RESUMO
Mucociliary clearance, which is conducted by beating cilia cooperating with the surface mucous layer, is a major host defense mechanism of the airway epithelium. Ezrin, a crosslinker between membrane proteins and the actin cytoskeleton, is located in microvilli and around the basal bodies in airway ciliary cells. It is also likely that ezrin plays an important role in apical localization of ß2 adrenergic receptor (ß2AR) in airway ciliary cells. Here, we studied the physiological roles of ezrin by using trachea and airway epithelial cells prepared from ezrin-knockdown (Vil2kd/kd) mice. The trachea and airway ciliary cells of Vil2kd/kd mice presented a normal morphology and basal body orientation, suggesting that ezrin is not directly involved in development and planar cell polarity of cilia. Procaterol stimulates ciliary beating (frequency and amplitude) via ß2AR in the airway ciliary cells. In the Vil2kd/kd mice, airway ciliary beating stimulated with procaterol was partly inhibited due to the impairment of cell surface expression of ß2AR. These results suggest that ezrin regulates the beating of airway ciliary cells by promoting the apical surface localization of ß2AR. This article has an associated First Person interview with the first author of the paper.
Assuntos
Cílios , Procaterol , Animais , Cílios/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Humanos , Camundongos , Procaterol/metabolismo , Procaterol/farmacologia , Traqueia/metabolismoRESUMO
Background: Gastric cancer remains the second leading cause of cancer-related death, and the third in mortality due to lack of effective therapeutic targets for late stage cancer patients. This study aims to identify potential druggable target biomarkers as potential therapeutic options for patients with gastric cancer. Methods: Immunohistochemistry of human gastric tumor tissues was conducted to determine the expression level of cyclin-dependent kinase 12 (CDK12). Multiple in vitro and in vivo assays such as RNAi, mass spectrometry, computer docking models, kinase assays, cell xenograft NU/NU mouse models (CDXs) and patient-derived xenograft NOD/SCID mouse models (PDXs) were conducted to study the function and molecular interaction of CDK12 with p21 activated kinase 2 (PAK2), as well as to find CDK12 inhibitors as potential treatment options for human gastric cancer. Results: Here we identified that CDK12 is a driver gene in human gastric cancer growth. Mechanistically, CDK12 directly binds to and phosphorylates PAK2 at T134/T169 to activate MAPK signaling pathway. We further identified FDA approved clinical drug procaterol can serve as an effective CDK12 inhibitor, leading to dramatic restriction of cancer cell proliferation and tumor growth in human gastric cancer cells and PDXs. Conclusions: Our data highlight the potential of CDK12/PAK2 as therapeutic targets for patients with gastric cancer, and we propose procaterol treatment as a novel therapeutic strategy for human gastric cancer.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Procaterol/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Quinases Ativadas por p21/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Broncodilatadores/farmacologia , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Fosforilação , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/metabolismoRESUMO
Sei-hai-to (TJ-90, Qing Fei Tang), a Chinese traditional medicine, increases ciliary beat frequency (CBF) and ciliary bend angle (CBA) mediated via cAMP (3',5'-cyclic adenosine monophosphate) accumulation modulated by Ca2+-activated phosphodiesterase 1 (PDE1A). A high concentration of TJ-90 (≥40 µg/mL) induced two types of CBF increases, a transient increase (an initial increase, followed by a decrease) and a sustained increase without any decline, while it only sustained the CBA increase. Upon inhibiting increases in intracellular Ca2+ concentration ([Ca2+]i) by 10 µM BAPTA-AM (Ca2+-chelator, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or Ca2+/calmodulin-dependent PDE1 by 8MmIBMX (a selective PDE1 inhibitor), TJ-90 (400 µg/mL) induced only the sustained CBF increase without any transient CBF increase. The two types of the CBF increase (the transient increase and the sustained increase) induced by TJ-90 (≥40 µg/mL) were mimicked by the stimulation with both procaterol (100 pM) and ionomycin (500 nM). Thus, TJ-90 stimulates small increases in the intracellular cAMP concentration ([cAMP]i) and [Ca2+]i in airway ciliary cells of mice. These small increases in [cAMP]i and [Ca2+]i cause inducing a transient CBF increase or a sustained CBF increase in an airway ciliary cells, depending on the dominant signal, Ca2+-signal, or cAMP-signal.
