Manosalva Carolina, Mena Jaqueline, Velasquez Zahady, Colenso Charlotte K, Brauchi Sebastian, Burgos Rafael A, Hidalgo Maria A
Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Science, Universidad Austral de Chile, Valdivia, Chile.
Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Science, Universidad Austral de Chile, Valdivia, Chile; Department of Biology, Universidad de Nariño, Pasto, Colombia.
PLoS One. 2015 Mar 19;10(3):e0119715. doi: 10.1371/journal.pone.0119715. eCollection 2015.
Long chain fatty acids (LCFAs), which are ligands for the G-protein coupled receptor FFAR1 (GPR40), are increased in cow plasma after parturition, a period in which they are highly susceptible to infectious diseases. This study identified and analyzed the functional role of the FFAR1 receptor in bovine neutrophils, the first line of host defense against infectious agents. We cloned the putative FFAR1 receptor from bovine neutrophils and analyzed the sequence to construct a homology model. Our results revealed that the sequence of bovine FFAR1 shares 84% identity with human FFAR1 and 31% with human FFAR3/GPR41. Therefore, we constructed a homology model of bovine FFAR1 using human as the template. Expression of the bovine FFAR1 receptor in Chinese hamster ovary (CHO)-K1 cells increased the levels of intracellular calcium induced by the LCFAs, oleic acid (OA) and linoleic acid (LA); no increase in calcium mobilization was observed in the presence of the short chain fatty acid propionic acid. Additionally, the synthetic agonist GW9508 increased intracellular calcium in CHO-K1/bFFAR1 cells. OA and LA increased intracellular calcium in bovine neutrophils. Furthermore, GW1100 (antagonist of FFAR1) and U73122 (phospholipase C (PLC) inhibitor) reduced FFAR1 ligand-induced intracellular calcium in CHO-K1/bFFAR1 cells and neutrophils. Additionally, inhibition of FFAR1, PLC and PKC reduced the FFAR1 ligand-induced release of matrix metalloproteinase (MMP)-9 granules and reactive oxygen species (ROS) production. Thus, we identified the bovine FFAR1 receptor and demonstrate a functional role for this receptor in neutrophils activated with oleic or linoleic acid.
长链脂肪酸（LCFAs）是G蛋白偶联受体FFAR1（GPR40）的配体，在奶牛产后血浆中含量升高，而这一时期奶牛极易感染传染病。本研究鉴定并分析了FFAR1受体在牛中性粒细胞（宿主抵御感染因子的第一道防线）中的功能作用。我们从牛中性粒细胞中克隆了假定的FFAR1受体，并分析其序列以构建同源模型。我们的结果显示，牛FFAR1的序列与人类FFAR1的同一性为84%，与人类FFAR3/GPR41的同一性为31%。因此，我们以人类为模板构建了牛FFAR1的同源模型。牛FFAR1受体在中国仓鼠卵巢（CHO）-K1细胞中的表达增加了由长链脂肪酸、油酸（OA）和亚油酸（LA）诱导的细胞内钙水平；在短链脂肪酸丙酸存在的情况下，未观察到钙动员增加。此外，合成激动剂GW9508增加了CHO-K1/bFFAR1细胞内钙水平。OA和LA增加了牛中性粒细胞内钙水平。此外，GW1100（FFAR1拮抗剂）和U73122（磷脂酶C（PLC）抑制剂）降低了FFAR1配体诱导的CHO-K1/bFFAR1细胞和中性粒细胞内钙水平。此外，抑制FFAR1、PLC和PKC可降低FFAR1配体诱导的基质金属蛋白酶（MMP）-9颗粒释放和活性氧（ROS）生成。因此，我们鉴定了牛FFAR1受体，并证明了该受体在被油酸或亚油酸激活的中性粒细胞中的功能作用。
