Protocols for Regulation and Study of Diphosphoinositol Polyphosphates

The roles of diphosphoinositol polyphosphates (DIPs) in mammalian cell biology have been difficult to determine because of the lack of tools known to regulate their levels. I have determined a series of protocols that regulate these DIPs, and these can be used to further our understanding of these molecules. Sorbitol and sucrose significantly raised levels of bis-diphosphoinositol tetrakisphosphate ([PP] 2 -InsP 4 ) but slightly lowered levels of diphosphoinositol pentakisphosphate (PP-InsP 5 ) in DDT 1 MF-2 cells. These effects correlate with the ability of hyperosmotic stress to interfere with protein trafficking described previously and suggest that [PP] 2 -InsP 4 specifically impedes protein trafficking. The effects on [PP] 2 -InsP 4 were not regulated by extracellular signal-regulated kinase or phospholipase D, as exemplified by the lack of effect of U0126 and butan-1-ol. I have also found that genistein potently and rapidly lowers levels of [PP] 2 -InsP 4 , whereas a similar inhibitor, herbimycin, was without effect. Thapsigargin, a sarcoplasmic-endoplasmic reticulum Ca 2+ -ATPase pump inhibitor previously shown to selectively lower PP-InsP 5 after short-term treatment, also selectively raises PP-InsP 5 after a longer treatment. The calmodulin inhibitors N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and chlorpromazine significantly lowered all higher inositol phosphates, as well as DIPs, whereas the calmodulin-dependent kinase inhibitors methyl 9-( S )-12-( R )-epoxy-1H-diindolo[1,2,3- fg :3′,2′,1′- kl ]pyrrolo[3,4- i ][1,6]benzodiazocine-2,3,9,10,11,12-hexahydro-10-( R )hydroxy-9-methyl-1-oxo-10-carboxylate (K-252a) and 2-[ N -(2-hydroxyethyl)- N -(4-methoxybenzenesulfonyl)]amino- N -(4-chlorocinnamyl)- N -methylbenzylamine (KN-93) were without effect. W-7 and chlorpromazine also lowered levels of phosphatidylinositol 4,5-bisphosphate and ATP but greatly increased levels of phosphatidylinositol 4-phosphate. Trypan blue exclusion deemed that these doses were not cytotoxic. These results identify an increasing number of reagents that regulate DIP levels. Using these tools, and those described previously, we can further understand the roles of the DIPs in cell biology.