Assuntos
Cálcio/metabolismo , Cílios/efeitos dos fármacos , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Feminino , Camundongos , Nigericina/análogos & derivados , Nigericina/farmacologia , Procaterol/farmacologiaRESUMO
BACKGROUND: Genetic variation in the ß2-adrenergic receptor (ADRB2) gene has been thought to have an important role in the differential response to ß2-agonist therapy for asthma. However, previous studies have shown little evidence for an association between these ADRB2 variants and the bronchial dilator response (BDR) in chronic obstructive pulmonary disease (COPD) patients. This discrepancy could be explained by differences in the distribution and heterogeneity of pulmonary emphysema in COPD patients, since emphysema distribution and heterogeneity are thought to have a role in pulmonary function in COPD patients. We hypothesized that differences in emphysema distribution and heterogeneity may have masked significant alterations of the bronchodilator response among ADRB2 genotypes in COPD patients in previous studies. METHODS: The BDR (induced by 20 µg of procaterol) was measured in 211 patients who had a smoking history of more than 10 pack/years and had undergone chest high resolution computed tomography examination. A low attenuations area (<960 Hounsfield Units) was identified and the emphysema heterogeneity index (EHI%) was calculated with a range in value from -100% to 100%. ADRB2 Arg16Gly genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: The BDR was augmented in patients with homogenous emphysema compared with those with upper-dominant emphysema. In patients carrying the AA genotype of ADRB2, the BDR was significantly increased in patients with upper-dominant emphysema, but not in patients with lower-dominant emphysema. CONCLUSION: Combination analysis of ADRB2 Arg16Gly polymorphism and EHI% may predict the effectiveness of ß2-adrenergic receptor agonist treatment in patients with COPD and emphysema.
Assuntos
Broncodilatadores/farmacologia , Procaterol/farmacologia , Enfisema Pulmonar/tratamento farmacológico , Receptores Adrenérgicos beta 2/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Enfisema Pulmonar/fisiopatologia , Tomografia Computadorizada por Raios XRESUMO
This study demonstrated that PDE1 (phosphodiesterase 1) existing in the ciliary beat frequency (CBF)-regulating metabolon regulates CBF in procaterol-stimulated lung airway ciliary cells of mouse. Procaterol (an ß2-agonist) increased the ciliary bend angle (CBA) and CBF via cAMP accumulation in the ciliary cells of mice: interestingly, the time course of CBF increase was slower than that of CBA increase. However, IBMX (3-isobutyl-1-methylxanthine, an inhibitor of PDE) increased CBA and CBF in an identical time course. Lowering an intracellular Ca2+ concentration ([Ca2+]i) caused by switching to an EGTA-containing Ca2+-free solution from normal one elevated the procaterol-induced increasing rate of CBF. These observations suggest that Ca2+-dependent PDE1 controls cAMP-stimulated CBF increase. Either application of 8MmIBMX (8-methoxymethyl-IBMX, a selective PDE1 inhibitor), BAPTA-AM (an intracellular Ca2+ chelator), or calmidazolium (an inhibitior of calmodulin) alone increased CBA and CBF in the lung airway ciliary cells and increased cAMP contents in the isolated lung cells, and like IBMX, each application of the compound made the time courses of CBA and CBF increase stimulated by procaterol identical. The immunoelectron microscopic examinations revealed that PDE1A exists in the space between the nine doublet tubules ring and plasma membrane in the lung airway cilium, where the outer dynein arm (a molecular motor regulating CBF) functions. In conclusion, PDE1A is a key factor slowing the time course of the procaterol-induced increase in CBF via degradation of cAMP in the CBF-regulating metabolon of the mouse lung airway cilia.
Assuntos
Cálcio/farmacologia , Cílios/efeitos dos fármacos , Cílios/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/antagonistas & inibidores , Animais , Calmodulina/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Procaterol/farmacologiaRESUMO
OBJECTIVE: It has been hypothesized that some patients with chest tightness of unknown origin can be successfully treated with a bronchodilator and that they should be diagnosed with chest pain variant asthma. We conducted a prospective study to characterize newly diagnosed patients with chest tightness relieved with bronchodilator use and without characteristic bronchial asthma attacks. METHODS: Eleven patients were registered following recurrent positive responses of chest tightness to inhalation of a ß2-agonist. These patients underwent assessments of airway responsiveness to methacholine, bronchial biopsy and bronchial lavage under fiber-optic bronchoscopy before receiving treatment. RESULTS: For the patients with chest tightness relieved with bronchodilator use, the bronchial biopsy specimens exhibited significant increases in lymphocyte and macrophage infiltration (p < 0.05) and no significant increase in eosinophils (p = 0.2918) compared with the control subjects. The bronchial responsiveness to methacholine was increased in two of the patients with chest tightness, and it was not increased in seven; in addition, increased percentages of eosinophils were detected in bronchial lavage fluid (5% or more) from two patients, but no increase was detected in eight patients. CONCLUSIONS: We suspect that the chest tightness was induced by airway constriction in these patients, but further study is necessary to validate this hypothesis. We propose that the chest tightness relieved with bronchodilator use was attributed to airway constriction resulting from inflammation with lymphocytes and macrophages and/or that the chest tightness was directly attributed to airway inflammation. This clinical trial is registered at www.umin.ac.jp (UMIN13994 and UMIN 16741).
