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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
1R01ES003572-01A1
Application #
3250974
Study Section
Toxicology Study Section (TOX)
Project Start
1985-08-26
Project End
1988-07-31
Budget Start
1985-08-26
Budget End
1986-07-31
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Type
Overall Medical
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Kern, M; Audesirk, G (2000) Stimulatory and inhibitory effects of inorganic lead on calcineurin. Toxicology 150:171-8
Kern, M; Wisniewski, M; Cabell, L et al. (2000) Inorganic lead and calcium interact positively in activation of calmodulin. Neurotoxicology 21:353-63
Ferguson, C; Kern, M; Audesirk, G (2000) Nanomolar concentrations of inorganic lead increase Ca2+ efflux and decrease intracellular free Ca2+ ion concentrations in cultured rat hippocampal neurons by a calmodulin-dependent mechanism. Neurotoxicology 21:365-78
Shakarjian, M P; Carchman, R A (1990) Alteration of human granulocyte functional responses by menadione. Arch Biochem Biophys 283:1-11
Coles, R B; Ranney, R R; Freer, R J et al. (1989) Thermal regulation of FMLP receptors on human neutrophils. J Leukoc Biol 45:529-37
Strnad, C F; Lin, W Q; Carchman, R A (1989) Interactive effects of pertussis toxin and the phorbol ester tumour promotor, phorbol dibutyrate, on T-lymphocyte mitogenesis and the expression of phenotypic determinants. Immunology 66:539-45
Kessler, F K; Fisher, B J; Bechard, D E et al. (1988) Responsiveness of guinea pig alveolar cells. Lung 166:33-46
McCrady, C W; Ely, C M; Westin, E et al. (1988) Coordination and reversibility of signals for proliferative activation and interleukin-2 mRNA production in resting human T lymphocytes by phorbol ester and calcium ionophore. J Biol Chem 263:18537-44
Strnad, C F; Carchman, R A (1987) Human T lymphocyte mitogenesis in response to the B oligomer of pertussis toxin is associated with an early elevation in cytosolic calcium concentrations. FEBS Lett 225:16-20
Gaut, J R; Carchman, R A (1987) A correlation between phorbol diester-induced protein phosphorylation and superoxide anion generation in HL-60 cells during granulocytic maturation. J Biol Chem 262:826-34

Showing the most recent 10 out of 12 publications