磷酸化钙/钙调蛋白依赖性蛋白激酶Ⅱ在大鼠脊髓背角C-纤维诱发电位长时程增强的诱导和维持中的作用

Role of phospho-calcium/ calmodulin-dependent protein kinase Ⅱ in the induction and maintenance of long-term potentiation of C-fiber-evoked field potentials in spinal dorsal horn of the rat

实验旨在探讨钙/钙调蛋白依赖性蛋白激酶Ⅱ(calcium/calmodulin—dependent protein kinase Ⅱ,CaMKⅡ)在脊髓背角C-纤维诱发电位长时程增强(long-term potentiation,LTP)的诱导和维持中的作用。用Western blot技术分别检测LTP形成30 min和3 h脊髓背角(L4-L6)CaMK Ⅱ的含量及其磷酸化水平。同时观察脊髓局部给予CaMK Ⅱ选择性抑制剂KN-93后对脊髓背角LTP和CaMK Ⅱ磷酸化的影响。观察结果如下:(1)诱导LTP后30 min.CaMK Ⅱ的磷酸化水平明显高于对照组,而CaMKⅡ的总量无变化;诱导LTP后3 h CaMK Ⅱ的磷酸化水平进一步升高,而且CaMK Ⅱ的总量也明显增加(n=4);(2)强直刺激前30 min 于脊髓局部给予CaMK Ⅱ的特异性抑制剂KN-93(100μmol/L),可阻断LTP的诱导,同时明显抑制CaMK Ⅱ的磷酸化水平;(3)诱导LTP后30 min给予KN-93,可显著抑制LTP的维持,同时CaMKⅡ的磷酸化水平与未用药组相比也明显降低(n=3);(4)LTP 3 h后给予KN-93,LTP的幅值不受影响,磷酸化的CaMKⅡ的含量与用药前相比也无差别(n=3)。根据上述实验结果可以认为,CaMK Ⅱ的激活参与脊髓背角C-纤维诱发电位LTP的诱导和早期维持过程。

Our previous studies have shown that long-term potentiation (LTP) of C-fiber-evoked field potentials in the spinal dorsal horn is NMDA receptor dependent. It is known that elevation of Ca2+ in the postsynaptic neurons through NMDA receptor channels during high-frequency stimulation of the afferent fibers is crucial for LTP induction, but how this leads to a prolonged potentiation of synaptic transmission in the spinal dorsal horn is not clear. In the hippocampus, a rise of Ca2+ activates calcium/ calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) through autophosphorylation. Once this occurs, the kinase remains active, even when Ca2+ concentration returns to baseline level. Phosphorylated CaMK Ⅱ potentiates synaptic transmission by enhancement of AMPA receptor channel function via phosphorylation of GluR1 subunit of the receptor and the addition of AMPA receptors to synapses. Up to now, the role of CaMK Ⅱ in the induction and maintenance of LTP of the C-fiber-evoked field potentials in spinal dorsal horn has not been evaluated. In the present study, we examined the expression of CaMK Ⅱ and phospho-CaMK Ⅱ in the lumbar segments (L4-L6) of the rat spinal dorsal horn at 30 min and 3 h after the establishment of LTP induced by tetanic electrical stimulation of the sciatic nerve (40 V, 0.5 ms pulses at 100 Hz for 1 s repeated four times at 10 s intervals) by using Western blot and eletrophysiological techniques. To determine the role of the phospho-CaMK Ⅱ in the induction and maintenance of the spinal LTP, a selective CaMK Ⅱ inhibitor KN-93 (100 μmol/L) was applied directly onto the spinal cord at the recording segments before and after LTP induction. We found that (1) the protein level of phospho-CaMK Ⅱ increased at both 30 min and 3 h after LTP induction, while the total protein level of CaMK Ⅱ increased at 3 h but not at 30 min after LTP induction. (2) Spinal application of KN-93 at 30 min prior to the tetanus blocked both LTP induction and the increase in phospho-CaMK Ⅱ. (3) 30 min after LTP induction, spinal application of KN-93 depressed LTP and the level of phospho-CaMK Ⅱ (n=3). (4) Spinal application of KN-93 at 3 h after LTP, however, affected neither the amplitude of the spinal LTP nor the level of phospho-CaMK Ⅱ in the spinal dorsal horn. These results suggest that activation of CaMK Ⅱ is probably crucial for the induction and the early-phase maintenance of LTP of C-fiber-evoked field potentials in the spinal dorsal horn.