Department of Cellular and Molecular Physiology, Yale University School of Medicine, PO Box 208026, New Haven, CT 06520, USA.
J Exp Biol. 2010 May;213(Pt 9):1558-66. doi: 10.1242/jeb.039644.
Euryhaline teleosts such as Atlantic killifish (Fundulus heteroclitus) are able to acclimate to changing environmental salinity by tightly regulating NaCl absorption and secretion across their gills. Many studies have examined the mechanisms responsible for long-term (days) salinity acclimation; however, much remains unknown about the mechanisms of acute (hours) salinity acclimation. In this study, we tested the hypotheses that phosphorylation of the Na(+)-K(+)-Cl(-) cotransporter (NKCC1) located in the basolateral membrane of the gill plays a role in acute salinity acclimation and that changes in NKCC1 phosphorylation are mediated by a cAMP-protein kinase A (cAMP-PKA) pathway. Using a phospho-specific antibody, we determined the time course of changes in total and phosphorylated NKCC1 protein during acclimation to water of various salinities. Long-term (>or=14 days) acclimation of killifish to seawater (SW) and 2x SW resulted in 4- to 6-fold and 5- to 8-fold increases, respectively, in total gill NKCC1 protein relative to fish maintained in freshwater (FW). NKCC1 was found to be between 20% and 70% activated in fish, with lower average activation in fish acclimated to SW and 2x SW compared with FW fish. Increases and decreases in the fractional level of NKCC1 phosphorylation were seen within 1 h of transfer of fish to water of higher and lower salinity, respectively, consistent with a regulatory role of phosphorylation prior to an increase in the biosynthesis of NKCC1; large changes in protein expression of NKCC1 were observed over periods of hours to days. We found that NKCC1 phosphorylation is acutely regulated in the killifish gill in response to changing environmental salinity and that phosphorylation in excised gills increases in response to forskolin stimulation of the cAMP-PKA pathway. The role of phosphorylation is further underscored by the observation that mRNA expression of sterile 20 (Ste20)-related proline-alanine-rich kinase (SPAK) changes with salinity acclimation, being 2.7-fold greater in SW-acclimated killifish relative to FW fish. Overall, these results demonstrate an important role of NKCC1 phosphorylation in the gill of Atlantic killifish during acute salinity acclimation.
广盐性鱼类，如大西洋鲦鱼（Fundulus heteroclitus），能够通过严格调节其鳃部的 NaCl 吸收和分泌来适应环境盐度的变化。许多研究已经研究了负责长期（数天）盐度适应的机制；然而，对于急性（数小时）盐度适应的机制仍知之甚少。在这项研究中，我们测试了以下假设：位于鳃基底外侧膜上的 Na(+)-K(+)-Cl(-)共转运蛋白（NKCC1）的磷酸化在急性盐度适应中起作用，并且 NKCC1 磷酸化的变化是由 cAMP-蛋白激酶 A（cAMP-PKA）途径介导的。使用磷酸特异性抗体，我们确定了在适应各种盐度的水中时 NKCC1 蛋白总磷酸化和磷酸化的时间过程。大西洋鲦鱼长期（>或=14 天）适应海水（SW）和 2xSW 分别导致总鳃 NKCC1 蛋白增加 4 到 6 倍和 5 到 8 倍，与在淡水（FW）中维持的鱼相比。发现 NKCC1 在鱼中被激活 20%到 70%之间，与 FW 鱼相比，在适应 SW 和 2xSW 的鱼中，平均激活水平较低。在将鱼转移到盐度更高和更低的水中后 1 小时内，观察到 NKCC1 磷酸化的分数水平分别增加和减少，这与在 NKCC1 生物合成增加之前磷酸化的调节作用一致；在数小时到数天的时间内观察到 NKCC1 蛋白表达的大变化。我们发现，在适应环境盐度变化时，大西洋鲦鱼鳃中的 NKCC1 磷酸化受到急性调节，并且在 forskolin 刺激 cAMP-PKA 途径时，离体鳃中的磷酸化增加。磷酸化的作用进一步强调了无菌 20（Ste20）相关脯氨酸-丙氨酸丰富激酶（SPAK）的 mRNA 表达随盐度适应而变化的观察结果，在 SW 适应的大西洋鲦鱼中相对 FW 鱼增加 2.7 倍。总的来说，这些结果表明 NKCC1 磷酸化在大西洋鲦鱼急性盐度适应过程中在鳃中起着重要作用。
