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1.
Mol Pharmacol ; 60(6): 1201-9, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11723227

RESUMO

The epithelial or endothelial cells that line the human bronchi and the aorta express nicotinic acetylcholine receptors (nAChRs) of alpha3 subtypes. We report here that human bronchial epithelial cells (BEC) and aortic endothelial cells (AEC) express also the nAChR alpha7 subunit, which forms functional nAChRs. Polymerase chain reaction and in situ hybridization experiments detected alpha7 subunit mRNA in cultured human BEC and AEC and in sections of rat trachea. The binding of radiolabeled alpha-bungarotoxin revealed a few thousand binding sites per cell in cultured human BEC and human and bovine AEC. Western blot and immunohistochemistry experiments demonstrated that cultured BEC and AEC express a protein(s) recognized by anti-alpha7 antibodies. Whole-cell patch-clamp studies of cultured human BEC demonstrated the presence of fast-desensitizing currents activated by choline and nicotine that were blocked reversibly by methyllycaconitine (1 nM) and irreversibly by alpha-bungarotoxin (100 nM), consistent with the expression of functional alpha7 nAChRs. In some cells, choline activated also slowly decaying currents, confirming previous reports that BEC express functional alpha3beta4 nAChRs. Exposure of cultured BEC to nicotine (1 microM) for 3 days up-regulated functional alpha7 and alpha3 nAChRs, as indicated by the increased number of cells responding to acetylcholine and choline, with both fast-desensitizing currents, which were blocked irreversibly by alpha-bungarotoxin, and with slowly desensitizing currents, which are alpha-bungarotoxin-insensitive currents. The presence of alpha7 nAChRs in BEC and AEC suggests that some toxic effects of tobacco smoke could be mediated through these nicotine-sensitive receptors.


Assuntos
Brônquios/metabolismo , Endotélio Vascular/metabolismo , Receptores Nicotínicos/biossíntese , Animais , Especificidade de Anticorpos , Sítios de Ligação , Western Blotting , Brônquios/citologia , Bungarotoxinas/metabolismo , Bovinos , Clonagem Molecular , Eletrofisiologia , Células Epiteliais/metabolismo , Imunofluorescência , Humanos , Hibridização In Situ , Radioisótopos do Iodo , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Receptores Nicotínicos/genética , Receptores Nicotínicos/imunologia , Receptores Nicotínicos/fisiologia , Traqueia/metabolismo , Transcrição Gênica , Receptor Nicotínico de Acetilcolina alfa7
2.
Eur J Pharmacol ; 393(1-3): 279-94, 2000 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-10771024

RESUMO

The nicotinic acetylcholine receptors are prototypic ionotropic receptors that mediate fast synaptic transmission. However, also non-excitable cells, and particularly the tegumental cells that line external and internal body surfaces, express acetylcholine receptors of neuronal type sensitive to nicotine. Bronchial epithelial cells, endothelial cells of blood vessels and skin keratinocytes express neuronal nicotinic receptors composed of alpha(3), alpha(5), beta(2) and beta(4) subunits, similar to those expressed in sympathetic ganglia, and neuronal nicotinic receptors composed of alpha(7) subunits. Neuronal nicotinic receptors in tegumental cells are involved in modulating cell shape and motility, and therefore in maintaining the integrity of the surfaces lined by those cells. Neuronal nicotinic receptors in non-neuronal tissues may modulate other functions, including cell proliferation and differentiation. Acetylcholine is synthesized, secreted and degraded by a variety of cells, including the tegumental cells that express neuronal nicotinic receptors. Thus, acetylcholine may function as a local "hormone" that is able to modulate cell functions that require fast adaptation to new conditions. The presence of neuronal nicotinic receptors sensitive to nicotine in tissues known to be involved in tobacco toxicity, like bronchi and blood vessels, raises the possibility that they mediate some of the toxic effects of smoking.


