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Originally published In Press as doi:10.1194/jlr.M500408-JLR200 on January 16, 2006
Papers In Press, published online ahead of print April 1, 2006
J. Lipid Res., doi:10.1194/jlr.M500408-JLR200
Journal of Lipid Research, Vol. 47, 705-714, April 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology
Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland
Mikel Garcia-Marcos*,
Encarnación Pérez-Andrés*,
Séverine Tandel ,
Unai Fontanils*,
Alain Kumps ,
Elie Kabré**,
Antonio Gómez-Muñoz*,
Aida Marino*,
Jean-Paul Dehaye1, and
Stéphanie Pochet
* Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad del Pais Vasco, 48080 Bilbao, Spain
Laboratoire de Biochimie et de Biologie Cellulaire, Institut de Pharmacie C.P. 205/3, Université libre de Bruxelles, B-1050 Brussels, Belgium
Laboratoire de Chimie Médicale, Institut de Pharmacie C.P. 205/3, Université libre de Bruxelles, B-1050 Brussels, Belgium
** Laboratoire de Biochimie et d'Immunologie, Unité de Formation et de Recherche des Sciences de la Santé, Université de Ouagadougou, Burkina Faso
The online version of this article (available at http://www.jlr.org) contains supplementary figure.
Published, JLR Papers in Press, January 16, 2006.
1 To whom correspondence should be addressed. e-mail: jdehaye{at}ulb.ac.be
The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated. The homogenate was centrifuged at high speed in a discontinuous sucrose gradient. Light fractions contained vesicles analogous to rafts: they were rich in cholesterol, they contained GM1 and caveolin-1, and P2X7 receptors were detected in these fractions. The location of the P2X7 receptors in rafts was abolished when cellular cholesterol was removed by methyl-ß-cyclodextrin (MCD). ATP activated neutral sphingomyelinase (N-SMase), which provoked a decrease of the cellular content of sphingomyelin and an increase of ceramide levels in these cells and in the rafts. Treatment with MCD and filipin (but not with -cyclodextrin) abolished the increase of the intracellular concentration of calcium ([Ca2+]i) in response to epinephrine but not to ATP. MCD and filipin also inhibited the activation by ATP of phospholipase A2 (PLA2). Inhibition of N-SMase with glutathione or GW4869 prevented the activation of PLA2 by P2X7 agonists without affecting [Ca2+]i levels. We conclude that P2X7 receptors are present in both raft and nonraft compartments of plasma membranes; the receptors forming a nonselective cation channel are located in the nonraft fraction. P2X7 receptors in the rafts are coupled to the activation of N-SMase, which increases the content of ceramides in rafts. This may contribute to the activation of PLA2 in response to P2X7 receptor occupancy.
Supplementary key words calcium methylcyclodextrin rafts ceramides purinergic salivary glands sphingomyelinase Abbreviations: ACD, -cyclodextrin; A-SMase, acidic sphingomyelinase; ß-COP, ß-coatomer protein; Bz-ATP, 2'-3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate; [Ca2+]i, intracellular concentration of calcium; HBS, HEPES-buffered saline; MCD, methyl-ß-cyclodextrin; N-SMase, neutral sphingomyelinase; PLA2, phospholipase A2; TfR, transferrin receptor; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1

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Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
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