Effects of neuropeptides on airway epithelial single ion channel current and involvement of signal transduction

以培养的犬气管上皮为材料，采用短路技术和膜片钳技术，测定其电学特性(短路电流、跨上皮电位差、细胞电导)和单离子通道电流。将气管上皮细胞接种在聚碳酸酯滤膜上，在CO2培养箱中孵育7天，然后将其安装在Lucite半腔体之间，连续测量生物电学特性。在膜片钳研究中，观察到细胞贴附和内向外两种贴片构型下的单通道活动。然而，在切除状态下，由内向外的贴片(贴片的细胞质一侧面向浴液中的0.9 mm钙)更能激活这些通道。在由内向外的样品中，检测到非选择性阳离子通道(NS通道)和K选择性通道。NS通道在正膜电位和负膜电位下均处于激活状态。在正常的Ringer Solu…中也观察到了类似的通道电流更多的氯化钠(140氯化钠)，无论是含140氯化钠、130纳葡糖酸盐/10氯化钠或70氯化钠/75Kc1的膜片吸管溶液。电流电压(I-V)关系是线性的，在膜电位(Vm)附近为0 mV处截断X轴。应用神经肽P物质和神经激肽B激活单个氯离子通道电流，用氯离子通道阻断剂二苯胺-2-羧酸盐阻断该电流；应用P物质、内皮素和血小板激活因子使气管上皮短路电流增加。这些效应是浓度依赖的，并被二苯胺-2-羧酸盐、氯转运抑制剂速尿或布美他尼和用不能跨呼吸道上皮转运的碘取代培养液中的氯而减弱。跨上皮电位差和细胞电导也增加。P物质引起的短路电流增加可被中性内肽酶的特异性抑制剂磷酰胺显著增强，但不能被卡托普利、亮肽素或贝斯汀改变。放射免疫分析显示，P物质对上皮细胞电学特性的刺激作用伴随着细胞内cAMP水平的升高。这些结果表明，多种神经肽可通过激活细胞内信号转导系统选择性地刺激通过呼吸道上皮的氯分泌，并可能在调节呼吸道粘液纤毛运输功能中发挥作用。较少

By using canine tracheal epithelium in culture, electrical properties (short-circuit current, transepithelial potential difference, cell conductance) and single ion channel current were estimated through Ussings short-circuited technique and patch clamp method. Tracheal epithelial cells were seeded on polycarbonate filters and incubated for seven days in a CO_2 incubator, which were then mounted between Lucite half-Chambers and bioelectric properties were continuously measured.In patch clamp studies, single cannel activity was observed in both cell-attached and inside-out patch configurations. However, these channels were more activated in excised status, inside-out patches (cytoplasmic side of the patch membrane was facing 0.9 mM Ca in the bath). In inside-out parches, nonselective cation chamels (NS channels) and K selective channels were detected. NS channels were active at both positive and negative membrane potentials. Similar channel currents were observed in a normal Ringer solu … More tion (140 NaCl) whether patch-pipette solution contained 140 NaCl, 130 Nagluconate/10 NaCl, or 70 NaCl/75 KC1. The current voltage (I-V) relationship was linear and intercepted the X-axis at near Vm (membrane potential) being 0 mV. Single Cl channel current was also detected and activated by application of neuropeptides including substance P and neurokinin B, which was abolished by the Cl channel blocker diphenylamine-2-carboxylate.Tracheal epithelial short-circuit current was increased by application of substance P, endothelin, and platelet-activating factor. These effects were concentration-dependent, and attenuated by pretreatment of cells by diphenylamine-2-carboxylate, the Cl transport inhibitor furosemide or bumetanide, and substitution of Cl in the medium by iodide that cannot be transported across airway epithelium. Transepithelial potential difference and cell conductance were also increased. The increase in short-circuit current induced by substance P was greatly potentiated by pretreatment of cells with phosphoramidon, a specific inhibitor of neutral endopeptidasse, whereas it was not altered by captopril, leupeptin, or bestatin. The stimulatory effect of substance P on the epithelial electrical properties was accompanied by the increase in intracellular cyclic AMP levels, as detected by radioimmunoassay. These results indicate the various neuropeptides can sellectively stimulate Cl secretion across airway epithelium through activation of intracellular signal transduction systems, and may play a role in regulating mucociliary transport function in the respiratory tract. Less