ATP In Mice Research Articles

Psychological stress induced bladder overactivity in female mice is associated with enhanced afferent nerve activity

Psychological stress has been linked to the development and exacerbation of overactive bladder symptoms, as well as afferent sensitisation in other organ systems. Therefore, we aimed to investigate the effects of water avoidance stress on bladder afferent nerve activity in response to bladder filling and pharmaceutical stimulation with carbachol and ATP in mice. Adult female C57BL/6J mice were exposed to either water avoidance stress (WAS) for 1 h/day for 10 days or normal housing conditions. Voiding behaviour was measured before starting and 24-h after final stress exposure and then animals were euthanised to measure afferent nerve activity in association with bladder compliance, spontaneous phasic activity, contractile responses, as well as release of urothelial mediators. WAS caused increased urinary frequency without affecting urine production. The afferent nerve activity at low bladder pressures (4–7 mmHg), relevant to normal physiological filling, was significantly increased after stress. Both low and high threshold nerves demonstrated enhanced activity at physiological bladder pressures. Urothelial ATP and acetylcholine release and bladder compliance were unaffected by stress as was the detrusor response to ATP (1 mM) and carbachol (1 µM). WAS caused enhanced activity of individual afferent nerve fibres in response bladder distension. The enhanced activity was seen in both low and high threshold nerves suggesting that stressed animals may experience enhanced bladder filling sensations at lower bladder volumes as well as increased pain sensations, both potentially contributing to the increased urinary frequency seen after stress.

Pannexin 1 channels control the hemodynamic response to hypoxia by regulating O2-sensitive extracellular ATP in blood.

Pannexin 1 (Panx1) channels export ATP and may contribute to increased concentration of the vasodilator ATP in plasma during hypoxia in vivo. We hypothesized that Panx1 channels and associated ATP export contribute to hypoxic vasodilation, a mechanism that facilitates the matching of oxygen delivery to metabolic demand of tissue. Male and female mice devoid of Panx1 (Panx1-/-) and wild-type controls (WT) were anesthetized, mechanically ventilated, and instrumented with a carotid artery catheter or femoral artery flow transducer for hemodynamic and plasma ATP monitoring during inhalation of 21% (normoxia) or 10% oxygen (hypoxia). ATP export from WT vs. Panx1-/-erythrocytes (RBC) was determined ex vivo via tonometer experimentation across progressive deoxygenation. Mean arterial pressure (MAP) was similar in Panx1-/- (n = 6) and WT (n = 6) mice in normoxia, but the decrease in MAP in hypoxia seen in WT was attenuated in Panx1-/- mice (-16 ± 9% vs. -2 ± 8%; P < 0.05). Hindlimb blood flow (HBF) was significantly lower in Panx1-/- (n = 6) vs. WT (n = 6) basally, and increased in WT but not Panx1-/- mice during hypoxia (8 ± 6% vs. -10 ± 13%; P < 0.05). Estimation of hindlimb vascular conductance using data from the MAP and HBF experiments showed an average response of 28% for WT vs. -9% for Panx1-/- mice. Mean venous plasma ATP during hypoxia was 57% lower in Panx1-/- (n = 6) vs. WT mice (n = 6; P < 0.05). Mean hypoxia-induced ATP export from RBCs from Panx1-/- mice (n = 8) was 82% lower than that from WT (n = 8; P < 0.05). Panx1 channels participate in hemodynamic responses consistent with hypoxic vasodilation by regulating hypoxia-sensitive extracellular ATP levels in blood.NEW & NOTEWORTHY Export of vasodilator ATP from red blood cells requires pannexin 1. Blood plasma ATP elevations in response to hypoxia in mice require pannexin 1. Hemodynamic responses to hypoxia are accompanied by increased plasma ATP in mice in vivo and require pannexin 1.

271 - Emerging role of glutathione in the activation of NLRP3 inflammasome

NLRP3 inflammasome is an intracellular protein complex, which is responsible for the maturation of precursor forms of interleukin (IL)-1β and IL-18 into active proinflammatory cytokines and initiation of pyroptotic cell death. Although generation of reactive oxygen species (ROS) has been implicated in the activation of NLRP3 inflammasome, detail mechanism how it is regulated remains unclear. Here we describe that glutathione (GSH), an antioxidant peptide abundantly present in cells, plays an important regulatory role in ATP-dependent inflammasome activation. In LPS-primed J774.1 cells, treatment of cells with ATP induced rapid GSH decline, which led to cellular redox imbalance with concomitant generation of ROS. Similar level of GSH were detected in the culture supernatants suggesting that ATP stimulated GSH efflux. Moreover, ATP treatment also induced efflux of polysulfurated derivatives of GSH such as GSSH and GSSSH. P2X7 receptor antagonist A-804598 treatment remarkably inhibited both IL-1β production and GSH efflux. Importantly, the activation of NLRP3 inflammasome in J774.1 cells was significantly suppressed by adding GSH or its oxidized form GSSG extracellularly, as assessed by IL-1β production. Interestingly, LC-MS/MS data showed that addition of GSH attenuated ATP-induced GSH efflux and induced remarkable increase in persulfide levels of the cell. Immunoprecipitation data showed that addition of GSH or GSSG extracellularly was negatively regulates the NLRP3 inflammasome complex formation. Furthermore, GSH reduced IL-1β production induced by LPS and ATP in mice. Our findings represent a novel model for NLRP3 inflammasome activation by GSH efflux-mediated ROS generation.

Use of luciferase probes to measure ATP in living cells and animals.

ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.

P–404 - Mechanisms of hyplralgesia induced by intrathecal administration of ATP analogue α, β-methylene ATP in mice.

P–404 - Mechanisms of hyplralgesia induced by intrathecal administration of ATP analogue α, β-methylene ATP in mice.

Sequence of a cDNA encoding mouse F1F0-ATP synthase g subunit.

A pregnant-induced clone was identified by differential screening from a cDNA library of mouse mammary gland. The clone was identified as a full-length cDNA encoding the F1F0-ATP synthase g subunit. Comparison of the deduced amino acid sequences of mouse ATP synthase g subunit with those of bovine species showed 86% identity. The high levels of ATP synthase g subunit mRNA were detected in heart and uterine tissues.

P-181 - Effect of stress on the plasma levels of catecholamines and ATP in mice

P-181 - Effect of stress on the plasma levels of catecholamines and ATP in mice