Application Of Adenosine Triphosphate Research Articles

ATP-Induced Contractile Response of Esophageal Smooth Muscle in Mice.

The tunica muscularis of mammalian esophagi is composed of striated muscle and smooth muscle. Contraction of the esophageal striated muscle portion is mainly controlled by cholinergic neurons. On the other hand, smooth muscle contraction and relaxation are controlled not only by cholinergic components but also by non-cholinergic components in the esophagus. Adenosine triphosphate (ATP) is known to regulate smooth muscle contraction and relaxation in the gastrointestinal tract via purinergic receptors. However, the precise mechanism of purinergic regulation in the esophagus is still unclear. Therefore, the aim of the present study was to clarify the effects of ATP on the mechanical responses of the esophageal muscle in mice. An isolated segment of the mouse esophagus was placed in a Magnus's tube and longitudinal mechanical responses were recorded. Exogenous application of ATP induced contractile responses in the esophageal preparations. Tetrodotoxin, a blocker of voltage-dependent sodium channels in neurons and striated muscle, did not affect the ATP-induced contraction. The ATP-evoked contraction was blocked by pretreatment with suramin, a purinergic receptor antagonist. RT-PCR revealed the expression of mRNA of purinergic receptor genes in the mouse esophageal tissue. The findings suggest that purinergic signaling might regulate the motor activity of mouse esophageal smooth muscle.

Exogenous ATP triggers antioxidant defense system and alleviates Cd toxicity in maize seedlings

The role of exogenous adenosine 5′-triphosphate (ATP) in the regulation of antioxidant response in plants under heavy metal stress is unclear. Here, we investigated the effects of exogenous ATP application on plant growth, antioxidant response, and Cd accumulation in maize seedlings. Treatment with 0.1 mM CdCl2 moderately reduced dry weight, decreased chlorophyll content, impaired photosynthesis, and increased lipid peroxidation in maize seedlings compared with controls. However, toxicity due to Cd was alleviated after 10–200 µM ATP treatment. Subsequently, the activity of Cd-regulated antioxidant enzymes, antioxidant metabolite accumulation, and total antioxidant capacity were drastically enhanced after 50 µM ATP treatment. Similar patterns were observed in the ADP-treated group but not in the AMP-treated group under Cd stress. However, the ATP-induced elevation in antioxidant defense ability was decreased by the inhibition of NADPH oxidase (NOX). ATP-induced elevation in NOX activity and H2O2 production was partly reversed by the inhibition of NOX in maize seedlings under Cd stress. Furthermore, ATP promoted Cd accumulation in the roots and shoots of maize seedlings. However, the ATP-induced increase in Cd accumulation was partly abolished by the inhibition of NOX. To our knowledge, this is the first report on the role and mechanism of exogenous ATP in regulating plant growth, antioxidant response, and heavy metal phytoextraction. The study provides a new method based on exogenous ATP for enhancing heavy metal tolerance in plants.

Label-Free and Bioluminescence-Based Nano-Biosensor for ATP Detection.

A bioluminescence-based assay for ATP can measure cell viability. Higher ATP concentration indicates a higher number of living cells. Thus, it is necessary to design an ATP sensor that is low-cost and easy to use. Gold nanoparticles provide excellent biocompatibility for enzyme immobilization. We investigated the effect of luciferase proximity with citrate-coated gold, silver, and gold-silver core-shell nanoparticles, gold nanorods, and BSA-Au nanoclusters. The effect of metal nanoparticles on the activity of luciferases was recorded by the luminescence assay, which was 3-5 times higher than free enzyme. The results showed that the signal stability in presence of nanoparticles improved and was reliable up to 6 h for analytes measurements. It has been suggested that energy is mutually transferred from luciferase bioluminescence spectra to metal nanoparticle surface plasmons. In addition, we herein report the 27-base DNA aptamer for adenosine-5'-triphosphate (ATP) as a suitable probe for the ATP biosensor based on firefly luciferase activity and AuNPs. Due to ATP application in the firefly luciferase reaction, the increase in luciferase activity and improved detection limits may indicate more stability or accessibility of ATP in the presence of nanoparticles. The bioluminescence intensity increased with the ATP concentration up to 600 µM with a detection limit of 5 µM for ATP.

