Effect of adenosine receptor subtype autoregulation on the inflammatory process.

<p>(A) Early expression of A₁R after bacterial inoculation decreases cAMP levels, enhances production of local pro-inflammatory cytokines and promotes leukocyte migration. (B) In a later phase of peritonitis A_2AR expression increase by A₁R which leads to increase in cAMP levels. High cAMP markedly decreases local pro-inflammatory cytokines and leukocyte recruitment, hence restraining inflammatory flames.</p

A₁R trigger the induction of A_2AR <i>in vitro</i>.

<p>(A) PMΦ or (B) PMC were exposed to increasing concentrations of adenosine or A₁R agonist (CHA), (0.1, 1 and 10 µM 3 hours intervals) in the presence or absence of A₁R antagonist (DPCPX, 50 nM, 30 min before treatment) (C) PMC were treated with PTX for 18 hr and then with increasing concentrations of CHA. (D) PMC were treated with increasing concentrations of adenosine, A₁R agonists (CHA and CCPA) or A_2AR agonist (CGS21680) in the presence or absence of A_2AR antagonist (ZM241385, 50 nM). Total RNA was extracted from cells and analyzed for A_2AR mRNA levels and normalized to β-actin. CT, non-treated cells. Data represent four experiments and are expressed as mean±SEM fold of control. ** <i>p</i><0.01, *** <i>p</i><0.001 from CT for B and D, <i>n</i> = 3 for each experiment.</p

The effect of A₁R agonist, in A_2AR^−/− and in the presence of A_2AR antagonist.

<p>Mice were administrated with A₁R agonist (CHA, 0.1 mg/kg) or vehicle 24 prior to bacterial inoculation. 30 min before inoculation the A_2AR antagonist (ZM241385, 1 mg/kg) or the A_2AR agonist (CGS21680, 1 mg/kg) were administered to the same animals or to untreated animals. (A) sera IL-6 and TNFα (12 hours) and (B) lavage fluids IL-6 and TNFα (12 hours). (C) A_2AR⁻^/− mice or their WT littermates were treated with the A₁R agonist (CHA, 0.1 mg/kg) i.p. or vehicle 24 hours prior to bacterial inoculation. 12 hours following inoculation sera were collected and analyzed for IL-6 and TNFα levels. Data are representative of three individual experiments and are expressed as mean±SEM. * <i>p</i><0.05, ** <i>p</i><0.01 between vehicle and CHA or CGS21680 and between CHA with or without ZM241385, <i>n</i> = 5 for each experiment.</p

Effect of A₁R agonist on A₁R and A_2AR levels <i>in vivo.</i>

<p>Mice were administered i.p. with the A₁R agonist (CHA, 0.1 mg/kg) or with vehicle. PMC were scraped from the peritoneal surface and analyzed for (A) A_2AR and A₁R mRNA levels at 4 hours or (B+C) A_2AR and A₁R protein levels at 24 hours. (B) Densitometry of protein blot depicted in (C). A₁R and A_2AR mRNA levels were normalized to GAPDH and protein levels were normalized to β-actin. Results are presented as fold change from vehicle-treated animals. Data represent three experiments and are expressed as mean±SEM. * <i>p</i><0.05 between conditions per receptor, <i>n</i> = 4 for each experiment.</p

A₁R and A_2AR expression in peritoneal leukocytes during inflammation <i>in vivo.</i>

<p>Peritonitis was induced in mice by <i>E. coli</i> inoculation at a sub-lethal dose. To examine the dynamic expression of the two high-affinity adenosine receptors, A₁R and A_2AR, peritoneal lavage was performed at indicated time points. A₁R and A_2AR mRNA levels in peritoneal leukocytes were analyzed by real time PCR and normalized to β-actin levels. Data represent three experiments and are expressed as mean±SEM. * <i>p</i><0.05, between expression levels of each receptor to expression at time 0, <i>n</i> = 5 for each experiment.</p

Effect of adenosine on A_2AR and A₁R levels <i>in vitro</i>.

<p>To simulate the gradual increase of adenosine that occurs during peritonitis, cultured primary PMC were treated with multiple and increasing concentrations of adenosine (0.1, 1 and 10 µM at 3 hour intervals). Total RNA was extracted after 9 hours and analyzed for A₁R and A_2AR mRNA levels. Results are normalized to β-actin. Data represent five experiments and are expressed as mean±SEM fold of control. * <i>p</i><0.05, ** <i>p</i><0.01 between expression levels of each receptor to expression at time 0, <i>n</i> = 3 for each experiment.</p

The anti-inflammatory effect of pretreatment with the A₁R agonist.

