RESUMO
Galectin-3 is a beta-galactoside-binding mammalian lectin and part of the 15 member galectin family that are evolutionarily highly conserved. It is the only chimeric protein with a C-terminal carbohydrate recognition domain (CRD) linked to a proline, glycine, and tyrosine rich additional N-terminal domain. Galectin-3 binds several cell surface glycoproteins via its CRD domain as well as undergoing oligomerization, via binding at the N-terminal or the CRD, resulting in the formation of a galectin-3 lattice on the cell surface. The galectin-3 lattice has been regarded as being a crucial mechanism whereby extracellular galectin-3 modulates cellular signalling by prolonging retention time or retarding lateral movement of cell surface receptors in the plasma membrane. As such galectin-3 can regulate various cellular functions such as diffusion, compartmentalization and endocytosis of plasma membrane glycoproteins and glycolipids and the functionality of membrane receptors. In multiple models of organ fibrosis, it has been demonstrated that galectin-3 is potently pro-fibrotic and modulates the activity of fibroblasts and macrophages in chronically inflamed organs. Increased galectin-3 expression also activates myofibroblasts resulting in scar formation and may therefore impact common fibrotic pathways leading to fibrosis in multiple organs. Over the last decade there has been a marked increase in the scientific literature investigating galectin-3 in a range of fibrotic diseases as well as the clinical development of new galectin-3 inhibitors. In this review we will examine the role of galectin-3 in fibrosis, the therapeutic strategies for inhibiting galectin-3 in fibrotic disease and the clinical landscape to date.
Assuntos
Endocitose , Fibrose/tratamento farmacológico , Galectinas/antagonistas & inibidores , Inflamação/prevenção & controle , Animais , Proteínas Sanguíneas/antagonistas & inibidores , Fibrose/metabolismo , Fibrose/patologia , Galectinas/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologiaRESUMO
Mesenchymal cells expressing platelet-derived growth factor receptor beta (PDGFRß) are known to be important in fibrosis of organs such as the liver and kidney. Here we show that PDGFRß+ cells contribute to skeletal muscle and cardiac fibrosis via a mechanism that depends on αv integrins. Mice in which αv integrin is depleted in PDGFRß+ cells are protected from cardiotoxin and laceration-induced skeletal muscle fibrosis and angiotensin II-induced cardiac fibrosis. In addition, a small-molecule inhibitor of αv integrins attenuates fibrosis, even when pre-established, in both skeletal and cardiac muscle, and improves skeletal muscle function. αv integrin blockade also reduces TGFß activation in primary human skeletal muscle and cardiac PDGFRß+ cells, suggesting that αv integrin inhibitors may be effective for the treatment and prevention of a broad range of muscle fibroses.
Assuntos
Integrina alfaV/metabolismo , Músculo Esquelético/patologia , Miocárdio/patologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Apoptose , Movimento Celular , Células Cultivadas , Colágeno/metabolismo , Fibrose , Genótipo , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Acute liver failure (ALF) is associated with significant morbidity and mortality. Studies have implicated the immune response, especially monocyte/macrophages as being important in dictating outcome. AIM: To investigate changes in the circulating monocytes and other immune cells serially in patients with ALF, relate these with cytokine concentrations, monocyte gene expression and patient outcome. METHODS: In a prospective case-control study in the Scottish Liver Transplant Unit, Royal Infirmary Edinburgh, 35 consecutive patients admitted with paracetamol-induced liver failure (POD-ALF), 10 patients with non-paracetamol causes of ALF and 16 controls were recruited. The peripheral blood monocyte phenotype was analysed by flow cytometry, circulating cytokines quantified by protein array and monocyte gene expression array performed and related to outcome. RESULTS: On admission, patients with worst outcomes after POD-ALF had a significant monocytopenia, characterised by reduced classical and expanded intermediate monocyte population. This was associated with reduced circulating lymphocytes and natural killer cells, peripheral cytokine patterns suggestive of a 'cytokine storm' and increased concentrations of cytokines associated with monocyte egress from the bone marrow. Gene expression array did not differentiate patient outcome. At day 4, there was no significant difference in monocyte, lymphocyte or natural killer cells between survivors and the patients with adverse outcomes. CONCLUSIONS: Severe paracetamol liver failure is associated with profound changes in the peripheral blood compartment, particularly in monocytes, related with worse outcomes. This is not seen in patients with non-paracetamol-induced liver failure. Significant monocytopenia on admission may allow earlier clarification of prognosis, and it highlights a potential target for therapeutic intervention.
Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Leucopenia/induzido quimicamente , Leucopenia/diagnóstico , Falência Hepática Aguda/induzido quimicamente , Monócitos/patologia , Adulto , Estudos de Casos e Controles , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/complicações , Doença Hepática Induzida por Substâncias e Drogas/mortalidade , Citocinas/metabolismo , Feminino , Humanos , Leucopenia/complicações , Leucopenia/mortalidade , Falência Hepática Aguda/sangue , Falência Hepática Aguda/diagnóstico , Falência Hepática Aguda/mortalidade , Transplante de Fígado/efeitos adversos , Transplante de Fígado/mortalidade , Masculino , Monócitos/efeitos dos fármacos , Prognóstico , Análise de Sobrevida , Resultado do TratamentoRESUMO
PURPOSE: Lung cancer is the leading cause of cancer deaths in the developed world. Small cell lung cancer (SCLC) has the worst prognosis due to the emergence of resistance to chemotherapy. This article will review recent work that has defined mechanisms of chemo-resistance focusing on the role of integrins. RESULTS: SCLC is surrounded by an extensive stroma of extracellular matrix (ECM) and high levels of expression correlate with poor prognosis. ECM protects SCLC cells against chemotherapy-induced cell death by activating beta1 integrins leading to activation of phosphoinositide-3-OH kinase (PI3-kinase), which prevents etoposide-induced caspase-3 activation and subsequent apoptosis. Engagement of ECM prevents etoposide and radiation induced G2/M cell cycle arrest in SCLC cells by blocking the up-regulation of p21Cip1/WAF1 and p27Kip1 and the down-regulation of cyclins E, A and B. These effects are abrogated by pharmacological and genetic inhibition of PI3-kinase signalling. CONCLUSIONS: Thus, ECM via beta1 integrin-mediated PI3-kinase activation allows SCLC cells to survive treatment induced cell cycle arrest and apoptosis with persistent DNA damage, providing a model to account for the emergence of acquired drug resistance. Novel therapeutic strategies may therefore be directed at inhibiting integrin-mediated cell survival signals improving response rates and cure in this devastating cancer.
Assuntos
Carcinoma de Células Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Matriz Extracelular/fisiologia , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma de Células Pequenas/patologia , Carcinoma de Células Pequenas/fisiopatologia , Carcinoma de Células Pequenas/radioterapia , Sobrevivência Celular , Humanos , Integrinas/fisiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Neoplasias Pulmonares/radioterapia , Modelos Biológicos , Prognóstico , Transdução de SinaisRESUMO
The emergence of resistance to chemotherapy remains a principle problem in the treatment of small-cell lung cancer (SCLC). We demonstrate that extracellular matrix (ECM) activates phosphatidyl inositol 3-kinase (PI3-kinase) signaling in SCLC cells and prevents etoposide-induced caspase-3 activation and subsequent apoptosis in a beta1 integrin/PI3-kinase-dependent manner. Crucially we show that etoposide and radiation induce G2/M cell cycle arrest in SCLC cells prior to apoptosis and that ECM prevents this by overriding the upregulation of p21(Cip1/WAF1) and p27(Kip1) and the downregulation of cyclins E, A and B. These effects are abrogated by pharmacological and genetic inhibition of PI3-kinase signaling. Importantly we show that chemoprotection is not mediated by altered SCLC cell proliferation or DNA repair. Thus, ECM via beta1 integrin-mediated PI3-kinase activation overrides treatment-induced cell cycle arrest and apoptosis, allowing SCLC cells to survive with persistent DNA damage, providing a model to account for the emergence of acquired drug resistance.
Assuntos
Apoptose , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Ciclo Celular , Matriz Extracelular/metabolismo , Integrina beta1/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Carcinoma de Células Pequenas/terapia , Adesão Celular , Linhagem Celular Tumoral , Dano ao DNA , Etoposídeo/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Laminina/metabolismo , Neoplasias Pulmonares/terapia , Mutação , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vanadatos/farmacologiaRESUMO
Small-cell lung cancer (SCLC) is a particularly aggressive cancer, which metastasises early. Despite initial sensitivity to radio- and chemo-therapy, it invariably relapses, so that the 2-year survival remains less than 5%. Neuropeptides particularly arginine vasopressin (AVP) and gastrin-releasing peptide (GRP) act as autocrine and paracrine growth factors and the expression of these and their receptors are a hallmark of the disease. Substance-P analogues including [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance-P (SP-D) and [Arg6,D-Trp7,9,NmePhe8]-substance-P (6-11) (SP-G) inhibit the growth of SCLC cells by modulating neuropeptide signalling. We show that GRP and V1A receptors expression leads to the development of a transformed phenotype. Addition of neuropeptide provides some protection from etoposide-induced cytotoxicity. Receptor expression also leads to an increased sensitivity to substance-P analogue-induced growth inhibition. We show that SP-D and SP-G act as biased agonists at GRP and V1A receptors causing blockade of Gq-mediated Ca2+ release while directing signalling to activate ERK via a pertussis toxin-sensitive pathway. This is the first description of biased agonism at V1A receptors. This unique pharmacology governs the antiproliferative properties of these agents and highlights their potential therapeutic potential for the treatment of SCLC and particularly in tumours, which have developed resistance to chemotherapy.