Cost-Effective Production of ATP and S-Adenosylmethionine Using Engineered Multidomain Scaffold Proteins.

Adenosine triphosphate (ATP) and S-adenosyl-L-methionine (SAM) are important intermediates that are widely present in living organisms. Large-scale preparation and application of ATP or SAM is limited by expensive raw materials. To lower the production costs for ATP/SAM, in this study we used strategies applying engineered multidomain scaffold proteins to synthesize ATP and SAM. An artificial scaffold protein containing CBM3 domain, IM proteins and CL-labeled proteins was assembled to form complex 1 for catalytic reactions to increase ATP production. The ATP synthesis system produced approximately 25 g/L of ATP with approximately 15 g/L of ADP and 5 g/L of AMP using 12.5 g/L of adenosine and 40 g/L of sodium hexametaphosphate reaction at 35 °C and a pH of 8.5 for 6 h. Based on the above ATP synthesis system, two CL-labeled methionine adenosyltransferases (CL9-MAT4 and CL9-MAT5) were applied to construct scaffold protein complex 2 to achieve SAM synthesis. Approximately 25 μg of MAT4 in a reaction system with 0.3 M MgCl2 catalyzed at 20 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 0.9 g/L of SAM. Approximately 25 μg of MAT5 in a reaction system with 0.7 M MgCl2 catalyzed at 35 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 1.2 g/L of SAM. Here, we showed that low-cost substrates can be efficiently converted into high-value additional ATP and SAM via multi-enzyme catalytic reactions by engineered multidomain scaffold proteins.

Metal-free colorimetric detection of pyrophosphate ions by the peroxidase-like activity of ATP

Pyrophosphate (P2O74−, PPi) plays a vital role in ecological environment. Its elevated levels in water bodies can lead to eutrophication. Hence, its detection is extremely significant. Whereas most of the existing methods for the actual detection of PPi may cause environmental pollution or suffer from operational complexity. In this study, we introduced a sensitive and selective method for detecting PPi based on the fact that PPi can inhibit the peroxidase-like activity of adenosine 5′-triphosphate (ATP). This strategy not only eliminated the complexity of material preparation (ATP is commercialized), but also addressed the general need for metal ions in detecting PPi. The dynamic range of PPi detection was 1.0–200 μM and the detection limit was 74 nM. In addition, this strategy had been successfully applied to the determination of PPi in tap water and lake water. This work extends the application of natural biological small molecule ATP in the analysis and provides an innovative thought for the metal-free detection of PPi.

Computational Model to Predict Tissue Concentration of Drugs Delivered via Intravital Fluid Exchange Devices

