Phospholipase A(2) and its products are involved in the purinergic receptor-mediated translocation of protein kinase C in CHO-K1 cells.
The signal transduction involved in the purinergic stimuli-induced activation of protein kinase C (PKC) in CHO-K1 cells was investigated. Purinergic stimuli such as adenosine triphosphate and uridine triphosphate induced a transient translocation of PKC epsilon, gamma, and delta from the cytoplasm to the plasma membrane. These translocations were blocked by an inhibitor of phosphatidylinositol-specific phospholipase C (PLC), but not by an inhibitor of phosphatidylcholine-specific PLC. A diacylglycerol (DAG) analogue also induced reversible translocations of PKC gamma, epsilon, and delta from the cytoplasm to the plasma membrane, while the calcium ionophore A23187 caused a similar translocation of only the gamma subtype. These results confirm that the hydrolysis of phosphatidylinositol-2-phosphate by PLC and the subsequent generation of DAG and increase in Ca(2+ )are involved in the purinergic stimuli-induced translocation of PKC. A DAG antagonist, 1-o-hexadecyl-2-o-acetyl-glycerol, blocked the DAG analogue-induced translocations of all PKC subtypes tested but failed to inhibit the purinergic stimuli-induced translocations of PKC epsilon and gamma. The DAG antagonist could not block the ATP- and UTP-induced translocation of PKC epsilon even in the absence of extracellular Ca(2+). Co-application of the DAG antagonist and a phospholipase A(2) (PLA(2)) inhibitor such as aristolochic acid, arachidonyltrifluoromethyl ketone, or bromoenol lactone inhibited the purinergic receptor-mediated translocation of PKC epsilon although each PLA(2) inhibitor alone did not block the translocation. In contrast to the epsilon subtype, ATP-induced translocation of PKC gamma was observed in the presence of both the PLA(2) inhibitor and the DAG antagonist. However, it is noteworthy that re-translocation of PKC gamma was hastened by the PLA(2) inhibitor. Furthermore products of PLA(2), such as lysophospholipids and fatty acids, induced the translocation of PKC gamma and epsilon in a dose dependent manner, but not delta. These results indicate that, in addition to PLC and DAG, PLA(2) and its products are involved in the purinergic stimuli-induced translocation of PKC epsilon and gamma in CHO-K1 cells. Each subtype of PKC in CHO-K1 cell is individually activated in response to a purinergic stimulation.
Shirai
Y
Y
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Nada-ku, Kobe 657-8501, Japan.
Kashiwagi
K
K
Sakai
N
N
Saito
N
N
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D010741:Phospholipases A / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011983:Receptors, Purinergic / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D004075:Diglycerides; D011983:Receptors, Purinergic; D011493:Protein Kinase C; D010741:Phospholipases A; D010738:Type C Phospholipases