RESUMO
BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare and essentially incurable malignancy most often linked with occupational exposure to asbestos fibres. In common with other malignancies, the development and progression of MPM is associated with extensive dysregulation of cell cycle checkpoint proteins that modulate cell proliferation, apoptosis, DNA repair and senescence. METHODS: The expression of cyclin-dependent kinase inhibitor p16/INK4A was evaluated by immunohistochemistry using tumour biopsy specimens from 88 MPM cases and a semi-quantitative score for p16/INK4A expression was obtained. Post-diagnosis survival and the survival benefit of chemotherapeutic intervention was correlated with p16/INK4A expression. RESULTS: A low, intermediate and high score for p16/INK4A expression was observed for 45 (51.1%), 28 (31.8%) and 15 (17.1%) of the MPM cases, respectively. Those cases with intermediate or high p16/INK4A tumour expression had a significantly better post-diagnosis survival than those cases whose tumours lost p16 expression (log-rank P<0.001). Those patients with sustained p16/INK4A expression who received chemotherapy also had a better survival than those treated patients whose tumours had lost p16/INK4A expression (log-rank P<0.001). CONCLUSIONS: Sustained p16/INK4A expression predicts better post-diagnosis survival in MPM and also better survival following chemotherapeutic intervention.
Assuntos
Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Mesotelioma/tratamento farmacológico , Neoplasias Pleurais/tratamento farmacológico , Linhagem Celular Tumoral , Estudos de Coortes , Humanos , Mesotelioma/metabolismo , Mesotelioma/patologia , Neoplasias Pleurais/metabolismo , Neoplasias Pleurais/patologiaRESUMO
In this paper, we study breast cancer screening policies using computer simulation. We developed a multi-state Markov model for breast cancer progression, considering both the screening and treatment stages of breast cancer. The parameters of our model were estimated through data from the Canadian National Breast Cancer Screening Study as well as data in the relevant literature. Using computer simulation, we evaluated various screening policies to study the impact of mammography screening for age-based subpopulations in Canada. We also performed sensitivity analysis to examine the impact of certain parameters on number of deaths and total costs. The analysis comparing screening policies reveals that a policy in which women belonging to the 40-49 age group are not screened, whereas those belonging to the 50-59 and 60-69 age groups are screened once every 5 years, outperforms others with respect to cost per life saved. Our analysis also indicates that increasing the screening frequencies for the 50-59 and 60-69 age groups decrease mortality, and that the average number of deaths generally decreases with an increase in screening frequency. We found that screening annually for all age groups is associated with the highest costs per life saved. Our analysis thus reveals that cost per life saved increases with an increase in screening frequency.
Assuntos
Neoplasias da Mama/diagnóstico , Análise Custo-Benefício/estatística & dados numéricos , Detecção Precoce de Câncer/economia , Mamografia/economia , Programas de Rastreamento/economia , Adulto , Fatores Etários , Idoso , Canadá , Simulação por Computador , Análise Custo-Benefício/métodos , Detecção Precoce de Câncer/mortalidade , Feminino , Humanos , Mamografia/mortalidade , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Modelos Teóricos , Fatores de TempoRESUMO
BACKGROUND: The aim of screening is to detect a cancer in the preclinical state. However, a false-positive or a false-negative test result is a real possibility. METHODS: We describe invasive breast cancer progression in the Canadian National Breast Screening Study and construct progression models with and without covariates. The effect of risk factors on transition intensities and false-negative probability is investigated. We estimate the transition rates, the sojourn time and sensitivity of diagnostic tests for women aged 40-49 and 50-59. RESULTS: Although younger women have a slower transition rate from healthy state to preclinical, their screen-detected tumour becomes evident sooner. Women aged 50-59 have a higher mortality rate compared with younger women. The mean sojourn times for women aged 40-49 and 50-59 are 2.5 years (95% CI: 1.7, 3.8) and 3.0 years (95% CI: 2.1, 4.3), respectively. Sensitivity of diagnostic procedures for older women is estimated to be 0.75 (95% CI: 0.55, 0.88), while women aged 40-49 have a lower sensitivity (0.61, 95% CI: 0.42, 0.77). Age is the only factor that affects the false-negative probability. For women aged 40-49, 'age at entry', 'history of breast disease' and 'families with breast cancer' are found to be significant for some of the transition rates. For the age-group 50-59, 'age at entry', 'history of breast disease', 'menstruation length' and 'number of live births' are found to affect the transition rates. CONCLUSION: Modelling and estimating the parameters of cancer progression are essential steps towards evaluating the effectiveness of screening policies. The parameters include the transition rates, the preclinical sojourn time, the sensitivity, and the effect of different risk factors on cancer progression.
