Inhibition of phosphoinositide-specific phospholipase C by manoalide.
Manoalide is a novel sesterterpenoid which has previously been shown to be a potent inhibitor of venom phospholipases A2. To determine whether manoalide inhibited other phospholipases, the sensitivity of phosphoinsitide-specific phospholipase C (PI-PLC) to inactivation by manoalide was examined using crude cytosolic PI-PLC and a PI-PLC purified to homogeneity from guinea pig uterus cytosol (PI-PLC I). Manoalide inhibited both cytosolic and purified PI-PLC I in a concentration-dependent fashion, exhibiting an IC50 of 3-6 microM. Inactivation of PI-PLC I was calcium- and pH-dependent, with greater inactivation occurring at alkaline pH. Manoalide inhibited hydrolysis of all three phosphoinositides by purified PI-PLC I. The substrate kinetics of PI-PLC I suggest that manoalide does not inhibit purified PI-PLC I by simple competitive or noncompetitive inhibition. Enzyme activity was not recovered after dialysis of manoalide-treated PI-PLC I, indicating that inactivation of PI-PLC I was irreversible. To determine whether manoalide inhibited PI-PLC in cells, the effects of manoalide on norepinephrine (NE)-stimulated phosphoinositide hydrolysis and calcium mobilization were investigated in a smooth muscle-like cell line, DDT1MF-2. Manoalide inhibited NE-induced inositol 1,4,5-trisphosphate and inositol 1-phosphate formation in a concentration-dependent manner. The IC50 for inhibition of inositol 1-phosphate formation was 1.5 microM. Manoalide also inhibited NE-induced calcium transients in DDT1MF-2 cells, exhibiting an IC50 of 2 microM. These data suggest that inhibition of PI-PLC may account, in part, for the anti-inflammatory actions of manoalide.
Bennett
C F
CF
Department of Molecular Pharmacology, Smith Kline & French Laboratories, Philadelphia, Pennsylvania 19101.
Mong
S
S
Wu
H L
HL
Clark
M A
MA
Wheeler
L
L
Crooke
S T
ST
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D005260:Female; D006168:Guinea Pigs; D007700:Kinetics; D010741:Phospholipases A / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
D013729:Terpenes; C045673:manoalide; D010741:Phospholipases A; D010738:Type C Phospholipases; D002118:Calcium