The microcirculation is capable of precise blood flow regulation enabling fine control over the distribution of oxygen (O2) to tissues in the body. The objective is to determine the role of capillaries in sensing and reporting local O2 concentration within tissue to upstream arterioles. It is hypothesized that red blood cells (RBCs) release ATP into the vessel lumen following O2 desaturation; ATP in the plasma binds to purinergic receptors on the endothelium triggering a signalling cascade that leads to a conducted vasodilatory response. Luminal application of ATP is known to initiate conducted dilation in arterioles yet whether or not this conducted response is present in capillaries has yet to be demonstrated.To investigate this mechanism of vascular control, we developed gas and liquid microfluidic devices for use in intravital video microscopy experiments to enable the local control of gases and agonists in skeletal muscle. These devices consist of a microfluidic flow channel interfaced with a glass slide with a laser cut micro‐outlet to allow exchange between the device and skeletal muscle during in vivo experiments. Experimentally, the gas exchange device was used to control the spatial distribution of O2 in the tissue to cause a highly localized hypoxic condition, to induce the release of ATP from RBCs. The liquid device was used to introduce ATP to a localized region of the capillary bed to stimulate ATP mediated mechanisms downstream. Further, we used acetylcholine (ACh) and phenylephrine (PE) as positive and negative controls, respectively. All animal protocols were approved by Memorial University’s Institutional Animal Care Committee.To quantify the local distribution of solutes in skeletal muscle we developed a transient, 3D transport model to predict the concentration of solutes applied experimentally in skeletal muscle. The transport equation was discretized and solved in parallel on a graphics processing unit. The computational model predicts highly localized spatial distributions of these solutes. As expected, the highest concentration of simulated solutes was at the surface of the tissue in contact with the exchange window, with steep decreases in concentration with increasing distance from the exchange window, falling by two orders of magnitude within 240 μm of the edge of the window in both the x and y directions, and by 300 μm in the z direction (see Fig. 1& 2). Logarithmic increases in applied drugs within the simulated microfluidic channel raise relative tissue concentrations proportionally. Predicted steady state concentrations of ACh, PE, and ATP varied due to the assumed rate of enzymatic degradation and efflux into the vasculature.In conclusion, our novel intravital video microscopy devices allow the exchange of gas and drugs to tissue to investigate the mechanisms of regulation in the microcirculation. Further, we quantified the localization of our devices using computational models of molecular transport, confirming the highly‐localized concentration gradients.Support or Funding InformationProject funded through NSERC Discovery Grant to GM Fraser.Tissue model results for 15 mmHg oxygen. The colour map shows the predicted tissue concentrations with 15 mmHg oxygen at the micro‐outlet. The top panel shows the gradients at the surface of the tissue closest to the gas‐based microfluidic device, in the x–y plane. The bottom panel illustrates the tissue partial pressure of oxygen perpendicular to the x–z plane centred at the middle of the micro‐outlet.Figure 1Tissue model results for adenosine triphosphate 10−4 M. The log scale colour map shows predicted tissue concentrations with 10−4 M adenosine triphosphate (ATP) at the micro‐outlet. The top panel shows the gradients at the surface of the tissue at the micro‐outlet. The bottom panel illustrates tissue ATP levels in the x–z plane centred on the micro‐outlet. ATP levels within the volume were above 3.47 × 10−7 M due to the initial assumption of blood ATP concentration.Figure 2

Purinergic modulation of neuronal gap junction circuits in the CNS of the leech.

Gap junctions (GJs) are widely distributed in brains across the animal kingdom. To visualize the GJ- coupled networks of two major mechanosensory neurons in the ganglia of medicinal leeches, we injected these cells with the GJ-permeable tracer Neurobiotin. When diffusion time was limited to only 30min, tracer coupling was highly variable for both cells, suggesting a possible modulation of GJ permeability. In invertebrates the innexins (homologs of vertebrate pannexins) form the GJs. Because extracellular adenosine triphosphate (ATP) modulates pannexin and leech innexin hemichannel permeability and is released by leech glial cells following injury, we tested the effects of bath application of ATP after the injection of Neurobiotin and observed a significant increase in the number of neurons tracer coupled to the sensory neurons. This effect required the elevation of intracellular Ca2+ and could be produced by bath application of caffeine. Conversely, scavenging endogenous extracellular ATP with the ATPase apyrase decreased the number of coupled cells. ATP also increased electrical conductance and tracer permeability between the bilateral Retzius neurons. This modulatory effect of ATP on GJ coupling was blocked by siRNA knockdown of a P1-like adenosine receptor. Finally, exposure of leech ganglia to extracellular ATP induced a characteristic low frequency (<0.3Hz) rhythmic bursting activity that was roughly synchronous among multiple neurons, a behavior that was significantly attenuated by the GJ blocker octanol. These findings highlight the mediation by ATP of a robust physiological mechanism for modifying neuronal circuits by rapidly recruiting neurons into active networks and entraining synchronized bursting activity.

Development and Characterization of a Fluorescent Protein‐Based Biosensor for the Detection of Extracellular ATP

Extracellular adenosine 5′‐triphosphate (ATP) is a fundamental component of purinergic signaling that contributes to wide ranging physiological processes and pathophysiological conditions. New methods that enable direct measurement of extracellular ATP dynamics with high spatial and temporal resolution are expected to advance our understanding of purinergic signaling mechanisms in healthy and pathological states. Several distinct classes of fluorescent protein‐based biosensors have emerged as powerful molecular tools that report real‐time intracellular ATP dynamics with single‐cell and subcellular resolution, but there are no reports of leveraging such technology for the detection of extracellular ATP. Therefore, the focus of this study was to develop and characterize a series of fluorescent protein‐based biosensors for the detection of extracellular ATP. Our strategy was to anchor several candidate ATP biosensors to the plasma membrane and to position the biosensor in the extracellular space, allowing for the detection of extracellular ATP. We present the extensive characterization of one such cell surface‐tethered candidate biosensor. Live‐cell fluorescence microscopy was used to verify biosensor expression and localization at the cell surface and to measure sensor response to exogenous application of ATP when expressed by cell types including Neuro2A and HEK293 cells. Furthermore, we present progress using this sensor to measure extracellular ATP dynamics that are modulated by cellular processes, including ligand‐stimulated cellular release of ATP and degradation of ATP by ectonucleotidase activity. In conclusion, we are developing a genetically‐encoded fluorescent protein‐based biosensor that currently demonstrates the ability to measure dynamic changes in extracellular ATP levels in live‐cell proof‐of‐concept studies.Support or Funding InformationWe acknowledge funding support from National Institutes of Health grant NS092010