Assuntos
Transformação Celular Neoplásica , Peptídeo Liberador de Gastrina/farmacologia , Animais , Arginina Vasopressina/farmacologia , Divisão Celular , Cricetinae , Cricetulus , Etoposídeo/farmacologia , Peptídeo Liberador de Gastrina/genética , Peptídeo Liberador de Gastrina/metabolismo , Humanos , Fragmentos de Peptídeos/farmacologia , Receptores de Neuropeptídeos/metabolismo , Substância P/genética , Substância P/metabolismo , Substância P/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
[Arg(6),D-Trp(7,9),N(me)Phe(8)]-substance P (6-11) (SP-G) is a novel anticancer agent that has recently completed phase I clinical trials. SP-G inhibits mitogenic neuropeptide signal transduction and small cell lung cancer (SCLC) cell growth in vitro and in vivo. Using the SCLC cell line series GLC14, 16 and 19, derived from a single patient during the clinical course of their disease and the development of chemoresistance, it is shown that there was an increase in responsiveness to neuropeptides. This was paralleled by an increased sensitivity to SP-G. In a selected panel of tumour cell lines (SCLC, non-SCLC, ovarian, colorectal and pancreatic), the expression of the mitogenic neuropeptide receptors for vasopressin, gastrin-releasing peptide (GRP), bradykinin and gastrin was examined, and their sensitivity to SP-G tested in vitro and in vivo. The tumour cell lines displayed a range of sensitivity to SP-G (IC(50) values from 10.5 to 119 microM). The expression of the GRP receptor measured by reverse transcriptase-polymerase chain reaction, correlated significantly with growth inhibition by SP-G. Moreover, introduction of the GRP receptor into rat-1A fibroblasts markedly increased their sensitivity to SP-G. The measurement of receptor expression from biopsy samples by polymerase chain reaction could provide a suitable diagnostic test to predict efficacy to SP-G clinically. This strategy would be of potential benefit in neuropeptide receptor-expressing tumours in addition to SCLC, and in tumours that are relatively resistant to conventional chemotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , Receptores da Bombesina/metabolismo , Substância P/análogos & derivados , Substância P/uso terapêutico , Animais , Bradicinina/metabolismo , Cálcio/metabolismo , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Fibroblastos/metabolismo , Peptídeo Liberador de Gastrina/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Ratos , Receptores de Neuropeptídeos/metabolismo , Substância P/antagonistas & inibidores , Transplante Heterólogo , Células Tumorais Cultivadas , Vasopressinas/metabolismoRESUMO
Substance P analogues including [d-Arg1,d-Phe5,d-Trp7,9,Leu11]substance P (SpD) act as "broad spectrum neuropeptide antagonists" and are potential anticancer agents that inhibit the growth of small cell lung cancer cells in vitro and in vivo. However, their mechanism of action is controversial and not fully understood. Although these compounds block bombesin-induced mitogenesis and signal transduction, they also have agonist activity. The mechanism underlying this agonist activity was examined. SpD binds to the ligand-binding site of the bombesin/gastrin-releasing peptide receptor and blocks the bombesin-stimulated increase in [Ca2+]i within the same concentration range that causes sustained activation of c-Jun N-terminal kinase and extracellular signal-regulated protein kinase (ERK). The activation of c-Jun N-terminal kinase by SpD and bombesin is blocked by dominant negative inhibition of G(alpha12). The ERK activation by SpD is pertussis toxin-sensitive in contrast to ERK activation by bombesin, which is pertussis toxin-insensitive but dependent on epidermal growth factor receptor phosphorylation. SpD does not simply act as a partial agonist but differentially modulates the activation of the G-proteins G(alpha12), G(i), and G(q) compared with bombesin. This unique ability allows the bombesin receptor to couple to G(i) and at the same time block receptor activation of G(q). Our results provide direct evidence that SpD is acting as a "biased agonist" and that this has physiological relevance in small cell lung cancer cells. This validation of the concept of biased agonism has important implications in the development of novel pharmacological agents to dissect receptor-mediated signal transduction and of highly selective drugs to treat human disease.
