RESUMO
Tiotropium is commonly used in the treatment of chronic obstructive pulmonary disease. Although largely considered to be a long-acting bronchodilator, its demonstrated efficacy in reducing the frequency of exacerbations and preliminary evidence from early studies indicating that it might slow the rate of decline in lung function suggested mechanisms of action in addition to simple bronchodilation. This hypothesis was examined in the recently published UPLIFT study and, although spirometric and other clinical benefits of tiotropium treatment extended to four years, the rate of decline in lung function did not appear to be reduced by the addition of tiotropium in this study. This article summarizes data from a variety of investigations that provide insights into possible mechanisms to account for the effects of tiotropium. The report summarizes the discussion on basic and clinical research in this field.
Assuntos
Broncodilatadores/farmacologia , Antagonistas Colinérgicos/farmacologia , Derivados da Escopolamina/farmacologia , Acetilcolina/fisiologia , Animais , Broncodilatadores/uso terapêutico , Antagonistas Colinérgicos/uso terapêutico , Tosse/tratamento farmacológico , Tosse/fisiopatologia , Humanos , Inflamação/patologia , Pulmão/inervação , Pulmão/fisiologia , Muco/metabolismo , Sistema Nervoso Parassimpático/efeitos dos fármacos , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/patologia , Derivados da Escopolamina/uso terapêutico , Brometo de TiotrópioRESUMO
Animal life is controlled by neurons and in this setting cholinergic neurons play an important role. Cholinergic neurons release ACh, which via nicotinic and muscarinic receptors (n- and mAChRs) mediate chemical neurotransmission, a highly integrative process. Thus, the organism responds to external and internal stimuli to maintain and optimize survival and mood. Blockade of cholinergic neurotransmission is followed by immediate death. However, cholinergic communication has been established from the beginning of life in primitive organisms such as bacteria, algae, protozoa, sponge and primitive plants and fungi, irrespective of neurons. Tubocurarine- and atropine-sensitive effects are observed in plants indicating functional significance. All components of the cholinergic system (ChAT, ACh, n- and mAChRs, high-affinity choline uptake, esterase) have been demonstrated in mammalian non-neuronal cells, including those of humans. Embryonic stem cells (mice), epithelial, endothelial and immune cells synthesize ACh, which via differently expressed patterns of n- and mAChRs modulates cell activities to respond to internal or external stimuli. This helps to maintain and optimize cell function, such as proliferation, differentiation, formation of a physical barrier, migration, and ion and water movements. Blockade of n- and mACHRs on non-innervated cells causes cellular dysfunction and/or cell death. Thus, cholinergic signalling in non-neuronal cells is comparable to cholinergic neurotransmission. Dysfunction of the non-neuronal cholinergic system is involved in the pathogenesis of diseases. Alterations have been detected in inflammatory processes and a pathobiologic role of non-neuronal ACh in different diseases is discussed. The present article reviews recent findings about the non-neuronal cholinergic system in humans.
Assuntos
Acetilcolina/metabolismo , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Expressão Gênica/fisiologia , Humanos , Inflamação/fisiopatologia , Neurônios/metabolismoRESUMO
In human skin both resident and transiently residing cells are part of the extra- or non-neuronal cholinergic system, creating a highly complex and interconnected cosmos in which acetylcholine (ACh) and choline are the natural ligands of nicotinic and muscarinic receptors with regulatory function in both physiology and pathophysiology. ACh is produced in keratinocytes, endothelial cells and most notably in immune competent cells invading the skin at sites of inflammation. The cholinergic system is involved in basic functions of the skin through autocrine, paracrine, and endocrine mechanisms, like keratinocyte proliferation, differentiation, adhesion and migration, epidermal barrier formation, pigment-, sweat- and sebum production, blood circulation, angiogenesis, and a variety of immune reactions. The pathophysiological consequences of this complex cholinergic "concert" are only beginning to be understood. The present review aims at providing insight into basic mechanisms of this highly complex system.
