The monocyte is a blood borne component which plays an important host defensive role as well as mediating certain histiolytic and cell proliferative reactions. Phorbol diesters (PDE) exert highly potent and specific effects on the activity of these cells (e.g. chemotaxis, histiolytic enzyme secretion, generation of reactive oxygen species - ROS). These PDE responses are receptor mediated and intimately associated with a calcium dependent phospholipid requiring protein kinase (PKC). Recent reports support the idea that PDEs are mimicking diacylglycerols (DAG), which are the endogenous activators of this enzyme.
The specific aims are to study the PDE-receptor interactions and their role in modulating functional activities. The experimental approach will be to use purified monocytes (e.g. counterflow centrifugation) as either intact or broken cell preparations. Monoclonal antibodies directed against PKC will be used to study the PDE receptor and PKC activities. Receptor activity (e.g. Kd, density) and cellular distribution will be studied and correlated with PKC activity. Structure activity relationships for PDE's with respect to receptor and PKC activities will be compared with functional responses (i.e. chemotaxis, generation of ROS, lysosomal enzyme secretion, and production of IL-1). The result of the PKC enhancement is the phosphorylation of protein species which may be of relevance for the functional responses. Endogenous protein phosphorylation mediated by PDE's and DAG (e.g. OAG) will be monitored and contrasted (i.e. quantitatively and temporally) to their behavior with respect to receptor, calcium, PKC, and functional activities. Because many of these responses and activiies have a calcium dependency, the metabolism of this ion will be studied under similar experimental conditions in order to more clearly define its role in this system. The overall methods of approach for evaluating PDE's impact in human monocytes will include the following 1) 3H-PDBu as the radioligand in the receptor studies, 2) chemotaxis in blind well Boyden chambers, 3) IL-1 production as determined by bioassay in culture, 4) 32P-ATP, 32P and substrate phosphorylation in PKC studies in intact and broken cell preparations, 5) 45Ca2+ and quin-2 fluorescence and atomic absorption in the calcium metabolism studies, and spectrophotometric determinations for 6) super oxide anion, 7) lysosomal enzyme secretion, 8) lipid peroxidation, and 9) lactic dehydrogenase release.
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