The long term objective of this study is to determine if phosphatidylinositol bisphosphate (PIP2) hydrolysis leads to Cs influx in stimulated neutrophils. Preliminary studies demonstrated that 20.0 MuM sodium fluoride caused, after a 1 1/2 -2 minute lag period, a slow but sustained increase in intracellular free Cs levels which eventually resulted in functional activation as measured by superoxide release. This increase was predominantly due to influx of Ca++ across the plasma membrane. Prior to the initiation of intracellular Ca++ increases, 20 mM NaF effected rapid disappearance of cellular PIP2. NaF also effected hydrolysis of PIP2 in isolated neutrophil plasma membranes. In the initial studies proposed in this application, the pathways by which F- effects PIP2 hydrolysis in intact cells and isolated plasma membranes will be defined and compared. Experiments will be undertaken with the goal of determining whether or not hydrolysis was catalyzed enzymatically. By carefully analyzing both the lipid and water-soluble products of hydrolysis, we will determine whether or not F- possibly exerts its effects by activating a cellular PIP2 specific phospholipase C. By separating inositol phosphates using anion-exchange high performance liquid chromatography and by quantitating diacylglycerol mass levels, we will attempt to determine if F- exerts its effect on PIP2 in isolated plasma membranes and in intact cells by the same mechanism. Experiments are also designed to investigate the potential role of a F- activated guanine nucleotide regulatory protein in hydrolysis of PIP2. Subsequent experiments will be undertaken in an attempt to determine if PIP2 hydrolysis is linked to Ca++ influx. Experiments will be undertaken to confirm that PIP2 hydrolysis is not a result of Ca++ influx promoted by F-. Finally, we will attempt to define how PIP2 hydrolysis leads to Ca++ influx by investigating possible pathways linking these events. It is hoped that these studies will clarify the mechanism and consequences of PIP2 hydrolysis in stimulated neutrophils. They may also provide important information relating to the mechanisms of Ca++ influx in other systems and provide new methods which will facilitate investigations of neutrophil Ca++ influx effected by physiologically relevant metabolic agonists.
| Dominguez, J H; Garcia, J G; Rothrock, J K et al. (1991) Fluoride mobilizes intracellular calcium and promotes Ca2+ influx in rat proximal tubules. Am J Physiol 261:F318-27 |
| Mock, B H; English, D (1990) Granulocyte-macrophage colony-stimulating factor enhances exudation of neutrophils to sites of inflammatory challenge in vivo. J Lipid Mediat 2 Suppl:S137-41 |
| Mock, B H; Schauwecker, D S; English, D et al. (1988) In vivo kinetics of canine leukocytes labeled with technetium-99m HM-PAO and indium-111 tropolonate. J Nucl Med 29:1246-51 |