Assuntos
Broncodilatadores/farmacologia , Broncodilatadores/uso terapêutico , Dor no Peito/tratamento farmacológico , Dor no Peito/imunologia , Administração por Inalação , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Adulto , Idoso , Obstrução das Vias Respiratórias/tratamento farmacológico , Obstrução das Vias Respiratórias/imunologia , Asma/tratamento farmacológico , Asma/imunologia , Hiper-Reatividade Brônquica , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Broncoscopia , Doença Crônica , Eosinófilos/metabolismo , Feminino , Fluticasona/farmacologia , Fluticasona/uso terapêutico , Humanos , Linfócitos/metabolismo , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Procaterol/farmacologia , Procaterol/uso terapêutico , Estudos Prospectivos , Testes de Função RespiratóriaRESUMO
OBJECTIVE: Our investigation was designed to examine the signaling pathway involved in the enhancement of vascular endothelial growth factor (VEGF) release by ß-adrenoceptor agonists. MATERIALS AND METHODS: Human U937 cells differentiated into macrophages were primed with lipopolysaccharide (LPS) in the absence or presence of ß-adrenoceptor agonists and antagonists. The VEGF released and the intracellular cyclic adenosine monophosphate (cAMP) generated were assayed by ELISA. Where necessary, differences between mean values were tested for significance using Student's t test. RESULTS: Isoprenaline, procaterol and salbutamol concentration-dependently enhanced the release of VEGF induced by LPS in U937 cells. R*,R*-(±)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344), a selective ß3-adrenoceptor agonist, did not enhance VEGF release. Using isoprenaline as an agonist, propranolol, ICI 118551 and atenolol produced a parallel rightward shift of the concentration-response curve with no reduction in the maximum response. The -logKB values were 8.12 ± 0.17, 8.03 ± 0.05 and 7.23 ± 0.05 for propranolol, ICI 118551 and atenolol, respectively, indicating the possible involvement of both ß1- and ß2-adrenoceptor subtypes. Isoprenaline and prostaglandin E2 concentration-dependently increased cAMP generation in U937 cells. Isoprenaline, db-cAMP and 6-Bnz-cAMP, a protein kinase A (PKA) activator, all enhanced VEGF release induced by LPS, and this effect was abolished by KT 5720 and Rp-cAMPS, which are both selective PKA inhibitors, suggesting that PKA is the downstream effector of cAMP activity. 8-CPT-cAMP, a selective activator of the Epac system, had no effect on VEGF release induced by LPS, indicating that the Epac pathway played no role in the release process. CONCLUSION: In this study, we established that ß1- and ß2- but not ß3-adrenoceptors mediated cAMP-dependent enhancement of VEGF release induced by LPS in differentiated U937 cells, and that PKA was the downstream effector of cAMP activity.
Assuntos
Antagonistas Adrenérgicos/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , AMP Cíclico/biossíntese , Macrófagos/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/biossíntese , Albuterol/farmacologia , Atenolol/farmacologia , Carbazóis/farmacologia , Relação Dose-Resposta a Droga , Etanolaminas/farmacologia , Humanos , Isoproterenol/farmacologia , Lipopolissacarídeos , Procaterol/farmacologia , Pirróis/farmacologiaRESUMO
BACKGROUND AND OBJECTIVE: Procaterol is a potent ß2-agonist frequently used for the management of asthma and chronic obstructive pulmonary disease. The efficacy and adverse effects of ß2-agonists are heterogeneous in individual patients, which may be partly caused by genetic variations in metabolizing enzymes and receptor molecules. The present study was designed to analyze the relationship between gene polymorphisms and physiological effects of procaterol in healthy subjects. METHODS: Ninety-two non-smoking healthy volunteers were given 1 µg/kg body weight (max 50 µg) of procaterol as a dry syrup preparation, and the serum concentrations of procaterol, serum K(+), and the physical responses were monitored for 240 min. We genotyped ß2-adrenergic receptor (ADRB2) (Arg16Gly and Gln27Glu), cytochrome P450 3A4 (rs2246709, rs4646437), and uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) (rs4148323 [allele A, *6], rs12479045, rs4148328, rs4663971, rs12052787, rs4148329, A (TA)6/7 TAA [seven-repeat allele, *28]). Procaterol concentrations in serum were measured by liquid chromatography-tandem mass spectrometry. RESULTS: No gene polymorphisms affected serum procaterol concentrations. Meanwhile, overall serum K(+) level changes were significantly lower in carriers of UGT1A1*28 than in non-carriers after correcting for strong effects of serum procaterol concentrations and baseline K(+) levels. No other polymorphisms were associated with serum K(+) levels. None of polymorphisms of ADRB2 were associated with any physical responses. CONCLUSION: The present study indicates that significant hypokalemia may occur in carriers of UGT1A1*28 by systemic administration of procaterol and potentially by other ß2-agonists metabolized in the liver.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Asma/genética , Broncodilatadores/farmacologia , Potássio/sangue , Procaterol/farmacologia , Adulto , Citocromo P-450 CYP3A/genética , Feminino , Genótipo , Glucuronosiltransferase/genética , Humanos , Japão , Masculino , Polimorfismo Genético , Receptores Adrenérgicos beta 2/genéticaRESUMO
