Nmr Studies Of The Mechanisms Of Cell Injury

Environmental Health Sciences

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Abstract

The goals of this project are to investigate the of altered ion homeostasis and ion signaling in cell injury and protective adaptation. Cells have an inherent ability to protect themselves from injury caused by environmental stress and other types of injury. Brief intermittent periods of ischemia and reflow, termed ischemic preconditioning (PC), have been shown to protect the myocardium against injury produced by a subsequent sustained period of ischemia. PC has been shown to significantly reduce infarct size, arrhythmias, and postischemic contractile dysfunction. Current studies to elucidate the signaling pathways that are responsible for PC have shown the following. 1. Protein kinase C (PKC) is involved in PC. Activators of PKC mimic preconditioning and inhibitors of PKC block PC. Furthermore, hearts from mice that overexpress PKC-epsilon exhibit less injury, as if they were constitively preconditioned. 2. Phosphoinositide-3-kinase (PI3-kinase) is upstream of PKC. PC causes phosphorylation of PKB, the kinase directly downstream of PI3-kinase and PC induced phosphorylation of PKB is blocked by wortmannin, an inhibitor of PI3-kinase. Wortmannin also blocks the protective effects of preconditioning, but does not block the protection induced by DOG, a direct activator of PKC. DOG does not cause phosphorylation of PKB. Furthermore, wortmannin blocks the PC induced translocation of PKC-epsilon. 3. PC via PI3-kinase activation leads to activation of nitric oxide synthase. PC leads to an increase in nitric oxide production, as measured by an increase in total nitrate + nitrite, and this increase is blocked by wortmannin Immunohistochemistry shows that preconditioning leads to an increase in phospho-eNOS in the myocytes and this increase is blocked by wortmannin. One of our goal was to determine downstream targets of PI3-kinase. PI3-kinase has been reported to enhance cell survival via phosphorylation of GSK-3-beta (GSK-3). We therefore examined the role of GSK-3 in PC. Our results suggest that phosphorylation and subsequent inactivation of GSK-3b play an important role in the protection afforded by PC in the heart.. In perfused rat hearts, PC (4 cycles of 5 minutes ischemia and 5 minutes reflow) increased phosphorylation of GSK-3, a downstream target of PI3-kinase and protein kinase B (PKB), an effect which was blocked by wortmannin. Since phosphorylation inactivates GSK-3?, we examined whether PC-induced phosphorylation and inhibition of GSK-3b is important in PC by employing two inhibitors of GSK-3?, lithium and SB216763. Pretreatment of perfused rat hearts with lithium or SB216763, prior to ischemia, mimicked the protective effects of PC; hearts treated with either lithium or SB216763 had improved postischemic function and reduced infarct size. These findings indicate that inhibition of GSK-3b is protective and that this PI3-kinase dependent signaling pathway may play an important role in ischemic preconditioning. We also examined the effects of COX deficiency on baseline functional characteristics, and on recovery of left ventricular pressure (LVDP) after 20 minutes of global ischemia and 40 minutes of reperfusion in untreated and preconditioned hearts. Compared to hearts from WT and COX-2-/- mice, baseline cardiac PGE2 and 6-keto PGF1-alpha levels were significantly decreased in hearts from COX-1-/- mice. Following ischemia, cardiac PGE2 levels increased in WT, COX-1-/-, and COX-2-/- mice (p<0.05). Recovery of function (LVDP) after global ischemia in hearts from COX-1-/- and COX-2-/- mice was significantly less than WT hearts. Pretreatment of WT mice with indomethacin for 2 days prior to ischemia significantly decreased LVDP recovery; however, perfusion of WT hearts with indomethacin for 40 minutes prior to ischemia did not significantly alter LVDP recovery. Postischemic recovery of LVDP in COX-1-/- and COX-2-/- was unchanged by perfusion with 5?M PGE2, PGD2, PGF2a or carbaprostacyclin. Hearts from COX-2-/- mice showed an increase in ischemic contracture compared to hearts from WT and COX-1-/- mice; however hearts did not differ in intracellular pH, ATP or inorganic phosphate during ischemia. Ischemic preconditioning significantly improved postischemic LVDP recovery in COX-1-/-, COX-2-/-, and WT mice. Thus, genetic disruption or 2-day chemical inhibition of COX-1 and COX-2 decreases recovery of LVDP after ischemia; however acute perfusion with indomethacin is not detrimental. These data are consistent with protection due to the altered expression of some protein that is modulated by COX or its metabolites. In an attempt to study the relative roles of the environmental and genetic components of heart disease we have undertaken a collaborative effort with the NIEHS microarray center to study diseased heart tissue that has been removed from patients who are receiving new heart transplants. Preliminary data suggested altered expression of genes involved in the GSK-3? pathway, cell redox regulation (genes regulated by the antioxidant response element), and genes involved in fatty acid metabolism (genes regulated by PPAR).We also have initiated a projected to examine the effects of the bio-available constituents of oil combustion particles on perfused rat heart; preliminary data show dose dependent alterations in contractility and a decrease in high energy phosphates.