Assuntos
Nicotiana/efeitos adversos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Plantas Tóxicas , Receptores Nicotínicos/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Neurônios , Receptores Nicotínicos/efeitos dos fármacos , Nicotiana/metabolismo
3.
Mol Pharmacol ; 54(5): 779-88, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9804613

RESUMO

We demonstrated previously that human skin keratinocytes express acetylcholine receptors (AChRs) sensitive to acetylcholine and nicotine, which regulate cell adhesion and motility. We demonstrate here that human and rodent bronchial epithelial cells (BECs) express AChRs similar to those expressed by keratinocytes and by some neurons. Patch-clamp experiments demonstrated that the BEC AChRs are functional, and they are activated by acetylcholine and nicotine. They are blocked by kappa-bungarotoxin, a specific antagonist of the AChR isotypes expressed by neurons in ganglia. Their ion-gating properties are consistent with those of AChR isotypes expressed in ganglia, formed by alpha3, alpha5, and beta2 or beta4 subunits. Reverse transcription-polymerase chain reaction and in situ hybridization experiments demonstrated the presence in BECs of mRNA transcripts for all those AChR subunits, both in cell cultures and in tissue sections, whereas we could not detect transcripts for the alpha2, alpha4, alpha6, and beta3 AChR subunits. The expression of alpha3 and alpha5 proteins in BEC in vivo was verified by the binding of subunit-specific antibodies to sections of trachea. Mecamylamine and kappa-bungarotoxin, which are cholinergic antagonists able to block the ganglionic alpha3 AChRs, caused a reversible change of the cell shape of cultured, confluent human BECs. This resulted in a reduction of the area covered by the cell and in cell/cell detachment. The presence of AChRs sensitive to nicotine on the lining of the airways raises the possibility that the high concentrations of nicotine resulting from tobacco smoking will cause an abnormal activation, a desensitization, or both of the bronchial AChRs. This may mediate or facilitate some of the toxic effects of cigarette smoking in the respiratory system.


Assuntos
Brônquios/ultraestrutura , Receptores Nicotínicos/fisiologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Brônquios/citologia , Brônquios/fisiologia , Adesão Celular/fisiologia , Tamanho Celular/fisiologia , Células Cultivadas , Antagonistas Colinérgicos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Imunofluorescência , Humanos , Hibridização In Situ , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Nicotina/farmacologia , Agonistas Nicotínicos/metabolismo , Agonistas Nicotínicos/farmacologia , Piridinas/metabolismo , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Coelhos , Ratos , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Especificidade da Espécie , Traqueia/metabolismo , Traqueia/fisiologia , Traqueia/ultraestrutura , Trítio
4.
J Pharmacol Exp Ther ; 287(1): 435-9, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9765366

RESUMO

ACh receptors sensitive to nicotine (nAChR) are present in human skin keratinocytes and in bronchial epithelial cells. They are stimulated by ACh secreted by the same cells that express them, and they modulate cell motility and shape. A variety of non-neuronal tissues, including endothelial cells, synthesize ACh, which raises the possibility that they are sensitive to nicotine. We demonstrate here that endothelial cells that line blood vessels express functional nAChRs. Their structure and ion-gating properties are similar to those of the nAChRs expressed by ganglionic neurons and by skin keratinocytes and bronchial epithelial cells. In situ hybridization experiments using primary cultures of endothelial cells from human aorta demonstrated the presence in these cells of the subunits believed to contribute to ganglionic ACh receptors (AChRs) of the alpha3 subtype: alpha3, alpha5, beta2 and beta4. Binding of radiolabeled epibatidine-a high-affinity specific ligand of certain neuronal AChRs, including the alpha3 subtypes-revealed the presence of approximately 900 specific binding sites per cell. We assessed the presence of functional AChRs by patch-clamp experiments. Cultured human endothelial cells express ion channels that are opened by (+)-anatoxin-a and are blocked by dihydro-beta-erythroidine. These are specific agonist and antagonist, respectively, of neuronal AChRs of the alpha3 subtype. The ion-gating properties of the endothelial AChRs were similar to those of neuronal ganglionic AChRs. The presence of AChRs sensitive to nicotine in endothelial cells may be related to the toxic effects of nicotine on the vascular system.


Assuntos
Endotélio Vascular/química , Receptores Nicotínicos/análise , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Bovinos , Células Cultivadas , Humanos , Hibridização In Situ , Ativação do Canal Iônico , Técnicas de Patch-Clamp , Piridinas/metabolismo , RNA Mensageiro/análise , Receptores Nicotínicos/genética , Receptores Nicotínicos/fisiologia
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