Assuntos
Neoplasias da Mama/patologia , Detecção Precoce de Câncer , Mamografia , Modelos Estatísticos , Adulto , Neoplasias da Mama/prevenção & controle , Canadá , Progressão da Doença , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Prognóstico , Fatores de RiscoRESUMO
BACKGROUND: Pseudomonas aeruginosa airway infection is associated with a high mortality rate in cystic fibrosis. Lipopolysaccharide (LPS), a main constituent of the outer membrane of P. aeruginosa, is responsible for activation of innate immune response but its role on airway epithelium ion transport, is not well known. The aim of this study was to determine the role for P. aeruginosa LPS in modulating chloride secretion and intracellular calcium in the human bronchial epithelial cell line, 16HBE14o-. METHODS: We used intracellular calcium imaging and short-circuit current measurement upon exposure of cells to P. aeruginosa LPS. RESULTS: Apical LPS stimulated intracellular calcium release and calcium entry and enhanced chloride secretion. This latter effect was significantly inhibited by CFTR(inh)-172 and BAPTA-AM (intracellular Ca(2+) chelator). CONCLUSIONS: Our data provides evidence for a new role of P. aeruginosa LPS in stimulating calcium entry and release and a subsequent chloride secretion via CFTR in human bronchial epithelium.
Assuntos
Brônquios/citologia , Cálcio/metabolismo , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Células Epiteliais/fisiologia , Lipopolissacarídeos/fisiologia , Pseudomonas aeruginosa , Transporte Biológico Ativo/efeitos dos fármacos , HumanosRESUMO
BACKGROUND: Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR. OBJECTIVES: The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects. METHODS: Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function. RESULTS: Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway. CONCLUSIONS: This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.
Assuntos
Peroxidase de Eosinófilo/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Mucosa Nasal/metabolismo , Rinite Alérgica Sazonal/metabolismo , Canais de Sódio/biossíntese , Canais Iônicos Sensíveis a Ácido , Adolescente , Adulto , Biópsia , Células Cultivadas , Células Epiteliais/patologia , Feminino , Humanos , Ácido Láctico/farmacologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mucosa Nasal/patologia , Reação em Cadeia da Polimerase , Rinite Alérgica Sazonal/patologiaRESUMO
BACKGROUND: The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A(2)α (cPLA(2)α) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA(2)α expression correlated with EGFR/HER2 over-expression in a small number of breast cancer cell lines. METHODS: The importance of differential cPLA(2)α activity in clinical breast cancer was established by relating the expression of cPLA(2)α in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters. RESULTS: High cPLA(2)α mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA(2)α expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA(2)α expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up. CONCLUSION: This study shows a role of cPLA(2)α in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker.
Assuntos
Neoplasias da Mama/enzimologia , Citosol/enzimologia , Receptores ErbB/metabolismo , Fosfolipases A2/metabolismo , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Estudos de Coortes , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fosfolipases A2/genética , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Aldosterone plays an important role in the regulation of blood pressure. The effects of this hormone have classically been described in terms of the transcriptional regulation of genes that facilitate electrolyte transport, particularly across high-resistance epithelia. The protein kinase signalling cascades that are rapidly activated in response to aldosterone are emerging as important modulators of the transcriptional response, and may serve to prime cells for the subsequent transcriptional changes. The activation of protein kinase D through an epidermal growth factor receptor transactivation pathway by aldosterone in renal cells has the potential to impact on cell trafficking events that regulate transporter activity.
Assuntos
Aldosterona/fisiologia , Proteína Quinase C/metabolismo , Transdução de Sinais , Receptores ErbB/genética , Fosforilação , Transcrição GênicaRESUMO
Glucocorticoids are anti-inflammatory molecules classically described as acting through a genomic pathway. Similar to many steroid hormones, glucocorticoids also induce rapid non-genomic responses. The present paper provides a general overview of the rapid non-genomic effects of glucocorticoids in airway and will be mainly focused on a retrospective of the authors work on rapid effects of glucocorticoids in airway epithelial cell transport. Using fluorescence microscopy, short circuit current measurements in human bronchial epithelial (16HBE14o(-)) cells, we reported rapid non-genomic effects of dexamethasone on cell signalling and ion transport. Dexamethasone (1 nM) rapidly stimulated Na(+)/H(+) exchanger activity and pH(i) regulation in 16HBE14o(-) cells. Dexamethasone also produced a rapid decrease of intracellular [Ca(2+)](i) to a new steady state concentration and inhibited the large and transient [Ca(2+)](i) increase induced by apical adenosine tri-phosphate (ATP). Dexamethasone also reduced by 1/3 the Ca(2+)-dependent Cl(-) secretion induced by apical ATP. The rapid effects of dexamethasone on intracellular pH and Ca(2+) were not affected by inhibitors of transcription, cycloheximide or by the classical glucocorticoid and mineralocorticoid receptors antagonists, RU486 and spironolactone, respectively. The rapid responses to glucocorticoid were reduced by the inhibitors of adenylated cyclase, cAMP-dependent protein kinase (PKA) and mitogen-activated protein kinase (ERK1/2). Our results demonstrate, that dexamethasone at low concentrations, rapidly regulates intracellular pH, Ca(2+) and PKA activity and inhibits Cl(-) secretion in human bronchial epithelial cells via a non-genomic mechanism which neither involve the classical glucocorticoid nor mineralocorticoid receptor.
Assuntos
Glucocorticoides/fisiologia , Mucosa Respiratória/metabolismo , Cálcio/antagonistas & inibidores , Cálcio/fisiologia , Dexametasona/farmacologia , Humanos , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
A non-genomic antisecretory role for dexamethasone at low concentrations (0.1 nM to1 microM) is described in monolayers of human bronchial epithelial cells in primary culture and in a continuous cell line (16HBE14o- cells). Dexamethasone produced a rapid decrease of [Ca(2+)](i) (measured with fura-2 spectrofluorescence) to a new steady-state concentration. After 15 min exposure to dexamethasone (1 nM), [Ca(2+)](i) was reduced by 32 +/- 11 nM (n = 7, P < 0.0001) from a basal value of 213 +/- 36 nM (n = 7). We have shown previously that aldosterone (1 nM) also produces a rapid fall in [Ca(2+)](i); however, after the decrease in [Ca(2+)](i) induced by dexamethasone, subsequent addition of aldosterone did not produced any further lowering of [Ca(2+)](i). The rapid response to dexamethasone was insensitive to pretreatment with cycloheximide and unaffected by the glucocorticoid type II and mineralocorticoid receptor antagonists RU486 and spironolactone, respectively. The rapid [Ca(2+)](i) decrease induced by dexamethasone was inhibited by the Ca(2+)-ATPase pump inhibitor thapsigargin (1 microM), the adenylate cyclase inhibitor MDL hydrochloride (500 microM) and the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphorothioate (200 microM), but was not affected by the protein kinase C inhibitor, chelerythrine chloride (0.1 microM). Treatment of 16HBE14o- cell monolayers with dexamethasone (1 nM) inhibited the large and transient [Ca(2+)](i) increase induced by apical exposure to ATP (10(-4) M). Dexamethasone (1 nM) also reduced by 30 % the Ca(2+)-dependant Cl(-) secretion induced by apical exposure to ATP (measured as the Cl(-)-sensitive short-circuit current across monolayers mounted in Ussing chambers). Our results demonstrate, for the first time, that dexamethasone at low concentrations inhibits Cl(-) secretion in human bronchial epithelial cells. The rapid inhibition of Cl(-) secretion induced by the synthetic glucocorticoid is associated with a rapid decrease in [Ca(2+)](i) via a non-genomic mechanism that does not involve the classical glucocorticoid or mineralocorticoid receptor. Rather, it is a result of rapid non-genomic stimulation of thapsigargin-sensitive Ca(2+)-ATPase, via adenylate cyclase and protein kinase A signalling.
