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

Abstract

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. 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. 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. 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.