PLD activation in Chinese hamster ovary (CHO) cells transfected with PGF2 alpha receptor cDNA.
Pertussis toxin-insensitive GTP-binding protein was observed to be involved in prostaglandin F2 alpha (PGF2 alpha)-induced phosphoinositide metabolism in Chinese hamster ovary (CHO) cells transfected with PGF2 alpha receptor cDNA (CHO-PGF2 alpha R cells) (Ito, S. et al. Biochem. Biophys. Res. Commun. 200: 756,1994). In the present study, we investigated PGF2 alpha-induced PLD activation in CHO-PGF2 alpha R cells. PLD activation was examined by measuring the production of [3H]phosphatidylbutanol ([3H]PBut), a specific product of the PLD-catalyzed transphosphatidylation reaction. PGF2 alpha-induced [3H]PBut formation was concentration-dependent with the maximal level obtained at 1 microM PGF2 alpha. The maximal [3H]PBut formation was observed at 2 min after addition of PGF2 alpha. Depletion of extracellular Ca2+ with EGTA suppressed PGF2 alpha-induced PLD activation by 50%. PKC inhibitors Ro31-8425 and calphostin C inhibited PGF2 alpha-induced [3H]PBut formation by 50%. PTK inhibitors genistein and herbimycin A failed to inhibit PGF2 alpha-induced PLD activation. A combination of maximal effective concentrations of PGF2 alpha (1 microM) and PMA (100 nM) enhanced PLD activation in an additive manner. Pretreatment of the cells with PMA for 2 h down-regulated PKC alpha and decreased PGF2 alpha-induced PLD activation. These results suggest that PLD activation by PGF2 alpha is mediated by both PKC-dependent and -independent pathways and that PKC alpha is involved in the former pathway.
Liu
B
B
Department of Biochemistry, Gifu University School of Medicine, Japan.
Nakashima
S
S
Ito
S
S
Nozawa
Y
Y
D000818:Animals; D016466:CHO Cells / Q000166:cytology / Q000201:enzymology*; D002118:Calcium / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006224:Cricetinae; D015237:Dinoprost / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D010739:Phospholipase D / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011982:Receptors, Prostaglandin / Q000235:genetics* / Q000502:physiology; D014162:Transfection
D011982:Receptors, Prostaglandin; C034671:prostaglandin F2alpha receptor; D015237:Dinoprost; D011505:Protein-Tyrosine Kinases; D010739:Phospholipase D; D002118:Calcium