The zinc chelator 1,10-phenanthroline enhances the stimulatory effects of protein kinase C activators and staurosporine, but not sphingosine and H2O2, on phospholipase D activity in NIH 3T3 fibroblasts.
Protein kinase C (PKC), an enzyme which is believed to mediate the stimulatory effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on phospholipase D (PLD) activity, has a zinc-dependent structure required for phorbol ester binding. Accordingly, zinc or zinc chelators would be expected to promote or inhibit, respectively, the stimulatory effects of PMA on PLD-mediated phospholipid hydrolysis. Instead, treatment of [14C]choline- and [14C]ethanolamine-labelled NIH 3T3 fibroblasts with the high-affinity zinc chelator 1,10-phenanthroline (0.2-1 mM) for 20-30 min was found to enhance the stimulatory effects of PMA on PLD-mediated hydrolysis of phosphatidylcholine and phosphatidylethanolamine. In [14C]palmitic acid-labelled fibroblasts, in the presence of ethanol, phenanthroline also enhanced the stimulatory effect of PMA on the synthesis of phosphatidylethanol, a marker of PLD activity. Addition of zinc (250 microM) to phenanthroline-treated fibroblasts reversed the stimulatory effects of the chelator. The potentiating effects of phenanthroline were also partially reversed by cadmium, whereas iron, lead, copper, magnesium and calcium were without effects. Of the other activators of PLD tested, phenanthroline also enhanced the stimulatory effects of platelet-derived growth factor and staurosporine, but not that of sphingosine and H2O2, on the hydrolysis of both phospholipids. These results suggest that regulation of PLD by PKC activators and staurosporine involves a common intermediate step, which is inhibited by a chelatable cellular pool of zinc.
Kiss
Z
Z
Hormel Institute, University of Minnesota, Austin 55912.
D016475:3T3 Cells; D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D004789:Enzyme Activation; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006868:Hydrolysis; D051379:Mice; D010618:Phenanthrolines / Q000494:pharmacology*; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013110:Sphingosine / Q000494:pharmacology*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism*
D000470:Alkaloids; D002614:Chelating Agents; D010618:Phenanthrolines; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine; D015032:Zinc; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; C025205:1,10-phenanthroline