Assuntos
Acetilcolina/metabolismo , Colina/metabolismo , Pele/metabolismo , Acetilcolina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Humanos , Sistema Imunitário/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Melanócitos/efeitos dos fármacos , Modelos Biológicos , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/fisiologia , Pigmentação da Pele/fisiologia , Cicatrização/fisiologiaRESUMO
Certain female reproductive tissues are known to express the non-neuronal cholinergic system. Using different experimental approaches, we tested the hypothesis that acetylcholine (ACh) in the porcine oviduct may also be derived from non-neuronal structures. Immunohistochemistry was performed to detect acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) in different segments of the oviduct of cyclic and pregnant sows. Immunohistochemical experiments revealed strong immunoexpression of ChAT in the entire oviductal epithelium at metoestrus. Thereby, a particular pronounced staining was found in the supranuclear region of almost all epithelial cells. Immunostaining of ChAT decreased markedly during dioestrus and prooestrus stages, respectively. At prooestrus, ChAT immunoreactivity was confined to ciliated cells. Furthermore, we found elevated level of staining intensity of ChAT in the pregnant oviduct at day 13. Using the same ChAT antibody for Western blot analyses, we detected immunoreactive bands of MW 69,000 and 46,000 mainly in ampulla, while MW 58,000 and 30,000 forms were present mainly in infundibulum and isthmus. Furthermore ACh was detected by HPLC and fluorimetric methods in oviductal epithelium. In conclusion, we show expression of ChAT in oviductal epithelial cells at different stages of the oestrus cycle and pregnancy, indicating that these cells can synthesize ACh in a cycle-dependent manner. These results suggest as yet unexplored roles of epithelial ACh in the oviduct.
Assuntos
Acetilcolina/biossíntese , Colina O-Acetiltransferase/metabolismo , Células Epiteliais/metabolismo , Ciclo Estral/metabolismo , Tubas Uterinas/metabolismo , Prenhez/metabolismo , Acetilcolina/análise , Animais , Western Blotting , Diestro/metabolismo , Tubas Uterinas/citologia , Feminino , Imuno-Histoquímica , Metestro/metabolismo , Gravidez , Proestro/metabolismo , SuínosRESUMO
PURPOSE: The new beta2 radioligand (R,R)(S,S) 5-(2-(2-[4-(2-[18F]fluoroethoxy)phenyl]-1-methylethylamino)-1-hydroxyethyl)-benzene-1,3-diol ([18F]FE-fenoterol; [18F]FEFE), a fluoroethylated derivative of racemic fenoterol, was evaluated in vivo and ex vivo using a guinea pig model. METHODS: Dynamic PET studies over 60 min with [(18)F]FEFE were performed in nine Hartley guinea pigs in which a baseline (group 1, n=3), a predose (group 2, n=3; 2 mg/kg fenoterol 5 min prior to injection of [18F]FEFE) or a displacement study (group 3, n=3; 2 mg/kg fenoterol 5 min post injection of [18F]FEFE) was conducted. RESULTS: In all animal groups, the lungs could be visualised and semi-quantified separately by calculating uptake ratios to non-specific binding in the neck area. Premedication with non-radioactive fenoterol and displacement tests showed significant reduction of lung uptake, by 94% and 76%, respectively. CONCLUSION: These data demonstrate specific binding of the new radioligand to the pulmonary beta2-receptors in accordance with ex vivo measurements. Therefore, [18F]FEFE seems to be suitable for the in vivo visualisation and quantification of the pulmonary beta2-receptor binding in this animal model.
Assuntos
Fenoterol/análogos & derivados , Pulmão/diagnóstico por imagem , Pulmão/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animais , Estudos de Viabilidade , Fenoterol/farmacocinética , Cobaias , Taxa de Depuração Metabólica , Modelos Animais , Especificidade de Órgãos , Cintilografia , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
Since 12(th) of August 2004 the EU Directive 2001/20/EG has been implemented into the national law. The 12th AMG amendment of 30 July 2004 and the good clinical practice decree on the conduct of clinical trials on drugs for human use of 9 August 2004 have been authorized and must be considered for new clinical trials with investigational medical products (drugs). The scope of the changes are to increase the quality of clinical trials and to continue the process of harmonization within the European Community. Based on the new law the sponsor has to apply for approval by the competent authority and for a favourable opinion by the responsible ethics committee. Both procedures are independent; a favourable opinion of the responsible ethics committee is a necessary condition before starting the trial. Thus, the role of the ethics committees has been changed; the committees are considered as an institution comparable to an authority to protect the rights and safety of human subjects involved in clinical trials. The permanent working group of the medical ethics committees in Germany has established a procedure to meet these requirements, particularly in the case of multicentre clinical trials, where only a single opinion shall be given for each member state. This article describes this procedure (application, process of ethical consideration among the leading and local ethics committees in multicentre trials, responsibilities during the trial).
