title: Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland.
abstract: 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 (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 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 (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.
authors.affiliation: Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad del Pais Vasco, 48080 Bilbao, Spain.
authors.forename: Mikel
authors.initials: M
authors.lastname: Garcia-Marcos
authors.forename: Encarnación
authors.initials: E
authors.lastname: Pérez-Andrés
authors.forename: Séverine
authors.initials: S
authors.lastname: Tandel
authors.forename: Unai
authors.initials: U
authors.lastname: Fontanils
authors.forename: Alain
authors.initials: A
authors.lastname: Kumps
authors.forename: Elie
authors.initials: E
authors.lastname: Kabré
authors.forename: Antonio
authors.initials: A
authors.lastname: Gómez-Muñoz
authors.forename: Aida
authors.initials: A
authors.lastname: Marino
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Dehaye
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Pochet
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D008297:Male; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction / Q000502:physiology*; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D013363:Submandibular Gland / Q000166:cytology / Q000378:metabolism*
chemical_list: C546470:P2rx7 protein, rat; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D013109:Sphingomyelins; D000255:Adenosine Triphosphate; D013108:Sphingomyelin Phosphodiesterase