Laboratorio de Neurobiología, Centro Investigación Príncipe Felipe , Eduardo Primo-Yufera 3, 46012 Valencia, Spain.
ACS Chem Neurosci. 2016 Dec 21;7(12):1753-1759. doi: 10.1021/acschemneuro.6b00263. Epub 2016 Oct 5.
Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.
细胞外蛋白激酶，包括环腺苷酸依赖性蛋白激酶（PKA），调节神经元功能，包括 N-甲基-D-天冬氨酸（NMDA）受体依赖性长时程增强。NMDA 受体的激活增加钙，钙与钙调蛋白结合并激活一氧化氮合酶（NOS），增加一氧化氮（NO），NO 激活鸟苷酸环化酶，增加 cGMP，cGMP 释放到细胞外液，通过微透析在体内分析谷氨酸-NO-cGMP 途径。该途径在高氨血症大鼠中功能受损。本工作的目的是评估（1）细胞外 PKA 是否在体内调节小脑的谷氨酸-NO-cGMP 途径，（2）磷酸化和钙/钙调蛋白依赖性蛋白激酶 II（CaMKII）和 NOS 的活性在细胞外 PKA 对途径的调节中的作用，以及（3）这些作用在高氨血症和对照大鼠中是否不同。通过体内微透析分析该途径。通过使用膜不可渗透的抑制剂抑制细胞外 PKA 来分析其作用。从对照和高氨血症大鼠的新鲜分离的小脑切片中分析涉及的机制。在对照大鼠中，抑制细胞外 PKA 降低体内谷氨酸-NO-cGMP 途径的功能。这是由于 CaMKII 磷酸化和活性的降低，降低 NOS 在 Ser1417 的磷酸化和 NOS 活性，导致鸟苷酸环化酶激活和 cGMP 形成减少。在高氨血症大鼠中，在基础条件下，CaMKII 磷酸化和活性增加，NOS 在 Ser847 的磷酸化减少，NOS 活性降低，鸟苷酸环化酶激活和 cGMP 减少。在高氨血症大鼠中抑制细胞外 PKA 可使 CaMKII 磷酸化和活性、NOS 磷酸化、NOS 活性和 cGMP 正常化，恢复途径的正常功能。