Assuntos
Ensaios Clínicos como Assunto/legislação & jurisprudência , Comissão de Ética/organização & administração , União Europeia , Cooperação Internacional , Drogas em Investigação/uso terapêutico , Comissão de Ética/legislação & jurisprudência , Europa (Continente) , Alemanha , Fidelidade a Diretrizes , Humanos , Legislação de Medicamentos/organização & administração , Garantia da Qualidade dos Cuidados de Saúde/legislação & jurisprudência , Garantia da Qualidade dos Cuidados de Saúde/organização & administração , Projetos de PesquisaRESUMO
1. The release of acetylcholine was investigated in the human placenta villus, a useful model for the characterization of the non-neuronal cholinergic system. 2. Quinine, an inhibitor of organic cation transporters (OCT), reduced acetylcholine release in a reversible and concentration-dependent manner with an IC(50) value of 5 microM. The maximal effect, inhibition by 99%, occurred at a concentration of 300 microM. 3. Procaine (100 microM), a sodium channel blocker, and vesamicol (10 microM), an inhibitor of the vesicular acetylcholine transporter, were ineffective. 4. Corticosterone, an inhibitor of OCT subtype 1, 2 and 3 reduced acetylcholine in a concentration-dependent manner with an IC(50) value of 2 microM. 5. Substrates of OCT subtype 1, 2 and 3 (amiloride, cimetidine, guanidine, noradrenaline, verapamil) inhibited acetylcholine release, whereas carnitine, a substrate of subtype OCTN2, exerted no effect. 6. Long term exposure (48 and 72 h) of villus strips to anti-sense oligonucleotides (5 microM) directed against transcription of OCT1 and OCT3 reduced the release of acetylcholine, whereas OCT2 anti-sense oliogonucleotides were ineffective. 7. It is concluded that the release of non-neuronal acetylcholine from the human placenta is mediated via organic cation transporters of the OCT1 and OCT3 subtype.
Assuntos
Acetilcolina/metabolismo , Proteínas de Transporte de Cátions Orgânicos/fisiologia , Placenta/metabolismo , Amilorida/farmacologia , Cimetidina/farmacologia , Corticosterona/farmacologia , DNA Antissenso/farmacologia , Relação Dose-Resposta a Droga , Feminino , Guanidina/farmacologia , Humanos , Técnicas In Vitro , Norepinefrina/farmacologia , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/antagonistas & inibidores , Transportador 1 de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/fisiologia , Transportador 2 de Cátion Orgânico , Piperidinas/farmacologia , Gravidez , Procaína/farmacologia , Quinina/farmacologia , Fatores de Tempo , Verapamil/farmacologiaRESUMO
The synthesis and release of non-neuronal acetylcholine, a widely expressed signaling molecule, were investigated in the human placenta. This tissue is free of cholinergic neurons, i.e. a contamination of neuronal acetylcholine can be excluded. The villus showed a choline acetyltransferase (ChAT) activity of 0.65 nmol/mg protein per h and contained 500 nmol acetylcholine/g dry weight. In the absence of cholinesterase inhibitors the release of acetylcholine from isolated villus pieces amounted to 1.3 nmol/g wet weight per 10 min corresponding to a fractional release rate of 0.13% per min. The following substances did not significantly modify the release of acetylcholine: oxotremorine (1 microM), scopolamine (1 microM), (+)-tubocurarine (30 microM), forskolin (30 microM), ouabain (10 microM), 4alpha-phorbol 12,13-didecanoate (1 microM) and tetrodotoxin (1 microM). Removal of extracellular calcium, phorbol 12,13-dibutyrate (1 microM) and colchicine (100 microM) reduced the acetylcholine release between 30% and 50%. High potassium chloride (54 mM and 108 mM) increased the acetylcholine release slightly (by about 30%). A concentration of 10 microM nicotine was ineffective, but 100 microM nicotine enhanced acetylcholine release gradually over a 50-min period without desensitization of the response. The facilitatory effect of nicotine was prevented by 30 microM (+)-tubocurarine. Inhibitors of cholinesterase (physostigmine, neostigmine; 3 microM) facilitated the efflux of acetylcholine about sixfold, and a combination of both (+)-tubocurarine (30 microM) and scopolamine (1 microM) halved the enhancing effect. In conclusion, release mechanisms differ between non-neuronal and neuronal acetylcholine. Facilitatory nicotine receptors are present which are activated by applied nicotine or by blocking cholinesterase. Thus, cholinesterase inhibitors increase assayed acetylcholine by two mechanisms, protection of hydrolysis and stimulation of facilitatory nicotine receptors.