<p>Mice were treated with the A₁R agonist (CHA, i.p., 0.1 mg/kg) or vehicle 24 hours prior to bacterial inoculation. (A) Sera levels of IL-6 and TNFα at 12 hours. (B) Chemokine mRNA levels. 12 hours after inoculation PMC were scraped from the peritoneal membrane and total RNA was extracted, analyzed for MCP-1 and MIP-2 mRNA levels and normalized to β-actin. (C) Total cell count at 24 hours after inoculation. Cell exudates were collected from peritoneal lavage fluid. Data represent five experiments and are expressed as mean±SEM for serum cytokine levels and as mean±SEM fold of control for chemokine mRNA levels.* <i>p</i><0.05, ** <i>p</i><0.01, <i>n</i> = 5 for each experiment.</p

Treatment with A₁R antagonist blocked the anti-inflammatory effect of A₁R agonists.

<p>2 hours prior to administration of A₁R agonist, (A) CCPA (0.1 mg/kg) or (B) CHA (0.02 mg/kg), mice were injected with A₁R antagonist (DPCPX, 1 mg/kg) or vehicle. After 24 hours, peritonitis was induced by bacterial inoculation. At 12 hours from inoculation, IL-6 and TNFα were analyzed in sera and lavage fluids. Data represent two experiments and are expressed as mean±SEM. * <i>p</i><0.05, between vehicle and CHA or CCPA, <i>n</i> = 5 for each experiment.</p

Effect of NCLX on mitochondrial Ca²⁺ transport, metabolic rate in resting and high glucose dependent manner.

<p><b>A.</b> Knocked down of NCLX modulates mitochondrial calcium transport. Pancreatic primary β cells were infected with lenti-pericam viral particles and transfected with either siNCLX or siControl and superfused with the indicated high glucose Ringer solution. <b>Insert.</b> Representative image of pancreatic primary β cell infected with lenti-pericam. The scale bar is 10 µm. <b>B.</b> Averaged mitochondrial Ca²⁺ influx rates of pancreatic primary β cells of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g005" target="_blank">Fig. 5A</a>, n = 3 (*P<0.05). <b>C.</b> Averaged mitochondrial Ca²⁺ efflux rates of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g005" target="_blank">Fig. 5A</a>, n = 3 (*P<0.05). <b>D.</b> Effect of NCLX on respiratory chain activity determined by monitoring NAD(P)H intrinsic fluorescence in pancreatic primary β cells, transfected with either siNCLX or siControl before and after application of high glucose Ringer solution. FCCP or high glucose Ringer's solution was added where indicated.</p

NCLX is expressed in mitochondria of pancreatic β cells and mediates mitochondrial Ca²⁺ transport.

<p><b>A</b>. Immunoblot analysis of NCLX expression in total lysate and isolated mitochondria in MIN6 cells (20 µg). <b>B.</b> Immunoblot analysis of NCLX expression in siNCLX vs. siControl (20 µg) transfected MIN6 cell lysates. VDAC and β Actin were used as mitochondrial and cytosolic markers, respectively. <b>C.</b> Knock down of NCLX expression increases Ca²⁺ influx and inhibits mitochondrial Ca²⁺ efflux. At the indicated time, cells were superfused with high K⁺ Ringer solution while monitoring mitochondrial Ca²⁺ in MIN6 cells transfected with mito-pericam and either siNCLX or siControl. <b>D.</b> Dominant negative NCLX construct increases Ca²⁺ influx and inhibits mitochondrial Ca²⁺ efflux. Representative fluorescent traces of pancreatic MIN6 cells co-transfected with mito-pericam and either dnNCLX or control vector (pcDNA), while applying the same experimental paradigm described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1C</a>. <b>Insert.</b> Representative images of MIN6 cells co-transfected with mito-pericam. The scale bar is 10 µm. <b>E.</b> Averaged rates of mitochondrial Ca²⁺ influx of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1C</a>, D, n = 9 (*P<0.05). <b>F.</b> Averaged rates of mitochondrial Ca²⁺ efflux of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1C</a>, D, n = 9 (*P<0.05). <b>G.</b> Silencing of NCLX expression inhibits mitochondrial Ca²⁺ efflux following a metabotropic cytosolic Ca²⁺ response. Cells were co-transfected with mito-pericam and either siNCLX or siControl and superfused with Ca²⁺ free Ringer solution containing 50 µM ATP, while monitoring the Ca²⁺ response. <b>H.</b> Averaged rates of mitochondrial Ca²⁺ influx of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1G</a>, n = 7 (*P<0.05). <b>I.</b> Averaged rates of mitochondrial Ca²⁺ efflux of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046649#pone-0046649-g001" target="_blank">Fig. 1G</a>, n = 7 (*P<0.05).</p