Received for publication May 19, 2004.
Revised August 6, 2004.
Accepted for publication September 1, 2004.

Protocols for regulation and study of diphosphoinositol polyphosphates

Abstract

The roles of diphosphoinositol polyphosphates (DIPs) in mammalian cell biology have been difficult to determine, due to the lack of tools known to regulate their levels. I have determined a series of protocols which 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-InsP₄), whilst slightly lowering levels of diphosphoinositol pentakisphosphate (PP-InsP₅) in DDT₁ MF-2 cells. These effects correlate with the ability of hyperosmotic stress to interfere with protein trafficking previously described and suggest that [PP]₂-InsP₄ specifically impedes protein trafficking. The effects on [PP]₂-InsP₄ 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-InsP₄, whereas a similar inhibitor, herbimycin, was without effect. Thapsigargin, a SERCA pump inhibitor previously shown to selectively lower PP-InsP₅ following acute treatment also selectively raises PP-InsP₅ following a longer-term treatment. The calmodulin inhibitors W-7 and chlorpromazine significantly lowered all higher inositol phosphates, as well as DIPs, whereas the calmodulin-dependent kinase inhibitors, K-252a and KN-93, were without effect. W-7 and chlorpromazine also lowered levels of phosphatidylinositol 4,5-bisphosphate and adenosine 5'-triphosphate 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 previously described, we can further understand the roles of the DIPs in cell biology.

Key words: IP3/DAG, Regulation - physiological