Assuntos
Trifosfato de Adenosina/farmacologia , Brônquios/metabolismo , Cloretos/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Cálcio/metabolismo , Linhagem Celular , Cicloeximida/farmacologia , Epitélio/metabolismo , Antagonistas de Hormônios/farmacologia , Humanos , Membranas Intracelulares/metabolismo , Mifepristona/farmacologia , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Concentração Osmolar , Inibidores da Síntese de Proteínas/farmacologia , Transdução de Sinais/fisiologia , Espironolactona/farmacologia , Fatores de TempoRESUMO
The effect of external ATP on intracellular pH (pH(i)) was investigated using a pH imaging system in a human bronchial epithelial cell line (16HBE14o-) loaded with BCECF-AM. The steady-state pH(i) of 16HBE14o- epithelial monolayers was 7.137 +/- 0.027 (n = 46). Apical addition of ATP (10(-4) M) to epithelial monolayers induced a rapid and sustained pH(i) decrease of 0.164 +/- 0.024 pH units (n = 17; P < 0.001). The intracellular acidification was rapidly reversed upon removal of external ATP. In contrast, the non-hydrolysable ATP analogue AMP-PNP did not produce any significant change in pH(i). Inhibition of purinoreceptors by suramin did not affect the acidification induced by apical ATP. Inhibition of Na+-H+ exchange by apical Na+ removal or addition of amiloride (0.5 mM) reduced the apical ATP-induced pH(i) decrease, suggesting the involvement of a Na+-H+ exchanger or surface pH effects on the ATP-induced pH(i) response. Inhibitors of proton channels such as ZnCl2 (10(-4) M) also partially inhibited the ATP response. The pH(i) response to ATP was dependent on the external pH (pH(o)), with increasing acidification produced at lower pH(o) values. Neither the basal pH(i) nor the ATP-induced intracellular acidification was affected by thapsigargin (a Ca2+-ATPase inhibitor), chelerythrine chloride (a protein kinase C (PKC) inhibitor), RpcAMP (a protein kinase A (PKA) inhibitor) or PMA (a PKC activator). Therefore, the intracellular acidification of human bronchial epithelial cells induced by apical ATP does not involve signalling via Ca2+, PKC or PKA nor binding to a purinoreceptor. We interpret the effect of ATP to produce an intracellular acidification as a three step process: activation of H+ channels, inhibition of Na+-H+ exchange and influx of protonated ATP.
Assuntos
Trifosfato de Adenosina/farmacologia , Células Epiteliais/metabolismo , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Adenilil Imidodifosfato/farmacologia , Brônquios/citologia , Cálcio/metabolismo , Linhagem Celular , Polaridade Celular/fisiologia , Cloretos/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/citologia , Humanos , Proteína Quinase C/metabolismo , Receptores Purinérgicos/metabolismo , Mucosa Respiratória/citologia , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/metabolismo , Compostos de Zinco/farmacologiaRESUMO
BACKGROUND: Renin-angiotensin system (RAS) components have been identified in human ciliary body and aqueous humour, pointing to a role for the RAS in the regulation of aqueous humour dynamics. Here, the authors examine the functional expression of a RAS and the effects of angiotensin II (AII) on a signal transduction pathway and ion secretion mechanism in cultured human ciliary body non-pigmented epithelium (HNPE). METHODS: RAS expression was examined in cultured HNPE cells using polymerase chain reaction (PCR) analysis. Secretory function was determined using spectrofluorescence imaging microscopy to measure cell calcium (Ca(2+)(I)) and volume responses. Single channel patch clamp techniques were employed to investigate ion channel activity. RESULTS: PCR analysis demonstrated the expression of angiotensinogen and the AT(1b) receptor in HNPE cells. A large conductance potassium (BK) channel (mean 190 (SEM 5.6) pS, n = 22 cells), was observed in plasma membrane patches. This channel was calcium sensitive with channel open probability (Po) increasing with increasing Ca(2+)(I) (K(0.5) 10.79 (0.44) microM Ca(2+), Hill coefficient of 1.04 (0.04)). AII (100 nM) increased the number (N) of active BK channels in HNPE cells and also the probability of channel opening (Po). N.P(o) increased from 0.008 (0.002) to 1.38 (0.4) following the addition of AII (p=0.0064). AII also induced a rapid rise in Ca(2+)(I) from resting values of 112 (14) nM to a peak of 992 (106) nM (p<10(-4)). A simultaneous cell volume reduction of 24.70% (3.34%) (p<10(-4)) occurred during this calcium signal. Losartan (1 microM) significantly blocked the AII induced BK channel activation (p=0.0131), the Ca(2+)(I) response (p<10(-4)), and the AII induced volume effect (p=0.0046). CONCLUSION: It was demonstrated that AII activates a Ca(2+)(I) signalling system which subsequently increases potassium ion channel activity. These effects are accompanied simultaneously by cell volume loss, indicating that AII acts as receptor operated secretagogue in HNPE cells. The ability of an AT(1) receptor antagonist to inhibit these processes may thus offer a new family of pharmaceutical agents to the current armamentarium in the treatment of glaucoma.
