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

我们自己的研究表明，绵羊瘤胃上皮能够在mRNA和蛋白水平上表达游离脂肪酸受体FFAR2和GPR109A。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，改变细胞功能，包括cAMP敏感转运蛋白。在前胃中，短链脂肪酸大量产生并被直接吸收，使其成为动物的主要能量来源。然而，将SCFA摄取到瘤胃上皮会使细胞的细胞质酸化。因此，我们假设SCFA与上皮ffa受体的结合特别改变了参与SCFA转移和/或调节细胞内ph的转运蛋白。为了验证这一假设，我们想要阐明FFAR的刺激是否会导致细胞内cAMP水平的降低，并改变转运蛋白NHE3、NHE1、MCT1和MCT4的活性。所有这些转运蛋白都会影响细胞内ph值。此外，MCT1和MCT4参与了SCFA和/或其上皮内分解代谢物的跨膜转移。为了研究SCFA、FFAR、cAMP与转运蛋白调节之间的相互关系，我们计划对分离的绵羊瘤胃上皮细胞和培养的瘤胃上皮细胞进行研究。在上皮细胞和培养细胞中，细胞内cAMP水平应在有或没有用醋酸盐或丁酸盐或受体的药物激动剂刺激FFAR受体和细胞内cAMP水平的药理学变化的情况下测量。然后，在分离的上皮细胞和培养细胞中，通过各种技术，在有或没有上述方式刺激受体的情况下，测量NHE3、NHE1、MCT1和MCT4的活性和蛋白质表达。因此，该研究可能揭示动物对SCFA暴露升高的短期适应性反应，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.