Direct measurement of ATP in seawater and application of ATP to monitor bacterial growth potential in SWRO pre-treatment systems

The use of adenosine triphosphate (ATP) to monitor bacterial growth potential of seawater is currently not possible as ATP cannot be accurately measured at low concentration in seawater using commercially available luciferase-based ATP detection. The limitation is due to interference of salt with the luciferin–luciferase reaction, which inhibits light production. This research demonstrates that new reagents developed for (i) ATP extraction from microbial cells and (ii) ATP detection in seawater are able to reliably detect Microbial ATP as low as 0.3 ng L–1 in seawater. The luminescence signal of the new detection reagent is significantly higher (>20 times) than the luminescence signal of the freshwater reagent, when applied in seawater. ATP can now be used to monitor bacterial growth potential (BGP) through pre-treatment trains of seawater reverse osmosis (SWRO) plants. The level of detection of the new BGP test is significantly lower than the estimated threshold value required to prevent biofouling in SWRO systems. The new reagents have been used to monitor Microbial ATP in coastal North Sea water. Moreover, Microbial ATP has been applied to monitor the bacterial growth potential (using indigenous bacteria) through the pre-treatment train of an SWRO desalination plant. A significant reduction (>55%) of the bacterial growth potential was found through the dual media filtration with 4.5 mg-Fe(III) L–1 coagulant. Overall, the new reagents can detect low Microbial ATP concentrations in seawater and can be used to monitor bacterial growth potential in seawater desalination plants.

ATP-induced P2X Receptor-Dependent Large Pore Formation: How Much Do We Know?

P2X receptors are a group of homo/hetero-trimeric membrane protein complexes with an integral ion channel that opens upon extracellular adenosine triphosphate (ATP) binding (North, 2002; Khakh and North, 2006). There are seven P2X subunits (P2X1-P2X7), all having a membrane topology of cytosolic N- and C-termini, and two transmembrane segments (TM1 and TM2) connected by a large extracellular domain (Figure ​(Figure1A;1A; Jiang L.-H. et al., 2013). During application of ATP for a few seconds, P2X receptors function as classical ligand-gated ion channels selectively permeable to small physiological cations such as Ca2+, Na+, and K+, with the exception of the human P2X5 receptor which exhibits significant Cl− permeability (Bo et al., 2003). Site-directed mutagenesis and functional studies of mammalian P2X receptors, in addition to the determination of the crystal structures of zebrafish P2X4 receptors in the apo, closed state and ATP-bound, open state, have defined the structural basis for ATP binding, ion permeation and channel gating (Kawate et al., 2009; Browne et al., 2010; Hattori and Gouaux, 2012; Jiang L.-H. et al., 2013; Jiang R. et al., 2013). Three ATP-binding pockets are located at the subunit interfaces (Figure ​(Figure1B),1B), each consisting of highly conserved residues from two adjacent subunits. Occupation of these sites by ATP or its synthetic analog agonists induces conformational changes of the extracellular domain which open the ion-permeating pathway formed by three TM2s (Figures 1C,D). The narrowest part of the ion-permeating pathway or the physical gate is provided by A347 and L351 in the crystal structures of zebrafish P2X4 receptor (Hattori and Gouaux, 2012) or the corresponding residues S342 and L346 in the structural models of rat and human P2X7 receptors (Figure ​(Figure1D)1D) (Bradley et al., 2011; Browne et al., 2013; Jiang L.-H. et al., 2013).

