Misako Hayama

Inhibition of Ca2+-independent phospholipase A2 by bromoenol lactone attenuates prostaglandin generation induced by interleukin-1β and dibutyryl cAMP in rat mesangial cells

Cytokine‐induced prostaglandin generation in rat mesangial cells has been suggested to be dependent on the expression of secretory phospholipase A2 (sPLA2). In the present study, we investigated the possible involvement of Ca2+‐independent phospholipase A2 (iPLA2) in the generation. The results showed that bromoenol lactone, a relatively selective iPLA2 inhibitor, significantly attenuated prostaglandin E2 generation induced by interleukin‐1β and dibutyryl cAMP in parallel with the inhibition of iPLA2 activity. However, the inhibitor did not affect sPLA2 release upon stimulation, activities of sPLA2 or cytosolic phospholipase A2, or Ca2+ ionophore‐induced arachidonic acid liberation. These results suggest that prostaglandin E2 generation upon stimulation may be partially mediated by iPLA2 in addition to sPLA2.

Protein kinase Cα-dependent increase in Ca2+-independent phospholipase A2 in membranes and arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells

We previously reported that zymosan-stimulated, protein kinase C (PKC)-dependent arachidonic acid liberation occurs with association of Ca2+-independent phospholipase A2 (iPLA2) with the membranes of macrophage-like P388D1 cells. In the present study, the possible involvement of PKC isoforms (alpha, beta, delta, and epsilon) on the increase in iPLA2 was examined. Stimulation of P388D1 cells with zymosan induced increases in iPLA2 activity and protein in the membranes and liberation of arachidonic acid. In the stimulated cells, PKCalpha, PKCdelta, and PKCepsilon, but not PKCbeta, were increased in the membranes. The zymosan-induced increase in iPLA2 activity was suppressed by pretreatment with 4beta-phorbol 12-myristate 13-acetate for 10 hr, by which PKCalpha and PKCdelta, but not PKCbeta and PKCepsilon, were depleted, and by Gö6976, a PKCalpha inhibitor, but not rottlerin, a PKCdelta inhibitor. The zymosan-induced release of arachidonic acid was also reduced by the PKC depletion and Gö6976. However, stimulation with 4beta-phorbol 12-myristate 13-acetate alone did not increase iPLA2 activity in the membranes. Furthermore, the depletion of intracellular Ca2+ also impaired the zymosan-induced increase in iPLA2 activity in the membranes. However, no increase in iPLA2 activity was observed upon stimulation with Ca2+-mobilizing agents (ionomycin or thapsigargin). Cytochalasin D, an inhibitor of actin polymerization, suppressed the zymosan-induced increases in iPLA2 activity and protein in the membranes and the release of arachidonic acid. These results suggest that zymosan stimulates an increase in iPLA2 in the membranes of P388D1 cells probably through activation of PKCalpha in concert with cytochalasin D-sensitive events.

Transforming growth factor-α stimulates prostaglandin generation through cytosolic phospholipase A2 under the control of p11 in rat gastric epithelial cells

The regulatory effects of transforming growth factor (TGF)‐α on phospholipase A2 (PLA2) isozymes contributing to prostaglandin generation in rat gastric epithelial RGM1 cells were examined. Stimulation with TGF‐α for 24 h time‐dependently induced prostaglandin E2 generation with an increase in cyclo‐oxygenase‐2 protein. The TGF‐α‐induced prostaglandin E2 generation was suppressed by NS‐398, a cyclo‐oxygenase‐2 inhibitor. TGF‐α stimulated the activity and the protein synthesis of cytosolic PLA2 (cPLA2). A time‐dependent increase in cPLA2 protein occurred in parallel with PGE2 generation, which was inhibited by methyl arachidonyl fluorophosphonate (MAFP), a cPLA2 inhibitor. However, no change in activity of secretory PLA2 or Ca+2‐independent PLA2 was observed in the TGF‐α‐stimulated cells. Stimulation with the Ca2+ ionophore A23187 for 10 min induced MAFP‐sensitive arachidonic acid liberation. Interestingly, preincubation with TGF‐α for 24 h diminished A23187‐stimulated arachidonic acid liberation despite the increase in cPLA2 protein. Under the conditions, TGF‐α was found to increase p11, an endogenous cPLA2 suppressor, also known as annexin II light chain. The TGF‐α‐induced increase in p11 was suppressed by tyrphostin AG1478, an inhibitor of tyrosine kinase of epidermal growth factor receptor, which was also found to restore the inhibition by TGF‐α of A23187‐stimulated arachidonic acid liberation. However, TGF‐α did not alter protein levels of annexin II heavy chain. These results suggest that TGF‐α stimulates prostaglandin generation through an increase in cPLA2, the hydrolytic action of which may be under the control of p11.

Inhibitory effect of cepharanthine on fibronectin production in growth factor-stimulated rat mesangial cells.

We examined the effect of cepharanthine, a biscoclaurine alkaloid, on extracellular matrix production in rat mesangial cells in response to platelet-derived growth factor (PDGF) or transforming growth factor-beta (TGF-beta). Stimulation of the cells with PDGF increased the amounts of fibronectin, one of extracellular matrix components. Pretreatment with cepharanthine (0.1-2 microM) suppressed the PDGF-stimulated increase in fibronectin in a dose-dependent manner. At a concentration of 2 microM, the alkaloid almost completely suppressed the production. Under the conditions, the alkaloid inhibited tyrosine phosphorylation of several proteins including PDGF beta receptor in PDGF-stimulated cells, and also tyrosine kinase activity of the receptor prestimulated with PDGF in a cell-free assay system. Furthermore, cepharanthine suppressed TGF-beta-stimulated fibronectin production at the same concentration ranges. Our results suggest that cepharanthine inhibits fibronectin production induced by growth factors, probably through suppression of receptor autophosphorylation.

Stimulation of cytosolic phospholipase A2-catalyzed arachidonic acid liberation by low dose tert-butyl hydroperoxide without an influence on the enzyme activity in rabbit platelets

The effect of lipid peroxide on the hydrolytic action of cytosolic phospholipase A2 (cPLA2) in rabbit platelets was investigated. Ionomycin‐stimulated arachidonic acid liberation and lysophosphatidylcholine formation were significantly potentiated when platelets were pretreated with tert‐butyl hydroperoxide (BHP) and FeSO4, and then washed. Under the conditions, oxidizing reagents did not enhance the increase in cPLA2 activity by ionomycin or the basal activity in unslimulated cells. Furthermore, the treatment of a platelet lysate with BHP and FeSO4 did not affect Ca2+‐induced translocation of cPLA2 to the membranes. However, with a membrane fraction, arachidonic acid liberation catalyzed by the partially purified cPLA2 was synergistically enhanced by BHP and FeSO4. These results suggest that oxidative stress may potentiate the hydrolytic action of cPLA2 on membrane phospholipids without an influence on the processes leading to the enzyme activation.