Department of Biomedical & Pharmaceutical Sciences, Chapman University, Irvine, CA 92618, USA.
Department of Medicine, Division of Nephrology, University of California Irvine, Irvine, CA 92697, USA.
Cells. 2019 Jul 11;8(7):704. doi: 10.3390/cells8070704.
Biosensors on the membrane of the vascular endothelium are responsible for sensing mechanical and chemical signals in the blood. Transduction of these stimuli into intracellular signaling cascades regulate cellular processes including ion transport, gene expression, cell proliferation, and/or cell death. The primary cilium is a well-known biosensor of shear stress but its role in sensing extracellular pH change has never been examined. As a cellular extension into the immediate microenvironment, the cilium could be a prospective sensor for changes in pH and regulator of acid response in cells. We aim to test our hypothesis that the primary cilium plays the role of an acid sensor in cells using vascular endothelial and embryonic fibroblast cells as in vitro models. We measure changes in cellular pH using pH-sensitive 2',7'-biscarboxyethy1-5,6-carboxyfluorescein acetoxy-methylester (BCECF) fluorescence and mitogen-activated protein kinase (MAPK) activity to quantify responses to both extracellular pH (pH) and intracellular pH (pH) changes. Our studies show that changes in pH affect pH in both wild-type and cilia-less cells and that the kinetics of the pH response are similar in both cells. Acidic pH or pH was observed to change the length of primary cilia in wild-type cells while the cilia in remained absent. Vascular endothelial cells respond to acidic pH through activation of ERK1/2 and p38-mediated signaling pathways. The cilia-less cells exhibit delayed responsiveness to pH dependent and independent pH acidification as depicted in the phosphorylation profile of ERK1/2 and p38. Otherwise, intracellular pH homeostatic response to acidic pH is similar between wild-type and cells, indicating that the primary cilia may not be the sole sensor for physiological pH changes. These endothelial cells respond to pH changes with a predominantly K-dependent pH recovery mechanism, regardless of ciliary presence or absence.
血管内皮细胞膜上的生物传感器负责感知血液中的机械和化学信号。这些刺激的转导进入细胞内信号级联调节细胞过程，包括离子转运、基因表达、细胞增殖和/或细胞死亡。初级纤毛是剪切力的已知生物传感器，但它在感知细胞外 pH 变化中的作用从未被研究过。作为细胞向临近微环境的延伸，纤毛可能是 pH 变化的潜在传感器，也是细胞内酸响应的调节剂。我们旨在通过血管内皮细胞和胚胎成纤维细胞作为体外模型来检验我们的假说，即初级纤毛在细胞中作为酸传感器发挥作用。我们使用 pH 敏感的 2',7'-双羧乙基-5,6-羧基荧光素乙酰氧基甲酯（BCECF）荧光和丝裂原激活的蛋白激酶（MAPK）活性来测量细胞内 pH 的变化，以量化对细胞外 pH（pH）和细胞内 pH（pH）变化的响应。我们的研究表明，pH 的变化会影响野生型和纤毛缺失细胞的 pH 值，并且两种细胞的 pH 响应动力学相似。在野生型细胞中，酸性 pH 或 pH 值会改变初级纤毛的长度，而 细胞中的纤毛则保持缺失。血管内皮细胞通过激活 ERK1/2 和 p38 介导的信号通路对酸性 pH 作出反应。纤毛缺失的 细胞表现出对 pH 依赖性和非依赖性 pH 酸化的延迟反应性，这在 ERK1/2 和 p38 的磷酸化谱中可以看出。然而，在野生型和 细胞之间，细胞内 pH 对酸性 pH 的稳态反应是相似的，这表明初级纤毛可能不是生理 pH 变化的唯一传感器。这些内皮细胞对 pH 变化的反应主要是依赖 K 的 pH 恢复机制，无论纤毛的存在与否。