Assuntos
Acetilcolina/metabolismo , Colina O-Acetiltransferase/metabolismo , Inibidores da Colinesterase/farmacologia , Placenta/metabolismo , Acetilcolina/biossíntese , Colforsina/farmacologia , Interações Medicamentosas , Estimulação Elétrica , Feminino , Humanos , Placenta/efeitos dos fármacos , Placenta/enzimologia , Receptores Nicotínicos/efeitos dos fármacosRESUMO
The presence of muscarinic receptors (MR) in the ovary of different species has been recognized, but the identity of these receptors as well as ovarian sources of their natural ligand, acetylcholine (ACh), have not been determined. Because luteinized human granulosa cells (GC) in culture express functional MR, we have determined whether the group of the related MR subtypes, M1R, M3R, and M5R, are present in vivo in human and rhesus monkey ovaries. To this end, ribonucleic acids (RNAs) of different human and monkey ovaries as well as RNAs from human GC and monkey oocytes were reverse transcribed and subjected to PCR amplification, followed by sequencing of the amplified complementary DNAs. Results obtained showed that M1R, M3R, and M5R messenger RNAs are present in adult human and monkey ovaries; oocytes express exclusively the M3R subtype, whereas GC express M1R and M5R. To determine the ovarian source(s) of the natural ligand of these ACh receptors, we attempted to localize the enzyme responsible for its synthesis with the help of a monoclonal antibody recognizing choline acetyltransferase for immunohistochemistry. In neither human nor monkey sections did we detect immunoreactive choline acetyltransferase-positive fibers or nerve cells, but, surprisingly, GC of antral follicles showed prominent staining. To determine whether GC can produce ACh, human cultured GC derived from preovulatory follicles were analyzed using a high pressure liquid chromatography technique. The results showed that these cells contained ACh in concentrations ranging from 4.2-11.5 pmol/10(6) cells. Samples of a rat granulosa cell line likewise contained ACh. Thus, the ovary contains multiple MR, and GC of antral follicles are able to synthesize ACh, the ligand of MR. We propose that ACh may serve as an as yet unrecognized factor involved in the complex regulation of ovarian function in the primate, e.g. regulation of cell proliferation or progesterone production.
Assuntos
Acetilcolina/biossíntese , Ovário/metabolismo , Receptores Muscarínicos/metabolismo , Acetilcolina/metabolismo , Adulto , Animais , Encéfalo/metabolismo , Carnitina O-Acetiltransferase/metabolismo , Células Cultivadas , Colina O-Acetiltransferase/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Macaca mulatta , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptores Muscarínicos/genéticaRESUMO
Acetylcholine, one of the most exemplary neurotransmitters, has been detected in bacteria, algae, protozoa, tubellariae and primitive plants, suggesting an extremely early appearance in the evolutionary process and a wide expression in non-neuronal cells. In plants (Urtica dioica), acetylcholine is involved in the regulation of water resorption and photosynthesis. In humans, acetylcholine and/or the synthesizing enzyme, choline acetyltransferase, have been demonstrated in epithelial (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium), endothelial, muscle and immune cells (granulocytes, lymphocytes, macrophages, mast cells). The widespread expression of non-neuronal acetylcholine is accompanied by the ubiquitous expression of cholinesterase and acetylcholine sensitive receptors (nicotinic, muscarinic). Both receptor populations interact with more or less all cellular signalling pathways. Thus, non-neuronal acetylcholine can be involved in the regulation of basic cell functions like gene expression, proliferation, differentiation, cytoskeletal organization, cell-cell contact (tight and gap junctions, desmosomes), locomotion, migration, ciliary activity, electrical activity, secretion and absorption. Non-neuronal acetylcholine also plays a role in the control of unspecific and specific immune functions. Future experiments should be designed to analyze the cellular effects of acetylcholine in greater detail and to illuminate the involvement of the non-neuronal cholinergic system in the pathogenesis of diseases such as acute and chronic inflammation, local and systemic infection, dementia, atherosclerosis, and finally cancer.
Assuntos
Acetilcolina/fisiologia , Fenômenos Fisiológicos Vegetais , Receptores Colinérgicos/fisiologia , Acetilcolina/análise , Colina O-Acetiltransferase/análise , Colina O-Acetiltransferase/metabolismo , Antagonistas Colinérgicos/farmacologia , Células Epiteliais/química , Humanos , Magnoliopsida/efeitos dos fármacos , Magnoliopsida/fisiologia , Fotossíntese , Receptores Colinérgicos/análise , Transdução de SinaisRESUMO
An increasing body of knowledge indicates that the cholinergic system is not confined to the nervous system, but is practically ubiquitous. The present paper will address the question of the non-neuronal cholinergic system in vascular endothelial cells (EC). In tissue sections of human skin, immunohistochemical studies using confocal laser scanning microscopy showed ChAT (choline acetyltransferase) activity in the EC of dermal blood vessels. Positive ChAT immunoreactivity was also demonstrated in monolayer cultures of human umbilical vein EC (HUVEC) and a human angiosarcoma EC line (HAEND). That the synthesizing enzyme is not only present in EC, but also active was shown by measuring ChAT activity. Thus, in HUVEC cultures, ChAT activity amounted to 0.78 +/- 0.15 nmol x mg protein(-1) x h(-1) (n = 3), but was only partially (about 50%) inhibited by the ChAT inhibitor bromoacetylcholine (30 microM). In HPLC measurements, a concentration of 22 +/- 2 pmol acetylcholine (ACh) per 10(6) cells was found (n = 6). However, using a cholinesterase-packed analytical column to check the identity of the acetylcholine peak, the peak height was found to be reduced, although a significant peak still remained, indicating the existence of a compound closely related to ACh. Further immunocytochemical experiments indicated that EC in vitro also express the vesicular acetylcholine transporter (VAChT) system. Preliminary immunoelectron microscopic studies suggest a topographical association of VAChT with endothelial endocytotic vesicles. The presented experiments clearly demonstrate the existence of essential elements of the cholinergic system (ChAT, VAChT, ACh) in the human endothelium. The biological functions of ACh synthesized by endothelial cells are the focus of ongoing research activity.
