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A more recent version of this article appeared on April 1, 2006

Papers In Press, published online ahead of print January 16, 2006
J. Lipid Res., doi:10.1194/jlr.M500408-JLR200
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Submitted on September 15, 2005
Revised on January 13, 2006
Accepted on January 16, 2006

Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland

Mikel García-Marcos, Encarnación Pérez-Andres, Séverine Tandel, Unai Fontanils, Alain Kumps, Elie Kabré, Antonio Gómez-Munoz, Aida Marino, Jean-Paul Dehaye, and Stéphanie Pochet

Biochemistry Dept., Institute of Pharmacy C.P. 205/3, Bruxelles B1050

Corresponding Author: 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-beta -cyclodextrin (MCD). ATP activated neutral sphingomyelinase which provoked a decrease of the cellular content of sphingomyelin and an increase of ceramide levels in these cells and in the rafts. A treatment with MCD and filipin (but not with alpha -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. Inhibition of neutral sphingomyelinase with glutathione or GW4869 prevented the activation of PLA2 by P2X7 agonists without affecting [Ca2+]i levels. It can be concluded that P2X7 receptors are present both in raft and non-raft compartments of plasma membranes; the receptors forming a non-selective cation channel are located in the non-raft fraction. P2X7receptors in the rafts are coupled to the activation of neutral sphingomyelinase which increases the content of ceramides in rafts. This may contribute to the activation of PLA2in response to P2X7 receptor occupancy.


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