Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, USA.
Allergan plc, Irvine, California, USA.
Pharm Res. 2017 Dec;34(12):2488-2497. doi: 10.1007/s11095-017-2234-z. Epub 2017 Aug 22.
Studies were conducted in primary cultured rat alveolar epithelial cell monolayers to characterize peptide transporter expression and function.
Freshly isolated rat lung alveolar epithelial cells were purified and cultured on permeable support with and without keratinocyte growth factor (KGF). Messenger RNA and protein expression of Pept1 and Pept2 in alveolar epithelial type I- and type II-like cell monolayers (±KGF, resp.) were examined by RT-PCR and Western blotting. H-Glycyl-sarcosine (H-gly-sar) transmonolayer flux and intracellular accumulation were evaluated in both cell types.
RT-PCR showed expression of Pept2, but not Pept1, mRNA in both cell types. Western blot analysis revealed presence of Pept2 protein in type II-like cells, and less in type I-like cells. Bi-directional transmonolayer H-gly-sar flux lacked asymmetry in transport in both types of cells. Uptake of H-gly-sar from apical fluid of type II-like cells was 7-fold greater than that from basolateral fluid, while no significant differences were observed from apical vs. basolateral fluid of type I-like cells.
This study confirms the absence of Pept1 from rat lung alveolar epithelium in vitro. Functional Pept2 expression in type II-like cell monolayers suggests its involvement in oligopeptide lung disposition, and offers rationale for therapeutic development of di/tripeptides, peptidomimetics employing pulmonary drug delivery.
本研究在原代培养的大鼠肺泡上皮细胞单层中进行，旨在研究肽转运蛋白的表达和功能。
新鲜分离的大鼠肺肺泡上皮细胞在有或没有角质细胞生长因子（KGF）的可渗透载体上进行纯化和培养。通过 RT-PCR 和 Western blot 检测肺泡上皮 I 型和 II 型细胞单层（分别为±KGF）中 Pept1 和 Pept2 的信使 RNA 和蛋白表达。评估两种细胞类型中 H-甘氨酰-丝氨酸（H-gly-sar）的跨单层通量和细胞内积累。
RT-PCR 显示两种细胞类型均表达 Pept2 mRNA，但不表达 Pept1 mRNA。Western blot 分析显示 Pept2 蛋白存在于 II 型细胞中，而在 I 型细胞中含量较少。两种细胞类型的 H-gly-sar 双向跨单层通量在转运中没有不对称性。II 型细胞从顶端液中的 H-gly-sar 摄取比从基底外侧液中的摄取高 7 倍，而在 I 型细胞中，从顶端液和基底外侧液中的摄取没有显著差异。
本研究证实了 Pept1 在体外大鼠肺肺泡上皮细胞中的缺失。功能性 Pept2 在 II 型细胞单层中的表达表明其参与了寡肽的肺分布，并为采用肺部药物输送的二肽/三肽、肽模拟物的治疗开发提供了依据。
