Perrot-Sinal T S, Sinal C J, Reader J C, Speert D B, McCarthy M M
Department of Psychology and Neuroscience Institute, Dalhousie University, Halifax, Nova Scotia, Canada.
J Neuroendocrinol. 2007 Apr;19(4):302-8. doi: 10.1111/j.1365-2826.2007.01530.x.
In immature neurones, high basal Cl(-) results in membrane depolarisation following GABA(A) receptor activation, which is critical for various developmental processes including steroid-mediated sexual differentiation of the hypothalamus. Previously, we demonstrated that oestradiol enhances GABA-mediated Ca(2+) influx in neonate hypothalamus and that Ca(2+) induced activation of the transcription factor, cyclicAMP response element binding protein (CREB), was higher in male (high oestradiol) relative to female neonate hypothalamus. Based on these results, we hypothesised that expression of developmentally regulated chloride cotransporters may be sexually dimorphic. Here, we investigate the expression of the chloride cotransporters, NKCC1 (Na-K-2Cl(-)) and KCC2 (K-Cl(-)) in neonate mediobasal hypothalamus of male and female rats. The NKCC1 transporter moves Cl(-) into cells and helps maintain depolarising GABA action while the KCC2 transporter has the opposite effect by moving Cl(-) out of cells. NKCC1 mRNA levels were higher in males than females on the day of birth (postnatal day 0; PND 0) and total NKCC1 protein levels were significantly higher in males than females on embryonic day (ED) 20 and PND0. Levels of activated phosphorylated NKCC1 (pNKCC1) were not sexually dimorphic. Females were treated with a masculinising dose of oestradiol benzoate (EB; 100 microg; EB-females) on PND0. Total NKCC1 protein levels in tissue processed on PND1 and PND2 were similar in EB-females and oil-treated PND0 males and females. However, pNKCC1 protein levels measured on PND2 (but not PND1) were significantly higher in EB-treated females relative to oil-treated males and females. By contrast, KCC2 mRNA levels were significantly lower in males relative to females on PND0. KCC2 protein was not detectable on ED20 or PND0 but was significantly lower in males relative to females on PND5. These results suggest a complex relationship between KCC2 and NKCC1 mRNA and protein in developing brain that is not easily linked to regulation by oestradiol.
在未成熟神经元中，较高的基础细胞内氯离子浓度（[Cl⁻]i）会导致γ-氨基丁酸A型（GABA(A)）受体激活后膜去极化，这对包括下丘脑类固醇介导的性别分化在内的各种发育过程至关重要。此前，我们证明雌二醇可增强新生下丘脑GABA介导的钙离子内流，并且与雌性新生下丘脑相比，雄性（高雌二醇水平）新生下丘脑内钙离子诱导的转录因子环磷酸腺苷反应元件结合蛋白（CREB）的激活程度更高。基于这些结果，我们推测发育调控的氯离子共转运体的表达可能存在性别差异。在此，我们研究了雄性和雌性大鼠新生中基底下丘脑氯离子共转运体钠-钾-2氯同向转运体1（NKCC1）和钾-氯同向转运体2（KCC2）的表达情况。NKCC1转运体将Cl⁻转运到细胞内，有助于维持去极化的GABA作用，而KCC2转运体则通过将Cl⁻转运出细胞产生相反的效果。出生当天（出生后第0天；PND 0）雄性的NKCC1 mRNA水平高于雌性，在胚胎期第20天（ED 20）和PND0时，雄性的总NKCC1蛋白水平显著高于雌性。激活的磷酸化NKCC1（pNKCC1）水平不存在性别差异。在PND0给雌性注射具有雄性化作用剂量的苯甲酸雌二醇（EB；100微克；EB-雌性）。在PND1和PND2处理的组织中，EB-雌性的总NKCC1蛋白水平与油处理的PND0雄性和雌性相似。然而，在PND2（而非PND1）检测到的EB处理雌性的pNKCC1蛋白水平显著高于油处理的雄性和雌性。相比之下，在PND0时雄性的KCC2 mRNA水平显著低于雌性。在ED20或PND0时未检测到KCC2蛋白，但在PND5时雄性的KCC2蛋白水平显著低于雌性。这些结果表明，发育中的大脑中KCC2和NKCC1的mRNA和蛋白之间存在复杂的关系，且这种关系不易与雌二醇的调节联系起来。