The elderly are at much higher risk for developing pneumonia than younger individuals. Pneumonia is a leading cause of death and is the third most common reason for hospitalization in the elderly. One reason that elderly people may be more susceptible to pneumonia is a breakdown in the lung's first line of defense, mucociliary clearance. Cilia beat in a coordinated manner to propel out invading microorganisms and particles. Ciliary beat frequency (CBF) is known to slow with aging, however, little is known about the mechanism(s) involved. We compared the CBF in BALB/c and C57BL/6 mice aged 2, 12, and 24 mo and found that CBF diminishes with age. Cilia in the mice at age 12 and 24 mo retained their ability to be stimulated by the ß2 agonist procaterol. To help determine the mechanism of ciliary slowing, we measured protein kinase C alpha and epsilon (PKCα and PKCε) activity. There were no activity differences in PKCα between the mice aged 2, 12, or 24 mo. However, we demonstrated a significantly higher PKCε activity in the mice at 12 and 24 mo than the in the mice 2 mo of age. The increase in activity is likely due to a nearly threefold increase in PKCε protein in the lung during aging. To strengthen the connection between activation of PKCε and ciliary slowing, we treated tracheas of mice at 2 mo with the PKCε agonist 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). We noted a similar decrease in baseline CBF, and the cilia remained sensitive to stimulation with ß2 agonists. The mechanisms for the slowing of baseline CBF have not been previously determined. In this mouse model of aging we were able to show that decreases in CBF are related to an increase in PKCε activity.
Assuntos
Envelhecimento/fisiologia , Pulmão/fisiopatologia , Depuração Mucociliar/fisiologia , Pneumonia/enzimologia , Proteína Quinase C-épsilon/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Fatores Etários , Animais , Caprilatos/farmacologia , Cílios/enzimologia , Cílios/fisiologia , Células Epiteliais/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , Procaterol/farmacologia , Proteína Quinase C-alfa/metabolismoRESUMO
Neuromodulators released during and after a fearful experience promote the consolidation of long-term memory for that experience. Because overconsolidation may contribute to the recurrent and intrusive memories of post-traumatic stress disorder, neuromodulatory receptors provide a potential pharmacological target for prevention. Stimulation of muscarinic receptors promotes memory consolidation in several conditioning paradigms, an effect primarily associated with the M1 receptor (M1R). However, neither inhibiting nor genetically disrupting M1R impairs the consolidation of cued fear memory. Using the M1R agonist cevimeline and antagonist telenzepine, as well as M1R knock-out mice, we show here that M1R, along with ß2-adrenergic (ß2AR) and D5-dopaminergic (D5R) receptors, regulates the consolidation of cued fear memory by redundantly activating phospholipase C (PLC) in the basolateral amygdala (BLA). We also demonstrate that fear memory consolidation in the BLA is mediated in part by neuromodulatory inhibition of the M-current, which is conducted by KCNQ channels and is known to be inhibited by muscarinic receptors. Manipulating the M-current by administering the KCNQ channel blocker XE991 or the KCNQ channel opener retigabine reverses the effects on consolidation caused by manipulating ß2AR, D5R, M1R, and PLC. Finally, we show that cAMP and protein kinase A (cAMP/PKA) signaling relevant to this stage of consolidation is upstream of these neuromodulators and PLC, suggesting an important presynaptic role for cAMP/PKA in consolidation. These results support the idea that neuromodulatory regulation of ion channel activity and neuronal excitability is a critical mechanism for promoting consolidation well after acquisition has occurred.
Assuntos
Medo/fisiologia , Canais de Potássio KCNQ/metabolismo , Memória/fisiologia , Receptor Muscarínico M1/fisiologia , Fosfolipases Tipo C/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Condicionamento Clássico/efeitos dos fármacos , Condicionamento Clássico/fisiologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica/efeitos adversos , Inibidores Enzimáticos/farmacologia , Medo/efeitos dos fármacos , Feminino , Canais de Potássio KCNQ/efeitos dos fármacos , Masculino , Moduladores de Transporte de Membrana/farmacologia , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Agonistas Muscarínicos/farmacologia , Procaterol/farmacologia , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inibidores , Receptor Muscarínico M1/deficiência , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Previously we have shown that chronic alcohol intake causes alcohol-induced ciliary dysfunction (AICD), leading to non-responsive airway cilia. AICD likely occurs through the downregulation of nitric oxide (NO) and cyclic nucleotide-dependent kinases, protein kinase G (PKG) and protein kinase A (PKA). Studies by others have shown that dietary supplementation with the antioxidants N-acetylcysteine (NAC) and procysteine prevent other alcohol-induced lung complications. This led us to hypothesize that dietary supplementation with NAC or procysteine prevents AICD. To test this hypothesis, C57BL/6 mice drank an alcohol/water solution (20% w/v) ad libitum for 6 weeks and were concurrently fed dietary supplements of either NAC or procysteine. Ciliary beat frequency (CBF) was measured in mice tracheas, and PKG/PKA responsiveness to ß-agonists and NOx levels were measured from bronchoalveolar lavage (BAL) fluid. Long-term alcohol drinking reduced CBF, PKG and PKA responsiveness to ß-agonists, and lung NOx levels in BAL fluid. In contrast, alcohol-drinking mice fed NAC or procysteine sustained ciliary function and PKG and PKA responsiveness to ß-agonists. However, BAL NO levels remained low despite antioxidant supplementation. We also determined that removal of alcohol from the drinking water for as little as 1 week restored ciliary function, but not PKG and PKA responsiveness to ß-agonists. We conclude that dietary supplementation with NAC or procysteine protects against AICD. In addition, alcohol removal for 1 week restores cilia function independent of PKG and PKA activity. Our findings provide a rationale for the use of antioxidants to prevent damage to airway mucociliary functions in chronic alcohol-drinking individuals.
