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.

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X(7)-KO mice.

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X(7)-invalidated (KO) mice was tested. Low concentrations (1-100 µM) of ATP transiently increased the intracellular concentration of calcium ([Ca(2+)](i)) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X(7) receptors. One millimolar ATP provoked a sustained increase in the [Ca(2+)](i) only in WT mice. The response to 10 µM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K(+)](i)) and stimulated the secretion of interleukin-1ß (IL-1ß) only in cells from WT mice. Ten micromolar ATP in combination with 3 µM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1ß was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 µM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 µM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 µM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X(4) receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K(+)](i) triggered the secretion of IL-1ß. Pore formation was also triggered by activation of P2X(4) receptors. Higher concentrations of ATP elicited similar responses after binding to P2X(7) receptors. The expression of the P2X(7) receptors was also coupled to a better response to P2Y receptors.

Contribution of two ionotropic purinergic receptors to ATP responses in submandibular gland ductal cells.

The effect of extracellular ATP on salivary gland function was compared in wild-type (WT) and P2X(7) knockout (KO) mice. The increase in the intracellular concentration of calcium ([Ca(2+)](i)) in response to carbachol was similar in submandibular ductal cells of WT and KO mice. ATP and its analog, benzoyl-ATP, induced a sustained increase in the [Ca(2+)](i) in WT animals. In KO mice, ATP slightly and transiently increased the [Ca(2+)](i) and benzoyl-ATP had no effect. The response to ATP of WT but not KO mice was blocked by KN-62, Coomassie blue and magnesium. The small response of ATP observed in KO mice was completely blocked in the absence of extracellular calcium, unchanged by U73122 and potentiated by ivermectin indicating the probable involvement of a P2X(4) receptor. A RT-PCR and a Western blot confirmed the presence of these receptors in ducts of both WT and KO mice. ATP increased the permeability of the cells to ethidium bromide and stimulated a phospholipase A(2) activity in WT but not KO mice. Mice submandibular gland cells secreted IL-1beta but this secretion was not modified by ATP and was similar in both groups of animals. The volume of saliva provoked by pilocarpine and the concentration of proteins, sodium and chloride in this saliva was similar in both groups of animals. The concentration of potassium was higher in KO mice. We can conclude that the major purinergic receptors expressed in mice submandibular ductal cells are P2X(7) receptors but that P2X(4) receptors are also involved in some ATP effects.

Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells.

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.

P2X7 and phospholipid signalling: the search of the "missing link" in epithelial cells.

The purinergic receptor P2X(7) is widely expressed in epithelial cells. This receptor shares in common with the other P2X receptors the ability to form a non-selective cation channel. On the other hand, the COOH terminus of P2X(7) seems to allow this receptor to couple to a spectrum of downstream effectors responsible for the regulation of cell death and pore formation among other functions. However, the coupling of P2X(7) to these downstream effectors, as well as the identity of possible adapters directly interacting with the receptor, remains poorly understood. Here we review the ability of P2X(7) to activate phospholipid signalling pathways in epithelial cells and propose this step as a possible link between the receptor and other downstream effectors. The P2X(7) ability to control the cellular levels of several lipid messengers (PA, AA, DAG, ceramide, etc.) through the modulation of phospholipases (C, A(2), D) and neutral sphingomyelinase is described. These pathways are sometimes regulated independently of the channel function of the receptor. Recent data concerning P2X(7) localization in lipid rafts is also discussed in relation to the coupling to these pathways and dissociation from channel function.

Regulation of phospholipase D by P2X7 receptors in submandibular ductal cells.

ATP (1 mM) increased the phospholipase D (PLD) activity of rat submandibular gland (RSMG) ductal cells in a concentration-dependent and calcium-sensitive manner. The response to ATP was reproduced by benzoylbenzoyl-ATP (Bz-ATP, 100 microM) and also partly by adenosine 5'-(gamma-thio)triphosphate (ATPgammaS, 1 mM). A similar stimulation was observed in control mice (P2X7R+/+ mice) but not in mice lacking the P2X7 receptors (P2X7R-/- mice). Oxidized ATP and Coomassie blue or the addition of magnesium or nickel to the incubation medium inhibited the response to ATP. The stimulation of PLD by purinergic agonist was inhibited by about 50% by calphostin C and chelerythrine, two protein kinase C (PKC) inhibitors. The stimulation of PLD by Bz-ATP and by o-tetradecanoylphorbol 13-acetate (TPA), a phorbol ester which activates PKC, were not additive. From these results we can conclude that the activation of P2X7 receptors in RSMG ductal cells is coupled to the activation of a PLD. This activation is partly mediated by protein kinase C.

Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells.

The muscarinic agonist carbachol stimulated phospholipase D (PLD) in rat submandibular gland (RSMG) ductal cells in a time and concentration-dependent manner. This effect was inhibited by chelation of extracellular calcium with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLD could also be activated by epinephrine and AlF(4)(-), two polyphosphoinositide-specific phospholipase C (PPI-PLC) activators, and by the phorbol ester o-tetradecanoylphorbol 13-acetate (TPA) which activates protein kinase C (PKC). Ionomycin and thapsigargin only slightly increased PLD activity. Ortho-vanadate, a tyrosine phosphatase inhibitor, also stimulated PLD activity. Both carbachol and o-vanadate increased the formation of inositol phosphates and the tyrosine phosphorylation of at least two proteins (55-60 and 120 kDa). Calphostin C (a PKC inhibitor), U73122 (a PPI-PLC inhibitor) and genistein (a tyrosine kinase inhibitor) blocked the activation of PLD, of PLC and the phosphorylation of tyrosyl residues in response to carbachol and vanadate. Taken together, these results suggest that rat submandibular gland ductal cells express a calcium-dependent PLD activity. This enzyme is regulated by carbachol via a PLC-PKC-tyrosine kinase pathway.

Sphingosine-1-phosphate stimulates cortisol secretion.

We show here for the first time that sphingosine-1-phosphate (Sph-1-P) stimulates cortisol secretion in zona fasciculata cells of bovine adrenal glands. This effect was dependent upon protein kinase C (PKC) and extracellular Ca2+, and was inhibited by pertussis toxin. Sph-1-P activated phospholipase D (PLD) through a pertussis toxin-sensitive mechanism, also involving extracellular Ca2+ and PKC. Primary alcohols, which attenuate formation of phosphatidic acid (the product of PLD), and cell-permeable ceramides, which inhibit PLD, blocked Sph-1-P-induced cortisol secretion. In conclusion, Sph-1-P stimulates cortisol secretion through a mechanism involving Gi/o protein-coupled receptors, extracellular Ca2+, PKC and PLD.

ATP antagonizes thrombin-induced signal transduction through 12(S)-HETE and cAMP.

In this study we have investigated the role of extracellular ATP on thrombin induced-platelet aggregation (TIPA) in washed human platelets. ATP inhibited TIPA in a dose-dependent manner and this inhibition was abolished by apyrase but not by adenosine deaminase (ADA) and it was reversed by extracellular magnesium. Antagonists of P2Y1 and P2Y12 receptors had no effect on this inhibition suggesting that a P2X receptor controlled ATP-mediated TIPA inhibition. ATP also blocked inositol phosphates (IP1, IP2, IP3) generation and [Ca(2+)]i mobilization induced by thrombin. Thrombin reduced cAMP levels which were restored in the presence of ATP. SQ-22536, an adenylate cyclase (AC) inhibitor, partially reduced the inhibition exerted by ATP on TIPA. 12-lipoxygenase (12-LO) inhibitors, nordihidroguaretic acid (NDGA) and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE), strongly prevented ATP-mediated TIPA inhibition. Additionally, ATP inhibited the increase of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) induced by thrombin. Pretreatment with both SQ-22536 and NDGA almost completely abolished ATP-mediated TIPA inhibition. Our results describe for the first time that ATP implicates both AC and 12-LO pathways in the inhibition of human platelets aggregation in response to agonists.

Membrane compartments and purinergic signalling: the role of plasma membrane microdomains in the modulation of P2XR-mediated signalling.

