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Papers In Press, published online ahead of print April 1, 2006
J. Lipid Res., doi:10.1194/jlr.M500408-JLR200

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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 Dehaye^1, 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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