Assuntos
Acetilcisteína/uso terapêutico , Antioxidantes/uso terapêutico , Transtornos da Motilidade Ciliar/induzido quimicamente , Transtornos da Motilidade Ciliar/prevenção & controle , Suplementos Nutricionais , Etanol/toxicidade , Ácido Pirrolidonocarboxílico/uso terapêutico , Tiazolidinas/uso terapêutico , Acetilcisteína/administração & dosagem , Acetilcisteína/análise , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/química , Cílios/efeitos dos fármacos , Cílios/fisiologia , Transtornos da Motilidade Ciliar/dietoterapia , Transtornos da Motilidade Ciliar/fisiopatologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Feminino , Camundongos , Procaterol/farmacologia , Ácido Pirrolidonocarboxílico/administração & dosagem , Espécies Reativas de Nitrogênio/análise , Tiazolidinas/administração & dosagem , Traqueia/metabolismoRESUMO
Airway hyperresponsiveness (AHR) and airway inflammation are key pathophysiological features of many respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). To evaluate the treatment responses of procaterol and CD38 inhibitors in an ozone-induced AHR mice model, we hypothesized that procaterol and two synthetic CD38 inhibitors (Compounds T and H) might have therapeutic effects on the ozone-induced AHR mice model, and the nuclear factor-kappaB (NF-κB) pathway and the CD38 enzymatic activity might be involved in the mechanisms. With the exception of the Control group, ozone exposure was used to establish an AHR model. Male Kunming mice in the Procaterol and CD38 inhibitors groups were treated with an emulsifier of procaterol hydrochloride, Compound T or H. Results indicated that (1) no drug showed severe toxicity in this study; (2) ozone exposure induced airway inflammation and AHR; (3) intragastric treatment with procaterol and Compound T achieved potent therapeutic effects, but Compound H did not show any therapeutic effect; (4) the NF-κB pathway was involved in both the pathogenic mechanisms of ozone and therapeutic mechanisms of procaterol and Compound T; (5) however, the in vivo effect of Compound T was not caused by its inhibitory activity on CD38. Taken together, procaterol and Compound T are potentially good drugs to treat asthma and COPD complicated with ozone exposure.
Assuntos
Antiasmáticos/uso terapêutico , Benzoatos/uso terapêutico , Hiper-Reatividade Brônquica/tratamento farmacológico , Indóis/uso terapêutico , Procaterol/uso terapêutico , ADP-Ribosil Ciclase 1/antagonistas & inibidores , Animais , Antiasmáticos/farmacologia , Benzoatos/farmacologia , Hiper-Reatividade Brônquica/induzido quimicamente , Hiper-Reatividade Brônquica/patologia , Hiper-Reatividade Brônquica/fisiopatologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Indóis/farmacologia , Contagem de Leucócitos , Pulmão/imunologia , Pulmão/patologia , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Cloreto de Metacolina , Camundongos , NF-kappa B/imunologia , Ozônio , Procaterol/farmacologiaRESUMO
BACKGROUND: It is a matter of great importance in a donation after cardiac death to attenuate ischemia-reperfusion injury (IRI) related to the inevitable warm ischemic time. METHODS: Donor dogs were rendered cardiac-dead and left at room temperature. The dogs were allocated into 2 groups: the ß-2 group (n = 5) received an aerosolized ß-2 adrenoreceptor agonist (procaterol, 350 µg) and ventilation with 100% oxygen for 60 minutes starting at 240 minutes after cardiac arrest, and the control group (n = 6) received an aerosolized control solvent with the ventilation. Lungs were recovered 300 minutes after cardiac arrest. Recipient dogs underwent left single-lung transplantation to evaluate the functions of the left transplanted lung for 240 minutes after the reperfusion. RESULTS: Oxygenation and dynamic compliance were significantly higher in the ß-2 group than in the control group. The ß-2 group revealed significantly higher levels of cyclic adenosine monophosphate and high-energy phosphates in the donor lung after the inhalation than before it. Histologic findings revealed that the ß-2 group had less edema and fewer inflammatory cells. CONCLUSION: Our results suggest that ß-2 adrenoreceptor agonist inhalation during the pre-procurement period may ameliorate IRI.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Morte , Transplante de Pulmão/métodos , Pulmão/efeitos dos fármacos , Procaterol/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Trifosfato de Adenosina/metabolismo , Administração por Inalação , Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Animais , AMP Cíclico/metabolismo , Citocinas/metabolismo , Cães , Pulmão/irrigação sanguínea , Pulmão/fisiologia , Modelos Animais , Procaterol/administração & dosagem , Ventiladores Mecânicos , Isquemia QuenteRESUMO