Assuntos
Acetilcolina/metabolismo , Endotélio Vascular/metabolismo , Proteínas de Membrana Transportadoras , Proteínas de Transporte Vesicular , Acetilcolina/fisiologia , Acetilcolinesterase/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Colina O-Acetiltransferase/metabolismo , Endotélio Vascular/fisiopatologia , Humanos , Receptores Colinérgicos/metabolismo , Transdução de Sinais , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
The non-neuronal cholinergic system is widely expressed in human airways. Choline acetyltransferase (ChAT) and/or acetylcholine are demonstrated in more or less all epithelial surface cells (goblet cells, ciliated cells, basal cells), submucosal glands and airway smooth muscle fibres. Acetylcholine is also demonstrated in the effector cells of the immune system (lymphocytes, macrophages, mast cells). Epithelial, endothelial and immune cells express nicotinic and muscarinic receptors. Thus the cytomolecule acetylcholine can contribute to the regulation of basic cell functions via auto-/paracrine mechanisms (proliferation, differentiation, ciliary activity, secretion of water, ions and mucus, organization of the cytoskeleton, cell-cell contact). Acetylcholine also modulates immune functions (release of cytokines; proliferation, activation and inhibition of immune cells). Preliminary experimental evidence suggests that mucosal inflammation may be associated with raised acetylcholine levels, impairing cell and organ homeostasis. It should be considered that anti-muscarinic drugs which are applied for the treatment of chronic airway diseases antagonize the effect of both neuronal and non-neuronal acetylcholine. Non-neuronal acetylcholine, however, is still active, possibly directly within the cell cytosol and also via nicotinic receptors localized on various non-neuronal cells. It is an essential task to clarify the pathophysiological role of the non-neuronal cholinergic system in more detail to develop new drugs which can target the synthesis, release, inactivation and cellular activity of non-neuronal acetylcholine.
Assuntos
Acetilcolina/antagonistas & inibidores , Acetilcolina/farmacologia , Colina O-Acetiltransferase/metabolismo , Pneumopatias/imunologia , Músculo Liso/fisiologia , Receptores Muscarínicos/fisiologia , Receptores Nicotínicos/fisiologia , Formação de Anticorpos , Células Epiteliais/fisiologia , Homeostase , Humanos , Imunidade Celular , Inflamação , Pneumopatias/tratamento farmacológico , Pneumopatias/patologiaRESUMO
Isolated human bronchi and rat tracheae were incubated in organ baths to measure histamine release. The calcium ionophore A23187, 3 micromol/L in rat trachea and 10 micromol/L in human bronchi, stimulated histamine release by 145 +/- 50% (n = 6) and 270 +/- 48% (n = 7) above the prestimulation level, respectively. Acetylcholine (100 pmol/L; human bronchi) or oxotremorine (1, 100, 10,000 nmol/L; rat trachea) did not affect the spontaneous histamine release. In rat tracheae neither acetylcholine nor oxotremorine inhibited A23187-evoked histamine release, whereas 100 pmol/L acetylcholine significantly suppressed the evoked histamine release in human bronchi by 86%. For receptor characterization the following subtype-specific muscarinic receptor antagonists were applied: pirenzepine (M1 subtype), para-fluorohexahydrosiladifendiol (pFHHSiD; similar affinities at human cloned M1-, M3-, and M4-receptors), AF-DX 116 (M2 subtype), and clozapine (antagonist at cloned M1-, M2-, M3-receptors; agonist at cloned M4-receptors). Pirenzepine, pFHHSiD, AF-DX 116, and clozapine (100 nmol/L each) antagonized the inhibitory effect of 100 pmol/L acetylcholine by 83 +/- 20% (n = 6), 83 +/- 9% (n = 8), 50 +/- 14% (n = 6), and 35 +/- 7% (6). In conclusion, a species difference exists in the cholinergic control of histamine release between human and rat airways. In human airways muscarinic receptors most likely of the M1 subtype are involved in the inhibitory control of mast cell function, whereas such an inhibitory pathway does not exist in the rat trachea.
