]AA in response to the purinergic agonists was still observed; this response was not affected by AACOCF 3 and completely blocked by bromoenol lactone. ATP and Bz-ATP stimulated a calcium-independent secretion of kallikrein, which could be blocked by BEL but which was enhanced by AACOCF 3 . It is concluded that the P2X 7 receptor in ductal cells is coupled to kallikrein secretion through a calcium-dependent cPLA 2 and a calcium-independent iPLA 2 .ATP plays an important role as an extracellular agonist that mediates its various effects by acting on specific membrane P2 receptor subtypes (1, 2). P2 receptors comprise receptors of the ligand-gated ion channel type, as well as of the G-proteinlinked superfamily, termed P2X and P2Y, respectively (3). At present, seven genes for P2X receptors have been cloned (4 -8), but their physiological significance has not been fully established yet. One of the most recently cloned P2X receptors (the P2X 7 ) has a pharmacological profile typical of the receptor previously termed P 2Z (9) with the photoactivable analog of ATP, Bz-ATP, 1 the most potent agonist. The P 2Z receptor induces the formation of pores when exposed to concentrations of extracellular ATP in the 100 M to 1 mM range (Refs. 10 -12, but see also Ref. 13). In contrast to other P2X receptors, the P2X 7 receptor has a long COOH-terminal intracellular chain, which is by itself not responsible for the lytic properties of this receptor (14) but probably induces the formation of a second messenger involved in the lysis (12). In summary, the P2X 7 (P 2Z )-receptors share with the other P2X receptors the ability to open a non-selective channel and with P 2Z receptors the induction of cell lysis by repeated applications of the agonist (8).Since the pioneering work of Gallacher (15), ATP has been recognized as a major non-adrenergic non-cholinergic stimulus of saliva secretion. Salivation, like other exocrine secretions, occurs in two steps; (a) acinar cells secrete an isotonic plasmalike fluid, and (b) the electrolyte composition of this primary secretion is modified during its transfer to the mouth by the ductal tree (16). The ducts reabsorb Na ϩ and Cl Ϫ and secrete K ϩ and HCO 3 Ϫ (17). The study of these two phases of the secretory process has been facilitated by the description of an improved technique to separate ducts and acini (18). It could be observed that extracellular ATP increased the intracellular

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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.

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