The beating cilia play a key role in lung mucociliary transport. The ciliary beating frequency (CBF) and ciliary bend amplitude (CBA) of isolated mouse bronchiolar ciliary cells were measured using a light microscope equipped with a high-speed camera (500 Hz). Procaterol (aß(2)-agonist) increased CBA and CBF in a dose dependent manner via cAMP. The time course of CBA increase is distinct from that of CBF increase: procaterol at 10 nM first increased CBA and then CBF. Moreover, 10 pM procaterol increased CBA, not CBF, whereas 10 nM procaterol increased both CBA and CBF. Concentration-response studies of procaterol demonstrated that the CBA curve was shifted to a lower concentration than the CBF curve, which suggests that CBA regulation is different from CBF regulation. Measurements of microbead movements on the bronchiole of lung slices revealed that 10 pM procaterol increased the rate of ciliary transport by 37% and 10 nM procaterol increased it by 70%. In conclusion, we have shown that increased CBA is of particular importance for increasing the bronchiolar ciliary transport rate, although CBF also plays a role in increasing it.
Assuntos
Bronquíolos/efeitos dos fármacos , Cílios/efeitos dos fármacos , Depuração Mucociliar , Procaterol/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Albuterol/farmacologia , Animais , Axonema/metabolismo , Axonema/fisiologia , Bronquíolos/metabolismo , Bronquíolos/fisiologia , Cálcio/farmacologia , Cílios/metabolismo , Cílios/fisiologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/fisiologia , Feminino , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Fatores de Tempo , Gravação em VídeoRESUMO
Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A(1), A(2A), A(2B), or A(3)). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A(1), A(2A), A(2B), or A(3), respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 µM) and/or procaterol (10 µM), in the presence or absence of 5'-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A(1), A(2A), A(3)); 10 µM (A(2B))], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 µM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 µM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ~3 Hz. Likewise, CBF significantly increased ~2-3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A(1), A(2A), and A(3) knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A(2B) knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A(2B) adenosine receptor activation, likely via a PKA-dependent pathway.
Assuntos
Adenosina/metabolismo , Brônquios/citologia , Cílios/fisiologia , Purinérgicos/farmacologia , Receptores Purinérgicos P1/metabolismo , Traqueia/citologia , Adenina/análogos & derivados , Adenina/farmacologia , Adenosina/deficiência , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Bovinos , Cílios/efeitos dos fármacos , Células Epiteliais/fisiologia , Feminino , Técnicas In Vitro , Isoproterenol/farmacologia , Camundongos , Camundongos Knockout , Movimento/efeitos dos fármacos , Procaterol/farmacologia , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/metabolismo , Receptores Purinérgicos P1/deficiênciaRESUMO
ß(2) agonists reduce the frequency of exacerbations in patients with bronchial asthma and chronic obstructive pulmonary disease caused by respiratory virus infection. ß(2) agonists reduce the production of pro-inflammatory cytokines. However, the inhibitory effects of ß(2) agonists on the infection of rhinovirus, the major cause of exacerbations, have not been well studied. To examine the effects of a ß(2) agonist, procaterol, on rhinovirus infection and rhinovirus infection-induced airway inflammation, human tracheal epithelial cells were infected with a major group rhinovirus, type 14 rhinovirus. Rhinovirus infection increased viral titers and the content of pro-inflammatory cytokines, including interleukin-1ß and interlukin-6, in supernatant fluids and rhinovirus RNA in the cells. Procaterol reduced rhinovirus titers and RNA, cytokine concentrations, and susceptibility to rhinovirus infection. Procaterol reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for type 14 rhinovirus, and the number of acidic endosomes in the cells from which rhinovirus RNA enters into the cytoplasm. Procaterol inhibited the activation of nuclear factor kappa-B (NF-κB) proteins including p50 and p65 in the nuclear extracts, while it increased the cytosolic amount of the inhibitory kappa B-α and intracellular cyclic AMP (cAMP) levels. A selective ß(2)-adrenergic receptor antagonist ICI 118551 [erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol] reversed the inhibitory effects of procaterol on rhinovirus titers and RNA, susceptibility to rhinovirus infection, pro-inflammatory cytokines production, ICAM-1 expression, acidic endosomes, and NF-κB. ICI 118551 also reversed the effects of procaterol on cAMP levels. Procaterol may inhibit rhinovirus infection by reducing ICAM-1 and acidic endosomes as well as modulate airway inflammation in rhinovirus infection.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Procaterol/farmacologia , Rhinovirus/efeitos dos fármacos , Rhinovirus/fisiologia , Traqueia/citologia , Células Cultivadas , AMP Cíclico/metabolismo , Citocinas/biossíntese , Replicação do DNA/efeitos dos fármacos , Endossomos/química , Endossomos/efeitos dos fármacos , Endossomos/virologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , NF-kappa B/metabolismo , RNA Viral/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhinovirus/genética , Rhinovirus/metabolismoRESUMO