Effects of Adenosine Triphosphate (ATP) Treatment on Postharvest Physiology, Quality and Storage Behavior of Longan Fruit

The effects of adenosine triphosphate (ATP) on postharvest physiology, quality, and storage behavior of harvested longan fruit were investigated. Fruit of “Fuyan” longan were dipped in 0.8 mM ATP solution or distilled water (control) for 20 min. The fruit were dried prior to packaging and then stored at 28 °C and 90 % relative humidity. The results showed that as compared with control fruit, application of ATP could significantly reduce fruit respiration rate; delay an increase of cell membrane permeability in pericarp; maintain higher contents of chlorophyll, carotenoid, anthocyanin, and flavonoid; and subsequently retard the change of apparent color on longans. In addition, lower polyphenol oxidase (PPO) activity and higher total phenolics content were also found in ATP-treated fruit, which contributed to lower browning index in longan pericarp. The application of ATP could effectively inhibit the decreases in contents of total soluble solids (TSS), total soluble sugars, and vitamin C with a limited increase of titratable acidity (TA) content in pulp, which maintain better quality and flavor in longan pulp. Furthermore, ATP treatment could keep a higher rate of commercially acceptable fruit, effectively prevent weight loss from longans, and retard the developments of aril breakdown and fruit disease in longan fruit. It could be concluded that 0.8 mM ATP treatment for 20 min could greatly delay longan fruit senescence, maintain higher fruit quality, and exhibit better storage behavior which might suggest a promising method for keeping quality and extending the shelf life of harvested “Fuyan” longan fruit.

Allosteric regulation of the P2X4 receptor channel pore dilation.

Allosteric modulators of ligand-gated receptor channels induce conformational changes of the entire protein that alter potencies and efficacies for orthosteric ligands, expressed as the half maximal effective concentration (EC50) and maximum current amplitude, respectively. Here, we studied the influence of allostery on channel pore dilation, an issue not previously addressed. Experiments were done using the rat P2X4 receptor expressed in human embryonic kidney 293T cells and gated by adenosine 5'-triphosphate (ATP) in the presence and absence of ivermectin (IVM), an established positive allosteric regulator of this channel. In the absence of IVM, this channel activates and deactivates rapidly, does not show transition from open to dilated states, desensitizes completely with a moderate rate, and recovers only fractionally during washout. IVM treatment increases the efficacy of ATP to activate the channel and slows receptor desensitization during sustained ATP application and receptor deactivation after ATP washout. The rescue of the receptor from desensitization temporally coincides with pore dilation, and the dilated channel can be reactivated after washout of ATP. Experiments with vestibular and transmembrane domain receptor mutants further established that IVM has distinct effects on opening and dilation of the channel pore, the first accounting for increased peak current amplitude and the latter correlating with changes in the EC50 and kinetics of receptor deactivation. The corresponding kinetic (Markov state) model indicates that the IVM-dependent transition from open to dilated state is coupled to receptor sensitization, which rescues the receptor from desensitization and subsequent internalization. Allosterically induced sensitization of P2X4R thus provides sustained signaling during prolonged and repetitive ATP stimulation.

The calcium-activated chloride channel Anoctamin 1 contributes to the regulation of renal function

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.

Nano-nutrition of chicken embryos. The effect of silver nanoparticles and ATP on expression of chosen genes involved in myogenesis

It has been suggested that the quantity and quality of nutrients stored in the egg might not be optimal for the fast rate of chicken embryo development in modern broilers, and embryos could be supplemented with nutrients by in ovo injection. Recent experiments showed that in ovo feeding reduces post-hatch mortality and skeletal disorders and increases muscle growth and breast meat yield. Adenosine triphosphate (ATP) is a “ready for use” energetic molecule, while nanoparticles of silver (Nano-Ag) may penetrate tissues as well as cells and localise inside cells. In this investigation, we hypothesised that silver nanoparticles could be used as a protective carrier for ATP as well as an active agent. ATP and/or an ATP complex with Nano-Ag would be delivered to the muscle cells as a gene expression regulator and promoter of growth and development of embryo breast muscle. A collection of 160 broiler eggs was randomly divided into a Control group without injection and injected groups with hydrocolloids of Nano-Ag, ATP or a complex of Nano-Ag and ATP (Nano-Ag/ATP). The embryos were evaluated on day 20 of incubation. The results indicate that the application of ATP to chicken embryos increases expression of fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF) and Na+/K+ transporting ATPase (ATP1A1), which may indicate that an extra energy source can enhance molecular mechanisms of muscle cell proliferation. Nano-Ag also up-regulated expression of FGF2, VEGF, ATP1A1 and, also up-regulated expression of myogenic differentiation 1(MyoD1), affecting cell differentiation. The results indicate that ATP and Nano-Ag may accelerate growth and maturation of muscle cells.

