Adenosine Triphosphate Treatment Research Articles

Phenolic components, antioxidant enzyme activities and anatomic structure of longan fruit pericarp following treatment with adenylate triphosphate

Fruit senescence and pericarp browning are strongly related to the antioxidant properties and are affected by the cellular energy status. In the present study, longan fruits were treated with 1mmolL−1 adenosine triphosphate (ATP) before storage for six days at 25°C. The phenolic contents and antioxidant enzyme activities in relation to pericarp browning were determined, and the fruit pericarp structure was observed under a confocal laser scanning microscope (CLSM) and transmission electron microscope. Increases in the browning index, membrane permeability, peroxidase (POD) activity and gallic acid content were slowed during storage following exogenous ATP application, whereas the flavonoids accumulation was increased significantly by ATP treatment. In addition, decrease in catechin, corilagin, epicatechin and gallocatechin gallate were accelerated by exogenous ATP treatment. The cuticle in the pericarp of the untreated longan fruit was incomplete, the organelle was swollen or disintegrated, the grana lamellae was destroyed and osmiophilic particles accumulated in the chloroplast, the cytoplasm was granulated and the fruiting body of a possible pathogenic organism was observed after storage. However, the pericarp structure maintained its relative integrity without any visible pathogenic organisms, as determined with CLSM in the ATP-treated fruit. It is suggested that ATP application protects longan fruit from pathogen attack, maintains the pericarp structure and delays senescence, which are closely related to the fruit's higher total phenolic levels and lower POD activity.

Quality deterioration of cut carnation flowers involves in antioxidant systems and energy status

Adenosine triphosphate (ATP) has played an important role in regulating senescence of horticultural crops. The physiological mechanism of exogenous ATP to affect the senescence of cut carnation during vase life was investigated. Fresh cut carnation flowers were treated with distilled water (control), 0.1mM adenosine triphosphate (ATP) or 0.5mM 2,4-dinitrophenol (DNP, an agent for uncoupling oxidative phosphorylation) and then held at 25°C up to nine days. Exogenous ATP supply increased flower size, maintained fresh weight, extended vase life, reduced ethylene production rate and maintained membrane integrity. ATP treatment also reduced activities of phospholipase D (LD) and lipoxygenase (LOX), enhanced activities of superoxide dismutase (SOD) and catalase (CAT) and increased endogenous contents of ATP and adenosine diphosphate (ADP) and energy charge level at the later vase life. In comparison to control, exogenous application of DNP accelerated ethylene production and reduced vase life, increased membrane permeability, MDA content and activities of PLD and LOX and reduced activities of SOD and CAT. Furthermore, contents of ATP and ADP and energy charge level decreased in the DNP-treated flowers during vase life. Thus, exogenous ATP supply could maintain membrane integrity, increase antioxidant system and improve endogenous energy level, thereby suppressing ethylene production and retarding the senescence of cut carnation flower during vase life.

The A1 adenosine receptor as a new player in microglia physiology

The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P2 X4,7 and P2 Y12 receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca(2+) influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive-specific neuron before and after application of resting or ATP-treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone.

Involvement of nitric oxide through endocannabinoids release in microglia activation during the course of CNS regeneration in the medicinal leech

The medicinal leech is notable for its capacity to regenerate its central nervous system (CNS) following mechanical trauma. Using an electrochemical nitric oxide (NO)-selective electrode to measure NO levels, we found that the time course of NO release in the injured leech CNS is partially under the control of endocannabinoids, namely, N-arachidonyl ethanolamide (AEA) and 2-arachidonyl glycerol (2-AG). Relative quantification of these endocannabinoids was performed by stable isotope dilution (2AGd8 and AAEd8) coupled to mass spectrometry in course of regeneration process or adenosine triphosphate (ATP) treatment. Data show that 2-AG levels rose to a maximum about 30 min after injury or ATP treatment, and returned to baseline levels 4 h after injury. In same conditions, AEA levels also rapidly (within 5 min) dropped after injury or ATP treatment to the nerve cord, but did not fully return to baseline levels within 4 h of injury. In correlation with these data, chemoattraction activities of endocannabinoids on isolated leech microglial cells have been shown in vitro and in vivo reflecting that control over NO production is accompanied by the controlled chemoattraction of microglia directed from the periphery to the lesion site for neuronal repair purposes. Taken together, our results show that in the leech, after injury concurrent with ATP production, purinergic receptor activation, NO production, microglia recruitment, and accumulation to lesion site, a fine imbalance occurs in the endocannabinoid system. These events can bring explanations about the ability of the leech CNS to regenerate after a trauma and the key role of endocannabinoids in this phenomenon.

