NA+-K+-2CL- COTRANSPORTERS IN CEREBRAL ISCHEMIA

Na+-K+-2Cl-协同转运体(NKCC)在重吸收和分泌上皮细胞的重吸收和分泌过程中起重要作用。NKCC还在维持和调节上皮和非上皮细胞的细胞体积方面发挥作用。然而，NKCC在中枢神经系统(CNS)中的作用尚未确定。本研究的长期目标是了解NKCC在脑缺血等生理和病理条件下的功能和调节。我们的初步研究表明，NKCC在维持星形胶质细胞K+稳态方面是必不可少的，它们似乎也参与了谷氨酸(Glu)介导的神经毒性。有四个具体的目标来测试我们的假肢。目的1：研究高[K+]o对原代培养星形胶质细胞NKCC活性的影响。目标是：1)。验证高[K+]o导致刺激NKCC；2)。以确定高[K+]o介导的NKCC刺激是否是由于细胞内[Ca++]升高。目的2：确定高[K+]o介导的NKCC刺激是否导致星形胶质细胞肿胀和Glu的非囊泡性释放。调查的目的是：1)。NKCC在[K+]o下是否导致细胞内Na+、C_1蓄积和细胞肿胀；抑制NKCC活性是否能显著增加[~3H]-L-Glu的摄取，同时阻断高[K+]诱导的星形胶质细胞Glu释放。目的3：探讨刺激NKCC是否参与了Glu介导的神经元毒性。调查的目的是：1)。NKCC是否参与了Glu介导的[c1-]i升高和神经元肿胀；抑制NKCC活性是否对谷氨酸诱导的细胞损伤有保护作用。目的4：确定脑内局部应用布美他尼一过性抑制NKCC活性时，缺血诱导的细胞损伤是否减轻。这项研究将在活体脑缺血模型中进行。

nA+-k+-2Cl-cotransporters (NKCC) are important in renal salt reabsorption and secretion by reabsorptive and secretory epithelia. The NKCC also function in maintenance and regulation of cell volume in both epithelial and non-epithelial cells. However, the role of the NKCC in the central nervous system (CNS) have not been defined. The long-term goal of this research is to understand function and regulation of the NKCC in the CNS under physiological as well as pathological conditions such as ischemia. Our pilot studies described in this proposal suggest that the NKCC are essential in maintenance of K+ homeostasis in astrocytes and they also appear to be involved in glutamate (Glu)-mediated neurotoxicity. There are four Specific Aims to test our hpotheses. Aim 1: Characterize high [K+]o-mediated stimulation of the NKCC activity in primary astrocyte cultures. The objectives are: 1). to verify that high [K+]o causes stimulation of the NKCC; 2). to determine whether the high [K+]o mediated stimulation of the NKCC is due to an increase in intracellular [Ca++]. Aim 2: Determine whether high [K+]o- mediated stimulation of the NKCC causes astrocyte swelling and non-vesicular release of Glu. The objectives are to investigate: 1). whether the NKCC contributes to accumulation of intracellular Na+, C1 and cell swelling under [K+]o; 2). whether inhibition of the NKCC activity could significantly increase [3H]-L-Glu uptake, while blocking of high [K+]-evoked release of Glu from astrocytes. Aim 3: Investigate whether stimulation of the NKCC contributes to Glu-mediated neuronal toxicity. The objectives are to investigate: 1). whether the NKCC contributes to Glu- mediated increase in [C1-]i and neuronal swelling; 2). whether inhibition of the NKCC activity could protect cells from Glu- induced damage. Aim 4: Determine whether ischemia-induced cell damage is reduced when the NKCC activity is transiently inhibited by a local administration of bumetanide in brain. This study will be performed in an in vivo cerebral ischemic model.