ATP‐mediated vasodilatation: all thanks to potassium?

ATP‐mediated vasodilatation: all thanks to potassium?

Mechanisms Underlying the Effects of Salicylic Acid and Its Salts on Helix Neurons

In experiments on Helix neurons, we found that calcium is not involved in the neurotropic effects of salicylic acid and cobalt and zinc salicylates. This conclusion is based on the fact that the addition of BaCl2 to the applied solutions of salicylates did not change the effects of the latter. Extracellular application of adenosine triphosphate (ATP) significantly modified the effects of salicylic acid and its salts. We believe that the inhibitory effect of salicylic acid on Helix neurons depends on the ability of salicylic acid to suppress ATP synthesis, while the activating effect of cobalt and zinc salicylates on neurons is related to the opposite effects of these salts on ATP synthesis.

Combined used of nerve growth factor and adenosine triphosphate for repair of peripheral nerve injury and its influence on the denervated muscle

Objective To investigate the unification effect of rat nerve growth factor(NGF)and adenosine triphosphate(ATP)on repair of the peripheral nerve injury and the denervated muscle.Methods Ninety-six healthy SD rats weighing(250±15)g were randomly divided into NGF+ATP group,NGF group,ATP group and NS group.The sciatic nerves were cut for 3 mm,and were connected with silicone tube to create the nerve regeneration room.Then,all rats were intraperitoneally injected with NS0.4ml,ATP0.4ml(1 mg/ml),NGF 0.4 ml(0.2 μg/ml),NGF+ATP 0.4 ml(ATP1 mg/ml,NGF 0.2 μg/ml)separately once every three days.Six rats from each group were selected at 2,4,6 and 8 weeks after operation for sciatic nerve index(SFI)detection,gross observation and electrophysiological test;the specimens were obtained from the sacrificed rats for nerve regeneration,gastrocnemius muscle wet weight,fiber diameter and cross-sectional area of the gastrocnemius muscle(by HE staining).ResultsAt 2,4,6 and 8 weeks after operation,every six rats were chosen to be tested.The SFI,nerve conduction velocity and wet weight of the muscle of NGF+ATP group were better than those of the other three groups(P ＜ 0.05).The swelling of the rat toes of NGF+ATP group was slighter than that of NGF group and ATP group,with no loss of toes found in NGF+ATP group.At the 2nd week after operation,new nerve fibers were observed in the regeneration room,with no connection of the nerve fibers.The nerve fibers crossed the regeneration room at 4,6 and 8 weeks after operation.Compared with other three groups,the nerve growth condition was better in NGF+ATP group,with slighter adhesion and inflammation of nerve fibers.The growth condition of the nerve fibers of NGF group was better than ATP group,and that of NGF group and ATP group was better than NS group.At 2,4,6 and 8 weeks after operation,the electrophysiological condition and wet weight of muscle in NGF group was better than those in ATP group.While the SFI,the cross area and diameter of the muscle in NGF group were better than ATP group at 4,6 sand 8 weeks(P ＜ 0.05).All the parameters in NGF group and ATP group were better than NS group at each time point(P ＜ 0.05).Conclusions(1)The unification effect of NGF and ATP exert apparent effect in repair of the injured nerve and on the denervated muscle.Combined application of NGF and ATP is superior than NGF or ATP used alone.(2)A certain effect of ATP in repair of the injured nerve and on the denervated muscle is detected,but it is inferior to NGF and NGF+ATP. Key words: Sciatic nerve; Nerve growth factor; Adenosine triphosphate