Assuntos
Bombesina/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Receptores da Bombesina/metabolismo , Substância P/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Western Blotting , Bombesina/química , Bombesina/farmacologia , Células COS , Cálcio/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Genes Dominantes , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Teóricos , Toxina Pertussis , Fosforilação , Ligação Proteica , Quinazolinas , Ratos , Receptores da Bombesina/química , Transdução de Sinais , Substância P/análogos & derivados , Substância P/química , Fatores de Tempo , Células Tumorais Cultivadas , Tirfostinas/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
[Arg(6), D-Trp(7,9), N(me)Phe(8)]-substance P (6-11) (antagonist G) inhibits small cell lung cancer (SCLC) growth and is entering Phase II clinical investigation for the treatment of SCLC. As well as acting as a neuropeptide receptor antagonist, antagonist G stimulates c-jun-N-terminal kinase (JNK) activity and apoptosis in SCLC cells. We extend these findings and show that the stimulation of JNK and apoptosis by antagonist G is dependent upon the generation of reactive oxygen species (ROS) being inhibited either by anoxia or the presence of N-acetyl cysteine (n-AC). Antagonist G is not intrinsically a free radical oxygen donor but stimulates free radical generation specifically within SCLC cells (6.2-fold) and increases the activity of the redox-sensitive transcription factor AP-1 by 61%. In keeping with this, antagonist G reduces cellular glutathione (GSH) levels (38% reduction) and stimulates ceramide production and lipid peroxidation (112% increase). At plasma concentrations achieved clinically in the phase I studies, antagonist G augments, more than additively, growth inhibition induced by etoposide. Our results suggest that antagonist G may be particularly effective as an additional treatment with standard chemotherapy in SCLC. These novel findings will be important for the clinical application of this new and exciting compound and for the future drug development of new agents to treat this aggressive cancer.
Assuntos
Antineoplásicos/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Oligopeptídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição AP-1/biossíntese , Acetilcisteína/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Células CHO , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/patologia , Divisão Celular/efeitos dos fármacos , Cricetinae , Sinergismo Farmacológico , Etoposídeo/farmacologia , Sequestradores de Radicais Livres , Glutationa/metabolismo , Inibidores do Crescimento/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MAP Quinase Quinase 4 , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oxirredução , Estimulação Química , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/fisiologia , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Resistance to chemotherapy is a principal problem in the treatment of small cell lung cancer (SCLC). We show here that SCLC is surrounded by an extensive stroma of extracellular matrix (ECM) at both primary and metastatic sites. Adhesion of SCLC cells to ECM enhances tumorigenicity and confers resistance to chemotherapeutic agents as a result of beta1 integrin-stimulated tyrosine kinase activation suppressing chemotherapy-induced apoptosis. SCLC may create a specialized microenvironment, and the survival of cells bound to ECM could explain the partial responses and local recurrence of SCLC often seen clinically after chemotherapy. Strategies based on blocking beta1 integrin-mediated survival signals may represent a new therapeutic approach to improve the response to chemotherapy in SCLC.
Assuntos
Apoptose/fisiologia , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Proteínas da Matriz Extracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Clorometilcetonas de Aminoácidos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bradicinina/farmacologia , Carcinoma de Células Pequenas/tratamento farmacológico , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Adesão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Colágeno/metabolismo , Ciclofosfamida/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Fibronectinas/metabolismo , Galanina/farmacologia , Humanos , Integrina beta1/metabolismo , Laminina/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Tenascina/metabolismo , Células Tumorais Cultivadas , Tirfostinas/farmacologiaRESUMO
[Arg6,D-Trp7,9,NmePhe8]-substance P (6-11) (antagonist G) is a novel class of anti-cancer agent that inhibits small-cell lung cancer (SCLC) cell growth in vitro and in vivo and is entering phase II clinical investigation for the treatment of SCLC. Although antagonist G blocks SCLC cell growth (IC50 = 24.5 +/- 1.5 and 38.5 +/- 1.5 microM for the H69 and H510 cell lines respectively), its exact mechanism of action is unclear. This study shows that antagonist G stimulates apoptosis as assessed by morphology (EC50 = 5.9 +/- 0.1 and 15.2 +/- 2.7 microM for the H69 and H510 cell lines respectively) and stimulates c-jun-N-terminal kinase (JNK) activity in SCLC cells (EC50 = 3.2 +/- 0.1 and 15.2 +/- 2.7 microM). This activity is neuropeptide-independent, but dependent on the generation of reactive oxygen species (ROS) and is inhibited by the free radical scavenger n-acetyl cysteine. Furthermore, antagonist G itself induces inflammation (59% increase in oedema volume compared to control) and potentiates (by 35-40%) bradykinin-induced oedema formation in vivo. In view of these results we show that, as well as acting as a 'broad-spectrum' neuropeptide antagonist, antagonist G stimulates basal G-protein activity in SCLC cell membranes (81 +/- 12% stimulation at 10 microM), thereby displaying a unique ability to stimulate certain signal transduction pathways by activating G-proteins. This novel activity may be instrumental for full anti-cancer activity in SCLC cells and may also account for antagonist G activity in non-neuropeptide-dependent cancers.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Carcinoma de Células Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno , Oligopeptídeos/farmacologia , Células 3T3 , Animais , Carcinoma de Células Pequenas/patologia , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Ativação Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Neoplasias Pulmonares/patologia , Camundongos , Neuropeptídeos/farmacologia , Coelhos , Espécies Reativas de Oxigênio/fisiologia , Células Tumorais CultivadasRESUMO
Stimulation of small cell lung cancer (SCLC) cells with neuropeptides bombesin, bradykinin, gastrin, and neurotensin resulted in increased tyrosine kinase activity and tyrosine phosphorylation of a number of polypeptides including a p120 kDa polypeptide identified by immunoblotting as focal adhesion kinase (p125FAK). The neuropeptides stimulated a rapid, concentration-dependent phosphorylation of p125FAK (EC50 of 1 nM, 5 nM, and 2 nM for bombesin, bradykinin, and gastrin, respectively), which was receptor mediated and inhibited by both specific and broad-spectrum neuropeptide receptor antagonists. Specific inhibition of protein tyrosine kinase activity by tyrphostin-25 inhibited both basal and neuropeptide-stimulated SCLC cell growth. These results identify a novel neuropeptide-stimulated growth signaling event in SCLC cells.
