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.

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication. In both cases a low density vesicular fraction was obtained after ultracentrifugation in a sucrose density gradient. These fractions contained about 20% of total cholesterol but less than 8% of total proteins, and were more rigid than bulk membranes. Fatty acid analyses revealed a similar composition of raft-like membranes isolated by the two different methods, which was characterized by an enrichment in saturated fatty acids in detriment of polyunsaturated acids when compared with the whole cell membranes. Protein profile of detergent resistant membranes or raft-like membranes prepared by sonication was assessed by silver staining after SDS-PAGE and by MALDI-TOF. Both analyses provided evidence of a different protein composition of the Triton X-100 and sonication preparations. Immunoblot experiments revealed that raft-like membranes prepared by detergent extraction or sonication were free of Golgi apparatus or endoplasmic reticulum protein markers (beta-COP and calnexin, respectively) and that they were not substantially contaminated by transferrin receptor (a non-raft protein). While caveolin-1 was highly enriched in raft-like membranes prepared by the two methods, the P2X(7) receptor was enriched in raft-like membrane fractions prepared by sonication, but almost undetectable in the detergent resistant membranes. It can be concluded that both methods can be used to obtain raft-like membranes, but that detergent may affect protein interactions responsible for their association with different membrane domains.

The interaction of mice submandibular gland cells with LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), a cationic peptide with immunomodulatory properties, was investigated. LL-37 at a concentration that did not affect the integrity of the cells increased the uptake of calcium and activated a calciuminsensitive phospholipase A 2 (PLA 2 ). The small release of ATP induced by LL-37 could not account for this stimulation because apyrase did not significantly block the response to LL-37. The divalent cation magnesium inhibited the response to LL-37, but this inhibition was probably nonspecific because it also inhibited the in vitro bacteriostatic effect of the peptide. The increase of calcium uptake by LL-37 was not affected by, a rather specific inhibitor of P2X 7 receptors in mice. LL-37 also increased [Ca 2ϩ ] i in cells from mice invalidated for these receptors. LL-37 had no effect on the response to carbachol. It inhibited the increase of [Ca 2ϩ ] i and the activation of phospholipase D by ATP. It potentiated the activation of the PLA 2 by the nucleotide. Finally, LL-37 increased the fluidity of the plasma membrane of submandibular gland cells. In conclusion, our results suggest that LL-37 is an autocrine regulator of submandibular gland cells. It does not stimulate mouse P2X 7 receptors but modulates their responses.Cathelicidins are proteins involved in the first phases of our defenses against pathogens. Their isolation relied on the analogy of their N-terminal proregions with cathelin, an inhibitor of cathepsin L originally isolated from bovine leukocytes (Ritonja et al., 1989). This highly conserved proregion (approximately 100 amino acids) shares many similarities with cystatins, inhibitors of the cysteine proteases. The Cterminal domain of cathelicidins varies among species both in terms of length (12-100 amino acids) and structure. The N-and C-terminal domains are separated by a sequence recognized by proteases. The digestion in the extracellular medium of the propeptides by elastase (Panyutich et al., 1997) or proteinase 3 (Sorensen et al., 2001) releases the C-terminal peptides originally described as antimicrobial but that now prove to be more immunomodulatory than antimicrobial in physiological conditions (Bowdish et al., 2005). Most of the studies on cathelicidins have focused on the C-terminal peptides. These peptides are ubiquitous. They are secreted by macrophages, lymphocytes, epithelial cells, keratinocytes, cells lining the upper respiratory tree, and vaginal cells (Bals and Wilson, 2003). LL-37, the only peptide from human origin, is derived from an antibacterial protein of 18 kDa. LL-37 and its only analog in mouse, the cathelinrelated antimicrobial peptide, are cationic and transform from a random coil in aqueous solution to an amphipathic ␣-helix at the contact of a membrane (Yeaman and Yount, 2003). The peptide binds to the bacteria by electrostatic interactions between the positive charges on one side of its

The sensitivity to cholesterol depletion of calcium handling by rat submandibular glands was investigated. The glands were digested with collagenase. After homogenization, the lysate was extracted at 4 degrees C with 0.5% Triton X-100 and the extract was submitted to an ultracentrifugation in a sucrose discontinuous gradient. A population of detergent-resistant membranes (DRM) was collected at the 5%-35% interface. The DRM had a higher content of cholesterol, saturated and long-chain fatty acids. Caveolin-1 and alpha(q/11) were located in these membranes. They were more ordered than vesicles from total cellular lysate as determined by anisotropy measurement. They disappeared after cholesterol extraction with methyl-beta-cyclodextrin (MCD). Exposure of the cellular suspension with MCD nearly abolished the response to carbachol, epinephrine, and substance P and inhibited the activation of phospholipase C (PLC) by these agonists and by sodium fluoride. MCD did not affect the mobilization of intracellular pools of calcium by thapsigargin. It increased the uptake of extracellular calcium or barium and did not inhibit the uptake of calcium after depletion of the intracellular stores of this ion. From these results, it is concluded that Triton X-100 can extract a fraction of membrane resistant to detergents. Treatment of the cells with MCD disrupts these membranes. The coupling between the heterotrimeric GTP-binding protein G(q/11) and poly-phosphoinositide-specific PLC is affected by disruption of these membrane fractions. At the opposite, the store-operated calcium channel (SOCC) is not affected by DRM-disruption.

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