Two conventional protein kinase C isoforms, α and βI, are involved in the ATP‐induced activation of volume‐regulated anion channel and glutamate release in cultured astrocytes

Abstract

Volume-regulated anion channels (VRACs) are activated by cell swelling and are permeable to inorganic and small organic anions, including the excitatory amino acids glutamate and aspartate. In astrocytes, ATP potently enhances VRAC activity and glutamate release via a P2Y receptordependent mechanism. Our previous pharmacological study identified protein kinase C (PKC) as a major signaling enzyme in VRAC regulation by ATP. However, conflicting results obtained with potent PKC blockers prompted us to re-evaluate PKC involvement in regulation of astrocytic VRAC using siRNA and pharmacological inhibitors that selectively target individual PKC isoforms. In primary rat astrocyte cultures, application of hypoosmotic medium (30% reduction in osmolarity) and 20 M ATP synergistically increased the release of excitatory amino acids, measured with non-metabolized analogue of L-glutamate, D-[ 3 H]aspartate. Both Go6976, the selective inhibitor of Ca 2+ -sensitive PKC , I/II, and , and MP-20-28, a cell permeable pseudosubstrate inhibitory peptide of PKC and I/II, reduced the effects of ATP on D-[ 3 H]aspartate release by ~45-55%. Similar results were obtained with a mixture of siRNAs targeting rat PKC and I. Surprisingly, downregulation of individual and I PKC isozymes by siRNA was completely ineffective. These data suggest that ATP regulates VRAC activity and volume-sensitive excitatory amino acid release via cooperative activation of PKC and I.

Activation of P2Y receptors causes strong and persistent shrinkage of C11-MDCK renal epithelial cells

“…The effects of 4␤-PMA on cell volume were Ca 2ϩ i sensitive (Table 2), indicating involvement of conventional Ca 2ϩ -sensitive, DAG-dependent PKC isoform(s). A similar mechanism involving conventional PKC-␣ and -␤I was reported for ATPinduced activation of VRACs in astrocytes (47).…”

L-Plastin Regulates Polarization and Migration in Chemokine-Stimulated Human T Lymphocytes

“…2D, top and middle panels). Because redundancy and/or synergy has been reported for PKCs in different cell types (including T lymphocytes), and thus may require inhibition of more than one isoform (33)(34)(35), we transfected cells with a combination of siRNAs to PKC isoforms (i.e., PKC a/b, PKC d/u, PKC ε/u, and PKC h/u), but again found no obvious role for these PKCs in L-plastin phosphorylation (Fig. 2D, bottom panel).…”

Extracellular osmolarity modulates G protein-coupled receptor-dependent ATP release from 1321N1 astrocytoma cells

“…1321N1 cells present an additional advantage for analysis of the signaling mechanisms that couple GPCR to VRAC/VSOAC activation (or other GPCR-regulated permeability pathways) because they lack endogenous expression of G protein-coupled P2Y receptors. This is germane because P2Y receptors also activate VRAC/VSOAC in primary astrocytes and other cell types (35,44,45,57). Thus, ATP released in response to hypotonic stress or other GPCR agonists can act as an autocrine modulator or amplifier of VRAC/VSOAC and volume regulatory responses.…”