Assuntos
Brônquios/metabolismo , Liberação de Histamina , Receptores Muscarínicos/fisiologia , Traqueia/metabolismo , Acetilcolina/farmacologia , Animais , Calcimicina/farmacologia , Clozapina/farmacologia , Feminino , Liberação de Histamina/efeitos dos fármacos , Humanos , Ionóforos/farmacologia , Masculino , Mastócitos/fisiologia , Antagonistas Muscarínicos/farmacologia , Técnicas de Cultura de Órgãos , Oxotremorina/farmacologia , Parassimpatolíticos/farmacologia , Piperidinas/farmacologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Muscarínicos/efeitos dos fármacos , Especificidade da EspécieRESUMO
Eosinophil-derived cationic proteins play an essential role in the pathogenesis of bronchial asthma. We tested whether cationic proteins interfere with the cationic amino-acid transport in alveolar macrophages (AMPhi) and tracheal epithelial cells, and whether L-arginine-dependent pathways were affected. The effect of cationic polypeptides on cellular uptake of [(3)H]-L-arginine, nitrite accumulation, and the turnover of [(3)H]-L-arginine by nitric oxide (NO) synthase and arginase (formation of [(3)H]-L-citrulline and [(3)H]-L-ornithine, respectively) were studied. Poly-L-arginine reduced [(3)H]-L-arginine uptake in rat AMPhi and tracheal epithelial cells in a concentration-dependent manner (at 300 microgram/ml by 70%). Poly-L-lysine, protamine, and major basic protein (each up to 300 microgram/ml) tested in rat AMPhi inhibited [(3)H]-L-arginine uptake by 35 to 50%. During 6 h incubation in amino acid-free Krebs solution, rat AMPhi, precultured in the absence or presence of LPS (1 microgram/ml), accumulated 1.4 and 3.5 nmol/10(6) cells nitrite, respectively. Addition of 100 microM L-arginine increased nitrite accumulation by 70 and 400% in control and lipopolysaccharide-treated AMPhi, respectively. Nitrite accumulation in the presence of L-arginine was reduced by poly-L-arginine and poly-L-lysine (100 and 300 microgram/ml) by 60 to 85% and 20 to 30%, respectively. Poly-L-arginine, but not poly-L-lysine, inhibited nitrite accumulation already in the absence of extracellular L-arginine. Poly-L-arginine (300 microgram/ml) inhibited [(3)H]-L-citrulline formation by AMPhi stronger than that of [(3)H]-L-ornithine. We conclude that cationic proteins can inhibit cellular transport of L-arginine and this can limit NO synthesis. Poly-L-arginine inhibits L-arginine uptake more effectively than other cationic proteins and exerts additional direct inhibitory effects on NO synthesis.
Assuntos
Arginina/farmacocinética , Células Epiteliais/metabolismo , Macrófagos Alveolares/metabolismo , Óxido Nítrico/biossíntese , Ribonucleases , Traqueia/metabolismo , Animais , Arginase/farmacologia , Proteínas Sanguíneas/farmacologia , Citrulina/farmacologia , Relação Dose-Resposta a Droga , Proteínas Granulares de Eosinófilos , Feminino , Fibrinolíticos/farmacologia , Expressão Gênica , Heparina/farmacologia , Lipopolissacarídeos/farmacologia , Pulmão/metabolismo , Lisina/metabolismo , Masculino , Nitritos/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
1. Acetylcholine (ACh) represents one of the most exemplary neurotransmitters. In addition to its presence in neuronal tissue, there is increasing experimental evidence that ACh is widely expressed in pro- and eukaryotic non-neuronal cells. Thus, ACh has been detected in bacteria, algae, protozoa, tubellariae and primitive plants, suggesting an extremely early appearance of ACh in the evolutionary process. 2. In humans, ACh and/or the synthesizing enzyme, choline acetyltransferase, has been demonstrated in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium) and endothelial and muscle cells. In addition, immune cells express the non-neuronal cholinergic system (i.e. the synthesis of ACh can be detected in human leucocytes (granulocytes, lymphocytes and macrophages)), as well as in rat microglia in vitro. 3. The widespread expression of non-neuronal ACh is accompanied by the ubiquitous expression of cholinesterase activity, which prevents ACh from acting as a classical hormone. 4. Non-neuronal ACh mediates its cellular actions in an auto- and paracrine manner via the activation of the widely expressed nicotinic and muscarinic acetylcholine receptors, which can interfere with virtually all cellular signalling pathways (ion channels and key enzymes). 5. Non-neuronal ACh appears to be involved in the regulation of basic cell functions, such as mitosis, cell differentiation, organization of the cytoskeleton, cell-cell contact, secretion and absorption. Non-neuronal ACh also plays a role in the regulation of immune functions. All these qualities together may mediate the so-called 'trophic property' of ACh. 6. Future experiments should be designed to analyse the cellular effects of ACh in greater detail. The involvement of the non-neuronal cholinergic system in the pathogenesis of chronic inflammatory diseases should be investigated to open up new therapeutic strategies.
