Molecular mechanism of osmosensing and ENaC gene expression by src kinase.

肾脏中的上皮Na^+转运对于控制血压和细胞外液量很重要，并受血浆渗透压和激素的调节。我们已经发现慢性细胞外低渗通过肾上皮A6细胞α-ENaC mRNA的表达刺激Na^+重吸收。然而，通过ENaC基因表达的Na^+重吸收的神经元感受机制和刺激机制还没有很好地理解。我们最近发现：1）低渗应激引起胞浆Cl^-浓度（[Cl^-]_c）的双相降低; 2）低渗应激调节在信号转导中起关键作用的src激酶的酪氨酸磷酸化。基于这些观察结果，我们假设细胞内CL^-作为信号分子调节低渗应激时src激酶的活性，src激酶通过ENaC基因表达在刺激肾上皮细胞Na^+重吸收中起关键作用。为了阐明胞浆中的Cl^-是否作为低渗应激的信号分子，我们研究了[Cl^-]_c对src激酶Tyr 416磷酸化的影响，src激酶Tyr 416被自磷酸化以激活酶。本研究发现：（1）在[Cl^-]_c降低时，PTP和src激酶活性均降低;（2）在[Cl^-]_c降低时，src激酶活性大于PTP。此外，我们还研究了慢性低渗应激刺激Na^+重吸收过程中src激酶的激活是否与[Cl^-]_c的减少有关。用PP 2（一种src激酶抑制剂）预处理可通过抑制ENaC mRNA的表达而显著抑制慢性低渗刺激的Na^+重吸收。基于这些结果，我们认为低渗应激通过降低肾上皮A6细胞中[Cl^-]_c，激活src激酶，诱导ENaC mRNA表达，从而刺激Na^+重吸收。

Epithelial Na^+ transport in the kidney is important for control of blood pressure and extracellular fluid volume and is regulated by plasma osmolality and hormones. We have indicated that chronic extracellular hypotonicity stimulates Na^+ reabsorption through alpha-ENaC mRNA expression in renal epithelial A6 cell. However, the osmosensing mechanism and the stimulatory mechanism of Na^+ reabsorption through ENaC gene expression are not well understood. We have recently indicated that : 1) the hypotonic stress causes a biphasic reduction of cytosolic Cl^- concentration ([Cl^-]_c), and 2) the hypotonic stress modulates tyrosine phosphorylation of src kinase playing a crucial role in signal transduction. Based on these observations, we hypothesized that cytosolic CL^-acts as a signal molecule to regulate src kinase activity in hypotonic stress and that src kinase plays a crucial role for stimulation of Na^+ reabsorption through ENaC gene expression in renal epithelia. To clarify if the cytosolic Cl^-acts as a signal molecule for the hypotonic stress, we studied the effects of ([Cl^-]_c on phosphorylation of src kinase at Tyr416 which is autophosphorylated for enzymatic activation. In the present study, we found that : 1) both activities of PTP and src kinase decrease at lowered ([Cl^-]_c, and 2) the activity of src kinase is larger than that PTP at lowered ([Cl^-]_c. Furthermore, we study if src kinase activation through reduction of ([Cl^-]_c is involved in stimulation of Na^+ reabsorption by chronic hypotonic stress. Pretreatment with PP2 (a src kinase inhibitor) significantly inhibited chronic hypotonicity-stimulated Na^+ reabsorption through suppression of ENaC mRNA expression. Based on these results, we suggest that hypotonic stress stimulates Na^+ reabsorption through induction of ENaC mRNA expression via src kinase activation by a reduction of ([Cl^-]_c in renal epithelial A6 cells.