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Free Cholesterol Alters Lipid Raft Structure and Function Regulating Neutrophil Ca²⁺ Entry and Respiratory Burst: Correlations with Calcium Channel Raft Trafficking¹

Kolenkode B. Kannan, Dimitrios Barlos and Carl J. Hauser^2,*,

^* Department of Surgery, Beth Israel-Deaconess Medical Center and Harvard Medical School, Boston, MA 02115; and Department of Surgery, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07101

Recent studies associate cholesterol excess and atherosclerosis with inflammation. The link between these processes is not understood, but cholesterol is an important component of lipid rafts. Rafts are thought to concentrate membrane signaling molecules and thus regulate cell signaling through G protein-coupled pathways. We used methyl -cyclodextrin to deplete cholesterol from polymorphonuclear neutrophil (PMN) rafts and thus study the effects of raft disruption on G protein-coupled Ca²⁺ mobilization. Methyl -cyclodextrin had no effect on Ca²⁺ store depletion by the G protein-coupled agonists platelet-activating factor or fMLP, but abolished agonist-stimulated Ca²⁺ entry. Free cholesterol at very low concentrations regulated Ca²⁺ entry into PMN via nonspecific Ca²⁺ channels in a biphasic fashion. The specificity of cholesterol regulation for Ca²⁺ entry was confirmed using thapsigargin studies. Responses to cholesterol appear physiologic because they regulate respiratory burst in a proportional biphasic fashion. Investigating further, we found that free cholesterol accumulated in PMN lipid raft fractions, promoting formation and polarization of membrane rafts. Finally, the transient receptor potential calcium channel protein TRPC1 redistributed to raft fractions in response to cholesterol. The uniformly biphasic relationships between cholesterol availability, Ca²⁺ signaling and respiratory burst suggest that Ca²⁺ influx and PMN activation are regulated by the quantitative relationships between cholesterol and other environmental lipid raft components. The association between symptomatic cholesterol excess and inflammation may therefore in part reflect free cholesterol- dependent changes in lipid raft structure that regulate immune cell Ca²⁺ entry. Ca²⁺ entry-dependent responses in other cell types may also reflect cholesterol bioavailability and lipid incorporation into rafts.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grant 2 R01 GM059179 from the National Institutes of Health (to C.J.H.).

² Address correspondence and reprint requests Dr. Carl J. Hauser, Department of Surgery, Lowry Medical Office Building, Room 2G, Beth Israel-Deaconess Medical Center, 110 Francis Street, Boston, MA 02215. E-mail address: cjhauser{at}bidmc.harvard.edu

³ Abbreviations used in this paper: [Ca²⁺]_i, cytosolic free calcium ion concentration; MCD, methyl--cyclodextrin; GPCR, G protein-coupled receptor; PAF, platelet-activating factor; PMN, polymorphonuclear neutrophil; ER, endoplasmic reticulum; S1P, sphingosine-1-phosphate; TRPC, transient receptor potential channel; CTB, cholera toxin B; DHR, 1,2,3-dihydrorhodamine; CalyA, calyculin A; Vmax, maximal reaction rate; fMLP/Tg, fMLP plus thapsigarin.

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