Assuntos
Acetilcolina/fisiologia , Animais , Colinesterases/metabolismo , Evolução Molecular , Humanos , Neurônios/fisiologiaRESUMO
1. The release of neuronal [3H]acetylcholine (ACh) from isolated human bronchi after labelling with [3H]choline was measured to investigate the effects of prostanoids. 2. A first period of electrical field stimulation (S1) caused a [3H]ACh release of 320+/-70 and 200+/-40 Becquerel (Bq) g(-1) in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (Sn, n=2, 3, and 4) released less [3H]ACh, i.e. decreasing Sn/ S1 values were obtained (0.76+/-0.09, 0.68+/-0.07 and 0.40+/-0.04, respectively). 3. Cumulative concentrations (1-1000 nM) of EP-receptor agonists like prostaglandin E2, nocloprost, and sulprostone (EP1 and EP3 selective) inhibited evoked [3H]ACh release in a concentration dependent manner with IC50 values between 4- 14 nM and maximal inhibition of about 70%. 4. The inhibition of evoked [3H]ACh release by prostaglandin E2, nocloprost and sulprostone was not affected by the DP-, EP1- and EP2-receptor antagonist AH6809 at a concentration of 3 microM, i.e. a 3-30 times greater concentration than its affinity (pA2 values) at the respective receptors. 5. Circaprost (IP-receptor agonist; 1-100 nM), iloprost (IP- and EP1-receptor agonist; 10-1000 nM) and U-46619 (TP-receptor agonist; 100-1000 nM) did not significantly affect [3H]ACh release. 6. Blockade of cyclooxygenase by 3 microM indomethacin did not significantly modulate evoked [3H]ACh release in epithelium-containing and epithelium-denuded bronchi. Likewise, the combined cyclo- and lipoxygenase inhibitor BW-755C (20 microM) did not affect evoked [3H]ACh release. 7. In conclusion, applied prostanoids appear to inhibit [3H]ACh release in epithelium-denuded human bronchi under the present in vitro conditions, most likely via prejunctional prostanoid receptors of the EP3 subtype.
Assuntos
Acetilcolina/metabolismo , Brônquios/efeitos dos fármacos , Prostaglandinas/farmacologia , Receptores de Prostaglandina E/metabolismo , 4,5-Di-Hidro-1-(3-(Trifluormetil)Fenil)-1H-Pirazol-3-Amina/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Brônquios/metabolismo , Humanos , Técnicas In Vitro , Indometacina/farmacologia , Receptores de Prostaglandina E/efeitos dos fármacos , TrítioRESUMO
The outflow of 5-hydroxytryptamine (5-HT) from isolated tracheae of newborn rabbits was determined by high pressure liquid chromatography with electrochemical detection. This 5-HT outflow reflects release from neuroendocrine epithelial cells of the airway mucosa, as previously shown. Phenylephrine, via alpha2B-adrenoceptors, caused a transient increase in 5-HT outflow, maximally by about 250%, an effect mediated by liberation of intracellular Ca2+, as previously shown. The non-selective phosphodiesterase inhibitor 2-isobutyl-1-methylxanthine (IBMX) concentration-dependently inhibited phenylephrine-induced 5-HT release (completely at 100 microM, IC50: 1.3 microM). Likewise, benzafentrine (inhibitor of phosphodiesterase 3 and 4) and siguazodan (inhibitor of phosphodiesterase 3) also almost completely inhibited phenylephrine-induced 5-HT release with IC50 values of 1.7 and 4.2 microM, respectively. Rolipram (inhibitor of phosphodiesterase 4), in a concentration of 10 microM, which exceeds more than 10-fold the reported IC50 for phosphodiesterase 4, did not significantly affect phenylephrine-induced 5-HT release. 5-HT release induced by depolarizing concentrations of K+ (45 mM), which largely depends on extracellular Ca2+, was not affected by IBMX. In conclusion, phosphodiesterases, with characteristics of phosphodiesterase 3, appear to play an important role in the control of cyclic nucleotide mediated inhibition of 5-HT release from neuroendocrine epithelial cells.
