title: Mobilization of diacylglycerol in intact HeLa cells by exogenous phospholipase C from Cl. perfringens is accompanied by release of fatty acids including arachidonic acid.
abstract: 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)
authors.affiliation: Department of Pathochemistry, German Cancer Research Center, Heidelberg.
authors.forename: K
authors.initials: K
authors.lastname: Schiess
authors.forename: M
authors.initials: M
authors.lastname: Kaszkin
authors.forename: P
authors.initials: P
authors.lastname: Jordan
authors.forename: L
authors.initials: L
authors.lastname: Seidler
authors.forename: V
authors.initials: V
authors.lastname: Kinzel
mesh_terms: 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*
chemical_list: 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