Department of Physiology and Anatomy, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas.
J Neuroendocrinol. 2019 Aug;31(8):e12752. doi: 10.1111/jne.12752. Epub 2019 Jun 14.
Salt-loading (SL) impairs GABA inhibition of arginine vasopressin (AVP) neurones in the supraoptic nucleus (SON) of the hypothalamus. Based on previous studies, we hypothesised that SL activates tyrosine receptor kinase B (TrkB), down-regulating the activity of K /Cl co-transporter2 (KCC2) and up-regulating Na /K /Cl co-transporter1 (NKCC1). These changes in chloride transport would result in increased [Cl ] in SON AVP neurones. The study combined virally-mediated chloride imaging with ClopHensorN with a single-cell western blot analysis. An adeno-associated virus with ClopHensorN and a vasopressin promoter (AAV2-0VP1-ClopHensorN) was bilaterally injected in the SON of adult male Sprague-Dawley rats that were either euhydrated (Eu) or salt-loaded (SL) for 7 days. Acutely dissociated SON neurones expressing ClopHensorN were tested for decreases or increases in [Cl ] in response to focal application of the GABA agonist muscimol (100 μmol L ). SON AVP neurones from Eu rats showed muscimol-induced chloride influx (P < 0.05;23/35). SON AVP neurones from SL rats either significantly increased chloride efflux (P < 0.05;27/39) or did not change chloride flux (12/39). The SON AVP neurones that responded to muscimol appeared to be viable and expressed KCC2 and β-actin. Neurones that did not respond during chloride imaging did not show KCC2 and β-actin protein expression. The KCC2 antagonist (VU0240551,10 μmol L ) significantly blocked the chloride influx in cells from Eu rats but did not affect cells from SL rats. A NKCC1 antagonist (bumetanide,10 μmol L ) significantly blocked the chloride efflux in cells from SL rats but had no effect on cells from Eu rats. Blocking NKCC1 using bumetanide had less of an effect on the muscimol-induced Cl influx in Eu rat neurones compared to the KCC2 antagonist. The TrkB antagonist (AnA-12) (50 μmol L ) and protein kinase inhibitor (K252a) (100 nmol L ) each significantly blocked chloride efflux in SON AVP neurones from SL rats. Salt-loading increases [Cl ] in SON AVP neurones via a TrKB-KCC2-NKCC1-dependent mechanism in rats.
盐负荷 (SL) 会损害下丘脑视上核 (SON) 中精氨酸加压素 (AVP) 神经元的 GABA 抑制作用。基于先前的研究，我们假设 SL 会激活酪氨酸受体激酶 B (TrkB)，下调 K / Cl 协同转运蛋白 2 (KCC2) 的活性并上调 Na / K / Cl 协同转运蛋白 1 (NKCC1)。氯离子转运的这些变化会导致 SON AVP 神经元中 [Cl-]增加。该研究将病毒介导的氯离子成像与 ClopHensorN 与单细胞western blot 分析相结合。携带 ClopHensorN 和加压素启动子 (AAV2-0VP1-ClopHensorN) 的腺相关病毒被双侧注射到成年雄性 Sprague-Dawley 大鼠的 SON 中，这些大鼠要么处于正常水合状态 (Eu)，要么接受 7 天盐负荷 (SL)。表达 ClopHensorN 的急性分离的 SON 神经元接受局部应用 GABA 激动剂 muscimol (100 μmol L) 时 [Cl-]的减少或增加。来自 Eu 大鼠的 SON AVP 神经元显示 muscimol 诱导的氯离子内流 (P < 0.05; 23/35)。来自 SL 大鼠的 SON AVP 神经元要么显著增加氯离子外排 (P < 0.05; 27/39)，要么氯离子流没有变化 (12/39)。对 muscimol 有反应的 SON AVP 神经元似乎是存活的，并表达 KCC2 和 β-肌动蛋白。在氯离子成像期间没有反应的神经元没有显示 KCC2 和 β-肌动蛋白蛋白表达。KCC2 拮抗剂 (VU0240551,10 μmol L ) 显著阻断来自 Eu 大鼠的细胞中的氯离子内流，但对来自 SL 大鼠的细胞没有影响。NKCC1 拮抗剂 (布美他尼,10 μmol L ) 显著阻断来自 SL 大鼠的细胞中的氯离子外排，但对来自 Eu 大鼠的细胞没有影响。与 KCC2 拮抗剂相比，使用布美他尼阻断 NKCC1 对 Eu 大鼠神经元中 muscimol 诱导的 Cl-内流的影响较小。TrkB 拮抗剂 (AnA-12) (50 μmol L ) 和蛋白激酶抑制剂 (K252a) (100 nmol L ) 均显著阻断来自 SL 大鼠的 SON AVP 神经元中的氯离子外排。盐负荷通过大鼠中的 TrkB-KCC2-NKCC1 依赖性机制增加 SON AVP 神经元中的 [Cl-]。