Purinergic signalling is implicated in virtually any cellular and physiological function. These functions are mediated through the activation of different receptor subfamilies, among which P2X receptors (P2XRs) are ligand-gated ion channels that respond mostly to ATP. In addition to forming a nonselective cation channel, these receptors engage with a complex network of signalling pathways, including protein kinase cascades, lipid signal mediators and proteases. It is poorly understood how P2XR stimulation couples to such a variety of intracellular pathways and how the outcome from this complex signalling network is tuned. In this context, segregation of receptors and other signalling components at the plasma membrane is an attractive explanation. Lipid rafts are microdomains of biological membranes with unique physicochemical properties that make them segregate from the bulk of the membrane, provoking the differential partition of receptors and signalling molecules among different domains of the plasma membrane. Here we give an overview of the properties of lipid rafts and how they are studied, along with recent advances in the understanding of their role in modulating P2XR-mediated signalling.

Pharmacological evidence for the stimulation of NADPH oxidase by P2X(7) receptors in mouse submandibular glands.

ATP in the 100 muM-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.

Modulation by LL-37 of the responses of salivary glands to purinergic agonists.

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 calcium-insensitive 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 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a rather specific inhibitor of P2X(7) receptors in mice. LL-37 also increased [Ca(2+)](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(2+)](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.

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-beta-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 alpha-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.

Ca(2+) influx through AMPA or kainate receptors alone is sufficient to initiate excitotoxicity in cultured oligodendrocytes.

Oligodendrocytes are vulnerable to excitotoxic insults mediated by AMPA receptors and by low and high affinity kainate receptors, a feature that is dependent on Ca(2+) influx. In the current study, we have analyzed the intracellular concentration of calcium [Ca(2+)](i) as well as the entry routes of this cation, upon activation of these receptors. Selective activation of either receptor type resulted in a substantial increase (up to fivefold) of [Ca(2+)](i), an effect which was totally abolished by the non-NMDA receptor antagonist CNQX or by removing Ca(2+) from the culture medium. Blockade of voltage-gated Ca(2+) channels with La(3+) or nifedipine, reduced the amplitude of the Ca(2+) current triggered by AMPA receptor activation by approximately 65%, but not that initiated by low and high affinity kainate receptors. In contrast, KB-R7943, an inhibitor of the plasma membrane Na(+)-Ca(2+) exchanger, solely attenuated the rise in [Ca(2+)](i) by approximately 25% due to activation of low affinity kainate receptors. However, oligodendroglial death by glutamate receptor overactivation was largely unaffected in the presence of La(3+) or KB-R7943. These findings indicate that Ca(2+) influx via AMPA and kainate receptors alone is sufficient to initiate cell death in oligodendrocytes, which does not require the entry of calcium via other routes such as voltage-activated calcium channels or the plasma membrane Na(+)-Ca(2+) exchanger.

Activation of phospholipase D-2 by P2X(7) agonists in rat submandibular gland acini.

Exogenous ATP stimulated phospholipase D (PLD), but not sphingomyelinase in rat submandibular gland (SMG) acini. PLD activation was dependent upon extracellular Ca(2+) and did not involve intracellular Ca(2+) mobilization or phosphoinositide-specific phospholipase C activation. ATP-stimulated PLD was attenuated by inhibition or downregulation of protein kinase C (PKC). PLD activation was fully blocked by the cytosolic phospholipase A(2) (PLA(2)) inhibitor ONO-RS-082 and partially attenuated by the selective Ca(2+)-dependent cytosolic PLA(2) inhibitor, arachidonyl trifluoromethylketone (AACOCF(3)), or by bromoenol lactone, an inhibitor of Ca(2+)-independent cytosolic PLA(2). Magnesium, which decreases the concentration of ATP(4-), and nickel, which blocks nonspecific cation channels coupled to purinergic receptors, inhibited PLD activation by ATP. Using reverse transcription-polymerase chain reaction and Northern blotting techniques, we demonstrated that the PLD isoform stimulated by ATP was PLD-2. Among various ATP analogs, only the P2Z/P2X(7) purinergic receptor agonist benzoyl-benzoyl ATP stimulated PLD-2. The response to ATP was inhibited by the nonselective P2X purinergic antagonist suramin and by oxidized ATP, a potent P2Z/P2X(7) receptor antagonist. It is concluded that in rat SMG acinar cells, PLD-2 is upregulated by exogenous ATP through a mechanism involving Ca(2+) influx, cytosolic PLA(2), and PKC. Also, the data suggest an involvement of P2X(7) receptors in PLD-2 stimulation by ATP.