LOCALIZATION OF SUBSTANCE P AND ACETYLCHOLINE IN THE PATHWAY MEDIATING MUCOCILI

P 物质和乙酰胆碱在介导粘液纤毛途径中的定位

• 批准号: 7381561
• 负责人: CARMEN R HERNANDEZ
• 金额: $ 18.52万
• 依托单位: UNIVERSITY OF PUERTO RICO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381561
• 关键词: LOCALIZATION; SUBSTANCE; ACETYLCHOLINE; PATHWAY; MEDIATING

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mucociliary activity is an important clearance mechanism in the respiratory system of air breathing vertebrates. An in depth knowledge of the regulation of mucociliary activity may assist in the development of new treatments for various clinical conditions such as asthma, chronic sinusitis, cystic fibrosis and bronchitis which are associated with impaired mucociliary activity. Increased mucociliary activity during these conditions is beneficial in the sense that pharmacological or physiotherapeutic manipulation of this reflex may be of therapeutic value. The administration of substance P (SP) and acetylcholine (ACh) as well as electrical stimulation of the palatine nerve were shown to increase mucociliary activity whereas dopamine and serotonin were shown to inhibit mucocitiary activity. Hernandez, Berrios and Chinapen (2003) found: SP-labeled cell bodies in the trigeminal ganglion from which the palatine nerve emerges; SP-labeled fibers innervating the palate; and SP-labeled fibers innervating the blood vessels of the palate. However, it remains to be established if SP-labeled cell bodies are the cells of origin of the palatine nerve? Once this question is addressed, the localization of SP, NK-1 receptors in this pathway will be determined and its interaction with SP fibers will be studied. Since ACh also regulates mucociliary activity it is necessary to determine whether it is localized in this pathway. Once this is established the identification and localization of the ACh muscarinic receptor subtype in the palate and its interaction with ChAT fibers will also be addressed. Therefore, the long term goal of this study is to elucidate the neuronal pathways mediating mucociliary activity using the palate of the frog Rana pipiens as an animal model. The results of this experiment will be compared with future studies in mammals. The experiments of this proposal are: I. Identification of SP in the pathway mediating mucociliary activity. - To determine whether the trigeminal ganglion contains the cells of origin of the palatine nerve. The retrograde tracer, Fluoro Gold and the anterograde tracer, Biotinylated Dextran Amine will be used to identify the cells and terminals respectively. It is hypothesized that the trigeminal ganglion contains the cells of origin of the palatine nerve. - To determine whether the cells of origin of the palatine nerve (identified in specific aim la) contain SP. This will be accomplished by double-labeling procedures combining retrograde and/or anterograde labeled sections with the immunofluorescent labeling for SP. It is hypothesized that the SP-labeled neurons of the trigeminal ganglion are the cells of origin of the palatine nerve. - To determine if BDA anterograde labeled nerve terminals contain SP and if so whether they synapse with mucus and epithelia cells of the palate using immunoelectron microscopy. It