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Determination of ATP and Its Metabolites Released from Rat Caudal Artery by Isocratic Ion-Pair Reversed-Phase High-Performance Liquid Chromatography
Abstract
A sensitive and selective assay method for adenine compounds (purines) using high-performance liquid chromatography with fluorescent detection was developed. The 1,N6-ethenoderivatives of adenine, adenosine, AMP, ADP, and ATP formed by reaction with chloroacetaldehyde at 80 degreesC were separated by ion-pair reversed-phase chromatography within 15 min under isocratic conditions. alpha,beta-Methylene adenosine 5'-diphosphate could be used as an internal standard for the determination of purines. The calibration graphs constructed with peak area ratios against amounts were linear between 0.1 and 10.0 pmol of each purine. The repeatability and intermediate precision were less than 6% (RSD, n = 5) and 8% (RSD, n = 3), respectively, at 0.5 pmol of each purine. A method for calculation of each purine amount which considers hydrolysis by derivatization is described. The optimized method was applied to determine the purines released from the rat caudal artery stimulated by noradrenaline.
... Currently, commercialized kits have also been developed for determination of intracellular ATP based on different enzymatic reactions. HPLC is widely used for determination of intracellular ATP (Kawamoto et al., 1998;Huang et al., 2003;Caruso et al., 2004;Zur Nedden et al., 2009;Zhou et al., 2012). Ionized reagents such as potassium phosphate are used as mobile phase. ...
... The wavelength for excitation and emission were set at 270/410 nm for ethenopurine derivatives and 285/395 nm for underivatized purines. The ethenopurine derivatives of ATP were separated within 15 min, and the lower limits of detection was 0.04 pmol per injection (Kawamoto et al., 1998). ...
... HPLC provides a more specific detection, but having trouble for determination of metabolites with low UV absorption. The LOQ range of HPLC with UV detector is 10-100 μmol/L, which would be much improved when using fluorescence detector (to 4-5 μmol/L) (Tong et al., 2013) (Kawamoto et al., 1998). HPLC is a good choice for quantitation of nucleotide sugars. ...
Food is essential for human survival. Nowadays, traditional agriculture faces challenges in balancing the need of sustainable environmental development and the rising food demand caused by an increasing population. In addition, in the emerging of consumers’ awareness of health related issues bring a growing trend towards novel nature-based food additives. Synthetic biology, using engineered microbial cell factories for production of various molecules, shows great advantages for generating food alternatives and additives, which not only relieve the pressure laid on tradition agriculture, but also create a new stage in healthy and sustainable food supplement. The biosynthesis of food components (protein, fats, carbohydrates or vitamins) in engineered microbial cells often involves cellular central metabolic pathways, where common precursors are processed into different proteins and products. Quantitation of the precursors provides information of the metabolic flux and intracellular metabolic state, giving guidance for precise pathway engineering. In this review, we summarized the quantitation methods for most cellular biosynthetic precursors, including energy molecules and co-factors involved in redox-reactions. It will also be useful for studies worked on pathway engineering of other microbial-derived metabolites. Finally, advantages and limitations of each method are discussed.
... Several analytical procedures utilizing either isocratic or gradient, reversed phase or ionexchange or ion-pairing HPLC in combination with UV or fluorescence detection are available for nucleotide analysis [5][6][7][8][9][10]. The method of choice is ion-pairing reversed phase HPLC due to its ability to provide stable and reproducible results [11]. ...
... The reaction scheme involves formation of an etheno-bridge between the 1 st nitrogen of the purine ring and amino group nitrogen on the 6 th carbon of the purine ring as described [12]. Variations of this method are used to quantify adenosine, adenine nucleotides and adenine analogues in different tissues, fluids, and cell culture systems [5][6][7][8][9][10]13]. However, the derivatization conditions described in these reports vary and depend on the tissue being analyzed making quantification of the nucleotides difficult to reproduce. ...
... However, the derivatization conditions described in these reports vary and depend on the tissue being analyzed making quantification of the nucleotides difficult to reproduce. Also, the derivatization conditions if not properly controlled leads to the hydrolysis of the adenine nucleotides adding yet more variation [6]. More importantly, a wide range of ion-pairing reagent concentrations (0.2-7.5 mM) are used to separate and quantify the 1, N 6 -etheno adenylate derivatives using reversed phase HPLC which is highly dependent on the amount of sample analyzed, and the specific HPLC conditions used to perform the separation [5,6,8]. ...
