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).
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