As indicated in the Global Initiative for Asthma guidelines, short-acting ß2-adrenoreceptor agonists (SABAs) are important relievers in asthma exacerbation. Interferon γ-inducible protein (IP)-10/CXCL 10 is a T-helper type 1 (Th1) cell-related chemokine which is important in the recruitment of Th1 cells involved in host immune defense against intracellular pathogens such as viral infection. Regulated on activation, normal T expressed and secreted (RANTES)/CCL 5 is a chemokine which plays a role in attractant of eosinophils, mast cells, and basophils toward the site of allergic inflammation. Bronchial epithelial cells are first-line barriers against pathogen invasion. However, whether SABAs have regulatory effects on the expression of IP-10 and RANTES in bronchial epithelial cells is unknown. BEAS-2B cells, the human bronchial epithelial cell lines, were pretreated with procaterol (one of the SABAs) or dibutyryl-cAMP (a cyclic AMP analog) at different doses for 1 h and then stimulated with poly I:C (10 µg/mL). Supernatants were collected 12 and 24 h after poly I:C stimulation to determine the concentrations of IP-10 and RANTES by ELISA. In some cases, the cells were pretreated with selective ß2-adrenoreceptor antagonist, ICI-118551, 30 min before procaterol treatment. To investigate the intracellular signaling, the cells were pretreated with mitogen-activated protein kinase (MAPK) inhibitors and a NF-κB inhibitor 30 min before procaterol treatment. Western blot was also used to explore the intracellular signaling. Procaterol significantly suppressed poly I:C-induced IP-10 and RANTES in BEAS-2B cells in a dose-dependent manner. ICI-118551, a selective ß2-adrenoreceptor antagonist, could significantly reverse the suppressive effects. Dibutyryl-cAMP could confer the similar effects of procaterol on poly I:C-induced IP-10 and RANTES expression. Data of Western blot revealed that poly I:C-induced p-ERK, p-JNK, and pp38 expression, but not pp65, were suppressed by procaterol. SABAs could suppress poly I:C-induced IP-10 and RANTES expression in bronchial epithelial cells, at least in part, via ß2-adrenoreceptor-cAMP and MAPK-ERK, JNK, and p38 pathways.
Assuntos
Brônquios/metabolismo , Quimiocina CCL5/biossíntese , Quimiocina CXCL10/biossíntese , Células Epiteliais/metabolismo , Procaterol/farmacologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Western Blotting , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Bucladesina/farmacologia , Linhagem Celular , Quimiocina CCL5/genética , Quimiocina CXCL10/genética , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/imunologia , NF-kappa B/antagonistas & inibidores , Poli I-C/farmacologia , Procaterol/metabolismo , Propanolaminas/farmacologia , Transdução de Sinais/efeitos dos fármacos , eIF-2 Quinase/biossínteseRESUMO
Procaterol is a ß2-adrenoceptor agonist used as a bronchodilator for the treatment of asthma; it also possesses an anti-inflammatory property. As chemokines play a pivotal role in inflammation and the pathogenesis of asthma, we investigated the effects of procaterol on type 2 helper T cell (Th2)-related [macrophage-derived chemokine (MDC) and I-309] and type 1 helper T cell (Th1)-related chemokines [monokine-induced by IFN-gamma (Mig) and interferon-inducible protein 10 (IP-10)] production of THP-1 cells and human primary monocytes. The effect on thymus- and activation-regulated chemokine (TARC) production in BEAS-2B cells was also evaluated. Nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) inhibitors were used to ascertain the intracellular signal pathways involved, and etazolate, a phosphodiesterase 4 inhibitor, was used to assess the correlation between the ß2-adrenoceptor-cAMP pathway and the effect on chemokines. In addition, chromatin immunoprecipitation assays (ChIPs) were performed to detect histone modification in the TARC promoter region. MDC and I-309 production of both THP-1 cells and primary human monocytes, as well as TARC expression of BEAS-2B cells, were significantly inhibited by procaterol (10(-10)-10(-7) M); however, procaterol did not suppress Mig and IP-10 expression by THP-1 cells. MDC secreted by monocytes is associated with the NF-κB and MAPK signaling pathways, in particular p38- and c-Jun N-terminal kinase (JNK) MAPKs. Etazolate blocked the expression of MDC by THP-1 cells and TARC by BEAS-2B cells. ChIP assay revealed decreased trimethylation of lysine 4 in histone 3 (H3K4) in the TARC promoter region of BEAS-2B cells. In conclusion, procaterol could inhibit Th2-related chemokines production in human monocytes and bronchial epithelial cells, an effect that may be mediated through not only the NF-κB, p38, and JNK-MAPK pathways, but also the ß2-adrenoceptor-cAMP pathway. Most importantly, the suppressive effect of Th2-related chemokines production by procaterol might be regulated via post-transcriptional modification by decreasing H3K4 trimethylation.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Células Epiteliais/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Procaterol/farmacologia , Células Th2/efeitos dos fármacos , Brônquios/citologia , Brônquios/efeitos dos fármacos , Quimiocinas/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Monócitos/metabolismoRESUMO
