This proposal aims a.) to study receptor-linked signal transduction mechanisms in various Ca2+-regulated cell types in sustained endotoxicosis and sepsis b.) to correlate the state of phosphorylation of cytoplasmic proteins associated with vasopressin (VP) stimulation, with the prevailing impairment in functions regulated by this hormone, c.) to define the link between altered immune competence and inositol lipid metabolism and free cytosolic [Ca2+] in spleen lymphocytes, and d.) to characterize some initial biochemical events induced by direct interaction between the hepatocyte cell membrane and endotoxin and lipid X and their relationship to Ca2+-linked hormonal responses. Two experimental models will be used: 1. endotoxicosis produced by long-term (5-7 days) i.v. infusion of endotoxin via an implanted osmotic pump, and 2. intra-abdominal sepsis induced by cecal ligation and puncture. Experiments are designed to test the hypothesis: that the protein kinase C system contributes significantly to the mediation of the deleterious effects of sepsis. Protein kinase C seems to play a role in signal transduction for protein phosphorylation and may modulate the responsiveness of hepatocytes and adipocytes to appropriate calcium-linked hormonal stimulation. Likewise, the altered immune responses of lymphocytes may be accompanied by perturbations in membrane inositol lipid turnover and protein kinase C activation. Protein kinase C activity will be measured in intact hepatocytes and adipocytes stimulated by vasopressin, phenylephrine and insulin, and in lymphocytes stimulated PHA, Con A and E. coli LPS. Phorbol esters will be used as a tool to study sepsis-induced modifications in the transduction mechanism of receptors linked to inositol lipid turnover. The role of guanine nucleotides in Ca2+ linked hormonal activation of hepatocytes and mitogenic activation of lymphocytes will be explored. Inositol lipid breakdown, PI turnover and free cytosolic [Ca2+]c will be studied in T and B lymphocytes in the course of continuous ET infusion and correlated with their blastogenic responsiveness and ability to produce immune mediators. These studies will contribute to our understanding of the nature of altered transmembrane control of cellular function in endotoxicosis and sepsis. Implicating such mechanisms in some functional impairments, we may provide a rational basis for novel therapeutic modalities using agents inhibiting protein kinase C (e.g. chlorpromazine, tetracaine and polyamines).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030312-07
Application #
3277979
Study Section
Surgery and Bioengineering Study Section (SB)
Project Start
1982-01-01
Project End
1991-12-31
Budget Start
1988-01-01
Budget End
1988-12-31
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Louisiana State University Hsc New Orleans
Department
Type
School of Medicine & Dentistry
DUNS #
782627814
City
New Orleans
State
LA
Country
United States
Zip Code
70112
Spitzer, J A (1994) Cytokine stimulation of nitric oxide formation and differential regulation in hepatocytes and nonparenchymal cells of endotoxemic rats. Hepatology 19:217-28
Etheredge, E E; Spitzer, J A (1993) Chronic endotoxemia reversibly alters respiratory burst activity of circulating neutrophils. J Surg Res 55:261-8
Hagar, A F; duSapin, K; Spitzer, J A (1993) Endotoxin infusion primes elicited neutrophils and Kupffer cells for platelet-activating factor-induced and tripeptide formyl-methionine-leucine-phenylalanine-induced basal free intracellular calcium concentration responses. Crit Care Med 21:1750-7
Hagar, A F; Spitzer, J A (1992) The effect of endotoxemia on concanavalin A induced alterations in cytoplasmic free calcium in rat spleen cells as determined with Fluo-3. Cell Calcium 13:123-30
Friedman, H; Newton, C; Pross, S et al. (1992) Continuous infusion of endotoxin depresses splenic blastogenesis. Immunopharmacol Immunotoxicol 14:689-706
Spitzer, J A; Deaciuc, I V (1992) Protein kinase C activity and lipogenesis from glucose in isolated adipocytes of endotoxemic rats. Circ Shock 37:111-6
Pross, S; Newton, C; Widen, R et al. (1992) Splenocyte blastogenesis suppressed in rats implanted with an osmotic pump. Life Sci 50:99-108
Rodriguez de Turco, E B; Spitzer, J A (1991) Hepatic phosphatidylinositol kinase activity in continuously endotoxemic rats. Biochim Biophys Acta 1093:216-22
Deaciuc, I V; Spitzer, J A (1991) Down-regulation of prostaglandin F2 alpha receptors in rat liver during chronic endotoxemia. Prostaglandins Leukot Essent Fatty Acids 42:191-5
Deaciuc, I V; Spitzer, J A (1991) Insulin-like, antilipolytic effect of 12-O-tetradecanoyl phorbol 13-acetate in rat white adipocytes. Proc Soc Exp Biol Med 197:193-6

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