Assuntos
Corpo Ciliar/citologia , Epitélio Pigmentado Ocular/citologia , Sistema Renina-Angiotensina/fisiologia , Angiotensina II/farmacologia , Cálcio/fisiologia , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Corpo Ciliar/metabolismo , Eletrofisiologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta , Técnicas de Patch-Clamp , Epitélio Pigmentado Ocular/metabolismo , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/fisiologia , Receptores de Angiotensina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
1. Using a Ca(2+) imaging system and fura-2 AM (5 microM) we showed that exposure of polarised monolayers of human bronchial epithelial cells (16HBE14o- cell line) to aldosterone produced a fast intracellular [Ca(2+)] ([Ca(2+)](i)) decrease, in 70 % of cells. Exposure to aldosterone (1 nM) reduced the [Ca(2+)](i) by 39 +/- 9 nM (n = 282, P < 0.0001) within 10 min, from a basal [Ca(2+)](i) of 131 +/- 19 nM (n = 282). 2. The effect of aldosterone on [Ca(2+)](i) was not affected by inhibitors of the classical genomic pathway, cycloheximide (1 microM) or spironolactone (10 microM). The aldosterone-induced [Ca(2+)](i) decrease was inhibited by thapsigargin (1 microM), pertussis toxin (24 h at 200 ng ml(-1)), the adenylate cyclase inhibitors 2',3'-dideoxyadenosine (200 microM) and MDL-12,330A hydrochloride (500 microM), and the protein kinase A inhibitor R(P)-adenosine 3',5'-cyclic monophosphorothioate (200 microM). In addition, treatment of 16HBE14o- monolayers with aldosterone (1 nM) inhibited by approximately 30 % the large and transient [Ca(2+)](i) increase induced by apical exposure to uridine triphosphate (UTP, 0.1 mM), a known secretagogue in airway epithelia. 3. Our results demonstrate for the first time that in human bronchial epithelial cells, aldosterone decreases [Ca(2+)](i) levels via a non-genomic mechanism. The hormone-induced changes to [Ca(2+)](i) involve stimulation of thapsigargin-sensitive Ca(2+)-ATPase, via G-protein-, adenylate cyclase- and protein kinase A-coupled signalling pathways.
Assuntos
Aldosterona/farmacologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Cálcio/metabolismo , Membranas Intracelulares/metabolismo , Adenilil Ciclases/fisiologia , Brônquios/citologia , Linhagem Celular Transformada , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Cicloeximida/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Glucocorticoides/farmacologia , Humanos , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Concentração Osmolar , Inibidores da Síntese de Proteínas/farmacologia , Sódio/farmacologia , Espironolactona/farmacologia , Tapsigargina/farmacologia , Fatores de Tempo , Uridina Trifosfato/farmacologia , Vanadatos/farmacologiaRESUMO
Recent evidence points to protein kinase C isoforms as highly specific receptors for aldosterone and estradiol in epithelia. The end targets of the kinase activation are Na(+)/H(+) exchange and K(+) and Ca(2+) channels. The physiological role of the nongenomic response is to increase electrolyte absorption and inhibit secretion in pluripotential epithelia.