BACKGROUND: The interaction between leukocytes and various parenchymal cells is the first step of inflammation. Therefore, the adhesion of eosinophils to lung fibroblasts is thought to be a crucial step in the inflammatory process of asthma. Procaterol, a beta(2)-selective full agonist, is currently prescribed for patients with asthma. In addition to its potent bronchodilatory action, the agonist has been reported to have anti-inflammatory actions. In this study, to examine whether procaterol can potentiate the anti-inflammatory action of glucocorticoids, the effect of procaterol on eosinophil adhesion to normal human lung fibroblasts (NHLF) was assessed in the presence and absence of budesonide, one of the most potent glucocorticoids. METHODS: Following pretreatment of NHLF with tumor necrosis factor-alpha (TNF-alpha) in the presence of various concentrations of procaterol and/or budesonide, the eotaxin-stimulated eosinophil adhesion was determined using the peroxidase activity of eosinophils. To investigate the mechanism of the inhibitory action of procaterol, TNF-alpha-induced expression of adhesion molecules, ICAM-1 and VCAM-1, in NHLF was also evaluated. RESULTS: Pretreatment with procaterol inhibited the adhesion of eosinophils to NHLF in a concentration-dependent manner, and shifted the concentration-response curve of budesonide to the left. Both procaterol and budesonide resulted in concentration-dependent inhibition of expression of ICAM-1 and VCAM-1 in NHLF, and an additive inhibitory effect was found when the agents were combined. CONCLUSIONS: Given the results of this study which indicated that procaterol exerted an additive action on the anti-inflammatory effect of budesonide, procaterol and glucocorticoids may provide better control for asthma when used together than when used separately.
Assuntos
Anti-Inflamatórios/farmacologia , Asma/tratamento farmacológico , Eosinófilos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Procaterol/farmacologia , Asma/imunologia , Asma/metabolismo , Asma/patologia , Budesonida/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Linhagem Celular , Quimiocina CCL11/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Eosinófilos/imunologia , Eosinófilos/metabolismo , Eosinófilos/patologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Pulmão/patologia , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND: Mammalian spermatozoa have been shown to have beta(1,2,3)- and alpha(2A)-adrenergic receptors, the former functioning only in uncapacitated spermatozoa and the latter only in capacitated cells. Cathine, an amphetamine-related metabolite of a compound found in Catha edulis leaves, accelerates capacitation and inhibits spontaneous acrosome loss by regulating cAMP production. This study tested the hypothesis that adrenergic receptors are involved in these responses. METHODS: Uncapacitated and capacitated mouse sperm suspensions were incubated with cathine +/- specific antagonists for alpha(2)- and beta-adrenergic receptors for 35 min, then assessed using chlortetracycline fluorescence. Reversibility of receptor accessibility was assessed by depleting suspensions of endogenous decapacitation factor (DF) and then adding crude DF with/without cathine and antagonists. Effects on tyrosine phosphorylation and calcium requirements for both ligand binding and biological responses were also evaluated. RESULTS: Cathine's acceleration of capacitation was blocked by a beta(1)-antagonist, whereas an alpha(2)-antagonist blocked inhibition of acrosome reactions. Cathine accelerated capacitation in decapacitated cells, a response inhibited by a beta(1)-antagonist; cathine also stimulated tyrosine phosphorylation. Although calcium was not required for binding, it was needed for responses. CONCLUSIONS: Cathine acts at beta(1)-adrenergic receptors in uncapacitated spermatozoa and at alpha(2A)-receptors in capacitated cells; biological activity requires calcium but binding does not. Adrenergic receptor-binding sites can be made reversibly accessible/inaccessible by changing the capacitation state of spermatozoa. These results suggest that amphetamine-related compounds might enhance chances of fertilization in vivo.