The working hypothesis of this proposal is that the quantity of superoxide produced by activated human neutrophils is dictated in part by the availability of NADPH to NADPH oxidase. In resting neutrophils the concentration of NADPH is insufficient to fuel optimal activity of the oxidase. Furthermore, the total concentration of NADP + NADPH in resting cells is such that even if it were all converted to the reduced form (NADPH) it would still be insufficient to allow appropriate expression of the oxidase. Therefore, in order to assure proper substrate availability for the oxidase, the total pool of NADP + NADPH is increased upon neutrophil activation. This increase in pool size is accomplished through the stimulation of NAD kinase which converts cellular NAD to NADP. Activation of the kinase occurs in response to a stimulus-induced increase in cytosolic calcium. The effect of these elevations in cytosolic calcium concentrations upon NAD kinase is mediated through the action of calmodulin, a calcium-dependent regulatory protein, or through the activation of the calcium-dependent neutral protease of neutrophils. To test this hypothesis the correlation between NADPH concentration and superoxide production will be examined in activated neutrophils whose intracellular NADPH concentration had been metabolically altered. Changes in nucleotide levels accompanying activation will be correlated to superoxide production, and the ability of calcium and calmodulin antagonists and calcium ionophores to affect these two parameters explored. Neutrophil NAD kinase will be purified, characterized, and its activation by calmodulin thoroughly investigated. And finally, the sensitivity of NAD kinase to irreversible activation by neutrophil calcium-dependent protease will be examined. The purpose then of this proposal is to explore the role of NAD kinase in the regulation of stimulus-associated metabolic changes in the human neutrophil. To that end we hope to show the importance of this enzyme in regulating superoxide production and how the activity of this enzyme is controlled in response to bacterial challenge. From a broader standpoint, we hope that these studies will further develop understanding of how calcium and calcium-regulated proteins serve to integrate various metabolic pathways in the bactericidal process. And finally, these findings may explain the metabolic defect underlying the compromised bactericidal action of neutrophils in some patients and enhanced superoxide-production in patients with autoimmune diseases such as arthritis.
| Legendre, J L; Jones, H P (1988) Purification and characterization of calpain from human polymorphonuclear leukocytes. Inflammation 12:51-65 |