In mono-layered primary cell cultures baseline AMP and ADP levels are found nominally in the mid to low picomolar range and are thus difficult to measure with conventional HPLC methods that often require the pooling of samples or require indirect detection methods using radiotracers or enzyme coupled assays. To address this issue, we developed a highly sensitive and selective ion-pairing HPLC method with fluorescence detection to quantify adenine nucleotides and the adenylate energy charge in primary astrocyte cell cultures. To accomplish this, we optimized the fluorescence derivatization conditions and the HPLC parameters to achieve baseline separation and quantification of all adenine nucleotides. Nucleotides were converted to their respective 1, N(6)-etheno derivatives by incubating with chloroacetaldehyde at pH 4.5 and 60°C for 60 min. Under these conditions, the loss of the adenine nucleotides due to hydrolysis was minimized with a derivatization yield of 94.1% for 1, N(6)-ethenoadenosine. The optimal concentration of tetrabutylammonium phosphate, the ion-pairing reagent, required to achieve a reproducible separation of the adenine nucleotides was found to be 0.8mM. Calibration curves of nucleotide standards were linear within the range of 0.16-10.4 pmol for adenosine, 0.16-20.6 pmol for AMP, 0.15-19.2 pmol for ADP, and 0.15-19.5 pmol for ATP. The limits of detection and quantification for all adenine nucleotides were approximately 0.08 and 0.16 pmol, respectively. The intra- and inter-day variability for this method was less than 5.1 and 3.4%, respectively. This method was successfully used to measure all adenine nucleotides and an adenylate energy charge of 0.92±0.02 in primary astrocyte cell cultures.
... The concentration of ATP, ADP, AMP, and adenosine was determined in the cerebrospinal fluid (CSF) by HPLC with a fluorescence detector according to the method of Kawamoto et al. (Kawamoto et al., 1998) with some modifications. Cerebrospinal fluid collection was performed aby direct CC puncture using a collection apparatus (consists of a 1 ml syringe, a disposable intravenous infusion needle and a clip) with negative pressure as described . ...
... 4 Moreover, ATP concentration has proven to be an important determinant for cell death. 5 Nowadays, the available techniques for sensing ATP are liquid chromatography, 6 and fluorescence, 7 but they are difficult to include in an easy-to-use and point-of-care monitoring system. On the other hand, electrochemistry is very suitable for portable user-friendly devices, such as the huge success of systems for automonitoring glucose clearly demonstrate. ...
Electrochemical detection based on cyclodextrin supramolecular complexes is founded on the competitive binding between electroactive probes and target molecules. This limits their versatility to be used for sensing a broad range of metabolites. In this work, we demonstrate the significant role of zinc ions as well as of β-cyclodextrins modified with dipicolylamine and of a phenylboronic acid-modified ferrocene probe to address a selective electrochemical detection of adenosin triphosphate (ATP). Our findings will definitively have an impact in oncological point-of-care systems, since a high level of extracellular ATP reveals the inflammatory response due to chemotherapeutic treatments.
... The supernatant was mixed with equal volume of 5% HClO 4. Acid extracts of membrane and incubated medium were mixed with 1/10 vol. of 4.2 M KOH to neutralize and deposit potassium perchlorate. Adenosine in the acid extracts were converted to 1,N 6etheno derivatives by treating with 1% chloroacetaldehyde at 80°C for 30 min [17]. The 1,N 6 -etheno adenine nucleotides were separated using a JASCO HPLC system equipped with an analytical YMC-Pack ODS-A column (S-5, 4.6 × 100 mm, YMC Inc., Kyoto, Japan) [18] and monitored by a fluorescence detection following excitation at 270 nm at the emission wavelength of 410 nm. ...
Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A1 receptor and G-protein was assessed by means of two [³⁵S]GTPγS binding assays, i.e., conventional filtration method and [³⁵S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A1 receptor-mediated Gαi-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [³⁵S]GTPγS binding determined with conventional assay derives from functional activation of Gαi/o proteins (not restricted only to Gαi-3) coupled to adenosine A1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [³⁵S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %Emax values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A1 receptor/G-protein interaction in postmortem human brain tissue.
