Regulation of the firing activity by PKA‐PKC‐Src family kinases in cultured neurons of hypothalamic arcuate nucleus

Abstract

The cAMP‐dependent protein kinase A family (PKAs), protein kinase C family (PKCs), and Src family kinases (SFKs) are found to play important roles in pain hypersensitivity. However, more detailed investigations are still needed in order to understand the mechanisms underlying the actions of PKAs, PKCs, and SFKs. Neurons in the hypothalamic arcuate nucleus (ARC) are found to be involved in the regulation of pain hypersensitivity. Here we report that the action potential (AP) firing activity of ARC neurons in culture was up‐regulated by application of the adenylate cyclase activator forskolin or the PKC activator PMA, and that the forskolin or PMA application‐induced up‐regulation of AP firing activity could be blocked by pre‐application of the SFK inhibitor PP2. SFK activation also up‐regulated the AP firing activity and this effect could be prevented by pre‐application of the inhibitors of PKCs, but not of PKAs. Furthermore, we identified that forskolin or PMA application caused increases in the phosphorylation not only in PKAs at T197 or PKCs at S660 and PKCα/βII at T638/641, but also in SFKs at Y416. The forskolin or PMA application‐induced increase in the phosphorylation of PKAs or PKCs was not affected by pre‐treatment with PP2. The regulations of the SFK and AP firing activities by PKCs were independent upon the translocation of either PKCα or PKCβII. Thus, it is demonstrated that PKAs may act as an upstream factor(s) to enhance SFKs while PKCs and SFKs interact reciprocally, and thereby up‐regulate the AP firing activity in hypothalamic ARC neurons.

Involvement of CGRP and CGRP receptor in nociceptive modulation in arcuate nucleus of the hypothalamus in rats with neuropathic pain

“…ARC also participates in nociceptive modulation [20,23]. In addition, ARC has been reported to be involved in central sensitization in a variety of ways [24,25]. However, less information is available for the functional roles in nociceptive modulation of CGRP in ARC.…”

MicroRNA-547-5p-mediated interleukin-33/suppressor of tumorigenicity 2 signaling underlies the genesis and maintenance of neuropathic pain and is targeted by the therapy with bone marrow stromal cells

“…Western blotting experiments were performed as described previously. 18,19,32 In brief, tissues isolated from the DRG or SDH were homogenized in an ice-cold radioimmunoprecipitation assay buffer (Beyotime Biotechnology) supplemented with protease inhibitor cocktail (Bimake.cn), phosphatase inhibitor cocktail (Bimake.cn), phenylmethylsulfonyl fluoride (PMSF, Beyotime Biotechnology), and dithiothreitol (DTT, 0.5 mM, Beyotime Biotechnology). After the homogenates were centrifuged at 12000 g for 20 min at 4°C, protein concentrations were determined with bicinchoninic acid (BCA) assay (Beyotime Biotechnology).…”

“…Western blotting experiments were performed as described previously. 18,19,32 In brief, tissues isolated…”

“…Western blotting experiments were performed as described previously. 18,19,32 In brief, tissues isolated GenePharma, Shanghai, China; www. genepharma.com [25][26][27] Antagomir-547-5p 5 0 -UGGGUGGUACAUCCUGAAGUGA-3 0 GenePharma, Shanghai, China; www.…”

“…Western blotting experiments were performed as described previously. 18,19,32 In brief, tissues isolated 5 0 -UCACUUCAGGAUGUACCACCCA-3 0 3 0 -GGUGGUACAUCCUGAAGUGAUU-5 0 GenePharma, Shanghai, China; www. genepharma.com Agomir-NC 5 0 -UUCUCCGAACGUGUCACGUTT-3 0 3 0 -ACGUGACACGUUCGGAGAATT-5 0 GenePharma, Shanghai, China; www.…”