Assuntos
Aldosterona/fisiologia , Estradiol/fisiologia , Caracteres Sexuais , Animais , Epitélio/fisiologia , Humanos , Transdução de Sinais/fisiologiaRESUMO
Oestrogen plays an essential role in regulating growth and differentiation in the human endometrium which undergoes dynamic morphological and functional changes during the menstrual cycle in preparation for implantation. In this tissue, it has been suggested that intracellular calcium could be a key signal in transducing early responses to steroid hormones. Here, we have investigated the rapid effects of 17beta-oestradiol on [Ca2+]i in a human endometrial cell line (RL95-2). Using confocal imaging microscopy, we show that physiological concentrations of 17beta-oestradiol trigger rapid and transient increases in [Ca2+]i. Our results demonstrate that 17beta-oestradiol-induced [Ca2+]i variations are critically dependent on calcium influx via lanthanum-sensitive calcium channels. Moreover, the 17beta-oestradiol-induced Ca2+ influx is significantly increased by the depletion of intracellular stores by thapsigargin and decreased by chelerythrine chloride, an inhibitor of protein kinase C. These data indicate a non-genomic action of 17beta-oestradiol to stimulate capacitative Ca2+ entry through store-operated calcium channels via a PKC-sensitive pathway.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Estradiol/farmacologia , Alcaloides , Benzofenantridinas , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Endométrio/enzimologia , Feminino , Humanos , Microscopia Confocal , Fenantridinas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Tapsigargina/farmacologiaRESUMO
In this study we used the short circuit current (ISC) technique to measure the non-genomic effects of the female sex steroid 17beta-oestradiol (E2) on electrogenic transepithelial ion transport in rat distal colonic epithelium. Basal ISC was largely composed of a transepithelial Cl- secretory component with minimal electrogenic Na+ movement. E2 (1-100 nM) caused a significant decrease in basal ISC after 15 min. In addition, pre-treating colonic epithelial tissues with E2 (0.1-100 nM) for 10 min significantly reduced forskolin (20 microM)-induced Cl- secretion. E2 also down-regulated Cl- secretion which was pre-stimulated by forskolin. Cl- secretory responses to the Ca2+-dependent secretagogue carbachol (10 microM) were also significantly reduced in the presence of E2 (10- 100 nM). However, E2 had no effect on amiloride-sensitive Na+ absorption. The rapid anti-secretory effect of E2 was abolished in the presence of the intracellular Ca2+ chelator BAPTA (50 microM) or the protein kinase C (PKC) inhibitor chelerythrine chloride (1 microM). However, in the presence of the nuclear oestrogen receptor antagonist tamoxifen (10 microM), E2 still produced an inhibition of Cl- secretion. Testosterone, progesterone and 17alpha-oestradiol had no significant effect on colonic Cl- secretion. Also, E2 (100 nM) did not alter Cl- secretion in colonic epithelia isolated from male rats. We conclude that E2 inhibits colonic Cl- secretion via a non-genomic pathway that involves intracellular Ca2+ and PKC. It is possible that this gender-specific mechanism contributes to the salt and water retention associated with high E2 states.
Assuntos
Cloretos/metabolismo , Colo/metabolismo , Estradiol/farmacologia , Animais , Carbacol/farmacologia , Quelantes/farmacologia , Colforsina/farmacologia , Colo/enzimologia , Regulação para Baixo/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/enzimologia , Epitélio/metabolismo , Antagonistas de Estrogênios/farmacologia , Técnicas In Vitro , Masculino , Agonistas Muscarínicos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Tamoxifeno/farmacologiaRESUMO
PURPOSE: To examine the effects of extracellular adenosine 5-triphosphate (ATP) on intracellular pH ([pH](i)) in cultured human non-pigmented ciliary body epithelium (HNPE). METHODS: Intracellular pH was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. RESULTS: In 5%CO(2)/HCO(3)(-) buffered Ringer's the resting [pH](i) was 7.25 +/- 0.006 (mean +/- SEM). Application of 10 microM ATP significantly decreased [pH](i) to 7.00 +/- 0.007 (P < 10(-5), n = 14). In the presence of 1 mM suramin, a P(2) receptor inhibitor, this process was significantly blocked. This [pH](i) effect required the presence of Cl(-) and was significantly inhibited by 0.1 mM diisothiocyanatostilbene-2-2'-disulfonic acid or acetazolamide (500 microM), indicating the involvement of a Cl(-)/HCO(3)( +) exchange mechanism. This response exhibited little dependence on external Na(+) and remained unaffected by the addition of the Na(+)/H( +) exchanger inhibitor amiloride (1 mM). Clamping intracellular calcium levels by incubation in the cell permeable calcium chelator, the acetoxymethyl ester form of BAPTA (100 microM) in low extracellular calcium solution (pCa9) did not affect the ATP-induced [pH](i) signal. In addition, the vacuolar H(+)-ATPase (V-ATPase) inhibitor, bafilomycin A(1) (1 microM), failed to alter the [pH](i) transient. CONCLUSION: We have demonstrated that extracellular ATP leads to a sustained increase in [H(+)](i) in HNPE cells via a purinergic receptor activated pathway which is independent of the intracellular calcium signaling system. This study demonstrates that the ATP induced [pH]( i) transient is mediated through an upregulation in Cl(-)/HCO( 3)(-) exchange across the plasmamembrane in HNPE cells.
