Two conventional protein kinase C isoforms, alpha and beta I, are involved in the ATP-induced activation of volume-regulated anion channel and glutamate release in cultured astrocytes.
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 receptor-dependent 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 the involvement of PKC in regulation of astrocytic VRACs by using small interfering RNA (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 microM ATP synergistically increased the release of excitatory amino acids, measured with a non-metabolized analog of L-glutamate, D-[(3)H]aspartate. Both Go6976, the selective inhibitor of Ca(2+)-sensitive PKCalpha, betaI/II, and gamma, and MP-20-28, a cell permeable pseudosubstrate inhibitory peptide of PKCalpha and betaI/II, reduced the effects of ATP on D-[(3)H]aspartate release by approximately 45-55%. Similar results were obtained with a mixture of siRNAs targeting rat PKCalpha and betaI. Surprisingly, down-regulation of individual alpha and betaI 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 PKCalpha and betaI.
Rudkouskaya
Alena
A
Center of Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY 12208.
Chernoguz
Artur
A
Center of Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY 12208.
Haskew-Layton
Renée E
RE
Burke/Cornell Medical Research Institute of Cornell University, White Plains, NY 10605.
Mongin
Alexander A
AA
Center of Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY 12208.
D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000201:enzymology* / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000201:enzymology / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D007424:Intracellular Fluid / Q000201:enzymology / Q000378:metabolism; D007527:Isoenzymes / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism*
D007527:Isoenzymes; D050994:Voltage-Dependent Anion Channels; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha