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.

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.

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.

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.

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.