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

J Neurosci Res. 2020 Feb;98(2):384-403. doi: 10.1002/jnr.24516. Epub 2019 Aug 12.

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.

Keywords: RRID:AB_10015289; RRID:AB_10865528; RRID:AB_141607; RRID:AB_1524202; RRID:AB_2168219; RRID:AB_2284224; RRID:AB_2298772; RRID:AB_2313567; RRID:AB_2535792; RRID:AB_2687938; RRID:AB_2750616; RRID:AB_777294; RRID:AB_779042; RRID:SCR_007369; RRID:SCR_014329; Src family kinases; action potential firing activity; hypothalamic arcuate nucleus; pain; phosphorylation; protein kinase A; protein kinase C.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / drug effects
  • Action Potentials / physiology*
  • Animals
  • Arcuate Nucleus of Hypothalamus / drug effects
  • Arcuate Nucleus of Hypothalamus / metabolism*
  • Cells, Cultured
  • Colforsin / pharmacology
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Female
  • Male
  • Neurons / drug effects
  • Neurons / metabolism*
  • Oligopeptides / pharmacology
  • Patch-Clamp Techniques
  • Phosphorylation / drug effects
  • Protein Kinase C / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Vasodilator Agents / pharmacology
  • src-Family Kinases / metabolism*

Substances

  • Oligopeptides
  • Vasodilator Agents
  • lysyl-arginyl-alanyl-lysyl-alanyl-lysyl-threonyl-threonyl-lysyl-lysyl-arginine
  • Colforsin
  • src-Family Kinases
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C