Expression of functional beta2-adrenergic receptors in the lung epithelial cell lines 16HBE14o(-), Calu-3 and A549.
Expression of functional beta2-adrenergic receptors in the lung epithelial cell lines 16HBE14o(-), Calu-3 and A549.
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功能性β2-肾上腺素能受体在肺上皮细胞系16HBE14o(-)、Calu-3和A549中的表达。
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发表时间：
2004
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通讯作者：
F. Ungemach
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作者：
G. Abraham;C. Kneuer;C. Ehrhardt;W. Honscha;F. Ungemach
肾上腺素能药物通过β（2）-肾上腺素受体（β（2）-AR）腺苷酸环化酶（AC）信号转导系统起作用，在哺乳动物气道上皮内引起多种反应;然而，其多种表型分化的细胞类型的组成使对该组织内的调节级联的理解复杂化。本研究应用逆转录聚合酶链反应（RT-PCR）、放射配体结合研究、[3 H]-放射免疫分析和免疫细胞化学染色等方法，研究了人气道上皮细胞系16 HBE 14 o（-）、Calu-3和A549中β 2-AR mRNA的表达、数量、亚型以及异丙肾上腺素（iso）对环腺苷酸（cAMP）的反应。在培养4-5天后，所有三种细胞类型都产生β（2）-AR mRNA和蛋白，其基因表达水平Calu-3>或= 16 HBE 14 o（-）>A549，而对照细胞Cos-1和Caco-2为阴性。β（2）-AR腺苷酸环化酶系统在人气道上皮细胞Calu-3和16 HBE 14 o（-）中高度表达并发挥功能。在Calu-3和16 HBE 14 o（-）细胞中，通过放射性配体结合评估的平均β（2）-AR密度（B（max））、平衡解离常数（K（D））和β-AR亚型百分比分别约为9908+/-1127和6423+/-895结合位点/细胞、32+/-2.7 pM和25+/-1.1 pM，约为100%。然而，在肺泡细胞型A549中，细胞表面β（2）-AR实际上无法通过（-）-[（125）I]-碘氰基吲哚酚（ICYP）结合检测到。用（-）-异丙肾上腺素刺激培养细胞，仅在Calu-3和16 HBE 14 o（-）细胞中增加了基础环AMP积累，这被β（2）-选择性拮抗剂ICI 118，551阻断，但不被β（1）-选择性拮抗剂CGP 20712 A阻断，证实了这些细胞中β（2）-AR与腺苷酸环化酶的功能偶联。免疫细胞化学染色结果显示，在Calu-3和16 HBE 14 o（-）细胞中，受体定位于细胞膜和细胞质，而在A549细胞中，受体仅定位于细胞质。总之，在人气道上皮细胞Calu-3和16 HBE 14 o（-）中，β（2）-AR表达及其与腺苷酸环化酶的功能偶联非常高，但在A549中不高，这表明细胞系Calu-3和16 HBE 14 o（-）为研究呼吸系统中β-肾上腺素受体信号传导的功能和调节提供了合适的模型。
Adrenergic drugs acting through the beta(2)-adrenoceptor (beta(2)-AR) adenylate cyclase (AC) signal transduction system elicit a variety of responses within the mammalian airway epithelium; however, its composition of multiple phenotypically differentiated cell types complicates the understanding of the regulation cascades within this tissue. The present study evaluates beta(2)-AR mRNA level, number, subtype and the cyclic adenosine-3',5'-monophosphate (cyclic AMP) response to isoproterenol (iso) in the human airway epithelial cell lines 16HBE14o(-), Calu-3 and A549, using reverse transcriptase polymerase chain reaction (RT-PCR), radioligand binding studies, [(3)H]-radioimmunoassay and immunocytochemical staining. After 4-5 days in culture, all three cell types produced beta(2)-AR mRNA and protein at a magnitude of gene expression levels Calu-3>or=16HBE14o(-)>A549, whereas control cells Cos-1 and Caco-2 were negative. The beta(2)-AR adenylate cyclase system was highly expressed and functional in the human airway epithelial cells Calu-3 and 16HBE14o(-). The mean beta(2)-AR density (B(max)), equilibrium dissociation constant (K(D)), and the percentage of beta-AR subtypes assessed by radioligand binding were approximately 9908+/-1127 and 6423+/-895 binding sites/cell, 32+/-2.7 pM and 25+/-1.1 pM, and approximately 100% in Calu-3 and 16HBE14o(-)cells, respectively. However, in the alveolar cell type A549 the cell surface beta(2)-AR was virtually undetectable by (-)-[(125)I]-iodocyanopindolol (ICYP) binding. Stimulation of cultured cells with (-)-isoproterenol enhanced the basal cyclic AMP accumulation only in Calu-3 and 16HBE14o(-) cells, which was blocked by the beta(2)-selective antagonist ICI 118,551, but not by the beta(1)-selective antagonist CGP 20712A, confirming functional coupling of the beta(2)-AR to adenylate cyclase in these cells. Immunocytochemical staining localised the receptor on the cell membrane and the cytoplasm in Calu-3 and 16HBE14o(-) cells, while it was confined to the cytoplasm only in A549 cells. In conclusion, the beta(2)-AR expression and its functional coupling to adenylyl cyclase was very high in the human airway epithelial cells Calu-3 and 16HBE14o(-), but not in A549, suggesting that the cell lines Calu-3 and 16HBE14o(-) present suitable models to study function and regulation of the beta-adrenoceptor signalling in the respiratory system.