Extracellular ATP excites ischemically sensitive cardiac sympathetic afferents

Cardiac sympathetic afferents are activated during myocardial ischemia, a complex process that leads to release of a number of mediators including extracellular adenosine 5′‐triphosphate (ATP). Also, ATP can elicit pain in animal models by stimulating somatic sensory nerve fibers. We therefore hypothesized that ATP stimulates ischemically sensitive cardiac afferents. Nerve activity of single unit cardiac afferents was recorded from the left sympathetic chain (T2–T5) in anesthetized cats. Receptive fields of 10 afferents (CV= 0.32–1.77 m/s) were identified in the left ventricle with a stimulating electrode. Five min of myocardial ischemia stimulated all 10 C‐fibers cardiac afferents, increasing their activity from 0.59±0.26 to 2.48±0.42 imp/s. Epicardial application of ATP (0.5–1 μmol), αβ‐meATP (0.2–0.5 μmol), and ADP (0.5–1 μmol) each stimulated 4 cardiac afferents, increasing the activity of these afferents by 356%, 435% and 178%, respectively. Administration of PPADS (30 mg/kg, iv), a P2 receptor antagonist, eliminated the responses of 3 other cardiac afferents to epicardial ATP, αβ‐meATP and ADP. PPADS also attenuated the ischemia‐related increase in activity of 3 separate afferents by 41%. These preliminary data suggest that extracellular ATP contributes to stimulation of cardiac sympathetic afferents during myocardial ischemia through a P2 receptor mechanism. (Supported by NIH HL66217)

Novel Excitatory Effects of Adenosine Triphosphate on Contractile and Pacemaker Activity in Rabbit Urethral Smooth Muscle

Adenosine triphosphate is thought to be an important neurotransmitter in urethral smooth muscle but its physiological role is still unclear. We characterized the effects of adenosine triphosphate on contractile and pacemaker activity in rabbit urethral smooth muscle. Tension recordings were made from strips of rabbit proximal urethral smooth muscle. Membrane currents from freshly isolated smooth muscle cells and interstitial cells of Cajal were recorded using the patch clamp technique. Intracellular Ca(2+) was measured using confocal microscopy. Exogenous application of adenosine triphosphate (10 microM) evoked robust contractions that were inhibited by the type 2 purinergic receptor blocker suramin (100 microM) and the selective type 2 purinergic Y1 receptor antagonist MRS2500 (Tocris Bioscience, Ellisville, Missouri) (100 nM). Application of the type 2 purinergic Y receptor agonist 2-MeSADP (1 microM) mimicked the effects of adenosine triphosphate. When smooth muscle cells were studied under voltage clamp at -60 mV, adenosine triphosphate evoked a large single inward current (greater than 1.2 nA) but 2-MeSADP produced a small current (about 16 pA). In contrast, when interstitial cells of Cajal were held at -60 mV, they showed spontaneous transient inward currents that were increased in frequency by adenosine triphosphate and 2-MeSADP. These excitatory effects were inhibited by suramin and MRS2500. Interstitial cells of Cajal showed spontaneous Ca(2+) waves that were increased in frequency by adenosine triphosphate and 2-MeSADP. These effects were also inhibited by suramin and MRS2500. Contractile effects of adenosine triphosphate in urethral smooth muscle are mediated by the activation of type 2 purinergic Y receptors on interstitial cells of Cajal.

ATP-regulation of antioxidant properties and phenolics in litchi fruit during browning and pathogen infection process

Abstract The impact of energy level on antioxidant properties in relation to pericarp browning and loss of disease resistance of litchi fruit was investigated. Litchi fruits were vacuum-infiltrated with distiled water (control), 1 mM adenosine triphosphate (ATP) and 0.5 mM 2,4-dinitrophenol (DNP) under 75 kPa for 3 min before being inoculated with Peronophythora litchi or not. ATP-treated fruits presented higher activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX). Higher activities of 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging, reducing power and contents of phenolic compounds were also observed in ATP-treated fruit during storage. In contrast, DNP treatment lowered the enzymes activities, scavenging ability and the contents of phenolic compounds. Higher levels of antioxidant enzymatic system and non-enzymatic system were observed in P. litchii-inoculated fruits than those in non-inoculated fruits. Application of ATP and DNP exhibited a similar change patterns and effects in inoculated fruits. When related to previously reported ATP levels, the results suggested that ATP levels could regulate the antioxidant system. Sufficient available energy is crucial for inhibiting browning and preventing the loss of disease resistance in harvested litchi fruit.