Liposomal NAD⁺prevents diminished O₂consumption by immunostimulated Caco-2 cells

Abstract

Accumulating data support the view that sepsis is associated with an acquired intrinsic derangement in the ability of cells to consume O 2, a phenomenon that has been termed "cytopathic hypoxia." We sought to use an in vitro "reductionist" model system using cultured cells stimulated with proinflammatory cytokines to test the hypothesis that cytopathic hypoxia is mediated, at least in part, by depletion of intracellular levels of NAD ϩ / NADH secondary to activation of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP). We measured O2 consumption by Caco-2 enterocytes growing on microcarrier beads after cells were incubated for 24 h under control conditions or with cytomix, a mixture of tumor necrosis factor-␣, interleukin-1␤, and interferon-␥. Immunostimulated cells consumed O2 at about one-half the rate of control cells, but this effect was largely prevented if any one of the following pharmacological agents was present during the period of incubation with cytomix: 4,5-dihydroxy-1,3-benzene disulfonic acid, a superoxide radical anion scavenger; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger; 5,10,15,20-HCl, a chemically dissimilar and more potent PARP inhibitor. The decrease in O 2 uptake induced by cytomix was associated with decreased cellular levels of NAD ϩ /NADH. The decrease in cellular NAD ϩ /NADH content and the decrease in O2 uptake induced by cytomix were completely abrogated if liposome-encapsulated NAD ϩ was added to the cultures during immunostimulation. Empty liposomes also increased O2 uptake by immunostimulated Caco-2 cells, but much less effectively than liposomes containing NAD ϩ . These data are consistent with the view that enterocytes exposed to proinflammatory cytokines consume less O2 due to NAD ϩ /NADH depletion secondary to activation of PARP by ONOO Ϫ or other oxidants.poly(ADP-ribose) polymerase; nitric oxide; peroxynitrite; enterocyte; epithelium; intestinal ACCUMULATING DATA SUPPORT the view that sepsis is associated with an acquired intrinsic derangement in the ability of cells to consume O 2 . This phenomenon has been termed "cytopathic hypoxia" (15). Direct and indirect evidence exists for the development of cytopathic hypoxia in sepsis (or related inflammatory conditions, such as endotoxemia) in experimental animals. For example, tissue PO 2 has been shown to increase in rats (33) and pigs (42) infused with purified lipopolysaccharide (LPS) and also in human patients with septic shock (5). In an indirect way, these data suggest that the ability of cells to extract O 2 from arterial blood is impaired in these conditions, which are associated with systemic inflammation. More direct evidence comes from a study performed by Kantrow et al. (22), where it was shown that the rate of O 2 consumption by hepatocytes isolated from septic rats was less than that by liver cells from control rats. More recently, King et al. (25), working in our laboratory, showed that ex vivo O 2 consumption by ileal mucosa was decreased (relative to the norm...

Sepsis, mitochondrial failure and multiple organ dysfunction

“…An excessive stimulation of PARP-1 can lead to depletion of NAD + /NADH and can therefore interfere with the synthesis of ATP [76], leading to a state of cytopathic hypoxia and cell death. is has been studied in various cells, including macrophages and smooth muscle cells stimulated with oxidizing molecules [77], and enterocytes stimulated with proin ammatory cytokines and oxidizing molecules [78].…”

Identification of a Novel Pathway of Transforming Growth Factor-β1 Regulation by Extracellular NAD+ in Mouse Macrophages

“…There is also extensive evidence that T-cell signaling is affected by NAD ϩ , with ultimate effects on proliferation and apoptosis (22)(23)(24)(25)(26)(27)(28). Furthermore, several studies have shown that NAD ϩ can protect against inflammatory stress both in vitro and in vivo (29,30), in part via reduced activity of the central inflammatory transcription factor NF-B. 5 We have also found that treatment of endotoxemic mice with NAD ϩ could protect from lethality in the case of mice injected with high dose LPSs and greatly reduced plasma TNF-␣ and NO b Ϫ / NO 3 Ϫ and elevated IL-10 in mice subjected to low-dose LPS.…”

NAD+Ameliorates Inflammation-Induced Epithelial Barrier Dysfunction in Cultured Enterocytes and Mouse Ileal Mucosa

“…Depletion of NAD ϩ , the precursor of cADPR, inhibits signaling via this pathway. Interestingly, we have reported that cytomix induces intracellular NAD ϩ depletion through an NO⅐-dependent process in Caco-2 cells (Khan et al, 2002). Whether cytomix-induced intracellular NAD ϩ depletion affects intracellular cADPR levels and [Ca 2ϩ ] i in Caco-2 cells remains to be investigated.…”

“…During experiments designed to explore the biological effects of decreased intracellular NAD ϩ content in immunostimulated human Caco-2 enterocyte-like cells (Khan et al, 2002), we serendipitously observed that adding NAD ϩ to the culture medium ameliorated derangements in epithelial barrier function caused by exposing these cells to a mixture of proinflammatory cytokines, called "cytomix". This observation prompted us to review a growing literature regarding the role of NAD ϩ and closely related compounds as signaling molecules.…”