Assuntos
Neuropeptídeos/farmacologia , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Bombesina/farmacologia , Bradicinina/farmacologia , Carcinoma de Células Pequenas , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Gastrinas/farmacologia , Humanos , Cinética , Neoplasias Pulmonares , Neurotensina/farmacologia , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Fosforilação , Células Tumorais Cultivadas , TirosinaRESUMO
1. RS-45041-190 (4-chloro-2-(imidazolin-2-yl)isoindoline) is an I2 imidazoline receptor ligand with the highest affinity and selectivity so far described; [3H]-RS-45041-190 has a tritium atom attached to the 7-position on the isoindoline ring. 2. [3H]-RS-45041-190 binding to rat kidney membranes was saturable (Bmax = 223.1 +/- 18.4 fmol mg-1 protein) and of high affinity (Kd = 2.71 +/- 0.59 nM). Kinetic studies revealed that the binding was rapid and reversible, with [3H]-RS-45041-190 interacting with two sites or two affinity states. 3. Competition studies showed that 60-70% of [3H]-RS-45041-190 binding (1 nM) was specifically to imidazoline binding sites of the I2 subtype, characterized by high affinity for idazoxan (pIC50 7.85 +/- 0.03) and cirazoline (pIC50 8.16 +/- 0.05). The remaining 30-40% was displaced specifically by the monoamine oxidase A inhibitors, clorgyline and pargyline. 4. alpha 1- and alpha 2-adrenoceptor, I1 imidazoline, histamine, 5-hydroxytryptamine or dopamine receptor ligands had low affinity suggesting that [3H]-RS-45041-190 did not label receptors of these classes. 5. In autoradiography studies, [3H]-RS-45041-190 labelled discrete regions of rat brain corresponding to the distribution of I2 subtypes, notably the subfornical organ, arcuate nucleus, interpeduncular nucleus, medial habenular nucleus and lateral mammillary nucleus, and additional sites in the locus coeruleus, dorsal raphe and dorsomedial hypothalamic nucleus. 6. [3H]-RS-45041-190 therefore labels I2 receptors with high affinity, and an additional site which has high affinity for some monoamine oxidase inhibitors.
Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Dioxanos/farmacologia , Imidazóis/farmacologia , Indóis/farmacologia , Rim/efeitos dos fármacos , Receptores de Droga/efeitos dos fármacos , Animais , Autorradiografia , Ligação Competitiva , Relação Dose-Resposta a Droga , Idazoxano , Receptores de Imidazolinas , Isoindóis , Masculino , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
1. RS-45041-190 (4-chloro-2-(imidazolin-2-yl)isoindoline) showed high affinity for I2 imidazoline receptors labelled by [3H]-idazoxan in rat (pKi = 8.66 +/- 0.09), rabbit (pKi = 9.37 +/- 0.07), dog (pKi = 9.32 +/- 0.18) and baboon kidney (pKi = 8.85 +/- 0.12), but had very low affinity for alpha 2-adrenoceptors in rat cerebral cortex (pKi = 5.7 +/- 0.09). 2. RS-45041-190 showed low affinity for other adrenoceptors, dopamine, 5-hydroxytryptamine, and muscarinic receptors and dihydropyridine binding sites (selectivity ratio > 1000). 3. RS-45041-190 showed moderate potency for the inhibition of monoamine oxidase A in vitro (pIC50 = 6.12), but had much lower potency for monoamine oxidase B (pIC50 = 4.47), neither of which equated with its affinity for I2 receptors. 4. RS-45041-190 (0.001 to 3 mg kg-1, i.v. and 1 ng-50 micrograms i.c.v.) had only small, transient effects on blood pressure and heart rate in anaesthetized rats. In conscious rats, RS-45041-190 had no effect on body core temperature or tail skin temperature (1 mg kg-1, s.c.) or on activity or rotarod performance (10 mg kg-1, i.p.). There were also no effects on barbiturate sleeping time in mice after doses of 1-10 mg kg-1, i.p. 5. RS-45041-190 (10 and 25 mg kg-1, i.p.) significantly increased food consumption in rats for up to 4 h after dosing, but unlike idazoxan (10 mg kg-1, i.p.) did not increase water consumption. RS-45041-190 is therefore a selective, high-affinity ligand at I2 imidazoline receptors and its hyperphagic effect may suggest a role for I2 imidazoline receptors in the modulation of appetite.However, in the absence of a selective agonist it is unclear whether this ligand is an agonist or an antagonist at I2 receptors.
Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Dioxanos/farmacologia , Imidazóis/farmacologia , Rim/efeitos dos fármacos , Receptores de Droga/efeitos dos fármacos , Animais , Apetite/efeitos dos fármacos , Ligação Competitiva , Pressão Sanguínea/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Idazoxano , Receptores de Imidazolinas , Masculino , Camundongos , Coelhos , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
1. [3H]-lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd = 10.7 +/- 2.9 nM; Bmax = 5.10 +/- 1.43 pmol mg-1 protein). 2. The binding of [3H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 microM had no effect on [3H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]-batrachotoxinin-A 20-alpha-benzoate (BTX) binding with moderate affinity (pIC50 7.31 +/- 0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [3H]-BTX and decreased both the affinity and density of sites labelled by [3H]-BTX, suggesting an allosteric interaction with toxin site 2. 3. The binding of [3H]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC50 for cold lifarizine = 7.99 +/- 0.09 in membranes and 6.68 +/- 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl increased the affinity of lifarizine almost 10 fold (pIC50 = 7.86 +/- 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4. There was negligible [3H]-lifarizine and [3H]-BTX binding to solubilized sodium channels, although [3H]-STX binding was retained under these conditions. 5. The potencies of a series of compounds in displacing [3H]-lifarizine from rat cerebrocortical membranes correlated well with their affinities for inactivated sodium channels estimated from whole-cell voltage clamp studies in the mouse neuroblastoma cell line, NIE-115 (r=0.96).6. These results show that [3H]-lifarizine is a high affinity ligand for neuronal sodium channels which potently and selectively labels a site, allosterically linked to toxin binding site 2, associated within activated sodium channels.
Assuntos
Imidazóis/farmacologia , Piperazinas/farmacologia , Canais de Sódio/efeitos dos fármacos , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Imidazóis/metabolismo , Masculino , Técnicas de Patch-Clamp , Piperazinas/metabolismo , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Saxitoxina/farmacologia , Canais de Sódio/metabolismo , Fatores de TempoRESUMO
The proliferation of receptor subtypes based on differences in amino acid sequence does not necessarily coincide with functional differences. The number of alpha 2-adrenoceptor subtypes, as defined by ligand-binding and molecular studies, has been increasing in the past few years, which suggests the possibility of distinct physiological and pathological pathways that could be targeted by new selective drugs. However, the evidence from functional studies has been less convincing. This could be due to the lack of sufficiently selective ligands or to the similarity between the activated state of receptor subtypes. Species differences and the local receptor environment are also important determinants of the pharmacological profile of a particular subtype. The pharmacology of the putative subtypes of alpha 2-adrenoceptors and their function are discussed in this review by Alison MacKinnon, Mike Spedding and Christine Brown.
Assuntos
Receptores Adrenérgicos alfa/classificação , Receptores Adrenérgicos alfa/fisiologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Clonagem Molecular , Humanos , Biologia Molecular , Receptores Adrenérgicos alfa/genética , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
1. The alpha 2-adrenoceptors on human platelets and neonatal rat lung were characterized with the agonist and antagonist ligands [3H]-adrenaline and [3H]-RS-15385-197 respectively. A correlation of affinities for 3H-antagonist binding showed the receptors to be of the alpha 2A-(platelet) and alpha 2B-(neonatal rat lung) adrenoceptor subtypes, whereas a correlation of affinities for 3H-agonist binding showed the receptors to have similar characteristics (r = 0.88). 2. NaCl (100 mM) had no effect on [3H]-RS-15385-197 binding in the human platelet, but increased the density of sites labelled with [3H]-RS-15385-197 in neonatal rat lung by 52%. NaCl increased the density of sites labelled by [3H]-adrenaline in neonatal rat lung, but there was a consequent 3.5 fold decrease in affinity. In the human platelet, no specific [3H]-adrenaline binding was observed in the presence of 100 mM NaCl. 3. In the neonatal rat lung, NaCl had no significant effect on the affinity of prazosin for [3H]-RS-15385-197 binding; however, imiloxan affinity was increased 13 fold. The affinity of the catecholamines, adrenaline and noradrenaline was significantly decreased, whereas the imidazolines, oxymetazoline and UK-14,304 were much less affected. The affinity of prazosin and imiloxan for [3H]-adrenaline binding was significantly increased in the presence of 10 and 100 mM NaCl. Conversely, the affinity of adrenaline and noradrenaline was decreased in the presence of NaCl, and there was no change in the affinity of the imidazoline agonists. 4. In the human platelet, NaCl had no effect on the affinity of prazosin for [3H]-RS-15385-197 binding but the affinity of imiloxan was significantly increased. NaCl significantly decreased the affinity of the catecholamines adrenaline and noradrenaline, whereas the affinity of UK-14,304 and oxymatazoline was much less affected. Competition experiments with [3H]-adrenaline in the presence of NaCl in platelets were difficult to characterize as there was no specific binding under these conditions.5. The results show that both the alpha2A- and alpha2B-adrenoceptor subtypes are allosterically regulated by Na+, but only the alpha2B-subtype showed a significant increase in density. Interestingly, there is a differential regulation of imidazoline (unchanged) and catecholamine (decreased affinity) agonist interactions with these subtypes. Na+ may therefore critically affect receptor subtype selectivity of drugs. The implications for receptor subclassification are discussed.
