Cell-based homologous expression system for in-vitro characterization of environmental effects on transmembrane peptide transport in fish

IF 2.1 Q3 PHYSIOLOGY Current research in physiology Pub Date : 2024-01-01 DOI:10.1016/j.crphys.2024.100118
Pazit Con , Jens Hamar , Jakob Biran , Dietmar Kültz , Avner Cnaani
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Abstract

All organisms encounter environmental changes that lead to physiological adjustments that could drive evolutionary adaptations. The ability to adjust performance in order to cope with environmental changes depends on the organism's physiological plasticity. These adjustments can be reflected in behavioral, physiological, and molecular changes, which interact and affect each other. Deciphering the role of molecular adjustments in physiological changes will help to understand how multiple levels of biological organization are synchronized during adaptations. Transmembrane transporters, which facilitate a cell's interaction with its surroundings, are prime targets for molecular studies of the environmental effects on an organism's physiology. Fish are subjected to environmental fluctuations and exhibit different coping mechanisms. To study the molecular adjustments of fish transporters to their external surrounding, suitable experimental systems must be established. The Mozambique tilapia (Oreochromis mossambicus) is an excellent model for environmental stress studies, due to its extreme salinity tolerance. We established a homologous cellular-based expression system and uptake assay that allowed us to study the effects of environmental conditions on transmembrane transport. We applied our expression system to investigate the effects of environmental conditions on the activity of PepT2, a transmembrane transporter critical in the absorption of dietary peptides and drugs. We created a stable, modified fish cell-line, in which we exogenously expressed the tilapia PepT2, and tested the effects of water temperature and salinity on the uptake of a fluorescent di-peptide, β-Ala-Lys-AMCA. While temperature affected only Vmax, medium salinity had a bi-directional effect, with significantly reduced Vmax in hyposaline conditions and significantly increased Km in hypersaline conditions. These assays demonstrate the importance of suitable experimental systems for fish ecophysiology studies. Furthermore, our in-vitro results show how the effect of hypersaline conditions on the transporter activity can explain expression shifts seen in the intestine of saltwater-acclimated fish, emphasizing the importance of complimentary studies in better understanding environmental physiology. This research highlights the advantages of using homologous expression systems to study environmental effects encountered by fish, in a relevant cellular context. The presented tools and methods can be adapted to study other transporters in-vitro.

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基于细胞的同源表达系统,用于体外鉴定环境对鱼类跨膜肽转运的影响
所有生物都会遇到环境变化,从而导致生理调整,进而推动进化适应。调整性能以应对环境变化的能力取决于生物的生理可塑性。这些调整可以反映在行为、生理和分子变化上,而这些变化是相互作用和相互影响的。破译分子调整在生理变化中的作用将有助于了解生物组织的多个层次在适应过程中是如何同步进行的。跨膜转运体可促进细胞与周围环境的相互作用,是研究环境对生物生理影响的分子研究的主要目标。鱼类会受到环境波动的影响,并表现出不同的应对机制。要研究鱼类转运体对外界环境的分子调节,必须建立合适的实验系统。莫桑比克罗非鱼(Oreochromis mossambicus)具有极强的耐盐性,是研究环境压力的绝佳模型。我们建立了一个基于细胞的同源表达系统和摄取检测方法,使我们能够研究环境条件对跨膜转运的影响。我们应用我们的表达系统研究了环境条件对 PepT2 活性的影响,PepT2 是一种跨膜转运体,对膳食肽和药物的吸收至关重要。我们创建了一个稳定的改良鱼细胞系,在其中外源表达罗非鱼 PepT2,并测试了水温和盐度对荧光二肽 β-Ala-Lys-AMCA 吸收的影响。温度只影响Vmax,而中盐度则具有双向影响,在低盐度条件下Vmax显著降低,而在高盐度条件下Km显著增加。这些实验证明了合适的实验系统对鱼类生态生理学研究的重要性。此外,我们的体外实验结果表明,低盐条件对转运体活性的影响可以解释盐水适应性鱼类肠道中的表达变化,强调了辅助研究对更好地理解环境生理学的重要性。这项研究强调了利用同源表达系统在相关细胞环境中研究鱼类所受环境影响的优势。介绍的工具和方法可用于体外研究其他转运体。
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62 days
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