Mobilization of diacylglycerol in intact HeLa cells by exogenous phospholipase C from Cl. perfringens is accompanied by release of fatty acids including arachidonic acid.
The second messenger diacylglycerol (DAG), chiefly derived from phosphatidylcholine (PC) or from phosphatidylinositol (PI), through the activation of specific phospholipases C (PLC), plays a key role in cellular stimulation. The activation of a particular PLC was simulated in intact HeLa cells by treatment with exogenous PC-PLC (Cl. perfringens) or with PI-PLC (B. cereus). Both enzymes rapidly mobilized DAG. However, only PC-PLC led, in Hela cells, to morphological changes (which were reversible on enzyme removal within the time frame of the experiments) and to an increase of intracellular calcium concentration with a lag of > 10 min. In cells prelabeled with [1-14C]arachidonic acid only PC-PLC but not PI-PLC induced the release of labeled fatty acid with a lag of > 10 min. Upon prelabeling of cells with [1-14C]oleic acid, PC-PLC led to a release of radioactive oleic acid. The release of arachidonic acid (AA) required a threshold dose of PC-PLC and a minimum time of treatment beyond which the AA release continued for a certain period, even in the absence of the exogenous enzyme. Under the conditions used, neither PLA2 nor DAG lipase activity were detectable in the PC-PLC preparation. Therefore, AA release was due to activation of a cellular enzyme, probably cellular PLA2 activity. The PC-PLC-induced AA release could be inhibited to a certain extent by EGTA and by quinacrine but not by the glucocorticoid fluocinolone acetonide. Only PC-PLC (but not PI-PLC) caused, in addition, an increase of the level of monoglycerol, which paralleled the appearance of AA. An increase of labeled monoglycerol was detectable in HeLa cells prelabeled with radioactive oleic acid or with 1-[1-14C]palmitoyl-lyso-PC but not in cells prelabeled with radioactive AA, thus indicating that the fatty acid originated from sn-2 position of the glycerol moiety. The 1-monoacylglycerol was probably generated from lysophospholipids by the bacterial PC-PLC. This enzyme preparation has been shown to catalyze such breakdown of lysophosphatidylcholine in vitro. PC-PLC-induced AA release occurred also after down-regulation of protein kinase C by an overnight pretreatment with phorbol ester TPA (TPA-pretreated cells, but not control cells, on treatment with PC-PLC, metabolized AA to prostaglandins).(ABSTRACT TRUNCATED AT 400 WORDS)
Schiess
K
K
Department of Pathochemistry, German Cancer Research Center, Heidelberg.
Kaszkin
M
M
Jordan
P
P
Seidler
L
L
Kinzel
V
V
D016718:Arachidonic Acid / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003016:Clostridium perfringens / Q000201:enzymology*; D004075:Diglycerides / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005227:Fatty Acids / Q000378:metabolism*; D005446:Fluocinolone Acetonide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011796:Quinacrine / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
D004075:Diglycerides; D005227:Fatty Acids; D005446:Fluocinolone Acetonide; D016718:Arachidonic Acid; D004533:Egtazic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D011796:Quinacrine