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 P2X 7 receptors were detected in these fractions. The location of the P2X 7 receptors in rafts was abolished when cellular cholesterol was removed by methyl-b-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 a-cyclodextrin) abolished the increase of the intracellular concentration of calcium ([Ca 21 ] i ) in response to epinephrine but not to ATP. MCD and filipin also inhibited the activation by ATP of phospholipase A 2 (PLA 2 ). Inhibition of N-SMase with glutathione or GW4869 prevented the activation of PLA 2 by P2X 7 agonists without affecting [Ca 21 ] i levels. We conclude that P2X 7 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. P2X 7 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 PLA 2 in response to P2X 7 receptor occupancy.-Garcia-Marcos, M., E. Pérez-Andrés, S. Tandel, U. Fontanils, A. Kumps, E. Kabré, A. Gómez-Muñoz, A. Marino, J-P. Dehaye, and S. Pochet. Coupling of two pools of P2X 7 receptors to distinct intracellular signaling pathways in rat submandibular gland. J. Lipid Res. 2006. 47: 705-714.

ATP in the 100 μM-1 mM concentration range provoked a calcium-independent increase of the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF) by mouse submandibular cells. 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (BzATP), a P2X 7 agonist, but not a muscarinic or an adrenergic agonist, reproduced the effect of ATP. The inhibition of phospholipase C by U73122 or the potentiation of P2X 4 receptor activation with ivermectin did not modify the response to ATP. ATP did not increase the oxidation of DCFH in cells isolated from submandibular glands of P2X 7 knockout mice or in cells pretreated with a P2X 7 antagonist. The inhibition of protein kinase C or of mitogen-activated protein kinase (MAP kinase) or of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocked the oxidation of DCFH without affecting the increase of the intracellular concentration of calcium or the uptake of ethidium bromide in response to extracellular ATP. From these results it is concluded that the activation of the P2X 7 receptors from submandibular glands triggers an intracellular signalling cascade involving protein kinase C and MAP kinase leading to the stimulation of NADPH oxidase and the subsequent generation of reactive oxygen species.