Administration of exogenous adenosine triphosphate to ischemic skeletal muscle induces an energy-sparing effect: Role of adenosine receptors

BackgroundIschemia–reperfusion injury is a devastating complication that occurs in allotransplantation and replantation of limbs. Over the years, several preservation strategies have been used to conserve the critical levels of intracellular adenosine triphosphate (ATP) during ischemia to sustain the ion gradients across the membranes and thus the tissue viability. The administration of exogenous ATP to ischemic tissues is known to provide beneficial effects during reperfusion, but it is unclear whether it provides protection during ischemia. The purpose of the present study was to determine the effect of ATP administration on high-energy phosphate levels in ischemic skeletal muscle and to examine the role of purinergic and adenosine receptors in mediating the response to exogenous ATP. MethodsThe extensor digitorum longus muscles of Fischer rats were subjected to ischemia and treated with different concentrations of ATP with or without purinergic and adenosine receptor blockers. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to measure the rate of decay of ATP, phosphocreatine (PCr), and the formation of adenosine monophosphate and acidification. Phosphorylated compounds were analyzed using a simple model of energy metabolism, and the PCr half-life was used as an index of internal depletion of ATP to distinguish between intracellular and extracellular ATP. ResultsPCr decay was rapid in all muscle groups and was followed by gradual ATP decay. The half-life of PCr was significantly longer in the ATP-treated muscles than in the vehicle controls and was maximally prolonged by treating with slow hydrolyzing adenosine 5′-O-(3-thio)triphosphate. Purinoceptor (P2X) blockade with ATP treatment significantly increased the half-life of PCr, and adenosine receptor blockers blunted the response. Administration of adenosine to ischemic muscles significantly increased the half-life of PCr compared with that in the vehicle controls. ConclusionsExogenous ATP administration to ischemic skeletal muscles appears to spare intracellular energy by acting primarily through adenosine receptors.

Effect of Human Muscle–derived Stem Cells on Cryoinjured Mouse Bladder Contractility

To investigate the effect of human muscle-derived stem cells (hMDSCs) on ameliorating impaired detrusor contractility in a cryoinjured bladder murine model. The hMDSCs were isolated and cultured by modified preplate technique, and only CD34-positive hMDSCs were extracted by Mini-MACS kits. Isolated hMDSCs were prelabeled with PKH26 and injected into the cryoinjured bladder to observe the pattern and characteristics. The nude mice were subdivided into three groups: normal group (N), cryoinjury bladder group with saline injection (C), and hMDSCs injection group after cryoinjury (M). At 2 weeks after injecting hMDSCs, we compared the contractility of bladder muscle strip stimulated by electrical field stimulation (EFS), acetylcholine (Ach.), and adenosine triphosphate (ATP), and the bladder smooth muscle tissue was examined by immunohistochemistry. The contractile powers of bladder muscle strip in the C group were more decreased than the N group after EFS, Ach, and ATP treatment (P < .05). The bladder contractility of the M group was more increased than in the C group (P < .05), but was lower than the N group after EFS and Ach treatment. However, there was no significant difference of contractile power between the C and M groups after ATP stimulation. In immunohistochemical staining, the thickness of the bladder smooth muscle layer in the M group was significantly increased compared with the C group, and PKH26-labeled implanted cells were positive for smooth muscle cell differentiation marker (α-SMA) in the injected region. hMDSCs injection increased cholinergic bladder contractile power but not the purinergic component of bladder contraction after cryoinjury.

Effect of adenosine triphosphate on changes of fatty acids in harvested litchi fruit infected by Peronophythora litchii

Recent investigations have shown that disease development of harvested horticultural crops may be attributed to a limited availability of energy or low energy production. In this study, litchi fruit were treated with 1.0 mM adenosine triphosphate (ATP) or 0.5 mM 2,4-dinitrophenol (DNP) and then half of the ATP-treated fruit were inoculated with Peronophythora litchii. The composition and contents of fatty acids (FAs) and esterase activity in litchi fruit during storage were investigated. Free fatty acids (FFAs) in all fruit increased over storage, especially in the P. litchii-inoculated fruit. In particular, the content of saturated FAs increased faster than unsaturated FAs. In polar lipids (PL), a decrease in the amount of C18:3 and an increase in the amount of C16:0 or C18:0 was found during storage, while the proportions of C16:0, C18:0 and C18:1 in neutral lipid (NL) gradually increased but the proportions of C18:3 decreased during storage. The proportion of C18:2 increased within the first four days and then decreased. Exogenous ATP treatment suppressed the release of FFAs and increased the contents of each FA in PL, indicating a slower hydrolysis of lipids. ATP treatment also delayed the increase in the proportion of C18:0 in NL. Further analysis showed that the double bond index (DBI) of litchi fruit decreased in all fractions of FAs and ATP treatment can slow the decrease in DBI. In addition, lower esterase enzyme activity was detected in all ATP-treated fruit. Treatment with DNP (a respiration uncoupler) increased esterase activity. P. litchii-inoculated fruit after ATP treatment also exhibited similar trends in delaying the release of FFAs. Enhanced disease resistance of litchi fruit by ATP could involve the levels of FAs and esterase activity.

