Calcium-dependent release of adenosine and uridine nucleotides from A549 cells

Extracellular nucleotides play an important role in lung defense, but the release mechanism and relative abundance of different nucleotide species secreted by lung epithelia are not well defined. In this study, to minimize cell surface hydrolysis, we used a low-volume, flow-through chamber and examined adenosine and uridine nucleotide concentrations in perfusate aliquots of human lung A549 cells challenged by 50% hypotonic shock. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (Ado) were quantified in high-performance liquid chromatography (HPLC) analysis of fluorescent etheno derivatives, and uridine triphosphate (UTP) and uridine diphosphate (UDP) were measured using HPLC-coupled radioenzymatic assays. After the onset of hypotonic shock, ATP, ADP, UTP, and UDP in the perfusates increased markedly and peaked at approximately 2.5 min, followed by a gradual decay in the next 15–20 min; peak changes in Ado and AMP were relatively minor. The peak concentrations and fold increment (in parentheses) were: 34 ± 13 nM ATP (5.6), 11 ± 5 nM ADP (3.7), 3.3 ± 1.2 nM AMP (1.4), 23 ± 7 nM Ado (2.1), 21 nM UTP (>7), and 11 nM UDP (27). Nucleotide release was almost completely abolished from cells loaded with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Under isotonic conditions, elevation of intracellular calcium with the calcium ionophore ionomycin (5 μM, 3 min) also released nucleotides with kinetics and relative abundance as above, albeit less robust. ADP:ATP (1:3) and UDP:UTP (1:2) ratios in perfusates from stimulated cells were markedly higher than the cytosolic ratios of these species, suggesting that a nucleotide diphosphate (NDP)-rich compartment, e.g., the secretory pathway, contributed to nucleotide release. Laser confocal microscopy experiments illustrated increased FM1-43 uptake into the plasma membrane upon hypotonic shock or ionomycin treatment, consistent with enhanced vesicular exocytosis under these conditions. In summary, our results strongly suggest that calcium-dependent exocytosis is responsible, at least in most part, for adenosine and uridine nucleotide release from A549 cells

Multiple P2Y receptors couple to calcium-dependent, chloride channels in smooth muscle cells of the rat pulmonary artery

BACKGROUND: Uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP) act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5'-triphosphate (ATP) acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems. METHODS: The perforated-patch clamp technique was used to record the Ca(2+)-dependent, Cl(- )current (I(Cl,Ca)) activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA) and large (LPA) intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student's t test or one way ANOVA. RESULTS: ATP, UTP and UDP (10(-4)M) evoked oscillating, inward currents (peak = 13–727 pA) in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P < 0.05). Subsequent currents tended to decrease in amplitude, with a variable time-course, to a level that was significantly smaller for ATP (P < 0.05), UTP (P < 0.001) and UDP (P < 0.05) in the SPA. The frequency of oscillations was similar for each agonist (mean≈6–11.min(-1)) and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10(-4)M) abolished currents evoked by ATP in SPA (n = 4) and LPA (n = 4), but pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (10(-4)M), also a non-selective P2 antagonist, had no effect (n = 4, 5 respectively). Currents elicited by UTP (n = 37) or UDP (n = 14) were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4) and abolished by suramin (n = 5). Both antagonists abolished the contractions in LPA. CONCLUSION: At least two P2Y subtypes couple to I(Cl,Ca )in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y(11 )receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes. These receptors likely play a significant role in nucleotide-induced vasoconstriction

Vaccines against toxoplasma gondii : challenges and opportunities

Development of vaccines against Toxoplasma gondii infection in humans is of high priority, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects. Rodent models have been used in research on vaccines against T. gondii over the past decades. However, regardless of the vaccine construct, the vaccines have not been able to induce protective immunity when the organism is challenged with T. gondii, either directly or via a vector. Only a few live, attenuated T. gondii strains used for immunization have been able to confer protective immunity, which is measured by a lack of tissue cysts after challenge. Furthermore, challenge with low virulence strains, especially strains with genotype II, will probably be insufficient to provide protection against the more virulent T. gondii strains, such as those with genotypes I or II, or those genotypes from South America not belonging to genotype I, II or III. Future studies should use animal models besides rodents, and challenges should be performed with at least one genotype II T. gondii and one of the more virulent genotypes. Endpoints like maternal-foetal transmission and prevention of eye disease are important in addition to the traditional endpoint of survival or reduction in numbers of brain cysts after challenge