... Heat-conjugation of chloroacetaldehyde to adenosine under these conditions produces a strongly fluorescent 1,N 6 -ethenopurine derivative. 10 The conjugated sample (55 µL) was injected onto a reversed-phase 150 x 3mm column (3 µm, ODS Hypersil C18 resin, Keystone Scientific, Bellefonte, Penn) and eluted at a flow rate of 0.6 mL/min (Shimadzu LC-10AS pumps, Shimadzu Corporation, Kyoto Japan). The gradient profile, controlled by a Shimadzu SCL-10A system controller, consisted of ramping from 100% low organic [sodium acetate (35 mmol/L), acetonitrile (2%), pH 5.0] and 0% high organic [sodium acetate (35 mmol/L), acetonitrile (84%), pH 5.85] mobile phase to 65% low organic/35% high organic in 3.5 minutes. ...
Study Objectives
To examine the pattern of extracellular adenosine in the human brain during sleep deprivation, sleep, and normal wake.
Design
Following recovery from implantation of clinical depth electrodes, epilepsy patients remained awake for 40 continuous hours, followed by a recovery sleep episode.
Setting
Neurology ward at UCLA Medical Center.
Patients or Participants
Seven male epilepsy patients undergoing depth electrode localization of pharmacologically refractory seizures.
Interventions
All subjects were implanted with depth electrodes, a subset of which were customized to contain microdialysis probes. Microdialysis samples were collected during normal sleep, sleep deprivation, and recovery sleep from human amygdalae (n=8), hippocampus (n=1), and cortex (n=1).
Measurements and Results
In none of the probes did we observe an increase in extracellular adenosine during the sleep deprivation. There was a significant, though very small, diurnal oscillation (2.5%) in 5 of the 8 amygdalae. There was no effect of epileptogenicity on the pattern of extracellular adenosine.
Conclusions
Our observations, along with those in animal studies, indicate that the role of extracellular adenosine in regulating sleep pressure is not a global brain phenomenon but is likely limited to specific basal forebrain areas. Thus, if energy homeostasis is a function of sleep, an increased rate of adenosine release into the extracellular milieu of the amygdala, cortex, or hippocampus is unlikely to be a marker of such a process.
Citation
Zeitzer JM; Morales-Villagran A; Maidment NT et al. Extracellular adenosine in the human brain during sleep and sleep deprivation: an in vivo microdialysis study.
... Modern standard methods of precise determination of ATP concentration, such as spectrophotometry [6] and liquid chromatography [7], require qualified personnel and sophisticated expensive equipment, need complex pretreatment of samples for analysis [8,9]. Fluorescent, bio-and chemiluminescent methods are free from the above drawbacks; however, often they do not correspond with the demands of ATP monitoring [10]. ...
... ATP is routinely measured with spectrophotometry [21], liquid chromatography [87], fluorescence [55], chemiluminescence [1], and bioluminescence [117]. ...
... The complete interpretation of the energetic homeostasis of RBCs under stressful conditions might be performed with a liquid chromatographic (LC) method using ultravio-let (UV) detection that measures all the compounds involved in the degradation pathway for adenine nucleotides alongside the uric acid (Anderson and Murphy 1976;Schweinsberg and Loo 1980;Harmsen et al. 1981;Crescentini and Stocchi 1984;Stocchi et al. 1985Stocchi et al. , 1987Bontemps et al. 1986;Werner et al. 1987;Maessen et al. 1988; Tekkanat and Fox 1988;Smolenski et al. 1990;Nishikawa et al. 1991;Guieu et al. 1994;Smoleńska et al. 1999;Caruso et al. 2004;Taniai et al. 2006;Coolen et al. 2008;Yeung et al. 2008;Contreras-Sanz et al. 2012). The commercial kits (ab65313, abcam; K255-200, BioVision; TB288, Promega; FL-AA, Sigma-Aldrich) and LC methods using fluorometric detection or mass spectrometry (Levitt et al. 1984;Ramos-Salazar and Baines 1985;Fujimori et al. 1990;Kawamoto et al. 1998;Katayama et al. 2001;Tuytten et al. 2002;Xing et al. 2004;Klawitter et al. 2007;Wang et al. 2009;Birkler et al. 2010;Pabst et al. 2010;Jiang et al. 2012) can only detect some of these compounds simultaneously. ...
