Unai Fontanils

Coupling of two pools of P2X7 receptors to distinct intracellular signaling pathways in rat submandibular gland.

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-β-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 α-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.

Regulation by CRAMP of the responses of murine peritoneal macrophages to extracellular ATP.

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.

Role of sodium in mitochondrial membrane depolarization induced by P2X7 receptor activation in submandibular glands.

It is concluded that the activation of P2X7 receptors depolarizes the mitochondrial membrane. The uptake of extracellular sodium is necessary but not sufficient to induce this response.

Stimulation by P2X7 receptors of calcium-dependent production of reactive oxygen species (ROS) in rat submandibular glands

BACKGROUND Agonists of P2X₇ receptors increase the production of reactive oxygen species (ROS) in immunocytes. In this work we tested this response and its effect on mitochondrial inner membrane potential (Deltapsi(m)) in exocrine glands. METHODS The production of ROS by rat submandibular glands was investigated by measuring the oxidation of dichlorodihydrofluorescein (DCFH), a fluorescent probe. The Deltapsi(m) was estimated with tetramethylrhodamine. RESULTS Activation of P2X₇ receptors by ATP or Bz-ATP increased the production of ROS. This response was not modified by inhibitors of phospholipase A2 or of various kinases. The effect of ATP was calcium-dependent and was blocked by diphenyliodonium, an inhibitor of flavoproteins. It was not affected by rotenone, an inhibitor of the complex I of the mitochondrial electron transfer chain. Scavengers of ROS had no effect on the dissipation of Deltaψ(m) by ATP. CONCLUSIONS We conclude that, in rat submandibular glands, P2X₇ receptors stimulate in a calcium-dependent manner an oxidase generating ROS, suggesting the involvement of the dual oxidase Duox2. The production of ROS does not contribute to the depolarization of mitochondria by purinergic agonists. GENERAL SIGNIFICANCE Purinergic receptors could be regulators of the bactericidal properties of saliva by promoting both the secretion of peroxidase from acinar cells and by activating Duox2.