Defining protein kinase/phosphatase isoenzymic regulation of mGlu₅ receptor-stimulated phospholipase C and Ca²⁺ responses in astrocytes

Br J Pharmacol. 2011 Sep;164(2b):755-71. doi: 10.1111/j.1476-5381.2011.01421.x.

Abstract

Background and purpose: Cyclical phosphorylation and dephosphorylation of a key residue within the C-terminal domain of the activated type 5 metabotropic glutamate (mGlu₅) receptor is believed to cause the synchronous, oscillatory changes in inositol 1,4,5-trisphosphate and Ca²⁺ levels observed in a variety of cell types. Here, we have attempted to better define the kinase and phosphatase enzymes involved in this modulation.

Experimental approach: Ca²⁺ and [³H]inositol phosphate ([³H]IP(x) ) measurements in astrocyte preparations have been used to evaluate the effects of pharmacological inhibition of protein kinase C (PKC) and protein phosphatase activities and small interfering RNA-mediated specific PKC isoenzymic knock-down on mGlu₅ receptor signalling.

Key results: Ca²⁺ oscillation frequency or [³H]IP(x) accumulation in astrocytes stimulated by mGlu₅ receptors, was concentration-dependently decreased by protein phosphatase-1/2A inhibition or by PKC activation. PKC inhibition also increased [³H]IP(x) accumulation two- to threefold and changed the Ca²⁺ response into a peak-plateau response. However, selective inhibition of conventional PKC isoenzymes or preventing changes in [Ca²⁺](i) concentration by BAPTA-AM loading was without effect on mGlu₅ receptor-stimulated [³H]IP(x) accumulation. Selective knock-down of PKCδ was without effect on glutamate-stimulated Ca²⁺ responses; however, selective PKCε knock-down in astrocytes changed Ca²⁺ responses from oscillatory into peak-plateau type.

Conclusion and implications: These data confirm the acute regulation of mGlu₅ receptor signalling by protein kinases and protein phosphatases and provide novel data pinpointing the isoenzymic dependence of this regulation in the native mGlu₅ receptor-expressing rat cortical astrocyte. These data also highlight a potential alternative mechanism by which mGlu₅ receptor signalling might be therapeutically manipulated.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Astrocytes / metabolism*
  • Calcium / metabolism*
  • Calcium Signaling
  • Gene Knockdown Techniques / methods
  • Inositol Phosphates / metabolism
  • Isoenzymes
  • Protein Kinase C / deficiency
  • Protein Kinase C / genetics
  • Protein Kinase C / metabolism*
  • Protein Kinase C-epsilon / deficiency
  • Protein Kinase C-epsilon / genetics
  • Protein Kinase C-epsilon / metabolism*
  • Protein Phosphatase 1 / antagonists & inhibitors
  • Protein Phosphatase 1 / metabolism*
  • Protein Phosphatase 2 / antagonists & inhibitors
  • Protein Phosphatase 2 / metabolism*
  • RNA, Small Interfering / genetics
  • Rats
  • Rats, Wistar
  • Receptor, Metabotropic Glutamate 5
  • Receptors, Metabotropic Glutamate / genetics
  • Receptors, Metabotropic Glutamate / metabolism*
  • Signal Transduction
  • Type C Phospholipases / metabolism*

Substances

  • Inositol Phosphates
  • Isoenzymes
  • RNA, Small Interfering
  • Receptor, Metabotropic Glutamate 5
  • Receptors, Metabotropic Glutamate
  • protein kinase C zeta
  • Protein Kinase C
  • Protein Kinase C-epsilon
  • Protein Phosphatase 1
  • Protein Phosphatase 2
  • Type C Phospholipases
  • Calcium