The R*-operation for Feynman graphs with generic numerators

Abstract The R *-operation by Chetyrkin, Tkachov, and Smirnov is a generalisation of the BPHZ R-operation, which subtracts both ultraviolet and infrared divergences of euclidean Feynman graphs with non-exceptional external momenta. It can be used to compute the divergent parts of such Feynman graphs from products of simpler Feynman graphs of lower loops. In this paper we extend the R *-operation to Feynman graphs with arbitrary numerators, including tensors. We also provide a novel way of defining infrared counterterms which closely resembles the definition of its ultraviolet counterpart. We further express both infrared and ultraviolet counterterms in terms of scaleless vacuum graphs with a logarithmic degree of divergence. By exploiting symmetries, integrand and integral relations, which the counterterms of scaleless vacuum graphs satisfy, we can vastly reduce their number and complexity. A FORM implementation of this method was used to compute the five loop beta function in QCD for a general gauge group. To illustrate the procedure, we compute the poles in the dimensional regulator of all top-level propagator graphs at five loops in four dimensional ϕ 3 theory

Kinetics of ATP release following compression injury of a peripheral nerve trunk

Compression and/or contusion of a peripheral nerve trunk can result in painful sensations. It is possible that release of ATP into the extracellular space may contribute to this symptom. In the present study, we used real-time measurements of ATP-induced bioluminescence together with electrophysiological recordings of compound action potentials to follow changes in the extracellular ATP concentration of isolated rat spinal roots exposed to mechanical stimuli. Nerve compression for about 8 s resulted in an immediate release of ATP into the extracellular space and in a decrease in the amplitude of compound action potentials. On average, a rise in ATP to 60 nM was observed when nerve compression blocked 50% of the myelinated axons. After the compression, the extracellular concentration of ATP returned to the resting level within a few minutes. The importance of ecto-nucleotidases for the recovery period was determined by exposure of isolated spinal roots to high concentrations of ATP and by use of inhibitors of ecto-nucleotidases. It was observed that spinal roots have a high capacity for ATP hydrolysis which is only partially blocked by βγ-methylene ATP and ARL 67156. In conclusion, acute nerve compression produces an increase in the extracellular concentration of ATP and of its metabolites which may be sufficient for activation of purinergic P2 and/or P1 receptors on axons of nociceptive afferent neurons

Gravitational waves from single neutron stars: an advanced detector era survey

With the doors beginning to swing open on the new gravitational wave astronomy, this review provides an up-to-date survey of the most important physical mechanisms that could lead to emission of potentially detectable gravitational radiation from isolated and accreting neutron stars. In particular we discuss the gravitational wave-driven instability and asteroseismology formalism of the f- and r-modes, the different ways that a neutron star could form and sustain a non-axisymmetric quadrupolar "mountain" deformation, the excitation of oscillations during magnetar flares and the possible gravitational wave signature of pulsar glitches. We focus on progress made in the recent years in each topic, make a fresh assessment of the gravitational wave detectability of each mechanism and, finally, highlight key problems and desiderata for future work.Comment: 39 pages, 12 figures, 2 tables. Chapter of the book "Physics and Astrophysics of Neutron Stars", NewCompStar COST Action 1304. Minor corrections to match published versio

A card game for the treatment of delusional ideas: A naturalistic pilot trial

BACKGROUND: "Michael's game" is a card game which aims at familiarizing healthcare professionals and patients with cognitive behavioral therapy of psychotic symptoms. This naturalistic study tests the feasibility and the impact of the intervention in various naturalistic settings. METHOD: Fifty five patients were recruited in seven centers. They were assessed in pre and post-test with the Peters Delusion Inventory – 21 items (PDI-21). RESULTS: Forty five patients completed the intervention significantly reducing their conviction and preoccupation scores on the PDI-21. CONCLUSION: This pilot study supports the feasibility and effectiveness of "Michael's game" in naturalistic setting. Additional studies could validate the game in a controlled fashion