Assuntos
Trifosfato de Adenosina/farmacologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloretos/metabolismo , Corpo Ciliar/citologia , Macrolídeos , Epitélio Pigmentado Ocular/efeitos dos fármacos , Antibacterianos/farmacologia , Cálcio/metabolismo , Células Cultivadas , Fluoresceínas , Corantes Fluorescentes , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Fluorescência , Epitélio Pigmentado Ocular/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , Receptores Purinérgicos/metabolismo , Espectrometria de Fluorescência , Suramina/farmacologia , Regulação para CimaRESUMO
PURPOSE: To determine the effects of extracellular ATP on calcium signaling in cultured human non-pigmented ciliary body epithelium (HNPE). METHODS: Intracellular calcium (Ca(2+)(i)) was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the fluorescent dye Fura-2. RESULTS: Nucleotides caused a transient oscillatory increase in Ca(2+)(i) with a potency order of ATP = UTP > ADP > AMP> alpha,beta-methylene-ATP. Treatment with thapsigargin (100 nM), an inhibitor of endoplasmic Ca(2+)-ATPase pumps, produced a sustained increase in Ca(2+)(i). Subsequent exposure to ATP caused a rapid reduction in Ca(2+)(i) and this effect was reduced by pre-exposure to vanadate and to a lesser extent in sodium free solution. Prolonged exposure to ATP in the presence of thapsigargin caused a transient spike increase in Ca(2+)(i) which was prevented by exposure to low extracellular Ca(2+) (1 nmol/l), verapamil, nifedipine or the microfilament disrupting agent, cytochalasin B. CONCLUSIONS: These results provide evidence for ATP mobilisation of Ca(2+) from intracellular stores via P2Y2 receptor activation in HNPE cells. ATP also primarily activates a vanadate-sensitive Ca(2+ )-ATPase pump, in addition to having a smaller effect on the Na( +)/ Ca(2+) exchanger in terminating the calcium signal. Capacitative calcium entry, possibly via an L-type Ca(2+) channel, is implicated in generating a calcium signal following emptying of intracellular stores and is sensitive to cytoskeleton disruption. ATP can thus regulate a potent intracellular signal for secretion, suggest-ing that purinergic receptors may provide a therapeutic target in glaucoma.
Assuntos
Trifosfato de Adenosina/farmacologia , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Corpo Ciliar/citologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Transdução de Sinais , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Células Cultivadas , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Humanos , Microscopia de Fluorescência , Epitélio Pigmentado Ocular/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2 , Trocador de Sódio e Cálcio/metabolismo , Espectrometria de Fluorescência , Tapsigargina/farmacologiaRESUMO
BACKGROUND: Oestrogens are important mitogens in epithelial cancers, particularly where tumours express complementary receptors. While the traditional model of oestrogen action involves gene-directed (genomic) protein synthesis, it has been established that more rapid, non-genomic steroid hormone actions exist. This study investigated the hypothesis that oestrogen rapidly alters cell membrane activity, intracellular pH and nuclear kinetics in a mitogenic fashion. METHODS: Crypts isolated from human distal colon and colorectal cancer cell lines were used as robust models. DNA replication and intracellular pH were measured by radiolabelled thymidine incorporation (12 h) and spectrofluorescence imaging respectively. Genomic protein synthesis, sodium-hydrogen exchanger (NHE) and protein kinase C (PKC) activity were inhibited with cycloheximide, ethylisopropylamiloride and chelerythrine chloride respectively. RESULTS: Oestrogen induced a rapid (less than 5 min) cellular alkalinization of crypts and cancer cells that was sensitive to NHE blockade (P < 0.01) or PKC inhibition (P < 0.01). Oestrogen increased thymidine incorporation by 44 per cent in crypts and by up to 38 per cent in cancer cells (P < 0.01), and this was similarly reduced by inhibiting the NHE (P < 0.01) or PKC (P < 0.05). CONCLUSION: Oestrogen rapidly activates cell membrane and nuclear kinetics by a non-genomic mechanism mediated by PKC but not gene-directed protein synthesis.
