NMDA receptors mediate contractile responses in human airway smooth muscle cells
NMDA receptors mediate contractile responses in human airway smooth muscle cells
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DOI：
10.1152/ajplung.00402.2014
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发表时间：
2015-06-15
影响因子：
4.9
通讯作者：
Moqbel, Redwan
中科院分区：
文献类型：
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作者：
Anaparti, Vidyanand;Ilarraza, Ramses;Moqbel, Redwan
人气道平滑肌（HASM）在哮喘中表现出增强的收缩性。炎症与气道过度收缩相关，但支持这些特征的因素尚未完全阐明。在气道炎症中观察到与血浆谷氨酸浓度增加相关的谷氨酸毒性，表明多亚基谷氨酸受体，N-甲基-D-天冬氨酸受体（NMDA-R）有助于气道高反应性。我们检验了HASM表达NMDA-R亚单位的假设，该亚单位可以形成功能性受体来介导对特定细胞外配体的收缩反应。在培养的HASM细胞中，我们通过定量PCR、免疫印迹、流式细胞术和荧光免疫细胞化学测定NMDA-R亚基mRNA和蛋白丰度。我们测量了一些NMDA-R亚基的mRNA，包括强制性NR 1亚基，我们证实它是以蛋白质的形式存在的。在体外和离体功能NMDA-R的激活HASM细胞测定使用细胞内钙流量（Fura-2 AM），胶原凝胶收缩试验，和小鼠薄切肺切片（TCLS）。NMDA，一种药理学谷氨酸类似物，诱导培养的HASM细胞胞浆钙动员。我们检测到三种不同的时间模式的钙反应，这表明存在异质性心肌细胞亚群。NMDA-R活化也诱导接种HASM细胞的小鼠TCLS和软胶原凝胶的气道收缩。细胞、肺切片和胶原凝胶中的反应由NMDA-R介导，因为它们可以被特异性NMDA-R抑制剂（2 R）-氨基-5-膦酰基戊酸阻断。总之，我们揭示了NMDA-R在HASM中的存在，其通过神经元能机制介导收缩反应。这些发现表明，与气道炎症相关的NMDA-R谷氨酸样配体的积累直接导致气道高反应性。
Human airway smooth muscle (HASM) exhibits enhanced contractility in asthma. Inflammation is associated with airway hypercontractility, but factors that underpin these features are not fully elucidated. Glutamate toxicity associated with increased plasma glutamate concentrations was observed in airway inflammation, suggesting that multisubunit glutamate receptors, N-methyl-D-aspartate receptors (NMDA-R) contribute to airway hyperreactivity. We tested the hypothesis that HASM expresses NMDA-R subunits that can form functional receptors to mediate contractile responses to specific extracellular ligands. In cultured HASM cells, we measured NMDA-R subunit mRNA and protein abundance by quantitative PCR, immunoblotting, flow cytometry, and epifluorescence immunocytochemistry. We measured mRNA for a number of NMDA-R subunits, including the obligatory NR1 subunit, which we confirmed to be present as a protein. In vitro and ex vivo functional NMDA-R activation in HASM cells was measured using intracellular calcium flux (fura-2 AM), collagen gel contraction assays, and murine thin-cut lung slices (TCLS). NMDA, a pharmacological glutamate analog, induced cytosolic calcium mobilization in cultured HASM cells. We detected three different temporal patterns of calcium response, suggesting the presence of heterogeneous myocyte subpopulations. NMDA-R activation also induced airway contraction in murine TCLS and soft collagen gels seeded with HASM cells. Responses in cells, lung slices, and collagen gels were mediated by NMDA-R, as they could be blocked by (2R)-amino-5-phosphonopentanoate, a specific NMDA-R inhibitor. In summary, we reveal the presence of NMDA-R in HASM that mediate contractile responses via glutamatergic mechanisms. These findings suggest that accumulation of glutamate-like ligands for NMDA-R associated with airway inflammation contributes directly to airway hyperreactivity.