Plasma Membrane Cholesterol Content Affects Nitric Oxide Diffusion Dynamics and Signaling

Michael Graham Espey,Bulent Mutus,Rodney Tweten,Ruchi Chaube,Sirinart Ananvoranich,Shane Miersch,Arzu Akarca

doi:10.1074/jbc.m800440200

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.

Highlights

As a small sized gaseous free radical, nitric oxide (NO)2 presents unique challenges toward understanding the nature of its Cholesterol, a major lipid component of the plasma membrane in eukaryotic cells, plays an essential role in maintaining membrane fluidity and architecture (9 –11)
These data show that plasma membrane cholesterol content modulates the level and type of NO signaling; pathological alterations in levels and distribution of dimyristoylphosphatidyl choline; DPTA, diethylenetriaminepentaacetic acid; bilayer membrane (BLM), bilayer lipid membrane; M␤CD, methyl-␤-cyclodextrin; Mn(III)TMPyP, Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride; NPC1, Niemann-Pick type C1; NHF, normal human fibroblast(s); PBS, phosphatebuffered saline; perfringolysin domain 4 (PFO-D4), perfringolysin-O domain 4; vasodilator-stimulated phosphoprotein (VASP), vasodilatorstimulated phosphoprotein; phospho-VASP, serine 239-phosphorylated VASP; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; SV, Stern-Volmer
Preparations of pyrene fluorochrome encapsulated with either dipalmitoylphosphatidyl choline (DPPC) and dimyristoylphosphatidyl choline (DMPC) liposomes were exposed to NO gas while sealed in cuvettes under anaerobic conditions

Summary

EXPERIMENTAL PROCEDURES

EDTA, phenylmethylsulfonyl fluoride, NGmonomethyl-L-arginine, diaminofluorescein diacetate, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, cholesterol, Dulbecco’s modified Eagle’s medium, diethylaminetriamine pentaacetic acid (DTPA), hemoglobin, sodium dithionite, malachite green, ammonium molybdate, and performic acid were obtained from Sigma. Suspensions of 106 trypsinized cells in PBS containing 500 ␮M DTPA were incubated with 100 ␮M adduct (determined on the basis of cholesterol by enzymatic assay) for 15 min with gentle nutation. 106 cells in suspension were incubated with either 5 mM M␤CD for 1 h or 2 units/ml cholesterol oxidase for 15 min at 37 °C with gentle rotation. Human red blood cells and platelets were isolated from whole blood by differential centrifugation and suspended in PBS containing 500 ␮M DTPA. Cyclodextrin, or enzyme was removed, and cells were suspended in a 10 ␮M DAF solution in PBS containing 0.5 mM DTPA and treated with the 250 nM DEA/NO for monitoring of evolved fluorescence over time in the wells of a 96-well microplate with shaking at 37 °C using an excitation/emission couple of 485/515 nm, unless otherwise indicated

RESULTS

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DISCUSSION