Modulation of pH-regulatory transport proteins by free fatty acid receptors in the ruminal epithelium

本课题组的研究表明，绵羊瘤胃上皮细胞能够在mRNA和蛋白水平上表达游离脂肪酸受体FFAR 2和GPR 109 A。在非瘤胃组织中，这些受体可以结合短链脂肪酸，导致细胞内cAMP水平降低和细胞功能改变，包括cAMP敏感性转运蛋白。在前胃中，SCFA大量产生并被直接吸收，这使得它们成为动物的主要能量来源。然而，瘤胃上皮细胞对SCFA的摄取使细胞的胞质溶胶酸化。因此，我们假设，在上皮FFA受体的SCFA结合，特别是修改涉及的SCFA转移和/或调节细胞内pH值的转运蛋白。为了检查的假设，我们要阐明，如果刺激的FFAR导致细胞内cAMP水平的降低和修改的转运蛋白NHE 3，NHE 1，MCT 1和MCT 4的活性。所有这些转运蛋白影响细胞内的pH值。此外，MCT 1和MCT 4参与SCFA和/或其上皮内catalysts的跨膜转移。为了研究SCFA，FFAR，cAMP和转运蛋白的调制之间的相互关系，我们计划进行研究，对分离的绵羊瘤胃上皮细胞和瘤胃上皮细胞保持在培养物中。在上皮细胞和培养的细胞中，细胞内cAMP水平应在有和没有通过乙酸盐或丁酸盐或通过受体的药理学激动剂刺激FFAR受体的情况下以及通过细胞内cAMP水平的药理学变化来测量。然后通过各种技术在分离的上皮细胞和培养的细胞中测量NHE 3、NHE 1、MCT 1和MCT 4的活性和蛋白质表达，并且以所述方式刺激和不刺激受体。因此，该研究可以揭示动物对升高的SCFA暴露的短期适应性反应，例如在摄入富含易发酵碳水化合物的比例后。

Our own studies have shown that the ovine ruminal epithelium is able to express the free fatty acid receptors FFAR2 and GPR109A on the mRNA and protein level. In non ruminal tissues these receptors can bind short chain fatty acids leading to a decreased intracellular level of cAMP and altered cell functions including cAMP sensitive transport proteins. In the forestomach SCFA are produced in large quantities and directly absorbed which makes them the main energy source for the animal. The uptake of SCFA into the ruminal epithelium, however, acidifies the cytosol of the cells. We thus assume that SCFA binding at the epithelial FFA-receptors particularly modifies the transport proteins involved in the SCFA transfer and/or regulation of intracellular pH. To check the assumption, we want to elucidate if stimulation of the FFAR leads to a decrease of the intracellular cAMP level and modifies the activity of the transport proteins NHE3, NHE1, MCT1 and MCT4. All these transport proteins influence the intracellular pH. Additionally, MCT1 and MCT4 are involved in the transmembranal transfer of SCFA and/or their intraepithelial catabolites. To examine the interrelationship between SCFA, FFAR, cAMP and modulation of the transport proteins, we plan to conduct studies on isolated ovine ruminal epithelia and ruminal epithelial cells kept in culture. In the epithelia and the cultured cells, the intracellular cAMP levels shall be measured both with and without stimulating the FFAR receptors by acetate or butyrate or by pharmacological agonist of the receptors and by pharmacological variation of the intracellular cAMP level. Activity and protein expression of NHE3, NHE1, MCT1 and MCT4 will then be measured in the isolated epithelial and cultured cells by various techniques and both with and without stimulating the receptors the way mentioned. The study may thus reveal short term adaptive responses of the animal to an elevated SCFA exposure for example after intake of a ratio rich in easily fermentable carbohydrates.