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While long-acting injectable treatments are gaining increasing interest in managing chronic diseases, the available drug delivery systems almost exclusively rely on hydrophobic matrixes, limiting their application to either hydrophobic drugs or large and hydrophilic molecules such as peptides. To address the technological lock for long-acting delivery systems tailored to small, hydrophilic drugs such as anticancer and antiviral nucleoside/nucleotide analogues, we have synthesized and characterized an original approach with a multi-scale structure: (i) a nucleotide (adenosine triphosphate, ATP) is first incorporated in hydrophilic chitosan-Fe(III) nanogels; (ii) these nanogels are then transferred by freeze-drying and resuspension into a water-free, hydrophobic medium containing PLGA and an organic solvent, N-methyl-2-pyrrolidone. We show that this specific association allows an injectable and homogeneous dispersion, able to form in situ implants upon injection in physiological or aqueous environments. This system releases ATP in vitro without any burst effect in a two-step mechanism, first as nanogels acting as an intermediate reservoir over a week, then as free drug over several weeks. In vivo studies confirmed the potential of such nanostructured implants for sustained drug release following subcutaneous injection to mice hock, opening perspectives for sustained and targeted delivery through the lymphatic system., (© 2024. Controlled Release Society.)

Introduction: Adenosine Triphosphate is a molecule with the function of generating energy, where high levels are directly related to cellular and muscular health. Oral supplementation appears as a possibility to guarantee such levels, and is normally consumed in capsules, which can be acute or chronic., Objectives: To verify the influence of a dose of ATP on muscular performance in adults., Methods: This is an acute, crossover, randomized, double-blind, placebo-controlled intervention study. 18 trained men were recruited, with an average age of 27.95 years. Two visits were made to the laboratory, where each of the protocols consisted of randomized supplementation of 400 mg of Peak ATP® or placebo, and 30 min later, five series were performed, where the first consisted of five repetitions and the following four of ten repetitions at 60° per second in knee extension and flexion on a Biodex® 4.0 isokinetic dynamometer, where the volunteers' muscle strength and resistance to fatigue were measured. For data analysis, data normality was assessed using Shapiro Wilk, the ANOVA repeated measures test with Bonferroni post hoc. To identify the size of the effect, the Cohen test was performed, and the statistical package used was SPSS 25 with an applied significance of p  < 0.05., Results: Acute supplementation was unable to achieve significant improvements in muscle strength indicators. The supplement delays drops in strength as the exercise progresses in knee extension compared to the placebo., Conclusions: An acute dose of 400 mg of ATP did not improve the volunteers' muscle strength indicators, but it was able to reduce fatigue levels as the exercise progressed, enabling greater performance for longer.

Calcium-dependent, neuronal adenylyl cyclase subtype 1 (AC1) is critical for cortical potentiation and chronic pain. NB001 is a first-in-class drug acting as a selective inhibitor against AC1. The present study delineated the pharmacokinetic (PK) properties of human-used NB001 (hNB001) formulated as immediate-release tablet. This first-in-human (FIH) study was designed as randomized, double-blind, placebo-controlled trial. hNB001 showed placebo-like safety and good tolerability in healthy volunteers. A linear dose-exposure relationship was demonstrated at doses between 20 mg and 400 mg. The relatively small systemic exposure of hNB001 in human showed low bioavailability of this compound through oral administration, which can be improved through future dosage research. Food intake had minimal impact on the absorption of hNB001 tablet. Animal experiments further confirmed that hNB001 had strong analgesic effect in animal models of neuropathic pain. In brain slice prepared from the anterior cingulate cortex (ACC), bath application of hNB001 blocked the induction of long-term potentiation (LTP). These results from both rodents and human strongly suggest that hNB001 can be safely used for the future treatment of different types of chronic pain in human patients.

Objective: The aim: This study was carried out to examine the presence of P2Y4 receptors in rat epidermal tissue and how their in vivo activation leads to histological and genetic changes., Patients and Methods: Materials and methods: Thirty-six Wistar rats were separated into six groups each of six rats, the control group and five injected groups with increasing concentrations of ATP intradermally (0.1, 5.0, 10.0, 50.0, 100.0 μg/ml). The histological and genetic examination was performed from excised tissues., Results: Results: Noticeable histological thickening of the epidermal layer in rats injected with high concentrations of ATP. No apparent histological damage was seen in all injected groups. The genetic expression seems to also increase following exposure to variable concentrations of ATP., Conclusion: Conclusions: Purinergic receptors activated by ATP molecules are highly involved in the development of adult tissues. Their precise location within the epidermal layer indicated their importance in cellular proliferation and differentiation of epidermal cells. Excessive exposure to ATP results in their robust genetic ectopic over expression indicative of increased cellular activity.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disorder that leads to progressive degeneration of motor neurons and severe muscle atrophy without effective treatment. Most research on the disease has been focused on studying motor neurons and supporting cells of the central nervous system. Strikingly, the recent observations have suggested that morpho-functional alterations in skeletal muscle precede motor neuron degeneration, bolstering the interest in studying muscle tissue as a potential target for the delivery of therapies. We previously showed that the systemic administration of the P2XR7 agonist, 2'(3')-O-(4-benzoylbenzoyl) adenosine 5-triphosphate (BzATP), enhanced the metabolism and promoted the myogenesis of new fibres in the skeletal muscles of SOD1G93A mice. Here we further corroborated this evidence showing that intramuscular administration of BzATP improved the motor performance of ALS mice by enhancing satellite cells and the muscle pro-regenerative activity of infiltrating macrophages. The preservation of the skeletal muscle retrogradely propagated along with the motor unit, suggesting that backward signalling from the muscle could impinge on motor neuron death. In addition to providing the basis for a suitable adjunct multisystem therapeutic approach in ALS, these data point out that the muscle should be at the centre of ALS research as a target tissue to address novel therapies in combination with those oriented to the CNS., (© 2021. The Author(s).)

The nervous system displays high energy consumption, apparently not fulfilled by mitochondria, which are underrepresented therein. The oxidative phosphorylation (OxPhos) activity, a mitochondrial process that aerobically provides ATP, has also been reported also in the myelin sheath and the rod outer segment (OS) disks. Thus, commonalities and differences between the extra-mitochondrial and mitochondrial aerobic metabolism were evaluated in bovine isolated myelin (IM), rod OS, and mitochondria-enriched fractions (MIT). The subcellular fraction quality and the absence of contamination fractions have been estimated by western blot analysis. Oxygen consumption and ATP synthesis were stimulated by conventional (pyruvate + malate or succinate) and unconventional (NADH) substrates, observing that oxygen consumption and ATP synthesis by IM and rod OS are more efficient than by MIT, in the presence of both kinds of respiratory substrates. Mitochondria did not utilize NADH as a respiring substrate. When ATP synthesis by either sample was assayed in the presence of 10-100 µM ATP in the assay medium, only in IM and OS it was not inhibited, suggesting that the ATP exportation by the mitochondria is limited by extravesicular ATP concentration. Interestingly, IM and OS but not mitochondria appear able to synthesize ATP at a later time with respect to exposure to respiratory substrates, supporting the hypothesis that the proton gradient produced by the electron transport chain is buffered by membrane phospholipids. The putative transfer mode of the OxPhos molecular machinery from mitochondria to the extra-mitochondrial structures is also discussed, opening new perspectives in the field of neurophysiology., (© 2021 Wiley Periodicals LLC.)

The pore-forming toxin streptolysin-O (SLO) enables intracellular delivery of molecules up to 100 kDa and has been used for short-term delivery of membrane-impermeable substances to assess their effects on cellular activities. A limitation of this technique is the loss of intracellular components and the potential unpredicted alterations of cellular metabolism and signaling. This protocol, optimized for primary mouse T lymphocytes, describes steps for SLO-mediated cell membrane permeabilization and substance supplementation, followed by immunoblotting and immunofluorescent microscopy for assessing cellular effects. For complete details on the use and execution of this protocol, please refer to Xu et al., 2021a, Xu et al., 2021b., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.

Aim: Whole dead tumor cells can be used as antigen source and the induction of protective immune response could be enhanced by damage-associated molecular patterns. Materials & methods: We generated whole dead tumor cells called B16-immunogenic cell bodies (ICBs) from B16 melanoma cells by nutrient starvation and evaluated the in vivo antitumor effect of B16-ICBs plus ATP and polymyxin B (PMB). Results: The subcutaneous immunization with B16-ICBs + PMB + ATP a 50% of tumor-free animals and induced a significant delay in tumor growth in a prophylactic approach. These results correlated with maturation of bone marrow-derived dendritic cells and activation of T CD8 + lymphocytes in vitro . Conclusion: Altogether, ICB + ATP + PMB is efficient in inducing the antitumor efficacy of the whole dead tumor cells vaccine.