Assuntos
Agonistas alfa-Adrenérgicos/metabolismo , Antagonistas Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Sódio/metabolismo , Animais , Animais Recém-Nascidos/fisiologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Membrana Celular/metabolismo , Epinefrina/metabolismo , Feminino , Humanos , Técnicas In Vitro , Isoquinolinas/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Naftiridinas/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
1. A high density of [3H]-PK 11195 binding sites was present in gerbil cortical membranes (Bmax [3H]-PK 11195 1360 +/- 71 fmol mg-1 protein) in comparison to rat cortical membranes (254 +/- 21 fmol mg-1 protein). This effect was species-specific as similar findings were obtained with hippocampal membranes (Bmax 1430 +/- 111 fmol mg-1 protein in gerbil, compared to 196 +/- 31 in rat). 2. RO 5-4864, also a peripheral type benzodiazepine compound, displayed low affinity for the [3H]-PK 11195 site in the gerbil (pKi 6.57 +/- 0.02 and 6.70 +/- 0.12 in hippocampus and cortex respectively) compared to rat (pKi 8.16 +/- 0.07 and 8.48 +/- 0.02). Central benzodiazepine compounds, diazepam and flunitrazepam, also displayed this trend. 3. RO 5-4864 displaced [3H]-PK 11195 binding from gerbil and rat cortical membranes through a competitive interaction with Hill slopes close to unity. In both tissues, saturation isotherms of [3H]-PK 11195 binding indicated that the presence of RO 5-4864 caused changes in Kd without any effect on Bmax. In kinetic experiments, the presence of RO 5-4864 failed to modify the rate of dissociation of [3H]-PK 11195 from equilibrium in both rat and gerbil cortical membranes. 4. Forebrain ischaemia in the Mongolian gerbil (5 min bilateral carotid artery occlusion) with 7 days recovery caused a significant (P<0.05) decrease in the density of hippocampal 5-HTlA binding sites labelled by [3H]-8-OH-DPAT (Bmax control, 393 +/- 33 fmol mg-1 protein; ischaemic, 289 +/- 21 fmol mg protein)and an increase (P<0.01) in [3H]-PK 11195 binding sites (Bmax control, 1430 +/- 111 fmol mg-1 protein; ischaemic, 2160 +/- 170 fmol mg-1 protein). Ischaemia and recovery had no effect on the affinity of either ligand.5. Autoradiography experiments in gerbil brain sections revealed that the ischaemia-induced increase in[3H]-PK 11195 binding was consistent and significant in the CA, subfield on the hippocampus (control,152 +/- 42 fmol mg-1 tissue; ischaemic, 314 +/- 43 fmol mg-1 tissue).
Assuntos
8-Hidroxi-2-(di-n-propilamino)tetralina/farmacocinética , Isquemia Encefálica/metabolismo , Isoquinolinas/farmacocinética , Prosencéfalo/metabolismo , Receptores de GABA-A/metabolismo , Animais , Autorradiografia , Benzodiazepinas/farmacologia , Benzodiazepinonas/farmacologia , Ligação Competitiva/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Convulsivantes/farmacologia , Gerbillinae , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Masculino , Membranas/efeitos dos fármacos , Membranas/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Prosencéfalo/citologia , Ratos , Receptores de GABA-A/efeitos dos fármacosRESUMO
In the presence of RS-15385-197 to preclude binding to alpha 2-adrenoceptors, [3H]p-aminoclonidine labelled a low affinity high capacity site, (Kd = 127.6 +/- 19.7 nM, Bmax 978 +/- 172 fmol/mg protein) whereas [3H]idazoxan labelled a high affinity low capacity site (Kd = 1.66 +/- 0.28 nM, Bmax 45.3 +/- 11.4 fmol/mg protein). Clonidine and p-aminoclonidine showed moderate affinity for the site labelled by [3H]p-aminoclonidine, but low affinity for the site labelled by [3H]idazoxan, whereas idazoxan showed high affinity for [3H]idazoxan and low affinity for [3H]p-aminoclonidine binding. Naphazoline inhibited [3H]idazoxan in a biphasic manner suggesting that [3H]idazoxan may label an heterogeneous population of imidazoline sites. GTP inhibited [3H]idazoxan but not [3H]p-aminoclonidine binding. These results suggest that [3H]idazoxan labelled imidazoline I2 binding sites, whereas [3H]p-aminoclonidine labelled a novel subtype which showed marked differences to the imidazoline I1 binding site reported in bovine and human brainstem.
