RESUMO
Synthesis of acetylcholine (ACh) by non-neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na(+) -dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non-neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non-neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter-like proteins, a five gene family choline-transporter like protein (CTL)1-5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na(+) -independent and CTL1-5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non-neuronal cell lines and presents a mechanism to target non-neuronal ACh synthesis without affecting neuronal ACh synthesis.
Assuntos
Acetilcolina/biossíntese , Colina/farmacocinética , Proteínas de Membrana Transportadoras/metabolismo , Acetilcolina/metabolismo , Atropina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo , Meios de Cultura/farmacologia , Humanos , Neoplasias Pulmonares , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Antagonistas Muscarínicos/farmacologia , RNA Interferente Pequeno/genética , Carcinoma de Pequenas Células do Pulmão , TrítioRESUMO
Our laboratory has previously reported that bronchial epithelial cells (BEC) express a regulatory cascade of classic neurotransmitters and receptors that communicate in an almost neuronal-like manner to achieve physiological regulation. In this paper we show that the similarity between neurotransmitter signaling in neurons and BEC extends to the level of transmitter receptor allosteric modulators. Lynx1 is a member of the ly-6/three-finger superfamily of proteins, many of which modulate receptor signaling activity. Lynx1 specifically has been shown to modulate nicotinic acetylcholine receptor (nAChR) function in neurons by altering receptor sensitivity and desensitization. We now report that lynx1 forms a complex with α7 nAChR in BEC and serves to negatively regulate α7 downstream signaling events. Treatment of primary cultures of BEC with nicotine increased levels of nAChR subunits and that increase was potentiated by lynx1 knockdown. Lynx1 knockdown also potentiated the nicotine-induced increase in GABA(A) receptors (GABA(A)R) and MUC5AC mRNA expression, and that effect was blocked by α7 antagonists and α7 knockdown. In parallel with the increases in nAChR, GABA(A)R, and mucin mRNA levels, lynx1 knockdown also increased levels of p-Src. Consistent with this, inhibition of Src signaling blocked the ability of the lynx1 knockdown to increase basal and nicotine-stimulated GABA(A)R and mucin mRNA expression. Thus lynx1 appears to act as a negative modulator of α7 nAChR-induced events by inhibiting Src activation. This suggests that lynx1 agonists or mimetics are a potentially important therapeutic target to develop new therapies for smoking-related diseases characterized by increased mucin expression.
Assuntos
Antígenos Ly/metabolismo , Proteínas Ligadas por GPI/metabolismo , Receptores Nicotínicos/metabolismo , Mucosa Respiratória/metabolismo , Transdução de Sinais/imunologia , Fumar/imunologia , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Asma/imunologia , Asma/metabolismo , Brônquios/citologia , Células Cultivadas , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Técnicas de Silenciamento de Genes , Macaca mulatta , Mucina-5AC/imunologia , Mucina-5AC/metabolismo , Nicotina/imunologia , Nicotina/metabolismo , Agonistas Nicotínicos/imunologia , Agonistas Nicotínicos/metabolismo , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , RNA Interferente Pequeno/genética , Receptores de GABA-A/imunologia , Receptores de GABA-A/metabolismo , Receptores Nicotínicos/imunologia , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Fumar/metabolismo , Receptor Nicotínico de Acetilcolina alfa7 , Quinases da Família src/metabolismoRESUMO
Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination appears to promote an epigenetic reprogramming of the male germ line that is associated with transgenerational adult-onset disease states. Transgenerational effects on the embryonic day 16 (E16) testis demonstrated reproducible changes in the testis transcriptome for multiple generations (F1-F3). The expression of 196 genes was found to be influenced, with the majority of gene expression being decreased or silenced. Dramatic changes in the gene expression of methyltransferases during gonadal sex determination were observed in the F1 and F2 vinclozolin generation (E16) embryonic testis, but the majority returned to control-generation levels by the F3 generation. The most dramatic effects were on the germ-line-associated Dnmt3A and Dnmt3L isoforms. Observations demonstrate that an embryonic exposure to vinclozolin appears to promote an epigenetic reprogramming of the male germ line that correlates with transgenerational alterations in the testis transcriptome in subsequent generations.
Assuntos
Antagonistas de Androgênios/toxicidade , DNA (Citosina-5-)-Metiltransferases/biossíntese , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Exposição Materna/efeitos adversos , Oxazóis/toxicidade , Efeitos Tardios da Exposição Pré-Natal/enzimologia , Testículo/enzimologia , Transcrição Gênica/efeitos dos fármacos , Animais , DNA Metiltransferase 3A , Feminino , Humanos , Isoenzimas/biossíntese , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Ratos , Ratos Sprague-Dawley , Processos de Determinação Sexual , Espermatozoides/enzimologia , Espermatozoides/patologia , Testículo/embriologia , Testículo/patologiaRESUMO
Exposure of gestating female rats to the anti-androgenic endocrine disruptor vinclozolin has been shown to induce transgenerational adult onset disease phenotypes. The current study, was designed to compare the actions of vinclozolin to the known anti-androgenic compound flutamide. The gestating female rats were exposed to intraperitoneal injections during embryonic day 8-14 (E8-E14) to 100mg/kg/day vinclozolin or flutamide at either 5mg or 20mg/kg/day. As previously observed, vinclozolin induced a transgenerational testis phenotype of increased spermatogenic cell apoptosis and decreased epididymal sperm number. In contrast, the flutamide exposures resulted in a testis phenotype of increased spermatogenic cell apoptosis and decreased epididymal sperm numbers in the F1 generation only, and not the F2 and F3 generation adult males. Interestingly, some of the low dose (5mg/kg) flutamide F2 generation offspring developed spinal agenesis and supernummery development (polymelia) of limbs. Although the actions of vinclozolin and flutamide appear similar in the F1 generation males, the transgenerational effects of vinclozolin do not appear to be acting through the same anti-androgenic mechanism as flutamide.
Assuntos
Inibidores da Angiogênese/toxicidade , Flutamida/toxicidade , Oxazóis/toxicidade , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Espermatogênese/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Masculino , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Ratos , Ratos Sprague-Dawley , Contagem de Espermatozoides , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/patologia , Testículo/efeitos dos fármacos , Testículo/crescimento & desenvolvimento , Testículo/patologiaRESUMO
Embryonic exposure to the endocrine disruptor vinclozolin at the time of gonadal sex determination was previously found to promote transgenerational disease states. The actions of vinclozolin appear to be due to epigenetic alterations in the male germline that are transmitted to subsequent generations. Analysis of the transgenerational epigenetic effects on the male germline (i.e. sperm) identified 25 candidate DNA sequences with altered methylation patterns in the vinclozolin generation sperm. These sequences were identified and mapped to specific genes and noncoding DNA regions. Bisulfite sequencing was used to confirm the altered methylation pattern of 15 of the candidate DNA sequences. Alterations in the epigenetic pattern (i.e. methylation) of these genes/DNA sequences were found in the F2 and F3 generation germline. Therefore, the reprogramming of the male germline involves the induction of new imprinted-like genes/DNA sequences that acquire an apparent permanent DNA methylation pattern that is passed at least through the paternal allele. The expression pattern of several of the genes during embryonic development were found to be altered in the vinclozolin F1 and F2 generation testis. A number of the imprinted-like genes/DNA sequences identified are associated with epigenetic linked diseases. In summary, an endocrine disruptor exposure during embryonic gonadal sex determination was found to promote an alteration in the epigenetic (i.e. induction of imprinted-like genes/DNA sequences) programming of the male germline, and this is associated with the development of transgenerational disease states.