In euryhaline teleosts, osmoregulatory mechanisms vary with osmotic stresses, and heat shock proteins (HSPs) play a central role in maintaining cellular homeostasis. The present study aimed to investigate the expression and potential roles of HSP70 and HSP90 in the gills of seawater (SW)- and freshwater (FW)-acclimated milkfish (Chanos chanos). Four HSP genes, including Cchsc70 (heat shock cognate 70), Cchsp70, Cchsp90α, and Cchsp90β, were analyzed in milkfish gills. Among these genes, only the mRNA abundance of branchial Cchsp90α was significantly lower in the FW-acclimated than in the SW-acclimated milkfish. Immunoblotting showed no significant difference in the relative protein abundance of branchial HSP70 and HSP90 between the two groups. The time-course experiments (from SW to FW) showed that the protein abundance of HSP70 and HSP90 at the 3 h and 6 h post-transfer and then declined gradually. To further illustrate the potential osmoregulatory roles of HSP70 and HSP90, their interaction with Na⁺, K⁺-ATPase (NKA, the primary driving force for osmoregulation) was analyzed using co-immunoprecipitation. The results showed the interaction between HSP70, HSP90 and NKA after acclimation to SW or FW increased within 3 h; and then returned to normal levels within 7 days. To our knowledge, the present study was the first to demonstrate that the interaction between HSP70, HSP90 and NKA changes with hypotonic stress in euryhaline teleosts. Before the transfer, no interaction was detected. When transferred to FW from SW, the interaction of HSP70 and HSP90 with NKA were detected. The results suggested that HSP70 and HSP90 participated in the acute responses of osmoregulatory mechanisms to protect branchial NKA from hypotonic stress in milkfish.

在鱼腥草硬骨鱼中，渗透调节机制随渗透压力而变化，并且热休克蛋白（HSP）在维持细胞稳态中起着核心作用。本研究旨在调查HSP70和HSP90在适应海水（SW）和淡水（FW）的milk鱼（Chanos chanos）g中的表达及其潜在作用。在遮目鱼g中分析了四个HSP基因，包括Cchsc70（热休克同源70），Cchsp70，Cchsp90α和Cchsp90β。在这些基因中，仅分支Cchsp90α的mRNA丰度FW适应性显着低于SW适应性遮目鱼。免疫印迹显示两组之间的HSP70和HSP90相对蛋白丰度没有显着差异。时程实验（从西南到华南）显示，HSP70和HSP90的蛋白质丰度在转移后3 h和6 h逐渐下降。为了进一步说明HSP70和HSP90的潜在渗透调节作用，以及它们与Na ⁺，K ^+的相互作用使用共免疫沉淀法分析-ATPase（NKA，渗透调节的主要驱动力）。结果表明，适应SW或FW后3h内HSP70，HSP90与NKA的相互作用增强; 然后在7天内恢复到正常水平。据我们所知，本研究是第一个证明HSP70，HSP90和NKA之间的相互作用随硬脂酸硬骨鱼低渗应激而变化的研究。传输之前，未检测到任何交互。当从SW转移到FW时，检测到HSP70和HSP90与NKA的相互作用。结果表明，HSP70和HSP90参与了渗透调节机制的急性反应，以保护milk鱼中的NKA免受低渗胁迫。