects at approximately 30 nM lysophosphatidic acid. Phosphatidic acid was effective but less potent whereas other lipids were ineffective. Sensitization by serum and by lysophosphatidic acid were almost completely inhibited by pertussis toxin pretreatment and partially inhibited by prolonged phorbol ester exposure to induce protein kinase C down-regulation. Among nine cell lines tested, those that exhibited sensitization with serum showed comparable sensitization with lysophosphatidic acid. The effects of both lysophosphatidic acid and serum were markedly inhibited by treatment with phospholipase B but only minimally altered with phospholipases A2, D, and C. Exposure of cells to phospholipase C alone induced approximately threefold sensitization, but both serum and lysophosphatidic acid were able to induce further three- to fourfold sensitization above that induced by phospholipase C alone. In contrast, the effects of serum and lysophosphatidic acid were not additive with each other. Together these results suggest that lysophosphatidic acid or a closely related compound present in serum is the factor responsible for sensitization of the cyclic AMP pathway.