ATP-evoked calcium signal stimulates protein phosphorylation/dephosphorylation in astrocytes.
Extracellular adenosine 5'-triphosphate (ATP)-evoked increases in intracellular calcium and the consequent stimulation of calcium-mediated protein phosphorylation systems were investigated in primary cultures of rat cerebral cortical astrocytes. Measurement of calcium responses in fura-2-loaded astrocytes indicated that extracellular ATP stimulated a transient calcium peak followed by a sustained increase in intracellular calcium which declined to baseline when external calcium was removed, thereby indicating that ATP evokes mobilization of internal calcium as well as influx of external calcium. Protein phosphorylation studies revealed that application of extracellular ATP resulted in increased phosphorylation of 55 and 52 kDa proteins (4-fold and 2-fold, respectively) and decreased phosphorylation of 24 and 21 kDa proteins (approximately 50% for each protein). These effects were time- and dose-dependent. The changes in phosphate incorporation were (a) inhibited by lanthanum, (b) reduced when calcium was omitted from the bath and (c) mimicked by ionomycin, thus suggesting that the ATP-induced changes in protein phosphorylation were dependent on increased levels of intracellular calcium. Adenosine diphosphate (ADP) gave similar, but reduced, effects while adenosine and guanosine triphosphate (GTP) were ineffective, findings consistent with activation of P2 purinergic receptors. The 52 kDa protein co-migrated with glial fibrillary acidic protein. These results support the premise that calcium-dependent protein kinases and phosphatases are transducing elements for the calcium signal brought about by activation of P2 purinergic receptors in astrocytes. Since ATP is released from neurons and endothelial cells, this signal transduction mechanism may be an important component of neuronal- and endothelial-astrocytic communication.
Neary
J T
JT
Laboratory of Neuropathology, Veterans Administration Medical Center, Miami, FL 33125.
Laskey
R
R
van Breemen
C
C
Blicharska
J
J
Norenberg
L O
LO
Norenberg
M D
MD
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D001345:Autoradiography; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats
D009419:Nerve Tissue Proteins; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D015759:Ionomycin; D000255:Adenosine Triphosphate; D002118:Calcium