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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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).

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).

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.