Remifentanil Decreases Mandibular Bone Marrow Blood Flow During Propofol or Sevoflurane Anesthesia in Rabbits

The purpose of this study was to investigate the effect of remifentanil on the blood flow in rabbit oral tissues during propofol or sevoflurane anesthesia. Thirty-two male tracheotomized Japanese white rabbits were anesthetized with propofol (P group) or sevoflurane (S group) under mechanical ventilation. Animals were further divided into 4 groups: adenosine triphosphate (ATP) treatment in the P group (P-ATP) (n = 8), remifentanil treatment in the P group (P-Remi) (n = 8), ATP treatment in the S group (S-ATP) (n = 8), and remifentanil treatment in the S group (S-Remi) (n = 8). Blood pressure, heart rate, common carotid artery blood flow (CABF), lingual mucosal blood flow (LMBF), and mandibular bone marrow blood flow (BMBF) were measured during each period before and during infusion of ATP or remifentanil. Mean arterial pressure (MAP), CABF, LMBF, and BMBF showed a significant decrease in all 4 groups during infusion of the drug. Similarly, LMBF and BMBF showed significant decreases in both the ATP and remifentanil groups, whereas the decreases in MAP and CABF measured as percent change were greater in the ATP groups than in the remifentanil groups. A significant correlation was observed between CABF and BMBF in the S-Remi group. In both the P and S groups, the correlations between MAP and LMBF or BMBF were relatively strong when ATP was administered. Our findings suggest that infusion of remifentanil during propofol or sevoflurane anesthesia reduces BMBF and LMBF, as does deliberate hypotension by use of ATP, without a major decrease in MAP.

Adenosine triphosphate increases the reactivity of the afferent arteriole to low concentrations of norepinephrine

Adenosine triphosphate (ATP) and norepinephrine (NE) interact in the control of blood flow in the kidney. A combined effect of NE and ATP has not been previously investigated at the level of the afferent arteriole (Af). We studied the effects of ATP on the contractile response of the Af to NE. Vascular reactivity to ATP, NE, and their combination was investigated in isolated perfused Af from mice. The roles of alpha-adrenoceptors and P2-ATP-receptors were investigated by use of specific agonists and antagonists. Cytosolic calcium was measured using the fluorescent calcium dye fura-2. ATP in concentrations from 10(-12) to 10(-4) mol/l induced transient contractions. NE constricted the Af in a dose-dependent manner and induced significant contractions at > 10(-7) mol/l. Treatment with ATP (10(-8) and 10(-6) mol/l) increased the NE response. Diameters were reduced by 20% already at 10(-11) mol/l NE during ATP treatment of 10(-6) mol/l. ATP increased the calcium response to NE significantly at 10(-8) and 10(-7)mol/l NE. The P2-type ATP receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10(-5) mol/l) abolished the sensitization of the NE response by ATP. The alpha(1)-blocker prazosin (10(-7) mol/l) inhibited the ATP effect, as did the alpha 2-blocker yohimbine (10(-7) mol/l). Neither the phenylephrine- nor clonidine-induced concentration response curves was affected by ATP in the bath solution. Costimulation with ATP enhances the response of the Af to NE. This effect is mediated by increased cytosolic calcium. The enhancing effect involves P2-type ATP receptors and both alpha (1)- and alpha 2-adrenoceptors.

Signaling Pathways Involved in Adenosine Triphosphate-Induced Endothelial Cell Barrier Enhancement

Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell-cell junctions. Increases in cytosolic Ca2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically Galphaq and Galphai2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of Galphaq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA.

ATP activates nuclear translocation of mitogen-activated protein kinases in human granulosa-luteal cells

To investigate the specific role of adenosine triphosphate (ATP) in the subsequent MAPK-induced signaling cascade in human granulosa-luteal cells (hGLCs). We have previously demonstrated an effect of extracellular adenosine triphosphate (ATP) on ERKs and gonadotropin-induced progesterone secretion, implicating the significance of ATP in the regulation of ovarian function. The present study was designed to examine the effect of ATP on the activation of the MAPK signaling pathway, including nuclear translocation in hGLCs. Human GLCs were collected from patients undergoing in vitro fertilization programs. Human GLCs were cultured in FBS-supplemented DMEM for 3 days prior to our studies. Cells were starved in serum-free medium for 4h prior to subsequent treatments. To examine the dose-response relationship, hGLCs were treated with increasing concentrations of ATP (100nM, 1μ, 10μ, 100μ) for 5 min. For time-course experiments, hGLCs were treated with 10μM ATP for 1, 5, 10, 20 min. To determine the translocation of MAPKs, hGLCs were fixed 5 min after 10μM ATP exposure. Western blot analysis was performed using antibodies against the phosphorylated forms of ERK1 and ERK2 (P-MAPK, p42mapk and p44mapk, respectively), p42/p44 MAPK (total MAPK, T- MAPK), phospho JNK and activated P38. To examine the translocation of activated ERKs, FITC-conjugated secondary antibody was used to detect the distribution of total and phosphorylated ERKs. Western blot analysis, which detected the total and phosphorylated forms of ERK1 and ERK2, demonstrated that exogenous ATP evoked ERKs in a dose- and time-dependent manner. In contrast, p38 and JNK were not significantly activated following ATP treatment. Immunofluorescent staining revealed that phosphorylated ERKs were translocated from cytoplasm into nucleus subsequent to 10μ ATP treatment. To our knowledge, this is the first demonstration of the ATP-induced nuclear translocation of MAPKs in the human ovary. These results suggest that the MAPKs signaling pathway plays a role in mediating ATP actions in the human ovary.