Background: This study evaluated whether caffeine abstention is required before fractional flow reserve (FFR) measurement by intravenous adenosine triphosphate (ATP) administration in Japanese patients., Methods: This study was a subanalysis of a previously published study and a total of 208 intermediate lesions that underwent FFR measurements were enrolled for this analysis. Hyperemia was induced by continuous intravenous ATP infusion at 150 μg/kg/min (IVATP150) and 210 μg/kg/min (IVATP210), and by intracoronary administration of nicorandil 2 mg (ICNIC2mg) as a reference standard., Results: The degree of change in the FFR value after ICNIC2mg and IVATP210 was similar between the caffeine and non-caffeine groups (0.00 ± 0.02 vs. 0.01 ± 0.02). In patients who consumed caffeine before the FFR measurement, the degree of FFR change was independent of the time interval (<12 h, 12-24 h, and 24-48 h) between caffeine intake and catheterization both after IVATP150 and ICNIC2mg and after IVATP210 and ICNIC2mg., Conclusion: When compared with the FFR value after ICNIC2mg, the degree of change in the FFR value after IVATP210 were similar regardless of caffeine intake. Strict caffeine abstention before intravenous ATP-induced FFR measurement may not be required in clinical practice., (Copyright © 2020. Published by Elsevier Ltd.)

Background: Myocardial perfusion single-photon emission computed tomography (SPECT) imaging with stress is a useful examination for detecting coronary artery disease. Since the presence of artifacts is remaining challenges, we aimed to define the minimum intensity of low-grade exercise stress levels combined with drug stress to reduce undesired artifacts and their related factors., Methods: We divided patients with suspicious coronary artery disease into 4 groups as follows: group A, adenosine triphosphate (ATP) for 6 min; group A + 25 W, ATP + 25 W exercise for 6 min; group A + 35 W, ATP + 35 W exercise for 6 min; group A + 45 W, ATP + 45 W exercise for 6 min) and enrolled only those whose summed stress scores were < 3. Undesired artifacts were evaluated on the basis of heart-to-liver activity (H/L) ratio and heart-to-10 pixels below the heart (H/below the H) ratio., Results: The logarithmic values of H/L and H/below the H ratios were significantly higher in groups A + 35 W and A + 45 W than in group A (p < 0.05, each). In all the patients, the logarithmic values of H/L and H/below the H ratios positively correlated with the increment of rate pressure product (RPP, p = 0.002 and p = 0.005, respectively) after stress in the univariate analysis. The left ventricular end-diastolic volume (LVEDV) after stress (p = 0.002) negatively correlated with the logarithmic value of H/below the H ratio, but not H/L ratio. Although the increment of RPP was independently associated with the logarithmic values of both H/L (p = 0.001) and H/below the H ratios (p = 0.005), LVEDV was also independently associated with the logarithmic value of H/below the H ratio (p < 0.001) in multivariate regression analysis under adjusting with age and sex., Conclusion: ATP plus ≥35 W exercise stress for 6 min was useful for reducing undesired artifacts after stress in myocardial perfusion SPECT. LVEDV after stress in addition to the increment of RPP was independently associated with the H/below the H ratio, but not the H/L ratio.

Background: The methods for calculating the optimal myocardial blood flow (MBF) relative parameters in stress dynamic myocardial CT perfusion (CTP) in the detection of hemodynamically significant coronary artery disease (CAD) are non-uniform and lack standards., Methods: A total of 86 patients who were prospectively recruited underwent APT stress dynamic myocardial CTP. The relative MBF perfusion parameters were calculated as av&#95;Ratio, Q3av&#95;Ratio and hi&#95;Ratio according to the three types of reference MBF values, respectively: (1) average segmental MBF value, (2) the third quartile of the average segmental MBF value, and (3) highest segmental MBF value. All the data were derived from both the endocardial and transmural layers of the myocardium. Invasive coronary angiography and fractional flow reserve (ICA/FFR) were used as the reference standards for myocardial ischemia evaluation., Results: A total of 151 vessels of 60 patients (43 men and 17 women; 61.38 ± 8.01 years) were enrolled in the analysis. The performance of the endocardial layer was superior to that of the transmural layer (all P < 0.05). The hi&#95;Ratio of the endocardial myocardium (AUC = 0.906, 95% CI: 0.857-0.954), for which the highest segmental value was selected as the reference MBF, was superior to both av&#95;Ratio and Q3av&#95;Ratio for ischemia detection (AUC, 0.906 vs.0.879, P < 0.05; 0.906 vs.0.891, P = 0.18), and the sensitivity, specificity, PPV, NPV and diagnostic accuracy were 74.1%, 93.6%, 87.8%, 85.3% and 86.1%, respectively. The cutoff value of hi&#95;Ratio was 0.675., Conclusions: The relative MBF parameter of the endocardial myocardium using the highest segmental MBF value as a reference provided optimal diagnostic accuracy for the detection of hemodynamically significant CAD., Competing Interests: Declaration of competing interest All the other authors have no conflict of interest to disclose., (Copyright © 2020 [The Author/The Authors]. Published by Elsevier Inc. All rights reserved.)

Chronic pain is a major health problem and the effective treatment for chronic pain is still lacking. The recent crisis created by the overuse of opioids for pain treatment has clearly shown the need for non-addictive novel pain medicine. Conventional pain medicines usually inhibit peripheral nociceptive transmission and reduce central transmission, especially pain-related excitatory transmission. For example, both opioids and gabapentin produce analgesic effects by inhibiting the release of excitatory transmitters and reducing neuronal excitability. Here, we will review recent studies of central synaptic plasticity contributing to central sensitization in chronic pain. Neuronal selective adenylyl cyclase subtype 1 (AC1) is proposed to be a key intracellular protein that causes both presynaptic and postsynaptic forms of long-term potentiation (LTP). Inhibiting the activity of AC1 by selective inhibitor NB001 blocks behavioral sensitization and injury-related anxiety in animal models of chronic pain. We propose that inhibiting injury-related LTPs will provide new mechanisms for designing novel medicines for the treatment of chronic pain and its related emotional disorders.

We hypothesized that in patients with QT prolongation, resistance might not decrease in the wave-free period, because QTU prolongation cannot be detected by instantaneous wave-free ratio (iFR) analysis software. We investigated whether corrected QTU (QTUc) prolongation affects the hyperemic iFR value. Forty-two consecutive patients with intermediate stenosis (≥ 50%) in the left anterior descending coronary artery (LAD) were analyzed. Fractional flow reserve (FFR) and hyperemic iFR were simultaneously and continuously recorded with intravenous adenosine triphosphate (ATP) and papaverine infusions. In 17 patients with stenosis in the proximal LAD, coronary flow was measured. Patients were divided into two groups according to the median absolute deviation of the QTUc by ATP administration/QTUc by papaverine administration. FFR, hyperemic iFR, and flow data were compared between each stimulus and group. Moreover, influences of pressure and electrocardiogram parameters on differences in iFR values under ATP and papaverine administration were compared between the following two groups (group 1: the absolute difference of hyperemic iFR values between ATP and papaverine administration is ≤ 0.05; group 2: that is > 0.05). The paired t test and t test were used in analysis. Hyperemic iFR values of patients under the use of papaverine were lower than those of patients under the use of ATP when QTUc was more prolonged by papaverine administration than by ATP administration (ATP 0.74 ± 0.14, papaverine 0.71 ± 0.15, P = 0.025). No significant differences were observed in the FFR value and flow data between the groups. Regarding QTU, QTUc, and QTUc by ATP/QTUc by papaverine, significant differences were observed between group 1 and group 2. Pressure parameters did not induce significant differences. QTUc prolongation induced by papaverine was associated with lower hyperemic iFR values. An iFR-based assessment might lead to inappropriate treatment of patients with QTUc prolongation.

