]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

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.

1 The permeabilizing e ect of P2X 7 agonists was tested in rat submandibular acinar cells using the uptake of ethidium bromide as an index. 2 The uptake of ethidium bromide by acini incubated at 378C in the presence of 1 mM ATP increased with time and reached after 5 min about 10% of maximal uptake measured in the presence of digitonin. 3 The response to ATP was dose-dependent (half-maximal concentration around 40 mM) and it was decreased when the temperature was lowered to 258C. 4 Benzoyl-ATP reproduced the response to ATP (half-maximal concentration around 10 mM). UTP or 2-methylthioATP had no e ect. 5 The permeabilization in response to ATP was blocked by oxidized ATP and by magnesium and inhibited by Coomassie blue. 6 ATP increased the activity of a calcium-insensitive phospholipase A 2 (iPLA 2 ). 7 Bromoenol lactone (BEL) inhibited the iPLA 2 stimulated by ATP but potentiated the uptake of ethidium bromide in response to the purinergic agonist. 8 From these results it is concluded that the activation of P2X 7 receptors permeabilizes rat submandibular acinar cells. The pore-forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA 2 by the receptor.

Peritoneal macrophages were isolated from wild type (WT) mice and from mice invalidated for the P2X(7) receptor (KO) which had been pretreated with thioglycolate. In cells from WT mice, 1 mM ATP increased the intracellular concentration of calcium ([Ca(2+)](i)), the uptake of ethidium bromide, the production of reactive oxygen species (ROS), the secretion of IL-1beta, the release of oleic acid and of lactate dehydrogenase; it decreased the intracellular concentration of potassium ([K(+)](i)). In KO mice, ATP transiently increased the [Ca(2+)](i) confirming that the P2X(7) receptor is a major receptor of peritoneal macrophages. WKYMVm, an agonist of receptors for formylated peptides (FPR) also increased the [Ca(2+)](i) in murine macrophages. The slight increase of the [Ca(2+)](i) was strongly potentiated by ivermectin confirming the expression of functional P2X(4) receptors by murine peritoneal macrophages. CRAMP, the unique antimicrobial peptide derived from cathelin in mouse inhibited all the responses coupled to P2X(7) receptors in macrophages from WT mice. Agonists for FPR had no effect on the increase of the [Ca(2+)](i) in response to ATP. CRAMP had no effect on the increase of the [Ca(2+)](i) evoked by a combination of ATP and ivermectin in macrophages from P2X(7)-KO mice. In summary CRAMP inhibits the responses secondary to the activation of the murine P2X(7) receptors expressed by peritoneal macrophages. This inhibition is not mediated by FPR receptors and is specific since CRAMP has no effect on the response coupled to P2X(4) receptors. It can thus be concluded that the interaction between P2X(7) receptors and cathelin-derived antimicrobial peptides is species-specific, in some cases (man) positive in others (mouse) negative.

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