Plasma membrane cholesterol content affects nitric oxide diffusion dynamics and signaling

Department of Chemistry and Biochemistry University of Windsor, Windsor Ontario N9B 3P4, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2008; 283(27):18513-21. DOI: 10.1074/jbc.M800440200
Source: PubMed

ABSTRACT Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO approximately 20-40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser(239) induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.

Download full-text


Available from: Shane Miersch, Sep 10, 2014
  • Source
    • "H 2 O 2 has a half-life of 1 ms, which allows it to react with several molecules or metals to produce the hydroxyl radicals (OH@BULLET) by Fenton-type reactions (Christine, 1995; Nappi & Vass, 1998; Reth, 2002). Nitric oxide (NO@BULLET), formed from L-arginine by the enzyme nitric oxide synthase (NOS), is a small hydrophobic molecule that freely diffuses across membranes (Miersch et al., 2008). NO@BULLET has been recognized to act as a paracrine signaling molecule playing an important role as second messenger in processes as diverse as cell survival (Patel et al., 2010), proliferation (Magalhães et al., 2006), apoptosis (Wei et al., 2000) and neuronal differentiation (Ciani et al., 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Oxidative stress is a common hallmark of neuronal cell death associated with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, as well as brain stroke/ischemia and traumatic brain injury. Increased accumulation of reactive species of both oxygen (ROS) and nitrogen (RNS) has been implicated in mitochondrial dysfunction, energy impairment, alterations in metal homeostasis and accumulation of aggregated proteins observed in neurodegenerative disorders, which lead to the activation/modulation of cell death mechanisms that include apoptotic, necrotic and autophagic pathways. Thus, the design of novel antioxidant strategies to selectively target oxidative stress and redox imbalance might represent important therapeutic approaches against neurological disorders. This work reviews the evidence demonstrating the ability of genetically encoded antioxidant systems to selectively counteract neuronal cell loss in neurodegenerative diseases and ischemic brain damage. Because gene therapy approaches to treat inherited and acquired disorders offer many unique advantages over conventional therapeutic approaches, we discussed basic research/clinical evidence and the potential of virus-mediated gene delivery techniques for antioxidant gene therapy.
    Pharmacology [?] Therapeutics 12/2013; 142. DOI:10.1016/j.pharmthera.2013.12.007 · 7.75 Impact Factor
  • Source
    • "The organic solubility of VP-16 may likewise localize and enhance interactions with reactive nitrogen oxides. The occurrence of both augmented iNOS catalysis (Ambs and Glynn, 2011) and alterations in cancer cell plasma membrane architecture (Zhuang et al., 2002; Moller et al., 2007; Miersch et al., 2008) will significantly influence iNOS-dependent detoxification of VP-16 and resistance toward cancer cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nitric oxide (NO) was originally identified as an innate cytotoxin. However, in tumors it can enhance resistance to chemotherapy and exacerbate cancer progression. Our previous studies have indicated that NO/NO-derived species react with etoposide (VP-16) in vitro and form products that show significantly reduced activity towards HL60 cells and lipopolysaccharide (LPS)-induced macrophages. Here, we have examined the interactions of NO with VP-16 in inducible nitric oxide synthase (iNOS)-expressing human melanoma A375 cells to further confirm the hypothesis that NO generation contributes to VP-16 resistance in cancer cells. We also examined the interactions of NO with another topoisomerase active drug, adriamycin. Inhibition of iNOS catalysis by N(6)-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) in human melanoma A375 cells reversed VP-16 resistance, leading to increased DNA damage and apoptosis. Furthermore, we found that co-culturing A375 melanoma cells with LPS-induced macrophage RAW cells also significantly reduced VP-16 cytotoxicity and DNA damage in A375 cells. In contrast, NO caused no significant modulation of cytotoxicity or adriamycin-dependent apoptosis, suggesting that NO did not interact with adriamycin. Our studies support the hypothesis that NO oxidative chemistry can detoxify VP-16 through direct nitrogen oxide radical attack and provide insights into the pharmacology and anticancer activities of VP-16 that may ultimately contribute to increased resistance, treatment failure, and induction of secondary leukemia in VP-16-treated patients.
    Journal of Pharmacology and Experimental Therapeutics 09/2013; 347(3). DOI:10.1124/jpet.113.207928 · 3.86 Impact Factor
  • Source
    • "PM cholesterol isolation was performed essentially as described in [33]. Briefly, 5 × 10 5 cells/10 cm plates were trypsinized and washed once with 100 mM Tris buffer, pH 7.4 and then with hypotonic 10 mM Tris buffer pH 7.4. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.
    Biochimica et Biophysica Acta 10/2011; 1821(2):313-23. DOI:10.1016/j.bbalip.2011.10.015 · 4.66 Impact Factor
Show more