持续酸中毒发生在缺血和多种神经系统疾病中。在以往的研究中,酸性刺激导致神经元细胞内钙的快速增加。然而,目前尚不清楚长期酸中毒如何改变神经元信号。在我们之前的研究中,我们发现GPR68介导的PKC活性对酸中毒引起的皮质切片损伤具有保护作用.这里,我们首先询问了同样的原理在器官型海马切片中是否成立。我们的数据显示,1小时pH6以GPR68依赖性方式诱导PKC磷酸化。在野生型(WT)中,PKC抑制剂Go6983恶化了酸中毒诱导的神经元损伤,但在GPR68-/-切片中没有作用。接下来,为了更深入地了解脑组织中的酸信号,我们用pH6处理器官型海马切片1小时,并通过Westernblot进行了kinome谱分析.酸中毒对细胞周期蛋白依赖性激酶(CDK)或酪蛋白激酶2活性影响不大,CMGC家族的两个成员,或共济失调毛细血管扩张突变(ATM)/ATM和RAD3相关(ATR)活性,但降低了MAPK/CDK底物的磷酸化。相比之下,酸中毒引起CaMKIIα的激活,PKA,Akt除了这些丝氨酸/苏氨酸激酶,酸中毒也诱导酪氨酸磷酸化。由于GPR68在大脑神经元中广泛表达,我们询问GPR68是否有助于酸中毒诱导的信号传导.删除GPR68对酸中毒诱导的CaMKII磷酸化没有影响,减弱了磷酸-Akt和磷酸-PKA底物,同时消除酸中毒诱导的酪氨酸磷酸化。这些数据表明,持续的酸中毒激活了信号级联的网络,由AGC激酶介导,CaMKII,和酪氨酸激酶。GPR68是酸中毒诱导的PKC活化和酪氨酸磷酸化的主要介质,而GPR68依赖性和非依赖性机制都有助于PKA和Akt的激活。
Persistent acidosis occurs in ischemia and multiple neurological diseases. In previous studies, acidic stimulation leads to rapid increase in intracellular calcium in neurons. However, it remains largely unclear how a prolonged acidosis alters neuronal signaling. In our previous study, we found that GPR68-mediated PKC activities are protective against acidosis-induced injury in cortical slices. Here, we first asked whether the same principle holds true in organotypic hippocampal slices. Our data showed that 1-h pH 6 induced PKC phosphorylation in a GPR68-dependent manner. Go6983, a PKC inhibitor worsened acidosis-induced neuronal injury in wild type (WT) but had no effect in GPR68-/- slices. Next, to gain greater insights into acid signaling in brain tissue, we treated organotypic hippocampal slices with pH 6 for 1-h and performed a kinome profiling analysis by Western blot. Acidosis had little effect on cyclin-dependent kinase (CDK) or casein kinase 2 activity, two members of the CMGC family, or Ataxia telangiectasia mutated (ATM)/ATM and RAD3-related (ATR) activity, but reduced the phosphorylation of MAPK/CDK substrates. In contrast, acidosis induced the activation of CaMKIIα, PKA, and Akt. Besides these serine/threonine kinases, acidosis also induced tyrosine phosphorylation. Since GPR68 is widely expressed in brain neurons, we asked whether GPR68 contributes to acidosis-induced signaling. Deleting GPR68 had no effect on acidosis-induced CaMKII phosphorylation, attenuated that of phospho-Akt and phospho-PKA substrates, while abolishing acidosis-induced tyrosine phosphorylation. These data demonstrate that prolonged acidosis activates a network of signaling cascades, mediated by AGC kinases, CaMKII, and tyrosine kinases. GPR68 is the primary mediator for acidosis-induced activation of PKC and tyrosine phosphorylation, while both GPR68-dependent and -independent mechanisms contribute to the activation of PKA and Akt.