Assuntos
Inibidores de Fosfodiesterase/farmacologia , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/fisiologia , Serotonina/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Animais Recém-Nascidos , Inibidores Enzimáticos/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Masculino , Oxidiazóis/farmacologia , Fenilefrina/farmacologia , Quinoxalinas/farmacologia , CoelhosRESUMO
1. The role of protein kinase C in controlling L-arginine transport in alveolar macrophages was investigated. 2. L-[3H]Arginine uptake in rabbit alveolar macrophages declined by 80 % after 20 h in culture. 4beta-Phorbol 12-myristate 13-acetate (PMA), but not 4alpha-phorbol 12-myristate 13-acetate (alpha-PMA), present during 20 h culture, enhanced L-[3H]arginine uptake more than 10-fold. Staurosporine and chelerythrine opposed this effect. 3. L-[3H]Arginine uptake was saturable and blockable by L-lysine. After PMA treatment Vmax was increased more than 5-fold and Km was reduced from 0.65 to 0.32 mM. 4. Time course experiments showed that PMA increased L-[3H]arginine uptake almost maximally within 2 h. This short-term effect was not affected by cycloheximide or actinomycin D. 5. L-[3H]Arginine uptake and its stimulation by PMA was also observed in sodium-free medium. 6. L-Leucine (0.1 mM) inhibited L-[3H]arginine uptake by 50 % in sodium-containing medium, but not in sodium-free medium. At 1 mM, L-leucine caused significant inhibition in sodium-free medium also. L-Leucine showed similar effects on PMA-treated cells. 7. N-Ethylmaleimide (200 microM, 10 min) reduced L-[3H]arginine uptake by 70 % in control cells, but had no effect on PMA-treated (20 or 2 h) cells. 8. In alveolar macrophages, multiple transport systems are involved in L-arginine uptake, which is markedly stimulated by protein kinase C, probably by modulation of the activity of already expressed cationic amino acid transporters.
Assuntos
Arginina/farmacocinética , Proteínas de Transporte/metabolismo , Macrófagos Alveolares/enzimologia , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , Sistemas de Transporte de Aminoácidos Básicos , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Carcinógenos/farmacologia , Proteínas de Transporte/genética , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Feminino , Cinética , Leucina/farmacologia , Macrófagos Alveolares/química , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/análise , Coelhos , Ratos , Ratos Sprague-Dawley , Sódio/farmacologia , Especificidade da Espécie , Acetato de Tetradecanoilforbol/farmacologia , TrítioRESUMO
The cholinergic system in rat and human airways and the effects of glucocorticoids were investigated by assay of choline acetyltransferase activity, by high-pressure liquid chromatography measurement of acetylcholine, and by anti-choline acetyltransferase immunocyto-/histochemistry. Human bronchi were obtained at surgery from patients with lung cancer. Group 1 patients did not suffer from additional lung diseases and had not been treated with glucocorticoids. Group 2 patients, who suffered in addition to lung cancer from chronic obstructive bronchitis, had been treated for at least 6 weeks before surgery with four puffs of flusinolid daily. Isolated bronchial epithelial cells as well as intact surface epithelium of human bronchi expressed choline acetyltransferase immunoreactivity and choline acetyltransferase enzyme activity (3 +/- 1 nmol/mg protein per h). Ciliated epithelial cells showed strong choline acetyltransferase immunoreactivity at the basal body and the roolet of cilia. Surface epithelium in group 1 and 2 bronchi contained 23 +/- 6 (n = 14) and 1.8 +/- 0.3 pmol/g acetylcholine) (n = 7, P < 0.001), respectively, whereas the transmural acetylcholine content did not differ significantly between both groups. The amount of choline acetyltransferase immunoreactivity appeared similar in the surface epithelium of both groups. In an animal (rat) study the effects of oral dexamethasone (3 mg/day, 1 week) on choline acetyltransferase activity and acetylcholine levels were investigated. Dexamethasone treatment reduced epithelial acetylcholine in the airways and small intestine by about 80% and inhibited epithelial choline acetyltransferase activity. In conclusion, epithelial cells of human airways possess components of the cholinergic system, i.e., contain the synthesizing enzyme choline acetyltransferase and store acetylcholine. The data obtained from the animal study indicate that glucocorticoids can inhibit epithelial acetylcholine.