Assuntos
Colo/efeitos dos fármacos , Estrogênios/farmacologia , Mitógenos/farmacologia , Receptores de Estrogênio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Colo/citologia , Replicação do DNA , Humanos , Receptores de Estrogênio/fisiologia , Espectrometria de Fluorescência/métodos , Células Tumorais CultivadasRESUMO
1. We investigated the effect of oestradiol on basolateral potassium channels in human colonic epithelium. 2. Ion transport was quantified using short circuit current (I:(sc)) measurements of samples mounted in Ussing chambers. Serosal K transport was studied using nystatin permeabilization of the apical membrane. Intracellular pH changes were quantified using spectroflouresence techniques. 3. Experiments were performed with either 10 nM or 1 microM Ca(2+) in the apical bathing solution. With 10 nM Ca(2+) in the apical bathing solution addition of oestradiol (1 nM) to the basolateral bath produced a rapid increase in current (delta I(K)=11.2+/-1.2 microA.cm(-2), n=6). This response was prevented by treatment of the serosal membrane with tolbutamide (1 microM). With 1 microM Ca(2+) in the apical bathing solution addition of oestradiol produced a rapid fall in current (delta I(K)=-12.8+/-1.4 microA.cm(-2)), this response was prevented by treatment of the basolateral membrane with tetra-pentyl-ammonium (TPeA). These responses were rapid and occurred independently of protein synthesis. 4. Inhibition of basolateral Na(+)/H(+) exchange with either amiloride or a low sodium bathing solution prevented this response. These responses were prevented by inhibition of protein kinase C (PKC) with bis-indolyl-maleimide. 5. Oestradiol (1 nM) produced a rapid intracellular alkanization (mean increase=0.11 pH units; n=6; P<0.01). 6. These results suggest that oestradiol rapidly modulates serosal K transport in human colon. These effects depend upon intact Na(+)/H(+) exchange and protein kinase C. We propose a non-classical, possibly membrane linked, mechanism for oestradiol action in human colonic epithelium.
Assuntos
Colo/efeitos dos fármacos , Estradiol/farmacologia , Potássio/farmacocinética , Cálcio/farmacologia , Colo/metabolismo , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Antagonistas de Estrogênios/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Compostos de Amônio Quaternário/farmacologia , Membrana Serosa/efeitos dos fármacos , Membrana Serosa/metabolismo , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/fisiologia , Tamoxifeno/farmacologia , Tolbutamida/farmacologiaRESUMO
In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl(-) secretion to secretagogues acting via cAMP. Using a Ca(2+) imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca(2+)](i) via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase in [Ca(2+)](i) in normal (16HBE14o(-) cell line and primary lung culture) and in cystic fibrosis (CFTE29o(-) cell line) human airway epithelia. The potency order of nucleotides on [Ca(2+)](i) variation was UTP >> ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca(2+)](i) response could be mimicked by activation of CFTR with forskolin (20 microm) in a temperature-dependent manner. In 16HBE14o(-) cells, the forskolin-induced [Ca(2+)](i) response increased with increasing temperature. In CFTE29o(-) cells, forskolin had no effect on [Ca(2+)](i) at body temperature-forskolin-induced [Ca(2+)](i) response in CF cells could only be observed at low experimental temperature (14 degrees C) or when cells were cultured at 26 degrees C instead of 37 degrees C. Pretreatment with CFTR channel blockers glibenclamide (100 microm) and DPC (100 microm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 microm), inhibited the forskolin [Ca(2+)](i) response. Together, these results demonstrate that once activated, CFTR regulates [Ca(2+)](i) by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia.