Objective The rostral anterior cingulate cortex(rACC)is implicated in processing the emotional component of pain.N-methyl-D-aspartate receptors(NMDARs)are highly expressed in the rACC and mediate painrelated affect by activating a signaling pathway that involves cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)and/or extracellular regulated kinase(ERK)/cAMP-response element-binding protein(CREB).The present study investigated the contributions of the NMDAR glycine site and GluN2B subunit to the activation of ERK and CREB both in vitro and in vivo in rat rACC.Methods Immunohistochemistry and Western blot analysis were used to separately assess the expression of phospho-ERK(pERK)and phospho-CREB(pCREB)in vitro and in vivo.Double immunostaining was also used to determine the colocalization of pERK and pCREB.Results Both bath application of NMDA in brain slices in vitro and intraplantar injection of formalin into the rat hindpaw in vivo induced significant up-regulation of pERK and pCREB in the rACC,which was inhibited by the NMDAR antagonist DL-2-amino-5-phospho-novaleric acid.Selective blockade of the NMDAR GluN2B subunit and the glycinebinding site,or degradation of endogenous D-serine,a co-agonist for the glycine site,significantly decreased the up-regulation of pERK and pCREB expression in the rACC.Further,the activated ERK predominantly colocalized with CREB.Conclusion Either the glycine site or the GluN2B subunit of NMDARs participates in the phosphorylation of ERK and CREB induced by bath application of NMDA in brain slices or hindpaw injection of 5% formalin in rats,and these might be fundamental molecular mechanisms underlying pain affect.
Hong CaoWen-Hua RenMu-Ye ZhuZhi-Qi ZhaoYu-Qiu Zhang
Objective Our previous study identified Threonine 161 (Thr-161), located in the second intracellular loop of the δ-opioid receptor (DOR), as the only consensus phosphorylation site for cyclin-dependent kinase 5 (Cdk5). The aim of this study was to assess the function of DOR phosphorylation by Cdk5 in complete Freund's adjuvant (CFA)-induced inflammatory pain and morphine tolerance. Methods Dorsal root ganglion (DRG) neurons of rats with CFA-induced inflammatory pain were acutely dissociated and the biotinylation method was used to explore the membrane localization of phosphorylated DOR at Thr-161 (pThr-161-DOR), and paw withdrawal latency was measured after intrathecal delivery of drugs orTat-peptide, using a radiant heat stimulator in rats with CFA-induced inflammatory pain. Results Both the total amount and the surface localization of pThr-161-DOR were significantly enhanced in the ipsilateral DRG following CFA injection. Intrathecal delivery of the engineered Tat fusion-interefering peptide corresponding to the second intracellular loop of DOR (Tat-DOR-2L) increased inflammatory hypersensitivity, and inhibited DOR- but not μ-opioid receptor-mediated spinal analgesia in CFA-treated rats. However, intrathecal delivery of Tat-DOR-2L postponed morphine antinociceptive tolerance in rats with CFA-induced inflammatory pain. Conclusion Phosphorylation of DOR at Thr-161 by Cdk5 attenuates hypersensitivity and potentiates morphine tolerance in rats with CFA-induced inflammatory pain, while disruption of the phosphorylation of DOR at Thr-161 attenuates morphine tolerance.
Hai-Jing ChenWei-Yan XieFang HuYing ZhangJun WangYun Wang
Dopamine D1 receptors(D1Rs) play a key role in cocaine addiction, and multiple protein kinases such as GRKs, PKA, and PKC are involved in their phosphorylation. Recently, we reported that protein kinase D1 phosphorylates the D1 R at S421 and promotes its membrane localization. Moreover, this phosphorylation of S421 is required for cocaineinduced behaviors in rats. In the present study, we generated transgenic mice over-expressing S421A-D1 R in the forebrain. These transgenic mice showed reduced phospho-D1R(S421) and its membrane localization, and reduced downstream ERK1/2 activation in the striatum. Importantly, acute and chronic cocaine-induced locomotor hyperactivity and conditioned place preference were significantly attenuated in these mice. These findings provide in vivo evidence for the critical role of S421 phosphorylation of the D1 R in its membrane localization and in cocaine-induced behaviors. Thus, S421 on the D1 R represents a potential pharmacotherapeutic target for cocaine addiction and other drug-abuse disorders.
Protein kinase D(PKD) is an evolutionarily-conserved family of protein kinases. It has structural, regulatory, and enzymatic properties quite different from the PKC family. Many stimuli induce PKD signaling, including G-protein-coupled receptor agonists and growth factors. PKD1 is the most studied member of the family. It functions during cell proliferation, differentiation, secretion, cardiac hypertrophy, immune regulation, angiogenesis, and cancer. Previously, we found that PKD1 is also critically involved in pain modulation. Since then, a series of studies performed in our lab and by other groups have shown that PKDs also participate in other processes in the nervous system including neuronal polarity establishment, neuroprotection, and learning. Here, we discuss the connections between PKD structure, enzyme function, and localization, and summarize the recent findings on the roles of PKD-mediated signaling in the nervous system.