Extracellular ATP inhibits apoptosis and maintains cell viability by inducing autocrine production of interleukin-4 in a myeloid progenitor cell line

Interleukin-3 (IL-3)-dependent myeloid progenitor cell FDC.P2 is induced to undergo apoptotic cell death upon IL-3 depletion. Extracellular adenosine triphosphate (ATP) was found to prevent apoptosis and maintain cell viability of FDC.P2 cells upon IL-3 withdrawal. The antiapoptotic effect of ATP required extracellular Ca 2+. Furthermore, FK506, a specific inhibitor of calcium/calmodulin-dependent protein phosphatase calcineurin, inhibited the antiapoptotic effect of ATP. As one of cytokines whose expression is dependent on the activation of calcineurin, interleukin-4 (IL-4) played a critical role in ATP-mediated cell survival of FDC.P2 cells because neutralizing antibody against IL-4 effectively abrogated the antiapoptotic activity of ATP. Moreover, ATP treatment induced a significant amount of secreted IL-4 that was sufficient to maintain cell viability. Taken together, our present results demonstrate that extracellular ATP triggers autocrine production of IL-4 through calcium-dependent activation of calcineurin and secreted IL-4 substitutes IL-3 in protecting FDC.P2 cells from apoptosis even in the absence of IL-3.

ATP-induced ATP release from astrocytes.

Propagation of interastrocyte Ca2+ waves is mediated by diffusion of extracellular adenosine triphosphate (ATP), and may require regenerative release of ATP. The ability of ATP to initiate release of intracellular ATP was assessed by labeling adenine nucleotide pools in astrocyte cultures with 14C-adenine. The 14C-purines released during exposure to ATP were then identified by thin-layer chromatography. ATP treatment caused a five-fold increase in release of 14C-ATP but not 14C-ADP or 14C-AMP, indicating selectivity for release of ATP. Other P2 receptor agonists also caused significant 14C-ATP release, and the P2 receptor antagonists suramin, reactive blue-2 and pyridoxalphosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) inhibited ATP-induced 14C-ATP release to varying degrees, suggesting the involvement of a P2 receptor. ATP-induced 14C-ATP release was not affected by chelation of intracellular Ca2+ with BAPTA-AM, or by blockers of Ca2+ release from intracellular stores or of extracellular Ca2+ influx, suggesting a Ca2+-independent response. ATP-induced 14C-ATP release was significantly inhibited by non-selective anion channel blockers but not by blockers of ATP-binding cassette proteins, gap junction hemichannels, or vesicular exocytosis. Release of adenine nucleotides induced by 0 Ca2+ was, in contrast, not selective for ATP, and was susceptible to inhibition by gap junction blockers. These findings indicate that astrocytes are capable of ATP-induced ATP release and support a role for regenerative ATP release in glial Ca2+ wave propagation.

Intra-amniotic injection of pyridine nucleotides or adenosine triphosphate as countertherapy for 6-aminonicotinamide (6-AN) teratogenesis.

AbstractDay‐15 rat uteri were exteriorized under ether anesthesia and 100 μg 6‐AN + 500 μg oxidized (NAD;NADP) or reduced (NADH;NADPH) coenzyme; 500 μg adenosine triphosphate (ATP); or 100 μg nicotinamide (NAM) were injected into amniotic cavities in one horn. Embryos in the opposite horn served as injection controls. Mothers were closed and returned to their stock diet until autopsy on day 21 of gestation (day 0 = day of finding spermatozoa); fetuses were weighed, fixed, and examined for congenital abnormalities. Coenzymes, ATP, and NAM appeared to correct fetal and placental weight loss observed in 6‐AN‐treated fetuses. Vitamin and coenzyme therapy but not ATP treatment reduced the frequency and severity of brain and skeletal malformations to the double puncture, solvent control value. The data suggest that malformation and mortality may involve separate mechanisms.