is hypothesized that there are SP-containing terminals snapping on the goblet and ciliated epithelial cells of the palate. - To localize SP, NK-1 receptors in the palate and medulla and demonstrate its interaction with SP-labeled BDA containing terminals. This will be accomplished with a triple labeling procedure combining BDA labeled sections with the immunocytochemical labeling for SP, NK-lreceptor and SP. It is hypothesized that SPlabeled BDA containing terminals interact with SP, NK-1 receptors in the palate and medulla. II. Identification of acetylcholine in the pathway mediating mucociliary activity. - To localize choline acetyl transferase (ChAT)-containing cell bodies in the trigeminal ganglion and in the fibers that innervate the palate. This will be accomplished with the immunocytochemical method to detect CHAT; the enzyme that catalyzes the synthesis of acetylcholine. It is hypothesized that there are ChAT containing cell bodies in the trigeminal ganglion and ChAT containing fibers in the palate of Rana pipiens. - To determine whether SP and ChAT -fibers in the palate are labeled with BDA injected into the trigeminal ganglion. This will be accomplished by a triple-labeling procedure combining anterograde labeled sections with the immunocytochemical labeling for SP and CHAT. It is hypothesized that SP, ChAT and/or BDA are colocalized in the same neurons. - To localize the ACh muscarinic receptor subtype (s) in the palate and demonstrate its interaction with CHATlabeled BDA fibers identified in the palate. This will be accomplished with a triple labeling procedure combining BDA labeled sections with the immunocytochemical labeling for ACh muscarinic receptor and CHAT. It is hypothesized that ChAT-labeled BDA containing terminals interact with ACh muscarinic receptor(s) in the palate and medulla.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。粘膜纤毛活性是空气呼吸脊椎动物呼吸系统中的重要清除机制。深入了解粘膜纤毛活性调节的知识可能有助于开发各种临床状况的新处理，例如哮喘，慢性鼻窦炎，囊性纤维化和支气管炎，这与粘膜助剂活性受损有关。在这些条件下，在这些反射的药理学或物理治疗操作的意义上，增加的粘钙活性可能具有治疗价值。施用P（SP）和乙酰胆碱（ACH）以及palatine神经的电刺激可增加粘膜纤毛活性，而多巴胺和5-羟色胺则显示抑制粘核活性。 Hernandez，Berrios和Chinapen（2003）发现：三叉神经神经的SP标记的细胞体出现。 Sp标记的纤维支配口感；和SP标记的纤维支配口感的血管。但是，如果SP标记的细胞体是palatine神经的起源细胞，仍然有待确定吗？一旦解决了这个问题，将确定SP，NK-1受体的定位，并研究其与SP纤维的相互作用。由于ACH还调节粘膜纤毛活性，因此有必要确定它是否位于该途径中。一旦确定了ACH毒蕈碱受体亚型中ACH的识别和定位，并且还将解决其与聊天纤维的相互作用。因此，这项研究的长期目标是阐明使用青蛙rana pipiens作为动物模型的味觉介导粘膜纤毛活性的神经元途径。该实验的结果将与未来在哺乳动物中的研究进行比较。 该提案的实验是：I。在介导粘液助剂活性的途径中识别SP。 - 确定三叉神经节是否包含palatine神经的起源细胞。逆行示踪剂，氟金和顺行示踪剂，生物素化的葡萄糖胺将分别用于鉴定细胞和末端。假设三叉神经节包含palatine神经的起源细胞。 - 确定palatine神经的起源细胞（在特定AIM LA中识别）是否包含sp。这将通过将逆行和/或顺行标记的切片与SP的免疫荧光标签结合的双标记程序来完成。假设三叉神经节的SP标记的神经元是palatine神经的起源细胞。 - 确定BDA顺行标记的神经末端是否包含SP，如果是使用免疫电子显微镜与pa的粘液和上皮细胞突触。假设在杯状上有含有SP的末端，并触及了pa的纤毛上皮细胞。 - 要定位SP，NK-1受体在口感和髓质中，并证明了其与含有SP标记的BDA末端的相互作用。这将通过将BDA标记的切片与SP，NK-LECEPTOR和SP的免疫细胞化学标记结合的三标记程序和SP。假设包含末端的滴定BDA与pa和髓质中的SP，NK-1受体相互作用。 ii。乙酰胆碱在介导粘膜助剂活性的途径中鉴定。 - 将胆碱乙酰基转移酶（CHAT）定位在三叉神经节中的含有含有的细胞体和神经化味的纤维中。这将通过免疫细胞化学方法来检测聊天。催化乙酰胆碱的合成的酶。假设三叉神经节中的聊天中包含细胞体的聊天，并在拉娜·索尼斯（Rana Pipiens）的口感中含有纤维。 - 确定口感中的SP和CHAT -fiber是否用注射到三叉神经节中的BDA标记。这将通过将标记的部分与SP和CHAT的免疫细胞化学标签相结合的三标签程序来实现。假设SP，CHAT和/或BDA在同一神经元中共定位。 - 将ACH毒蕈碱受体亚型（S）定位在口感中，并证明了其与口感中鉴定出的聊天标签的BDA纤维相互作用。这将通过将BDA标记部分与ACH毒蕈碱受体和聊天的免疫细胞化学标记结合的三标记程序和聊天结合使用。假设包含末端的聊天标记的BDA与pa和髓质中的ACH毒蕈碱受体相互作用。