The Anti-inflammatory Drug Indomethacin Alters Nanoclustering in Synthetic and Cell Plasma Membranes

Yong Zhou,Sarah J Plowman,Lenard M Lichtenberger,John F Hancock

doi:10.1074/jbc.m110.141200

Abstract

The nonsteroidal anti-inflammatory drug indomethacin exhibits diverse biological effects, many of which have no clear molecular mechanism. Membrane-bound receptors and enzymes are sensitive to their phospholipid microenvironment. Amphipathic indomethacin could therefore potentially modulate cell signaling by changing membrane properties. Here we examined the effect of indomethacin on membrane lateral heterogeneity. Fluorescence lifetime imaging of cells expressing lipid-anchored probes revealed that treatment of BHK cells with therapeutic levels of indomethacin enhances cholesterol-dependent nanoclustering, but not cholesterol-independent nanoclustering. Immuno-electron microscopy and quantitative spatial mapping of intact plasma membrane sheets similarly showed a selective effect of indomethacin on promoting cholesterol-dependent, but not cholesterol-independent, nanoclustering. To further evaluate the biophysical effects of indomethacin, we measured fluorescence polarization of the phase-sensitive probe Laurdan and FRET between phase-partitioning probes in model bilayers. Therapeutic levels of indomethacin enhanced phase separation in DPPC/DOPC/Chol (1:1:1) and DPPC/Chol membranes in a temperature-dependent manner, but had minimal effect on the phase behavior of pure DOPC at any temperature. Taken together, the imaging results on intact epithelial cells and the biophysical assays of model membranes suggest that indomethacin can enhance phase separation and stabilize cholesterol-dependent nanoclusters in biological membranes. These effects on membrane lateral heterogeneity may have significant consequences for cell signaling cascades that are assembled on the plasma membrane.

Highlights

IntroductionNon-steroidal anti-inflammatory drugs (NSAIDs) perturb membrane integrity resulting in altered red blood cell morphology [8] and reduced mechanical stability of phosphatidylcholine (PC) membranes [5]
Indomethacin Alters the Phase Behavior of Model Membranes—To verify that the observed effect of indomethacin is caused by a direct effect on the phospholipid bilayer, we examined the ability of indomethacin to alter membrane phase behavior in a mixed synthetic model membrane of dioleoyl phosphatidylcholine (DOPC)/
To better understand the underlying mechanism contributing to indomethacin-induced phase separation in a mixed DOPC/ DPPC/Chol membrane, and to ascertain which component(s) in the mixed membrane indomethacin targets, we examined the ability of indomethacin to alter the phase behavior of DPPC/Chol and DOPC bilayers

Summary

Introduction

NSAIDs perturb membrane integrity resulting in altered red blood cell morphology [8] and reduced mechanical stability of phosphatidylcholine (PC) membranes [5] Other amphiphilic drugs, such as local and general anesthetics, influence membrane biophysical properties which contribute, in part, to their diverse biological activities [9]. Extrapolating from these studies we hypothesize that NSAIDs intercalate in cell membranes and by perturbing lipid-lipid interactions modulate diverse membrane-scaffolded biological processes. We examined whether a potent NSAID, indomethacin, alters nanoclustering in baby hamster kidney cell plasma membrane The outcome of this in vivo study was correlated to the effect of indomethacin on phase behavior in model liposomes. The studies we describe here provide strong preliminary evidence in favor of this proposal

Objectives

Results

Conclusion