title: Defining protein kinase/phosphatase isoenzymic regulation of mGlu₅ receptor-stimulated phospholipase C and Ca²⁺ responses in astrocytes.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.
authors.forename: S J
authors.initials: SJ
authors.lastname: Bradley
authors.forename: R A J
authors.initials: RA
authors.lastname: Challiss
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D055785:Gene Knockdown Techniques / Q000379:methods; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes; D011493:Protein Kinase C / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism*; D000099221:Protein Kinase C zeta
chemical_list: D002118:Calcium; D007295:Inositol Phosphates; D007527:Isoenzymes; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D034741:RNA, Small Interfering; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; D010738:Type C Phospholipases; D000099221:Protein Kinase C zeta