Dynamics of macrophage polarization reveal new mechanism to inhibit IL-1β release through pyrophosphates

In acute inflammation, extracellular ATP activates P2X7 ion channel receptors (P2X7R) on M1 polarized macrophages to release pro-inflammatory IL-1β through activation of the caspase-1/nucleotide-binding domain and leucine-rich repeat receptor containing pyrin domain 3 (NLRP3) inflammasome. In contrast, M2 polarized macrophages are critical to the resolution of inflammation but neither actions of P2X7R on these macrophages nor mechanisms by which macrophages switch from pro-inflammatory to anti-inflammatory phenotypes are known. Here, we investigated extracellular ATP signalling over a dynamic macrophage polarity gradient from M1 through M2 phenotypes. In macrophages polarized towards, but not at, M2 phenotype, in which intracellular IL-1β remains high and the inflammasome is intact, P2X7R activation selectively uncouples to the NLRP3-inflammasome activation but not to upstream ion channel activation. In these intermediate M1/M2 polarized macrophages, extracellular ATP now acts through its pyrophosphate chains, independently of other purine receptors, to inhibit IL-1β release by other stimuli through two independent mechanisms: inhibition of ROS production and trapping of the inflammasome complex through intracellular clustering of actin filaments

Pharmacology of cloned P2X receptors

There are seven P2X receptor cDNAs currently known. Six homomeric (P2X1, P2X2, P2X3, P2X4, P2X5, P2X7) and three heteromeric (P2X2/P2X3, P2X4/P2X6, P2X1/P2X5) P2X receptor channels have been characterized in heterologous expression systems. Homomeric P2X1 and P2X3 receptors are readily distinguishable by their rapid desensitization, the agonist action of αβmethyleneATP, and the block by 2′,3′-O-(2,4,6-trinitrophenyl)-ATP. P2X2 receptors are unique among homomeric forms in their potentiation by low pH. Homomeric P2X4 receptors are much less sensitive to antagonism by suramin and pyridoxal 5-phosphate-6-azo-2′,4′-disulfonic acid. Homomeric P2X7 receptors are the only form in which 2′,3′-O-(4-benzoylbenzoyl)-ATP is more potent than ATP. The heteromeric P2X2/P2X3 receptor resembles P2X2 in slow desensitization kinetics and potentiation by low pH and is similar to P2X3 with respect to agonism by αβmethyleneATP and block by 2′,3′-O-(2,4,6-trinitrophenyl)-ATP. Other agonists, antagonists, and ions that can be used to differentiate among the receptors are discussed. </jats:p

Sustained depolarization and ADP-ribose activate a common ionic current in rat peritoneal macrophages

Abstract Phagocytosis is associated with large changes in the membrane potential of macrophages, but the functional significance of this is unknown. Whole cell recordings were made from rat peritoneal macrophages. Sustained (&amp;gt;30 s) depolarization of the cells progressively activated a conductance that remained high (several nanoSeimens) for several tens of seconds. This current: 1) was linearly dependent on potential between −100 and +50 mV; 2) reversed close to 0 mV in a physiological external solution; 3) could also be carried in part by N-methyl-d-glucamine (PNMDG/PNa 0.7), chloride (PCl/PNa 0.5), or calcium (PCa/PNa 1.3); and 4) was blocked by intracellular ATP (5 mM) or ADP (10 mM) and by extracellular lanthanum (half-maximal concentration 1 mM). A current with all the same properties was recorded in cells when the intracellular solution contained ADP-ribose (10–300 μM) or β-NAD (1 mM) (but not any other nucleotide analogs tested). The results suggest that prolonged depolarization leads to an increased intracellular level of ADP-ribose, which in turn activates this nonselective conductance(s).</jats:p

Different sensitivities to pH of ATP-induced currents at four cloned P2X receptors

Stoop, Ron, Annmarie Surprenant, and R. Alan North. Different sensitivities to pH of ATP-induced currents at four cloned P2X receptors. J. Neurophysiol. 78: 1837–1840, 1997. The effect of changing extracellular pH was studied on the currents induced by ATP or αβ-methylene-ATP in HEK293 cells transfected with different P2X receptor subunits. In cells expressing P2X1, P2X3, or P2X4 receptors, the effect of ATP was decreased by acidification. In cells expressing P2X2 receptors, acidification increased the ATP-induced current; this effect was also seen in cells expressing heteromeric P2X2 and P2X3 receptors. At P2X2 receptors, acidification caused a leftward shift in the ATP concentration-response curve, without change in maximum; the pKa for this effect was 7.3. At P2X4 receptors, acidification caused a rightward shift in the ATP concentration-response curve, without change in the maximum; the pKa for this effect was 6.8. The pH dependence of the action of ATP should be taken into account in studies of synaptic transmission, and it may provide a further tool to assign molecular identity to P2X receptors expressed by brain neurons. </jats:p

Pharmacological characterization of pannexin-1 currents expressed in mammalian cells

Pannexin (Panx) 1 is a widely expressed protein that shares structural, but not amino acid, homology with gap junction proteins, the connexins. Panx1 does not form gap junctions in mammalian cells, but it may function as a plasma membrane hemichannel. Little is known of the pharmacological properties of panx1 expression in mammalian cells. Here, we identify three variants in the human PANX1 gene. We expressed these variants and mouse Panx1 in mammalian cells and compared Panx1-induced currents. All human Panx1 variants and the mouse Panx1 showed identical protein expression levels, localization patterns, and functional properties, although the frequency of functional expression was species-dependent. Panx1 currents were independent of changes in extracellular or intracellular calcium or phospholipase C transduction. We found compounds that inhibited Panx1 currents with a rank order of potency: carbenoxolone > disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS) ≈ disodium 4-acetamido-4′-isothiocyanato-stilben-2,2′-disulfonate ≈ 5-nitro-2-(3-phenylpropylamino)benzoic acid > indanyloxyacetic acid 94 >> probenecid >> flufenamic acid = niflumic acid. Triphosphate nucleotides (ATP, GTP, and UTP) rapidly and reversibly inhibited Panx1 currents via mechanism(s) independent of purine receptors. When Panx1 was coexpressed with purinergic P2X(7) receptor (P2X(7)R), DIDS was found to act as a P2X(7)R antagonist to inhibit ATP-evoked currents, but none of the other compounds inhibited P2X(7)R currents. This is the first detailed pharmacological characterization of Panx1-mediated currents in mammalian cells and sheds new, although contradictory, light on the hypothesis that Panx1 acts as a hemichannel to allow passage of large molecules in response to P2X(7)R activation