Background: The term vascular anomalies include various vascular tumors and vascular malformations, among them infantile hemangiomas and capillary malformations are the most well-known associated diseases in early ages. Multiple drugs have been introduced for intervention, but susceptibility test in vitro were scarcely reported., Objective: To evaluate the inhibition effect of different drugs by adenosine triphosphate sensitivity assay in vitro before the treatment of infantile hemangiomas and capillary malformations., Methods: Specimens were selected from 5 cases of infantile hemangiomas and 11 cases of capillary malformations. Propranolol, rapamycin, sildenafil and itraconazole were tested for their growth inhibition effect by using the adenosine triphosphate sensitivity assay., Results: Propranolol demonstrated inhibitory effects on infantile hemangiomas cells. Rapamycin and itraconazole both showed inhibitory effects on infantile hemangiomas cells and capillary malformations cells. Sildenafil has no growth inhibitory effect on infantile hemangiomas cells or capillary malformations cells., Conclusion: Adenosine triphosphate sensitivity assay is a sensitive and useful testing method before the management of vascular anomalies, and individualized medication suggestions for the choice of therapeutic drugs were offered based on the testing result and together with a comprehensive evaluation of each infant.

trans-Fatty acids (TFAs) are unsaturated fatty acids with at least one carbon-carbon double bond in trans configuration. TFA consumption has been epidemiologically associated with neurodegenerative diseases (NDs) including Alzheimer's disease. However, the underlying mechanisms of TFA-related NDs remain unknown. Here, we show a novel microglial signaling pathway that induces inflammation and cell death, which is dramatically enhanced by elaidic acid (EA), the most abundant TFA derived from food. We found that extracellular ATP, one of the damage-associated molecular patterns (DAMPs) leaked from injured cells, induced activation of the apoptosis signal-regulating kinase 1 (ASK1)-p38 pathway, which is one of the major stress-responsive mitogen-activated protein (MAP) kinase signaling pathways, and subsequent caspase-3 cleavage and DNA ladder formation (hallmarks of apoptosis) in mouse microglial cell lines including BV2 and MG6 cells. Furthermore, we found that in these microglial cell lines, EA, but not its cis isomer oleic acid, facilitated extracellular ATP-induced ASK1/p38 activation and apoptosis, which was suppressed by pharmacological inhibition of either p38, reactive oxygen species (ROS) generation, P2X purinoceptor 7 (P2X 7 ), or Ca 2+ /calmodulin-dependent kinase II (CaMKII). These results demonstrate that in microglial cells, extracellular ATP induces activation of the ASK1-p38 MAP kinase pathway and ultimately apoptosis downstream of P2X 7 receptor and ROS generation, and that EA promotes ATP-induced apoptosis through CaMKII-dependent hyperactivation of the ASK1-p38 pathway, in the same manner as in macrophages. Our study may provide an insight into the pathogenesis of NDs associated with TFAs.

New Findings: What is the central question of this study? What is the effect of an elevated baseline blood flow, induced by high-dose intra-arterial infusion of either adenosine or ATP, on the rapid-onset vasodilatory response to a single forearm muscle contraction? What is the main finding and its importance? The peak response to a single contraction is unaffected by augmented baseline blood flow, and thus, is likely to be attributable to a feedforward vasodilatory mechanism., Abstract: The hyperaemic responses to single muscle contractions are proportional to exercise intensity, which, in turn, is proportional to tissue metabolic demand. Hence, we tested the hypothesis that the rapid-onset vasodilatory response after a single muscle contraction would be unaffected when baseline blood flow was increased via high-dose intra-arterial infusion of either adenosine (ADO) or ATP. Twenty-four healthy young participants (28 ± 1 years) performed a single forearm contraction (20% maximal voluntary contraction) 75 min after commencement of a continuous infusion of ADO (n = 6), ATP (n = 8) or saline (control; n = 10). Brachial artery diameter and blood velocity were measured using Doppler ultrasound. Resting forearm vascular conductance (FVC; in millilitres per minute per 100 mmHg per decilitre of forearm volume) was significantly higher during ADO (33 ± 17) and ATP infusion (33 ± 17) compared with the control infusion (8 ± 3; P < 0.05). The peak FVCs post-contraction during ADO and ATP infusions were significantly greater than during the control infusion (P < 0.05), but not different from one another. The peak change in FVC from baseline was similar in all three conditions (control, 14 ± 1; ADO, 24 ± 2; and ATP, 23 ± 6; P = 0.15). Total FVC (area under the curve) did not differ significantly between ADO and ATP (333 ± 69 and 440 ± 125); however, total FVC during ATP infusion was significantly greater compared with the control value (150 ± 19; P < 0.05). We conclude that the peak response to a single contraction is unaffected by augmented baseline blood flow and is therefore likely to be attributable to a feedforward vasodilatory mechanism., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

Background: Papaverine is used to induce maximal hyperemia for index of coronary microcirculatory resistance (IMR) measurement in animal experiments, although it can lead to polymorphic ventricular tachycardia and ventricular fibrillation., Objectives: This study investigated the effect of an intracoronary (IC) bolus of high adenosine triphosphate (ATP) and nicorandil doses for IMR measurement and explored the possibility of inducing maximal hyperemia with an IC alprostadil bolus., Material and Methods: Index of coronary microcirculatory resistance was measured in a hyperemic state induced by 7 experimental conditions in 21 pigs (IC bolus of papaverine (18 mg), ATP (40 μg, 80 μg, 160 μg, and 240 μg), and nicorandil (2 mg and 4 mg)). The 7 conditions were induced sequentially, and the average IMR was calculated. Because of the long-term hyperemic condition in the pilot experiments, the IMR was measured 1, 3, 5, 8, and 10 min after an IC bolus of alprostadil (10 μg) in another 7 pigs., Results: The IMR induced by 240 μg of ATP or 4 mg of nicorandil was not significantly different from that induced by 18 mg of papaverine (both p > 0.05). A strong linear correlation was observed between IMRs with papaverine (18 mg) and nicorandil (4 mg) (R2 = 0.936, p < 0.001) and with papaverine (18 mg) and ATP (240 μg) (R2 = 0.838, p < 0.05). The IC bolus of nicorandil (4 mg) produced the smallest changes, whereas papaverine caused the most significant changes in mean blood pressure and heart rate (p < 0.05). Tachypnea and transient ST depression were more common with increasing ATP dosages (especially 240 μg). Alprostadil (5 min) yielded a significant hyperemic response but reduced baseline blood pressure by almost 40% for a long time., Conclusions: Intracoronary bolus administration of 4 mg of nicorandil was better than 18 mg of papaverine or 240 μg of ATP for induction of maximal hyperemia and IMR measurement in a pig model, whereas alprostadil was not suitable for IMR measurement.

Exogenous adenosine triphosphate (ATP) treatment at 0, 250, 500, 750, and 1000 µM retarded cap browning in mushrooms by 0, 34, 26, 51 and 32 %, respectively, during storage at 4 °C for 18 days. Triggering signaling H 2 O 2 accumulation arising from elevating NADPH oxidase enzyme activity during 6 days of storage at 4 °C may be pivotal for promoting shikimate dehydrogenase enzyme activity in mushrooms treated with ATP during 18 days of storage at 4 °C. Promoting melatonin accumulation (390 µg kg -1 FW vs. 160 µg kg -1 FW) in mushrooms treated with ATP during cold storage may attribute to signaling H 2 O 2 accumulation. Higher DPPH scavenging capacity (72 % vs. 65 %) in mushrooms treated with ATP may attribute to higher phenols accumulation arising from higher phenylalanine ammonialyase/polyphenol oxidase enzymes activity concomitant with higher alternative oxidase gene expression during 18 days of storage at 4 °C., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Intracoronary (IC) administration of nicorandil has been proposed as an alternative choice of hyperemic agent for fractional flow reserve (FFR) measurements. This study evaluated the utility and validity of IC nicorandil administration alone to induce maximal hyperemia.Methods and Results:Two-hundred-seven patients with coronary artery disease listed for coronary angiography with FFR were prospectively enrolled. FFR was measured after (1) IC administration of nicorandil 2 mg (ICNIC2 mg); (2) continuous intravenous (IV) adenosine triphosphatase (ATP) infusion at 150 μg/kg/min (IVATP150); (3) IV ATP infusion at 210 μg/kg/min (IVATP210); (4) IC administration of 0.5 mg nicorandil during IVATP150 (ICNIC0.5 mg+IVATP150); (5) IC administration of 1 mg nicorandil during IVATP150 (ICNIC1 mg+IVATP150); and (6) IC administration of 2 mg nicorandil during IVATP150 (ICNIC2 mg+IVATP150). The average FFR values and the rate of achieving maximum hyperemia after ICNIC2 mg, IVATP150, IVATP210, ICNIC0.5 mg+IVATP150, ICNIC1 mg+IVATP150, and ICNIC2 mg+IVATP150 were 0.85±0.08, 0.89±0.08, 0.85±0.09, 0.84±0.08, 0.83±0.08, 0.83±0.08 (P<0.01), and 92%, 54%, 91%, 96%, 99%, 99% (P<0.01), respectively. The incidence of systolic aortic pressure drop, chest discomfort, and transient atrioventricular block increased in a dose-dependent manner after IV ATP infusion, but almost no adverse effects were observed after ICNIC2 mg., Conclusions: ICNIC2 mg produced a more pronounced hyperemia than continuous IV ATP, and might be the preferred method for assessment of FFR.

Sterile inflammation is a key determinant of myocardial reperfusion injuries. It participates in infarct size determination in acute myocardial infarction and graft rejection following heart transplantation. We previously showed that P2Y11 exerted an immunosuppressive role in human dendritic cells, modulated cardiofibroblasts' response to ischemia/reperfusion in vitro and delayed graft rejection in an allogeneic heterotopic heart transplantation model. We sought to investigate a possible role of P2Y11 in the cellular response of cardiomyocytes to ischemia/reperfusion. We subjected human AC16 cardiomyocytes to 5 h hypoxia/1 h reoxygenation (H/R). P2Y11R (P2Y11 receptor) selective agonist NF546 and/or antagonist NF340 were added at the onset of reoxygenation. Cellular damages were assessed by LDH release, MTT assay and intracellular ATP level; intracellular signaling pathways were explored. The role of P2Y11R in mitochondria-derived ROS production and mitochondrial respiration was investigated. In vitro H/R injuries were significantly reduced by P2Y11R stimulation at reoxygenation. This protection was suppressed with P2Y11R antagonism. P2Y11R stimulation following H 2 O 2 -induced oxidative stress reduced mitochondria-derived ROS production and damages through PKCε signaling pathway activation. Our results suggest a novel protective role of P2Y11 in cardiomyocytes against reperfusion injuries. Pharmacological post-conditioning targeting P2Y11R could therefore contribute to improve myocardial ischemia/reperfusion outcomes in acute myocardial infarction and cardiac transplantation.

Background: Recent studies have demonstrated that coronary microcirculation dysfunction (CMVD) is closely correlated with adverse clinical outcomes. In this study, quantitative stress myocardial contrast echocardiography (MCE) was used to evaluate the CMVD and to investigate its association with the prognosis of patients with nonobstructive coronary artery disease (CAD)., Material and Methods: From 2006 to 2014, 227 consecutive patients with chest pain and a diagnostic coronary angiography without significant coronary artery stenosis (<50%) who underwent adenosine triphosphate disodium (ATP) stress MCE were enrolled. Quantitative MCE measurements were analyzed using replenishment curves., Results: Median follow-up time of this study was 5.3 years. Predictors of impaired coronary flow reserve (CFR) were smoking, diabetes, high apolipoprotein B, high low-density lipoprotein, serum uric acid, and low apolipoprotein A. During follow-up, 22 patients were reported to have 30 cardiac events (21 unstable angina, 3 nonfatal myocardial infarctions, 6 percutaneous coronary interventions). Using multivariate analysis, abnormal β reserve (≤1.6), impaired CFR (≤2.0), and diabetes were independent predictors of primary endpoint events in patients with nonobstructive CAD (P < .05). Multivariate analysis showed that CFR ≤2.0 (odds ratio [OR] =  25.21, 95% confidence interval [CI]: 3.01-182.32; P = .003), β reserve ≤1.6 (OR = 29.96, 95% CI: 3.5-241.27; P = .002), and diabetic (OR = 33.11, 95% CI: 3.65-300.02; P = .002) significantly increased the risk of the primary endpoint events., Conclusions: ATP stress quantitative MCE is a feasible and effective method to evaluate microcirculation abnormalities in human coronary arteries and it can be used for the clinical analysis, risk stratification, and treatment of early CAD.

Introduction: Adenosine triphosphate (ATP)-provoked dormant conduction (DC) and pacing for unexcitability are used to identify conduction gaps along the ablation lines after circumferential pulmonary vein isolation (CPVI). We aim to determine whether ATP provocation and pacing are interchangeable as endpoints for ablation of paroxysmal atrial fibrillation (PAF)., Methods and Results: A total of 107 patients with PAF were randomly divided into two groups after completion of CPVI. In group I (A-P group, n = 53), ATP was administered first. If DC was uncovered, additional ablation was performed until ATP tests were negative. Bipolar pacing along the ablation line was performed subsequently. In group II (P-A group, n = 54), the same protocol was used, but the pacing and the ATP tests were performed in the opposite sequence. The 12-month ablation outcomes of all patients were compared with those of a historical control group of 107 patients with PAF in whom only ATP test was performed. Regardless of which test was performed first, the other modality still identified conduction gaps. In group I, pacing maneuvers identified gaps in 49% (n = 26) of patients who had negative ATP tests. In group II, ATP tests uncovered DC in 18.5% (n = 10) of patients in whom pacing identified no gaps. After 12 months, a higher proportion of patients (91.6%) were free from atrial tachyarrhythmias compared with the historical control group (81.3%; P = 0.031)., Conclusion: Pacing along the ablation lines and ATP provocation are complementary tests for evaluating the durability of CPVI and can lead to better long-term outcomes when used in combination., (© 2019 Wiley Periodicals, Inc.)

We report a case of atypical fast-slow atrioventricular nodal reentrant tachycardia (AVNRT) using a slow pathway variant extending to the superoanterior right atrium. The AVNRT diagnosis was confirmed by using standard electrophysiological criteria that exclude a diagnosis of atrial tachycardia and atrioventricular reentrant tachycardia. The earliest atrial activation during tachycardia was found in the superoanterior right atrium adjacent to the tricuspid annulus, where the first delivery of radiofrequency energy terminated and eliminated the inducibility of the tachycardia.

To evaluate the vasodilator effect of adenosine triphosphate (ATP) compared with adenosine in stress perfusion cardiac magnetic resonance (CMR) examinations. A total of thirty-three patients underwent clinically indicated stress/rest perfusion CMR examination following intravenous injection of a total dose of 0.2 mmol/kg of gadobutrol. Individuals were randomly assigned to ATP (160 mcg/kg/min) or adenosine (140 mcg/kg/min). The vasodilator effect of both drugs was analyzed by comparing differences in heart rate, symptoms during stress, and semiquantitative myocardial and splenic perfusion parameters, including time, time to peak, upslope, myocardial perfusion reserve index, tissue perfusion values, splenic and myocardial signal intensity ratios, and splenic-to-myocardial signal intensity ratios. No significant difference was found in heart rate variation between the stressors (26.1 ± 19.1 bpm for ATP vs. 21.7 ± 17.3 bpm for adenosine, p = 0.52). Patients receiving ATP referred less pronounced clinical symptoms. Semiquantitative myocardial perfusion parameters were comparable, and patients in the adenosine and ATP groups showed similar myocardial perfusion reserve index values (2.34 [1.62-2.73] vs 1.63 [1.29-2.10], p = 0.07). Splenic switch off was visually confirmed in all patients and estimated spleen to myocardium ratio was similar (0.92 [0.53-1.09] vs 0.81 [0.53-0.86] with ATP and adenosine, respectively, p = 0.12). Both ATP and adenosine are potent coronary vasodilators that can be safely employed in stress-CMR. Both stressor cause similar hyperemic response. Splenic switch-off can be used to assess stress adequacy in patients undergoing stress-CMR with either adenosine or ATP.

Introduction: Adenosine triphosphate (ATP) sensitive atrial tachycardia (AT) has been treated by radiofrequency catheter ablation. Cryofreezing energy has emerged as a novel energy source for catheter ablation. The aim of this study was to investigate the efficacy and safety of cryofreezing ablation for ATP-sensitive AT., Methods and Results: A total of six patients with ATP-sensitive ATs were included in this study. A single atrial extrastimulation was able to initiate and terminate these ATs in all six patients. The electrophysiological findings satisfied the diagnostic criteria of ATP-sensitive AT. The ablation catheter was located at the earliest activation site of atrial excitation during the AT, and cryofreezing energy was delivered through a cryoablation catheter to perform cryomapping at temperature of -30 or -80°C. When cryomapping successfully terminated the ATs, cryoablation at a temperature of -80°C was subsequently performed. The earliest atrial activation during AT was recorded at the Koch's triangle area associated with a distinct intra-atrial activation sequence from that recorded during ventricular pacing. Cryoablation was performed at successful cryomapping sites and resulted in the complete elimination of the AT in all six patients without affecting the bidirectional atrioventricular (AV) nodal conduction., Conclusion: Cryofreezing energy was safe and effective in treating ATP-sensitive ATs even in patients with its origins located in the vicinity of the AV node., (© 2019 Wiley Periodicals, Inc.)

