Marshall W S, Ossum C G, Hoffmann E K
Department of Biology, St Francis Xavier University, PO Box 5000 Antigonish, Nova Scotia, Canada B2G 2W5.
J Exp Biol. 2005 Mar;208(Pt 6):1063-77. doi: 10.1242/jeb.01491.
Hypotonic shock rapidly inhibits Cl(-) secretion by chloride cells, an effect that is osmotic and not produced by NaCl-depleted isosmotic solutions, yet the mechanism for the inhibition and its recovery are not known. We exposed isolated opercular epithelia, mounted in Ussing chambers, to hypotonic shock in the presence of a variety of chemicals: a general protein kinase C (PKC) inhibitor chelerythrine, Gö6976 that selectively blocks PKC alpha and beta subtypes, H-89 that blocks PKA, SB203580 that blocks p38 mitogen-activated protein kinase (MAPK), as well as serine/threonine protein phosphatase (PP1 and 2A) inhibitor okadaic acid, and finally tamoxifen, a blocker of volume-activated anion channels (VSOAC). Chelerythrine has no effect on hypotonic inhibition but blocked the recovery, indicating PKC involvement in stimulation. Gö6976 had little effect, suggesting that PKC alpha and PKC beta subtypes are not involved. H-89 did not block hypotonic inhibition but decreased the recovery, indicating PKA may be involved in the recovery and overshoot (after restoration of isotonic conditions). SB203580 significantly enhanced the decrease in current by hypotonic shock, suggesting an inhibitory role of p38 MAPK in the hypotonic inhibition. Okadaic acid increased the steady state current, slowed the hypotonic inhibition but made the decrease in current larger; also the recovery and overshoot were completely blocked. Hypotonic stress rapidly and transiently increased phosphorylated p38 MAPK (pp38) MAPK (measured by western analysis) by eightfold at 5 min, then more slowly again to sevenfold at 60 min. Hypertonic shock slowly increased p38 by sevenfold at 60 min. Phosphorylated JNK kinase was increased by 40-50% by both hypotonic and hypertonic shock and was still elevated at 30 min in hypertonic medium. By immunoblot analysis it was found that the stress protein kinase (SPAK) and oxidation stress response kinase 1 (OSR1) were present in salt and freshwater acclimated fish with higher expression in freshwater. By immunocytochemistry, SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK) were colocalized with NKCC at the basolateral membrane. The protein tyrosine kinase inhibitor genistein (100 micromol l(-1)) inhibited Cl(-) secretion that was high, increased Cl(-) secretion that was low and reduced immunocytochemical staining for phosphorylated FAK. We present a model for rapid control of CFTR and NKCC in chloride cells that includes: (1) activation of NKCC and CFTR via cAMP/PKA, (2) activation of NKCC by PKC, myosin light chain kinase (MLCK), p38, OSR1 and SPAK, (3) deactivation of NKCC by hypotonic cell swelling, Ca(2+) and an as yet unidentified protein phosphatase and (4) involvement of protein tyrosine kinase (PTK) acting on FAK to set levels of NKCC activity.
低渗性休克能迅速抑制氯细胞的Cl⁻分泌，这种作用是渗透性的，而非由低渗性等渗溶液引起，然而抑制及其恢复的机制尚不清楚。我们将安装在尤斯灌流小室中的分离鳃盖上皮暴露于低渗性休克环境中，并添加了多种化学物质：一种通用的蛋白激酶C（PKC）抑制剂白屈菜红碱、选择性阻断PKCα和β亚型的Gö6976、阻断PKA的H-89、阻断p38丝裂原活化蛋白激酶（MAPK）的SB203580，以及丝氨酸/苏氨酸蛋白磷酸酶（PP1和2A）抑制剂冈田酸，最后还有容积激活阴离子通道（VSOAC）阻断剂他莫昔芬。白屈菜红碱对低渗性抑制无影响，但阻断了恢复过程，表明PKC参与了刺激过程。Gö6976影响很小，提示PKCα和PKCβ亚型不参与其中。H-89未阻断低渗性抑制，但降低了恢复程度，表明PKA可能参与了恢复过程和超调（等渗条件恢复后）。SB203580显著增强了低渗性休克引起的电流下降，提示p38 MAPK在低渗性抑制中起抑制作用。冈田酸增加了稳态电流，减缓了低渗性抑制，但使电流下降幅度更大；此外，恢复过程和超调完全被阻断。低渗应激在5分钟时迅速且短暂地使磷酸化p38 MAPK（pp38）MAPK（通过蛋白质印迹分析测定）增加了八倍，然后在60分钟时再次缓慢增加至七倍。高渗性休克在60分钟时使p38缓慢增加了七倍。低渗和高渗休克均使磷酸化JNK激酶增加了40 - 50%，且在高渗培养基中30分钟时仍保持升高。通过免疫印迹分析发现，应激蛋白激酶（SPAK）和氧化应激反应激酶1（OSR1）在海水和淡水适应的鱼类中均有表达，在淡水中表达更高。通过免疫细胞化学方法，发现SPAK、OSR1和磷酸化粘着斑激酶（pFAK）在基底外侧膜与NKCC共定位。蛋白酪氨酸激酶抑制剂染料木黄酮（100 μmol l⁻¹）抑制高分泌状态下的Cl⁻分泌，增加低分泌状态下的Cl⁻分泌，并减少磷酸化FAK的免疫细胞化学染色。我们提出了一个关于氯细胞中CFTR和NKCC快速调控的模型，包括：（1）通过cAMP/PKA激活NKCC和CFTR；（2）通过PKC、肌球蛋白轻链激酶（MLCK）、p38、OSR1和SPAK激活NKCC；（3）通过低渗性细胞肿胀、Ca²⁺和一种尚未确定的蛋白磷酸酶使NKCC失活；（4）蛋白酪氨酸激酶（PTK）作用于FAK以设定NKCC活性水平。
