Role of protein kinase A in phosphorylation of NMDA receptor 1 subunits in dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats
Role of protein kinase A in phosphorylation of NMDA receptor 1 subunits in dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats
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DOI：
10.1016/s0306-4522(02)00490-6
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发表时间：
2002-12
期刊：
影响因子：
3.3
通讯作者：
X. Zou;Q. Lin;W. Willis
中科院分区：
文献类型：
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作者：
X. Zou;Q. Lin;W. Willis
蛋白磷酸化是N-甲基-D-天冬氨酸（NMDA）受体功能调节的主要机制。NMDA受体1亚基（NR 1）被蛋白激酶A（PKA）在丝氨酸890和897上磷酸化。我们最近报道了在大鼠皮内注射辣椒素（CAP）后，背角和脊髓丘脑束（STT）神经元中丝氨酸897上的NR 1磷酸化增强[Zou等人（2000）J. Neurosci. 20，6989-6997]。是否这种磷酸化，在CAP注射后的中枢致敏过程中发展，是由PKA介导的仍有待确定。在这项研究中，蛋白质印迹和免疫荧光染色，观察是否预处理PKA抑制剂，N-[2-（（对溴肉桂基）氨基）乙基]-5-异喹啉磺酰胺，盐酸（H89），阻断丝氨酸897上的NR 1的磷酸化后，CAP注射到麻醉大鼠的一个后爪的光滑的皮肤。Western印迹显示，H89预处理导致CAP诱导的脊髓L4-S1节段NR 1蛋白磷酸化减少。在免疫荧光染色实验中，注射CAP后，PKA抑制后，注射同侧L4-L5节段背角的磷酸化NR 1样免疫反应（p-NR 1-LI）神经元数量明显减少。用荧光金逆行标记STT细胞，发现CAP注射前鞘内注射H89可使注射侧L4-L5节段脊髓浅层p-NR 1-LI STT细胞比例明显减少。然而，p-NR 1-LI STT细胞在深层的比例是不变的，除非PKC抑制剂，白屈菜红碱氯化物，与H89共同管理。结合我们以前的研究结果，本研究结果表明，NR 1在脊髓背角神经元，包括浅背角STT细胞，磷酸化CAP注射后，这种磷酸化是由于PKA的作用。然而，深STT细胞的磷酸化涉及PKA和PKC。
Protein phosphorylation is a major mechanism for regulation of N-methyl-D-aspartate (NMDA) receptor function. The NMDA receptor 1 subunit (NR1) is phosphorylated by protein kinase A (PKA) on serine 890 and 897. We have recently reported that there is enhanced phosphorylation of NR1 on serine 897 in dorsal horn and spinothalamic tract (STT) neurons after intradermal injection of capsaicin (CAP) in rats [Zou et al. (2000) J. Neurosci. 20, 6989–6997]. Whether or not this phosphorylation, which develops during central sensitization following CAP injection, is mediated by PKA remains to be determined. In this study, western blots and immunofluorescence staining were employed to observe if pretreatment with a PKA inhibitor, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H89), blocks the enhanced phosphorylation of NR1 on serine 897 following injection of CAP into the glabrous skin of one hind paw of anesthetized rats. Western blots showed that pretreatment with H89 caused a decrease in CAP-induced phosphorylation of NR1 protein in spinal cord segments L4–S1. In experiments using immunofluorescence staining, the numbers of phospho-NR1-like immunoreactive (p-NR1-LI) neurons seen after CAP injection were significantly decreased in the dorsal horn of the L4–L5segments on the side ipsilateral to the injection after PKA was inhibited. When STT cells were labeled by microinjection of the retrograde tracer, fluorogold, we found that the proportion of p-NR1-LI STT cells on the side ipsilateral to the injection in the superficial laminae of spinal cord segments L4–L5was markedly reduced when H89 was administered intrathecally before CAP injection. However, the proportion of p-NR1-LI STT cells in deep laminae was unchanged unless the PKC inhibitor, chelerythrine chloride, was co-administered with H89. Combined with our previous findings, the present results indicate that NR1 in spinal dorsal horn neurons, including the superficial dorsal horn STT cells, is phosphorylated following CAP injection and that this phosphorylation is due to the action of PKA. However, the phosphorylation of deep STT cells involves both PKA and PKC.