人呼吸道上皮（Calu-3）细胞中促性腺激素释放激素（LHRH）受体表达的证据及其在LHRH激动剂转运中的作用。
Evidence for LHRH-receptor expression in human airway epithelial (Calu-3) cells and its role in the transport of an LHRH agonist.
作者信息
Koushik Kavitha, Bandi Nagesh, Sundaram Sneha, Kompella Uday B
机构信息
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
出版信息
Pharm Res. 2004 Jun;21(6):1034-46. doi: 10.1023/b:pham.0000029294.70707.74.
PURPOSE
To determine whether LHRH-receptor is expressed in Calu-3, a human bronchial epithelial cell line, and to further determine whether this receptor plays a role in the transport of deslorelin, an LHRH agonist.
METHODS
Using cultured monolayers of Calu-3 grown at air-interface, the presence and localization of LHRH-receptors in Calu-3 cells was determined using immunochemical methods. To determine the mechanisms of deslorelin transport, the directionality [apical-basolateral (A-B) and basolateral-apical (B-A)] of deslorelin transport across Calu-3 monolayers and the effects of temperature (37 degrees C and 4 degrees C) and an energy depletor (2,4-dinitrophenol) were investigated. To determine the role of LHRH-receptor in deslorelin transport across Calu-3 monolayers, the influence of an LHRH-receptor antisense oligonucleotide on the LHRH-receptor expression and deslorelin transport was studied. Also, the effect of a competing LHRH agonist, buserelin, on deslorelin transport was determined.
RESULTS
Immunofluorescence studies indicated the predominance of LHRH-receptor in Calu-3 cells at the apical and lateral surfaces. Western blot and RT-PCR studies further confirmed the expression of LHRH-receptor in Calu-3 cells. Deslorelin transport across Calu-3 monolayers was vectorial, with the cumulative A-B transport (1.79 +/- 0.29%) at the end of 240 min being higher than the B-A transport (0.34 +/- 0.11%). Low temperature as well as 2,4-dinitrophenol abolished this directionality. LHRH-receptor antisense oligonucleotide decreased the receptor expression at the mRNA and protein level and reduced the A-B deslorelin transport by 55 +/- 4%, without affecting the B-A transport, suggesting a role for LHRH-receptor in the vectorial transport of deslorelin. In addition, buserelin reduced the A-B deslorelin transport by 56 +/- 5% without affecting the B-A transport.
CONCLUSIONS
Taken together, our results provide evidence that deslorelin is transported across the respiratory epithelium via the LHRH-receptor.
目的
确定促黄体生成素释放激素（LHRH）受体是否在人支气管上皮细胞系Calu-3中表达，并进一步确定该受体是否在LHRH激动剂地洛瑞林的转运中发挥作用。
方法
使用在气液界面培养的Calu-3单层细胞，采用免疫化学方法确定Calu-3细胞中LHRH受体的存在和定位。为了确定地洛瑞林的转运机制，研究了地洛瑞林跨Calu-3单层细胞的转运方向性[顶侧-基底侧（A-B）和基底侧-顶侧（B-A）]以及温度（37℃和4℃）和能量耗尽剂（2,4-二硝基苯酚）的影响。为了确定LHRH受体在跨Calu-3单层细胞转运地洛瑞林中的作用，研究了LHRH受体反义寡核苷酸对LHRH受体表达和地洛瑞林转运的影响。此外，还确定了竞争性LHRH激动剂布舍瑞林对地洛瑞林转运的影响。
结果
免疫荧光研究表明，Calu-3细胞中LHRH受体主要位于顶侧和侧面。蛋白质印迹和逆转录-聚合酶链反应（RT-PCR）研究进一步证实了Calu-3细胞中LHRH受体的表达。地洛瑞林跨Calu-3单层细胞的转运是有方向性的，240分钟结束时的累积A-B转运（1.79±0.29%）高于B-A转运（0.34±0.11%）。低温以及2,4-二硝基苯酚消除了这种方向性。LHRH受体反义寡核苷酸降低了mRNA和蛋白质水平的受体表达，并使A-B地洛瑞林转运减少了55±4%，而不影响B-A转运，这表明LHRH受体在向量转运地洛瑞林中发挥作用。此外，布舍瑞林使A-B地洛瑞林转运减少了56±5%，而不影响B-A转运。
结论
综上所述，我们的结果提供了证据表明地洛瑞林通过LHRH受体跨呼吸上皮转运 .
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