Purpose: Low-dose adenosine triphosphate (LD-ATP) is useful for diagnosing ATP-sensitive atrial tachycardia. However, the clinical implications of the sensitivity of LD-ATP in atrioventricular nodal reentrant tachycardia (AVNRT) still remain unknown. This study aimed to evaluate the mechanism of LD-ATP sensitivity in slow-fast AVNRT., Methods: We estimated the sensitivity of LD-ATP in slow-fast AVNRT by a 2-4-mg ATP intravenous injection during the tachycardia. We evaluated the atrial-His (A-H) interval, tachycardia termination mode, prevalence of a lower common pathway (LCP), and successful ablation site in slow-fast AVNRT with LD-ATP sensitivity. LCPs were defined as His-atrial interval differences of at least 5 ms between that during ventricular pacing at the tachycardia cycle length and that during the tachycardia., Results: Twenty-eight patients (mean age = 58 ± 11 years old, 18 females) with slow-fast AVNRT, who underwent catheter ablation of the antegrade slow pathway, were enrolled. Seventeen of 28 (61%) patients had LD-ATP sensitivity defined as termination of the tachycardia and/or a prolongation of the A-H interval of over 30 ms after an LD-ATP injection. The patients with LD-ATP sensitivity had a significantly higher prevalence of an LCP than those without (15/17 vs0/11, P < 0.0001). The successful ablation site in the LD-ATP sensitive group was significantly closer to the His bundle area than that in the LD-ATP nonsensitive group (13.3 ± 3.8 vs 20.5 ± 5.4 mm; distance to His bundle area in the left anterior oblique fluoroscopic view, P < 0.0001)., Conclusions: LD-ATP sensitivity in slow-fast AVNRT may suggest the existence of an LCP. The successful ablation site in patients with LD-ATP sensitivity could be closer to the His bundle region., (© 2018 Wiley Periodicals, Inc.)

Background The basis of discordance between invasive coronary angiographic (ICA) anatomic stenosis and fractional flow reserve (FFR) is not fully understood. We analyzed coronary computed tomography angiography (CTA) characteristics of ICA-verified nonobstructive lesions in the proximal or midleft anterior descending artery with FFR ≤0.8, that is, anatomy-physiology mismatch. Methods and Results CTA and ICA were performed in 108 patients. FFR was measured during intravenous ATP (180 μg/[kg·min]) infusion. CTA-verified plaque characteristics between 53 consecutive ICA-FFR mismatch (ICA-quantitative coronary angiography <50%, FFR≤0.8) and 55 ICA-FFR match (ICA-quantitative coronary angiography<50%, FFR>0.8) vessels were compared. CTA-verified vessel area (20.7±6.7 versus 16.9±4.8 mm 2 ; P=0.0007), positive area remodeling index (ARI; 1.38±0.23 versus 1.06±0.11; P<0.0001), %plaque area (64.7±12.7 versus 57.4±8.5%; P<0.0007), jeopardized myocardial mass (46.2±18.5 versus 37.1±14.3 g; P= 0.006), and the prevalence of low attenuation plaque (45.3% versus 9.1%; P<0.0001) at the minimum lumen area were significantly higher in the ICA-FFR mismatch than the match group. By receiver operation curve analysis, the areas under the curve for positive area remodeling index, %plaque area and jeopardized myocardial mass were 0.921, 0.681, and 0.641, respectively, for the diagnosis of mismatch (cutoff values 1.13, 66% and 58.7 g, respectively). The sensitivity and specificity of area remodeling index >1.13 for predicting ICA-FFR mismatch were 88.7% and 78.2%, respectively. Conclusions In the absence of anatomically significant stenosis, abnormal FFR is more frequently encountered in patients with CTA-derived positive remodeling, larger plaque burden, and low attenuation plaque. These findings contribute to a better understanding of how FFR-based decision-making might translate into demonstrated superior clinical outcomes.

Background: Major depressive disorder (MDD) is a common psychiatric disorder. With systematic antidepressant treatment, 50-75% of patients have a treatment response but require 4-6 weeks to have their symptoms alleviated. Therefore, researchers anticipate the development of novel fast-acting antidepressants. Previous studies have revealed that the decrease of bio-energetic metabolism may contribute to the occurrence of depression, while our team has found adenosine triphosphate (ATP) and phosphocreatine (PCr) to be fast-acting antidepressants in the depressed-animal model. ATP and PCr have already been widely prescribed clinically as energy supplements for cells. This will be the first clinical attempt of the intravenous administration of ATP and PCr combined with orally administered fluoxetine in MDD., Methods: This is a single-center, randomized, double-blind, placebo-controlled pilot study. A total of 42 patients will be divided randomly into three groups. Patients will receive an intravenous administration of ATP or PCr or saline twice daily combined with orally administered fluoxetine (20 mg/day) for the first 2 weeks and fluoxetine monotherapy for the following 4 weeks. Follow-up assessment will be completed at week 10. Feasibility outcomes will include percentages of patient eligibility, intention to use medication, willingness to participate, drug adherence, completion of the scheduled assessment, retention, drop-out, etc. Physical examination results, Side Effect Rating Scale, adverse events, results from blood tests, electroencephalogram, and electrocardiograph will be recorded for safety evaluation of the augmentation therapy. The trends of efficacy will be evaluated by the reduction rate of the Hamilton Depression Rating Scale, the mean change of the Clinical Global Impression Scale, and the Patients Health Questionaire-9 items., Discussion: In our study, ATP and PCr will be given by intravenous infusion. Thus patients will be hospitalized for the initial 2 weeks for safety concern. Hospitalization will be an impact factor for the recruitment, participation, drop-out, efficacy, results, etc. The evaluation of our feasibility outcomes, study setting, safety of augmentation therapy and possible efficacy trends among groups, will facilitate a full-scale trial design and sample size calculation., Trial Registration: NCT03138681 . Registered on 3 May 2017. First patient: 4 May 2017.

A 53-year-old male underwent a pulmonary vein isolation (PVI) of atrial fibrillation (AF) with a second-generation cryoballoon (CB). Although the patient maintained sinus rhythm after the PVI, a superior vena cava (SVC) fibrillation was recorded by a circular-multipolar-electrode catheter positioned inside the SVC that suggested conduction block between the right atrium (RA)-SVC connection. An adenosine triphosphate intravenous injection induced a dormant reconnection of the SVC myocardial sleeve and converted sinus rhythm to an AF rhythm. This case demonstrated that a CB application for the isolation of a right superior pulmonary vein could induce an electrical conduction block between the RA-SVC connection., (© 2018 Wiley Periodicals, Inc.)

ATP in the suburothelial layer is released from the bladder urothelium by mechanical stimuli. ATP directly activates purinergic receptors that are expressed on primary bladder afferent neurons and induces the micturition reflex. Although ATP is also released to the bladder lumen from the bladder urothelium, the role of ATP in the bladder lumen is unknown. Recently, clinical studies have reported that urinary ATP levels are much higher in patients with an overactive bladder than healthy controls. These results suggest that ATP in the bladder lumen is also involved in the micturition reflex. In this study, we performed intravesical ATP instillation in the mouse bladder. We evaluated urinary function with novel reliable methods using improved cystometry and ultrasonography, which we previously established. We found that intravesical ATP instillation induced urinary frequency because of activation of bladder afferent nerves without inflammatory changes in the bladder or an increase in post-void residual urine. These results suggest that not only ATP in the suburothelial layer, but also ATP in the bladder lumen, are involved in enhancement of the micturition reflex., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Astrocytes are a glial cell type, which is indispensable for brain energy metabolism. Within cells, the NADH/NAD + redox state is a crucial node in metabolism connecting catabolic pathways to oxidative phosphorylation and ATP production in mitochondria. To characterize the dynamics of the intracellular NADH/NAD + redox state in cortical astrocytes Peredox, a genetically encoded sensor for the NADH/NAD + redox state, was expressed in cultured cortical astrocytes as well as in cortical astrocytes in acutely isolated brain slices. Calibration of the sensor in cultured astrocytes revealed a mean basal cytosolic NADH/NAD + redox ratio of about 0.01; however, with a broad distribution and heterogeneity in the cell population, which was mirrored by a heterogeneous basal cellular concentration of lactate. Inhibition of glucose uptake decreased the NADH/NAD + redox state while inhibition of lactate dehydrogenase or of lactate release resulted in an increase in the NADH/NAD + redox ratio. Furthermore, the NADH/NAD + redox state was regulated by the extracellular concentration of K + , and application of the neurotransmitters ATP or glutamate increased the NADH/NAD + redox state dependent on purinergic receptors and glutamate uptake, respectively. This regulation by K + , ATP, and glutamate involved NBCe1 mediated sodium-bicarbonate transport. These results demonstrate that the NADH/NAD + redox state in astrocytes is a metabolic node regulated by neuronal signals reflecting physiological activity, most likely contributing to adjust astrocytic metabolism to energy demand of the brain., (© 2018 Wiley Periodicals, Inc.)