Assuntos
Clonidina/análogos & derivados , Dioxanos , Imidazóis/metabolismo , Rim/metabolismo , Receptores de Droga/metabolismo , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Ligação Competitiva , Guanosina Trifosfato/farmacologia , Idazoxano , Receptores de Imidazolinas , Técnicas In Vitro , Isoquinolinas/farmacologia , Masculino , Nafazolina/farmacologia , Naftiridinas/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , TrítioRESUMO
1. RS-15385-197 ((8aR, 12aS, 13aS)-5,8,8a,9,10,11,12,12a,13,13a-decahydro- 3-methoxy-12-(methylsulphonyl)-6H-isoquino [2,1-g][1,6]-naphthyridine) was evaluated in a series of in vitro and in vivo tests as an antagonist at alpha 2-adrenoceptors. 2. RS-15385-197 had a pKi of 9.45 for alpha 2-adrenoceptors in the rat cortex (pA2 in the guinea-pig ileum of 9.72), whereas the 8aS, 12aR, 13aR enantiomer, RS-15385-198, had a pKi of only 6.32 (pA2 6.47) indicating a high degree of stereoselectivity. The racemate RS-15385-196 had a pKi of 9.18. 3. RS-15385-197 showed unprecedented alpha 2 vs. alpha 1 adrenoceptor selectivity in vitro. In the rat cortex, RS-15385-197 had a pKi of 9.45 in displacing [3H]-yohimbine and 5.29 in displacing [3H]-prazosin (alpha 2/alpha 1 selectivity ratio in binding experiments > 14000). The compound had a pA2 of 9.72 as a competitive antagonist of the inhibitory effects of UK-14,304 in transmurally-stimulated guinea-pig ileum and 10.0 against BHT-920-induced contractions in dog saphenous vein (DSV); this latter value was unaltered by phenoxybenzamine. An apparent pKB of 5.9 was obtained against cirazoline-induced contractions in DSV, whilst a pA2 of 6.05 was obtained against phenylephrine-induced contractions in the rabbit aorta (alpha 2/alpha 1 selectivity ratio in functional experiments > 4000). 4. RS-15385-197 was highly selective for alpha 2-adrenoceptors over other receptors: the compound showed low affinity for 5-HT1A (pKi 6.50) and 5-HT1D (pKi 7.00) receptor subtypes, and even lower affinity (pKi < or = 5) for other 5-HT receptor subtypes, dopamine receptors, muscarinic cholinoceptors, beta-adrenoceptors and dihydropyridine binding sites. RS-15385-197 was devoid of affinity for the non-adrenoceptor imidazoline binding site, labelled by [3H]-idazoxan, which provides further evidence that these sites are not related to alpha 2-adrenoceptors. In the DSV, contractile responses to 5-hydroxytryptamine (5-HT) were unaffected by a concentration of 1 microM RS-15385-197. 5. RS-15385-197 was non-selective for the alpha 2A- and alpha 2B-adrenoceptor subtypes in that the pKi for the alpha 2A-adrenoceptor in human platelets was 9.90 and the pKi for the alpha 2B-adrenoceptor in rat neonate lung was 9.70. However, RS-15385-197 showed lower affinity for the alpha 2-adrenoceptor subtype in hamster adipocytes (pKi 8.38). 6. In anaesthetized rats, RS-15385-197 was a potent antagonist of the mydriasis response induced by UK-14,304 or clonidine (AD50 5 and 7 microg kg-1, i.v., respectively; 96 microg kg-1, p.o.) and of UK-14,304-induced pressor responses in pithed rats (AD50 7 microg kg-1, i.v.); the compound therefore is both centrally and orally active. Even at a high dose (10 mg kg-1, i.v.), RS-15385-197 did not antagonize pressor responses to cirazoline in pithed rats, indicating that the selectivity for alpha2 vs. alpha1-adrenoceptors was maintained in vivo.8 RS-15385-197 is therefore a very potent, selective, competitive alpha2-adrenoceptor antagonist, both in vitro and in vivo, is orally active and readily penetrates the brain. It will thus be a powerful pharmacological tool for exploring the various physiological roles of alpha2-adrenoceptors.