Impairments in hepatocyte phosphoinositide metabolism in endotoxemia

Abstract

The status of phospholipid metabolism and inositol lipids-mediated transmembrane signaling in rat hepatocytes was analyzed during chronic, nonlethal endotoxemia. Rats were infused intravenously (IV) with Escherichia coli endotoxin (ET) via subcutaneously implanted osmotic pumps at a rate of 0.1 mg 100 g bw/day . The experiments were performed after 30 hours of ET or sterile saline (NaCl) infusion, in hepatocytes prelabelled “in vitro” with 32P (15 μCi/mL) and further stimulated with vasopressin (VP, 0.23 μmol/L). Similar experiments were done with food-restricted animals, whose food intake was matched with the voluntary intake of ET-infused rats. Uptake of 32P label into phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP 2) occurs rapidly in cells from pair-fed, saline and ET-infused animals, and reaches a plateau between 60 and 80 minutes of incubation. Labeling of phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) proceeds linearly after a ten-minute lag period for PI and 20 minutes for the two other lipids. The nutritional state greatly affects the distribution of 32P uptake into lipids, resulting in very low labeling of PA and PI and a high labeling of poly-PI as compared with control (taken from untreated rats) cells. In ET- v saline-infused rats, the labeling of PI and PE was depressed concomitantly with a proportional increase in the labeling of PIP and PC. The ability of VP to induce polyphosphoinositide (poly-PI) degradation in hepatocytes from saline-infused animals was similar to that observed in control cells. The rapid loss in PIP 2 and PIP labeling (50% and 35%, respectively, within 30 seconds) was paralleled by an increase in 32P uptake in PA and followed by a later increase in PI labeling (150% at five minutes), and a sustained labeling of poly-PI towards prestimulation levels. In ET-infused rats, the early poly-PI response to VP stimulation was inhibited by 40% with no subsequent recovery in the PIP 2 labeling up to the last sampling time (five minutes). Also, the stimulation of PI turnover was 50% lower than that observed in saline-infused animals. The well-known stimulating effect of VP on 32P uptake into PI and PA when present from the start of incubation of the cells with the precursor, was abolished in ET-infused rats, while the labeling of PIP 2 was inhibited by 50%. In cells of saline-infused animals, 32P uptake into PA and PI was greatly increased, as in hepatocytes from control and pair-fed animals, with no effect at the level of poly-PI labeling. The results support the concept that ET infusion significantly perturbs changes in inositol lipid metabolism elicited by VP, affecting PIP 2 degradation and resynthesis at the plasma membrane level and PI turnover and/or synthesis in the endoplasmic reticulum.