The phenothiazine methylene blue (MB) is attracting increasing attention because it seems to have beneficial effects in the pathogenesis of Alzheimer's disease (AD). Among other factors, the presence of neuritic plaques of amyloid-β peptide (Aβ) aggregates, neurofibrilar tangles of tau and perturbation of cytosolic Ca 2+ are important players of the disease. It has been proposed that MB decreases the formation of neuritic plaques due to Aβ aggregation. However, the molecular mechanism underlying this effect is far from clear. In this work, we show that MB stimulates the Ca 2+ -ATPase activity of the plasma membrane Ca 2+ -ATPase (PMCA) in human tissues from AD-affected brain and age-matched controls and also from pig brain and cell cultures. In addition, MB prevents and even blocks the inhibitory effect of Aβ on PMCA activity. Functional analysis with mutants and fluorescence experiments strongly suggest that MB binds to PMCA, at the C-terminal tail, in a site located close to the last transmembrane helix and also that MB binds to the peptide. Besides, Aβ increases PMCA affinity for MB. These results point out a novel molecular basis of MB action on Aβ and PMCA as mediator of its beneficial effect on AD., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The functions of purinergic P2 receptors (P2Rs) for extracellular adenosine triphosphate (ATP) are poorly understood. Here, for the first time, we show that activation of P2Rs in an important arousal region, the basal forebrain (BF), promotes wakefulness, whereas inhibition of P2Rs promotes sleep. Infusion of a non-hydrolysable P2R agonist, ATP-γ-S, into mouse BF increased wakefulness following sleep deprivation. ATP-γ-S depolarized BF cholinergic and cortically-projecting GABAergic neurons in vitro, an effect blocked by antagonists of ionotropic P2Rs (P2XRs) or glutamate receptors. In vivo, ATP-γ-S infusion increased BF glutamate release. Thus, activation of BF P2XRs promotes glutamate release and excitation of wake-active neurons. Conversely, pharmacological antagonism of BF P2XRs decreased spontaneous wakefulness during the dark (active) period. Together with previous findings, our results suggest sleep-wake regulation by BF extracellular ATP involves a balance between excitatory, wakefulness-promoting effects mediated by direct activation of P2XRs and inhibitory, sleep-promoting effects mediated by degradation to adenosine.

Background: Successful pulmonary vein isolation (PVI) is the most reliable predictor of success after ablation in patients with atrial fibrillation (AF). Adenosine triphosphate (ATP) unmasks the dormant conduction and can be used to improve the effectiveness of PVI. The impact of ATP guided PVI on clinical outcomes is discordant in various randomized controlled trials (RCTs)., Objectives: To delineate the incremental benefit of ATP during PVI in patients with AF through a meta-analysis., Methods and Results: Database searches through January 2017 identified 5 RCTs (enrolling 2839 patients) comparing ATP guided PVI versus standard PVI (non-ATP). Four trials exclusively studied paroxysmal AF while one trial included both paroxysmal and non-paroxysmal AF patients. Baseline characteristics, dose of adenosine and ablation strategies were clearly identified among all the trials. The risk ratio (RR) for AF episodes lasting >30 s after 3-month blanking period was calculated with random effects meta-analysis and showed no difference at a median follow up of 12 months [RR: 1.02, 95 % Confidence interval (CI): 0.85 to 1.25; p = 0.82]. Similarly, the number of repeat ablation was similar in both groups [RR: 1.02, 95 % CI: 0.63, 1.56; p = 0.98]., Conclusions: ATP guided PVI does not decrease the recurrence of AF or the need for repeat ablation at 12 months.

Background: Growing evidence indicates a wide array of cellular remodeling in the mucosal microenvironment during irritable bowel syndrome (IBS), which possibly contributes to pathophysiology and symptom generation. Here, we investigated whether enteric glial cells (EGC) may be altered, and which factors/mechanisms lead to these changes., Methods: Colonic mucosal biopsies of IBS patients (13 IBS-Constipation [IBS-C]; 10 IBS-Diarrhea [IBS-D]; 11 IBS-Mixed [IBS-M]) and 24 healthy controls (HC) were analyzed. Expression of S100β and GFAP was measured. Cultured rat EGC were incubated with supernatants from mucosal biopsies, then proliferation and Ca 2+ response to ATP were analyzed using flow cytometry and Ca 2+ imaging. Histamine and histamine 1-receptor (H1R) involvement in the effects of supernatant upon EGC was analyzed., Key Results: Compared to HC, the mucosal area immunoreactive for S100β was significantly reduced in biopsies of IBS patients, independently of the IBS subtype. IBS-C supernatants reduced EGC proliferation and IBS-D and IBS-M supernatants reduced Ca 2+ response to ATP in EGC. EGC expressed H1R and the effects of supernatant upon Ca 2+ response to ATP in EGC were blocked by pyrilamine and reproduced by histamine via H1R. IBS supernatants reduced mRNA expression of connexin-43. The S100β-stained area was negatively correlated with the frequency and intensity of pain and bloating., Conclusion and Inferences: Changes in EGC occur in IBS, involving mucosal soluble factors. Histamine, via activation of H1R-dependent pathways, partly mediates altered Ca 2+ response to ATP in EGC. These changes may contribute to the pathophysiology and the perception of pain and bloating in patients with IBS., (© 2017 John Wiley & Sons Ltd.)

We investigated the effects of caffeine intake on fractional flow reserve (FFR) values measured using intravenous adenosine triphosphate (ATP) before cardiac catheterization. Caffeine is a competitive antagonist for adenosine receptors; however, it is unclear whether this antagonism affects FFR values. Patients were evenly randomized into 2 groups preceding the FFR study. In the caffeine group (n = 15), participants were given coffee containing 222 mg of caffeine 2 h before the catheterization. In the non-caffeine group (n = 15), participants were instructed not to take any caffeine-containing drinks or foods for at least 12 h before the catheterization. FFR was performed in patients with more than intermediate coronary stenosis using the intravenous infusion of ATP at 140 μg/kg/min (normal dose) and 170 μg/kg/min (high dose), and the intracoronary infusion of papaverine. FFR was followed for 30 s after maximal hyperemia. In the non-caffeine group, the FFR values measured with ATP infusion were not significantly different from those measured with papaverine infusion. However, in the caffeine group, the FFR values were significantly higher after ATP infusion than after papaverine infusion (P = 0.002 and P = 0.007, at normal and high dose ATP vs. papaverine, respectively). FFR values with ATP infusion were significantly increased 30 s after maximal hyperemia (P = 0.001 and P < 0.001 for normal and high dose ATP, respectively). The stability of the FFR values using papaverine showed no significant difference between the 2 groups. Caffeine intake before the FFR study affected FFR values and their stability. These effects could not be reversed by an increased ATP dose.

Breathlessness during daily activities is the primary symptom in patients with heart failure (HF). Poor correlation between the hemodynamic parameters of left ventricular performance and perceived symptoms suggests that other factors, such as skeletal muscle function, play a role in determining exercise capacity. We investigated the effect of 6 wk of high-intensity, one-legged cycling (HIC; 8 × 4 at 90% one-legged cycling max) on 1) the ability to override sympathetic vasoconstriction (arterial infusion of tyramine) during one-legged knee-extensor exercise (KEE), 2) vascular function (arterial infusion of ACh, sodium nitroprusside, tyramine, and ATP), and 3) exercise capacity in HF patients with reduced ejection fraction ( n = 8) compared with healthy individuals ( n = 6). Arterial tyramine infusion lowered leg blood flow and leg vascular conductance at rest and during KEE before the training intervention in both groups ( P < 0.05) but not during KEE after the training intervention. There was no difference between groups. The peak vasodilatory response to ATP was blunted in HF patients ( P < 0.05), whereas there was no difference in ACh- and sodium nitroprusside-induced vasodilation between HF patients and healthy individuals. ACh-induced vasodilation increased in HF patients after the training intervention ( P < 0.05). HIC improved aerobic capacity in both groups ( P < 0.05), whereas only HF patients made improvements in the 6-min walking distance ( P < 0.05). These results suggest that exercise hyperemia and functional sympatholysis are not altered in HF patients and that functional sympatholysis is improved with HIC in both HF patients and healthy individuals. Moreover, these results suggest that the peak vasodilatory response to ATP is blunted in HF. NEW & NOTEWORTHY The ability to override sympathetic vasoconstrictor activity (by arterial tyramine infusion) during exercise is not different between heart failure patients and healthy individuals and is improved by high-intensity, one-legged cycling training. The peak vasodilatory response to ATP is reduced in heart failure patients.

Injury to the peripheral axons of sensory neurons strongly enhances the regeneration of their central axons in the spinal cord. It remains unclear on what molecules that initiate such conditioning effect. Because ATP is released extracellularly by nerve and other tissue injury, we hypothesize that injection of ATP into a peripheral nerve might mimic the stimulatory effect of nerve injury on the regenerative state of the primary sensory neurons. We found that a single injection of 6 μl of 150 μm ATP into female rat sciatic nerve quadrupled the number of axons growing into a lesion epicenter in spinal cord after a concomitant dorsal column transection. A second boost ATP injection 1 week after the first one markedly reinforced the stimulatory effect of a single injection. Single ATP injection increased expression of phospho-STAT3 and GAP43, two markers of regenerative activity, in sensory neurons. Double ATP injections sustained the activation of phospho-STAT3 and GAP43, which may account for the marked axonal growth across the lesion epicenter. Similar studies performed on P2X7 or P2Y2 receptor knock-out mice indicate P2Y2 receptors are involved in the activation of STAT3 after ATP injection or conditioning lesion, whereas P2X7 receptors are not. Injection of ATP at 150 μm caused little Wallerian degeneration and behavioral tests showed no significant long-term adverse effects on sciatic nerve functions. The results in this study reveal possible mechanisms underlying the stimulation of regenerative programs and suggest a practical strategy for stimulating axonal regeneration following spinal cord injury. SIGNIFICANCE STATEMENT Injury of peripheral axons of sensory neurons has been known to strongly enhance the regeneration of their central axons in the spinal cord. In this study, we found that injection of ATP into a peripheral nerve can mimic the effect of peripheral nerve injury and significantly increase the number of sensory axons growing across lesion epicenter in the spinal cord. ATP injection increased expression of several markers for regenerative activity in sensory neurons, including phospho-STAT3 and GAP43. ATP injection did not cause significant long-term adverse effects on the functions of the injected nerve. These results may lead to clinically applicable strategies for enhancing neuronal responses that support regeneration of injured axons., (Copyright © 2018 Wu, Lee et al.)

Sympathetic vasoconstriction is blunted in exercising muscle (functional sympatholysis) but becomes attenuated with age. We tested the hypothesis that functional sympatholysis is further impaired in chronic obstructive pulmonary disease (COPD) patients. We determined leg blood flow and calculated leg vascular conductance (LVC) during 1) femoral-arterial Tyramine infusion (evokes endogenous norepinephrine release, 1 µmol·min -1 ·kg leg mass -1 ), 2) one-legged knee extensor exercise with and without Tyramine infusion [10 W and 20% of maximal workload (WL max )], 3) ATP (0.05 µmol·min -1 ·kg leg mass -1 ) and Tyramine infusion, and 4) incremental ATP infusions (0.05, 0.3, and 3.0 µmol·min -1 ·kg leg mass -1 ). We included 10 patients with moderate to severe COPD and 8 age-matched healthy control subjects. Overall, leg blood flow and LVC were lower in COPD patients during exercise ( P < 0.05). Tyramine reduced LVC in both groups at 10-W exercise (COPD: -3 ± 1 ml·min -1 ·mmHg -1 and controls: -3 ± 1 ml·min -1 ·mmHg -1 , P < 0.05) and 20% WL max (COPD: -4 ± 1 ml·min -1 ·mmHg -1 and controls: -3 ± 1 ml·min -1 ·mmHg -1 , P < 0.05) with no difference between groups. Incremental ATP infusions induced dose-dependent vasodilation with no difference between groups, and, in addition, the vasoconstrictor response to Tyramine infused together with ATP was not different between groups (COPD: -0.03 ± 0.01 l·min -1 ·kg leg mass -1 vs., Controls: -0.04 ± 0.01 l·min -1 ·kg leg mass -1 , P > 0.05). Compared with age-matched healthy control subjects, the vasodilatory response to ATP is intact in COPD patients and their ability to blunt sympathetic vasoconstriction (functional sympatholysis) as evaluated by intra-arterial Tyramine during exercise or ATP infusion is maintained. NEW & NOTEWORTHY The ability to blunt sympathetic vasoconstriction in exercising muscle and ATP-induced dilation in chronic obstructive pulmonary disease patients remains unexplored. Chronic obstructive pulmonary disease patients demonstrated similar sympathetic vasoconstriction in response to intra-arterial Tyramine during exercise and ATP-induced vasodilation compared with age-matched healthy control subjects.

Purpose: Mutations in the gene encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final 2 steps of the purine de novo biosynthetic pathway, were identified in a subject referred for radiation sensitivity testing. Functional studies were performed to determine whether ATIC inhibition was radiosensitizing and, if so, to elucidate the mechanism of this effect and determine whether small molecule inhibitors of ATIC could act as effective radiosensitizing agents., Methods and Materials: Both small interfering RNA knockdown and small molecule inhibitors were used to inactivate ATIC in cell culture. Clonogenic survival assays, the neutral comet assay, and γH2AX staining were used to assess the effects of ATIC inhibition or depletion on cellular DNA damage responses., Results: Depletion of ATIC or inhibition of its transformylase activity significantly reduced the surviving fraction of cells in clonogenic survival assays in multiple cancer cell lines. In the absence of ionizing radiation exposure, ATIC knockdown or chemical inhibition activated cell cycle checkpoints, shifting cells to the more radiosensitive G2/M phase of the cell cycle, and depleted cellular adenosine triphosphate but did not result in detectable DNA damage. Cells in which ATIC was knocked down or inhibited and then treated with ionizing radiation displayed increased numbers of DNA double-strand breaks and a delay in the repair of those breaks relative to irradiated, but otherwise untreated, controls. Supplementation of culture media with exogenous adenosine triphosphate ameliorated the DNA repair phenotypes., Conclusions: These findings implicate ATIC as an effective, and previously unrecognized, target for chemoradiosensitization and, more broadly, suggest that purine levels in cells might have an underappreciated role in modulating the efficiency of DNA damage responses that could be exploited in radiosensitizing strategies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The process of skin wound healing involves the following three steps: inflammation, tissue formation and tissue remodelling. These optimal steps are required for the development of normal wound healing. Recent reports demonstrated that inflammasomes are involved in the innate immune response. In the present study, we examined whether the activation of inflammasomes affects the process of skin wound repair. The skin wound repair model was established using wild-type (WT), NACHT, LRR and PYD domains-containing protein 3 (NALP3) knockout (KO) and ASC-KO mice. The wounds were observed every other day, and changes in wound size over time were calculated using photography. Wound repair in NALP3-KO and ASC-KO mice was significantly impaired compared with WT mice. Isoliquiritigenin, an inhibitor of NALP3, decreased the rate of wound repair in WT mice. mRNA expression of pro-inflammatory cytokines in the wound sites of NALP3-KO mice was markedly decreased compared with WT mice. Treatment with adenosine triphosphate (ATP), a ligand of NALP3, upregulated the mRNA expression of pro-inflammatory cytokines at the wound site and accelerated wound healing in the WT mice. Scratch assay revealed that ATP accelerated wound closure in mouse embryonic fibroblasts from WT mice but not from NALP3-KO mice. In conclusion, the present study demonstrated that NALP3 pathway activation is involved in wound repair, and the topical use of ATP may be useful as an effective treatment for accelerating wound healing., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Postocclusional hyperemia caused by balloon occlusion is a potential alternative method of inducing hyperemia for measuring post-percutaneous coronary intervention fractional flow reserve (FFR). The aim of this study was to investigate postocclusional hyperemia as a method of inducing hyperemia., Methods and Results: FFR measured by postocclusional hyperemia (FFRoccl) caused by balloon occlusion after percutaneous coronary intervention was compared with FFR measured by drug-induced hyperemia (FFR measured by intravenous ATP; and FFR measured by intracoronary papaverine injection [FFRpap]) in 98 lesions from 98 patients. The hyperemia duration was also measured for FFRoccl and FFRpap. The correlation coefficient between FFRoccl, FFR measured by intravenous ATP ( r =0.973; P <0.01), and FFRpap ( r =0.975; P <0.01) showed almost identical values to those obtained for the correlation coefficient between FFR measured by intravenous ATP and FFRpap ( r =0.967; P <0.01). No clear difference was observed on Bland-Altman analysis. Hyperemia duration was significantly longer with FFRoccl than with FFRpap (70±22 versus 51±25 s; P <0.01)., Conclusions: Strong correlations were found between FFRoccl and FFR measured by intravenous ATP and FFRoccl and FFRpap. Hyperemia caused by FFRoccl was significantly longer than that caused by FFRpap., (© 2017 American Heart Association, Inc.)

Pregnancy-derived uterine artery endothelial cells (P-UAEC) express P2Y2 receptors and at high cell density show sustained and synchronous [Ca2+]i burst responses in response to ATP Bursts in turn require coupling of transient receptor potential canonical type3 channel (TRPC3) and inositol 1,4,5-triphosphate receptor type 2 (IP3R2), which is upregulated in P-UAEC in a manner dependent on connexin 43 (Cx43) gap junctions. While there is no known direct interaction of TRPC3 with Cx43, early descriptions of TRPC3 function showed it may also be influenced by altered membrane potential ( V m ). Herein, we ask if enhanced TRPC3 Ca2+ bursting due to enhanced Cx43 coupling may be coupled via dynamic alterations in V m in P-UAEC, as reported in some (HUVEC) but not all endothelial cells. Following basic electrical characterization of UAEC, we employed a high sensitivity cell imaging system to simultaneously monitor cell V m and [Ca2+]i in real time in continuous monolayers of UAEC Our findings show that while acute and sustained phase [Ca2+]i bursting occur dose-dependently in response to ATP, V m is not coregulated with any periodicity related to [Ca2+]i bursting. Only a small but significant progressive change in V m is seen, and this is more closely related to overall mobilization of Ca2+. Surprisingly, this is also most apparent in NP-UAEC > P-UAEC In contrast [Ca2+]i bursting is more synchronous in P-UAEC and even achieves [Ca2+]i waves passing through the P-UAEC monolayer. The relevance of these findings to mechanisms of pregnancy adaptation and its failure in hypertensive pregnancy are discussed., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Background: Adenosine 5'-triphosphate (ATP) stimulates pulmonary vagal slow conducting C-fibres and fast conducting Aδ-fibres with rapidly adapting receptors (RARs). Pulmonary C-fibres but not RARs are also sensitive to capsaicin, a potent tussigenic agent in humans. Thus, the aim of this study was to determine the effects of ATP and its metabolite adenosine (given as adenosine 5'-monophosphate, AMP) on capsaicin challenge in asthmatic patients., Methods: Cough (quantified as visual analogue scale, VAS), dyspnoea (quantified as Borg score), and FEV 1 were quantified following bronchoprovocation using capsaicin, adenosine and ATP in healthy non-smokers (age 40±4y, 6 males), smokers (45±4y, 5 males) and asthmatic patients (37±3y, 5 males); n = 10 in each group., Results: None of the healthy non-smokers responded to either AMP or ATP. AMP induced bronchoconstriction in one smoker and eight asthmatics, and ATP in two smokers and all ten asthmatics. The geometric mean of capsaicin causing ≥5 coughs (C5) increased from 134 to 203 μM in non-smokers and from 117 to 287 μM in asthmatics after AMP, whereas it decreased from 203 to 165 μM and 125 to 88 μM, respectively after ATP. AMP decreased C5 from 58 to 29 μM and ATP increased from 33 to 47 μM in smokers. However, due to intergroup variability, these effects of ATP and AMP were not statistically significant (0.125 ≤ p ≤ 0.998). That notwithstanding, in healthy and asthmatic subjects the effects of the ATP showed a tendency to be greater than those of AMP (p < 0.053). Dyspnea, assessed by Borg score, increased after ATP (p < 0.001) and AMP (p < 0.001) only in asthmatic patients. Intensity of cough assessed by VAS increased (p < 0.05) after second capsaicin challenges performed after AMP in all groups, but not after ATP., Conclusions: Asthmatic patients exhibit hypersensitivity to aerosolized AMP and ATP, but aerosolized AMP does not mimic the effects of ATP and the effects of ATP are not mediated by adenosine., (Copyright © 2017. Published by Elsevier Ltd.)

Aims: Atrial fibrillation recurrences after pulmonary vein isolation (PVI) are not uncommon and are frequently related to pulmonary vein reconnection. Adenosine/ATP can reveal dormant pulmonary vein conduction after PVI. Previous studies revealed that adenosine-guided Additional ablation could improve arrhythmia-free survival. We performed a meta-analysis to assess the impact of additional ablation to eliminate adenosine-induced transient pulmonary vein reconnection in terms of atrial fibrillation recurrence at follow-up., Methods: MEDLINE/PubMed, Cochrane Library and references reporting atrial fibrillation ablation and adenosine/ATP-following PVI were screened, and studies were included if they matched inclusion and exclusion criteria., Results: A total of 3524 patients were enrolled with a median follow-up of 13 (6-20) months. Overall, 70% (60-85) of patients in ATP-guided ablation vs. 63% (48-79) in no ATP-guided ablation were free of atrial fibrillation at follow-up. Pooled results revealed that ATP-guided ablation reduced the risk of atrial fibrillation recurrence of 42% [odds ratio (OR) 0.58, 0.41-0.81], but this result was primary because of the contribution of retrospective over-randomized studies [OR 0.48 (0.35-0.65) vs. 0.76 (0.42-1.40), respectively]. 3.2% of patients experienced an adverse event. ATP-guided ablation is related to a nonsignificant increase in fluoroscopy time (OR 1.71, 0.98-2.96) and to a significant increase in procedure time (OR 2.84, 1.32-6.09)., Conclusion: Additional ablation aiming to eliminate adenosine-induced transient pulmonary vein reconnection failed to reduce the risk of atrial fibrillation recurrence at follow-up. Moreover, although adenosine-guided PVI is not affected by an augmented risk of adverse events, it is associated with a NS increased fluoroscopy exposure and significantly longer procedure duration. Further studies are required to identify the actual role of adenosine in PVI.

Mechanical strain in neural tissues can lead to the up-regulation and release of multiple cytokines including interleukin 6 (IL-6). In the retina, the mechanosensitive release of ATP can autostimulate P2X7 receptors on both retinal ganglion cell neurons and optic nerve head astrocytes. Here, we asked whether the purinergic signaling contributed to the IL-6 response to increased intraocular pressure (IOP) in vivo, and stretch or swelling in vitro. Rat and mouse eyes were exposed to non-ischemic elevations in IOP to 50-60 mmHg for 4 h. A PCR array was used to screen cytokine changes, with quantitative (q)PCR used to confirm mRNA elevations and immunoblots used for protein levels. P2X7 antagonist Brilliant Blue G (BBG) and agonist (4-benzoyl-benzoyl)-ATP (BzATP) were injected intravitreally. ELISA was used to quantify IL-6 release from optic nerve head astrocytes or retinal ganglion cells. Receptor identity was confirmed pharmacologically and in P2X7 -/- mice, acute elevation of IOP altered retinal expression of multiple cytokine genes. Elevation of IL-6 was greatest, with expression of IL1rn, IL24, Tnf, Csf1, and Lif also increased more than twofold, while expression of Tnfsf11, Gdf9, and Tnfsf4 were reduced. qPCR confirmed the rise in IL-6 and extracellular ATP marker ENTPD1, but not pro-apoptotic genes. Intravitreal injection of P2X7 receptor antagonist BBG prevented the pressure-dependent rise in IL-6 mRNA and protein in the rat retina, while injection of P2X7 receptor agonist BzATP was sufficient to elevate IL-6 expression. IOP elevation increased IL-6 in wild-type but not P2X7R knockout mice. Application of mechanical strain to isolated optic nerve head astrocytes increased IL-6 levels. This response was mimicked by agonist BzATP, but blocked by antagonists BBG and A839977. Stretch or BzATP led to IL-6 release from both astrocytes and isolated retinal ganglion cells. The mechanosensitive up-regulation and release of cytokine IL-6 from the retina involves the P2X7 receptor, with both astrocytes and neurons contributing to the response., (© 2017 International Society for Neurochemistry.)