The ATP-related compounds in whole blood or red blood cells have been used to evaluate the energy status of erythrocytes and the degradation level of the phosphorylated compounds under various conditions, such as chronic renal failure, drug monitoring, cancer, exposure to environmental toxics, and organ preservation. The complete interpretation of the energetic homeostasis of erythrocytes is only performed using the compounds involved in the degradation pathway for adenine nucleotides alongside the uric acid value. For the first time, we report a liquid chromatographic method using a diode array detector that measures all of these compounds in a small human whole blood sample (125 μL) within an acceptable time of 20 min. The stability was evaluated for all of the compounds and ranged from 96.3 to 105.1% versus the day zero values. The measurement had an adequate sensitivity for the ATP-related compounds (detection limits from 0.001 to 0.097 μmol/L and quantification limits from 0.004 to 0.294 μmol/L). This method is particularly useful for measuring inosine monophosphate, inosine, hypoxanthine, and uric acid. Moreover, this assay had acceptable linearity (r > 0.990), precision (coefficients of variation ranged from 0.1 to 2.0%), specificity (similar retention times and spectra in all samples) and recoveries (ranged from 89.2 to 104.9%). The newly developed method is invaluable for assessing the energetic homeostasis of red blood cells under diverse conditions, such as in vitro experiments and clinical settings.
... The complete interpretation of the energetic homeostasis of RBCs under stressful conditions might be performed with a liquid chromatographic (LC) method using ultraviolet (UV) detection that measures all the compounds involved in the degradation pathway for adenine nucleotides alongside the uric acid (Anderson and Murphy 1976; Schweinsberg and Loo 1980; Harmsen et al. 1981; Crescentini and Stocchi 1984; Stocchi et al. 1985 Stocchi et al. , 1987 Bontemps et al. 1986; Werner et al. 1987; Maessen et al. 1988; Tekkanat and Fox 1988; Smolenski et al. 1990; Nishikawa et al. 1991; Guieu et al. 1994; Smoleńska et al. 1999; Caruso et al. 2004; Taniai et al. 2006; Coolen et al. 2008; Yeung et al. 2008; Contreras-Sanz et al. 2012). The commercial kits (ab65313, abcam; K255-200, BioVision; TB288, Promega; FL-AA, Sigma-Aldrich) and LC methods using fluorometric detection or mass spectrometry (Levitt et al. 1984; Ramos-Salazar and Baines 1985; Fujimori et al. 1990; Kawamoto et al. 1998; Katayama et al. 2001; Tuytten et al. 2002; Xing et al. 2004; Klawitter et al. 2007; Wang et al. 2009; Birkler et al. 2010; Pabst et al. 2010; Jiang et al. 2012 ) can only detect some of these compounds simultane- ously. To conserve the adenine nucleotides in RBCs, these residues should be immediately measured in whole blood (Stocchi et al. 1987). ...
The ATP-related compounds in whole blood or red blood cells have been used to evaluate the energy status of erythrocytes and the degradation level of the phosphorylated compounds under various conditions, such as chronic renal failure, drug monitoring, cancer, exposure to environmental toxics, and organ preservation. The complete interpretation of the energetic homeostasis of erythrocytes is only performed using the compounds involved in the degradation pathway for adenine nucleotides alongside the uric acid value. For the first time, we report a liquid chromatographic method using a diode array detector that measures all of these compounds in a small human whole blood sample (125 μL) within an acceptable time of 20 min. The stability was evaluated for all of the compounds and ranged from 96.3 to 105.1% versus the day zero values. The measurement had an adequate sensitivity for the ATP-related compounds (detection limits from 0.001 to 0.097 μmol/L and quantification limits from 0.004 to 0.294 μmol/L). This method is particularly useful for measuring inosine monophosphate, inosine, hypoxanthine, and uric acid. Moreover, this assay had acceptable linearity (r > 0.990), precision (coefficients of variation ranged from 0.1 to 2.0%), specificity (similar retention times and spectra in all samples) and recoveries (ranged from 89.2 to 104.9%). The newly developed method is invaluable for assessing the energetic homeostasis of red blood cells under diverse conditions, such as in vitro experiments and clinical settings.
... These methods require skilled personnel and sophisticated expensive equipment. 5,6 Another disadvantage of the above methods is the need for complicated sample preparation prior to the analysis. ...
Majority of biosensors for adenosine-5'-triphosphate (ATP) determination are based on cascades of enzymatic reactions, therefore they are sensitive to glucose or glycerol (depending on the enzymatic system) as well as to ATP. The presence of unknown concentrations of these substances in the sample greatly complicates the determination of ATP. To overcome this disadvantage of known biosensors, we developed a biosensor system consisting of two biosensors: the first one is based on glucose oxidase and is intended for measuring glucose concentration, and the second one is based on glucose oxidase and hexokinase and is sensitive towards both glucose and ATP. Using glucose concentration measured by the first biosensor, we can analyze the total response to glucose and ATP obtained by the second biosensor. Platinum disc electrodes were used as amperometric transducers. The polyphenilenediamine membrane was deposited onto the surface of platinum electrodes to avoid the response to electroactive substances. The effect of glucose concentration on biosensor determination of ATP was studied. The reproducibility of biosensor responses to glucose and ATP during a day was tested (R.S.D of responses to glucose was 3-6%, to ATP - 8-12%) as well as storage stability of the biosensors (no decrease of glucose responses and 43% drop of ATP responses during 50 days). The measurements of ATP and glucose in pharmaceutical vials (including mixtures of ATP and glucose) were carried out. It was shown that the developed biosensor system can be used for simultaneous analysis of glucose and ATP concentrations in water solutions.
... Electrical stimulation was applied to a lumber dorsal root and reflex potentials were recorded from a corresponding ipsilateral ventral root. The extracellular adenosine concentration was measured by making ethenoadenosine derivatives and using high-performance liquid chromatography (HPLC) according to the method described by Kawamoto et al. (1998). Adenosine kinase activity was assessed by measuring the in vivo phosphorylation of [U-14 C] adenosine. ...
Carbon dioxide (CO 2) has been used to euthanatize laboratory animals because of its rapid analgesic and sedative effects. However, the mechanism of these effects is unclear. To investigate the anesthetic effect of CO 2 , we used isolated spinal cords of neonatal rats as an alternative to living animals. In this preparation, stimulation of the dorsal root mainly evokes two types of reflex potentials, a monosynaptic reflex potential (MSR) and a slow ventral root potential (sVRP), at the ipsilateral ventral root. The sVRP was considered to reflect nociceptive responses because the order of inhibitory potency of analgesics for sVRP was quite similar to that for capsaicin-induced nociceptive responses in vivo. Acute hypercapnic acidosis (20% CO 2 , pH 6.7) depressed both reflex potentials in the isolated spinal cord. This depression was reversible and partly inhibited by a selective adenosine A 1 receptor antagonist. Accumulation of extracellular adenosine and inhibition of adenosine kinase activity were observed during hypercapnic acidosis. These results indicate that hypercapnic acidosis promptly depresses spinal nociceptive transmission through the activation of adenosine A 1 receptors. It is suggested that the accumulation of extracellular adenosine results from the inhibition of adenosine kinase during hypercapnic acidosis in the spinal cord of the neonatal rat. Introduction Exposure to carbon dioxide (CO 2) is widely used for euthanasia of laboratory animals. There are a number of merits for using CO 2 to kill animals, e.g. its immediate analgesic and sedative effect, cheapness, safety, ease of use and so on. On the other hand, there are also some concerns about side effects of CO 2 and thus for how CO 2 should be administered (Conlee et al., 2005). The most important question concering the usage of CO 2 is why CO 2 produces analgesia. The isolated spinal cord of the neonatal rat is useful for studying the spinal action of analgesics in vitro (Konishi and Otsuka, 1974). Stimulation of the dorsal root evokes reflex potentials at the corresponding ipsilateral ventral root. An early part of the reflex potential is a monosynaptic reflex potential (MSR), which is followed by a slow ventral root potential (sVRP). The sVRP is considered to reflect the nociceptive reflex at the spinal level (Faber et al., 1997). Therefore, it is expected that it could be useful for investigating analgesics as an alternative to living animals. First, in our experiments, the effects of analgesics, morphine and α 2 -adrenoceptor agonists, were examined to compare their potency between the isolated spinal cord in vitro and neonatal rats in vivo. To quantify the nociceptive behavior of immature rats, we used the elegant method developed by Kubota et al. (1996), which is suitable for objectively and easily investigating the effects of analgesics in small animals. Subsequently, we examined the effects of CO 2 on the isolated spinal cord in vitro.
... The remainder of the mixture was used for the measurement of inosine. The concentration of adenosine and adenine nucleotides was determined by HPLC with a fluorescence detector according to the method of Kawamoto et al. (1998) with some modifications as previously described (Otsuguro et al., 2009 ). The inosine con- BJP Adenosine and inosine release during hypoxia centration was determined according to the method described by Ferraris et al. (1991) with the following modifications: the samples were separated by reverse-phase HPLC with an ODS column (Cosmosil 5C18-MS, 4.6 ¥ 150 mm, Nacalai Tesque Inc., Kyoto, Japan) and monitored at 254 nm wavelength with a UV detector (UV-2075, JASCO, Tokyo, Japan). ...
Adenosine and inosine accumulate extracellularly during hypoxia/ischaemia in the brain and may act as neuroprotectants. In spinal cord, there is pharmacological evidence for increases in extracellular adenosine during hypoxia, but no direct measurements of purine release. Furthermore, the efflux pathways and origin of extracellular purines are not defined. To characterize hypoxia-evoked purine accumulation, we examined the effect of acute hypoxia on the extracellular levels of adenosine and inosine in isolated spinal cords from rats.
Extracellular adenosine and inosine concentrations were assayed in an in vitro preparation of the isolated spinal cord of the neonatal rat by HPLC.
The extracellular level of inosine was about 10-fold higher than that of adenosine. Acute hypoxia (10 min) caused a temperature-dependent increase in these two purines, which were inhibited by an increase in external Ca(2+), but not by several inhibitors of efflux pathways or metabolic enzymes of adenine nucleotides. Inhibitors of adenosine deaminase or the equilibrative nucleoside transporter (ENT) abolished the hypoxia-evoked increase in inosine but not adenosine. The inhibition of glial metabolism abolished the increase of both purines evoked by hypoxia but not by oxygen-glucose deprivation, hypercapnia or an adenosine kinase inhibitor.
Our data suggest that hypoxia releases adenosine itself from intracellular sources. Inosine formed intracellularly may be released through ENTs. During hypoxia, astrocytes appear to play a key role in purine release from neonatal rat spinal cord.
... The solvent system was the solution described above. The methodology used was modified from the original protocol described by Kawamoto et al. (1998). The nucleotides and adenosine were separated with a flow rate maintained at 1 mL min À1 (retention times, minimum: adenosine, 8.5 AE 0.1; AMP, 7.5 AE 0.07; ADP, 10.8 AE 0.2; ATP, 15.9 AE 0.2) and detected by UV spectroscopy at 254 nm. ...
In this work, we describe the ability of intact cells of Candida parapsilosis to hydrolyze extracellular ATP. ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. The ecto-ATPase activity was increased in the presence of 5 mM MgCl(2), with values of V(max) and apparent K(m) for Mg-ATP(2-) increasing to 33.80 +/- 1.2 nmol Pi h(-1) 10(-8) cells and 0.6 +/- 0.06 mM, respectively. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases and Na(+)-ATPases had no effect on the C. parapsilosis Mg(2+)-stimulated ATPase activity, but extracellular impermeant compounds, 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid and suramin, reduced enzyme activity in yeast living cells by 83.1% and 81.9%, respectively. ARL 67156 (6-N,N'-diethyl-d-beta-gamma-dibromomethylene ATP), a nucleotide analogue, also inhibited the ecto-ATPase activity in a dose-dependent manner. ATP was the best substrate for the yeast Mg(2+)-stimulated ecto-enzyme, but ADP, ITP, CTP, GTP and UTP were also hydrolyzed. A direct relationship between ecto-ATPase activity and adhesion to host cells was observed. In these assays, inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells. Based also on the differential expression of ecto-ATPase activities in the different isolates of C. parapsilosis, the possible role of this enzyme in fungal biology is discussed.
... The mixture was kept in a water bath (85 • C) for 2 min, and the pellet of islets was then lysed by mechanical stress and filtered through a 0.45 µm Millex filter (Millipore, Milford, MA, USA). Subsequently, 200 µL of filtrate was mixed with 20 µL of 2-chloroacetaldehyde solution and heated at 80 • C for 20 min [30,31]. An aliquot of 25 µL of the reaction mixture was then resolved by liquid chromatography. ...
Taurine (TAU), a naturally occurring sulfur-containing amino acid, is found at high concentrations in plasma and mammalian tissues and regulates osmolarity, ion channel activity, and glucose homeostasis. Several reports have shown that physiological plasma TAU levels seem to be important for adequate beta (beta)-cell function and insulin action, since low concentrations of TAU in the plasma have been reported in the pre-diabetic and diabetic states.
Glucose tolerance and insulin sensitivity were investigated in mice supplemented with 2% (w/v) TAU in their drinking water for 30 days, as well as the insulin secretion from isolated islets stimulated by glucose or L-leucine.
TAU-supplemented mice demonstrated improved glucose tolerance and higher insulin sensitivity, compared to controls (CTL). In addition, their islets secreted more insulin in response to high concentrations of glucose and L-leucine. L-[U-(14)C]leucine oxidation was higher in TAU than in CTL islets, whereas D-[U-(14)C]glucose oxidation, ATP levels, glucose transporter (GLUT) 2 and glucokinase (GCK) protein expressions were similar in both types of islets. The L-type beta(2) subunit voltage-sensitive Ca(2+) channel protein, as well as (45)Ca uptake, were significantly higher in TAU-supplemented than CTL islets. In addition, islets from TAU-supplemented mice secreted more glucagon than CTL islets at low glucose.
TAU supplementation improves glucose tolerance and insulin sensitivity in mice, as well as insulin secretion from isolated islets. The latter effect seems to be, at least in part, dependent on a better Ca(2+) handling by the islets.
... In some experiments, tissues were preincubated with drugs for at least 10 min before exposure to hypercapnia. The adenosine concentration was determined by HPLC according to the methods of Kawamoto et al. (1998) with some modifications. Each sample (250 mL) was mixed with 90 mL of 0.1 mol·L-1 citrate-phosphate buffer (pH 4.0), 10 mL of 40% chloroacetaldehyde and 25 mL of 4 mmol·L-1 a,b-methylene ADP (an internal standard), and then incubated at 80°C for 40 min. ...
The purine compounds, adenosine 5'-triphosphate (ATP) and adenosine, are known to accumulate in the extracellular space and to elicit various cellular responses during hypoxia/ischemia, whereas the roles of purines during hypercapnia are poorly understood. In this study, we examined the effects of various drugs affecting purine turnover on the responses to hypercapnia in the spinal cord.
Electrically evoked reflex potentials were measured in an in vitro preparation of the isolated spinal cord of the neonatal rat by extracellular recording. Extracellular adenosine concentrations were assayed by high performance liquid chromatography (HPLC) methods.
Hypercapnia (20% CO2) depressed the reflex potentials, which were partially reversed by an adenosine A1 receptor antagonist, 8-cyclopentyl theophylline, but not by a P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. Exogenous adenosine and ATP also depressed the reflex potentials via adenosine A1 receptors. The hypercapnia-evoked depression was not reversed by inhibitors of gap junction hemichannels, anion channels, P2X7 receptors or equilibrative nucleoside transporters, all of which might be involved in purine efflux pathways. The adenosine accumulation evoked by hypercapnia was not inhibited by tetrodotoxin, ethylene glycol-bis(beta-amino ethyl ether) tetraacetic acid (EGTA) or an ecto-ATPase inhibitor, ARL 67156. Homocysteine thiolactone, used to trap intracellular adenosine, significantly reduced extracellular adenosine accumulation during hypercapnia.
These results suggest that hypercapnia released adenosine itself from intracellular sources, using pathways different from the conventional exocytotic mechanism, and that this adenosine depressed spinal synaptic transmission via adenosine A1 receptors.
... Consequently, there is an actual demand for ATP assays. ATP concentration is usually analyzed using spectrophotometry [4], liquid chromatography [5], fluorescence [6], chemiluminescence [7], bioluminescence [8] methods, potentiometric [9][10][11] and amperometric [12][13][14][15][16][17][18] biosensors. Among these techniques, biosensors seem to be the most promising tools owing to their characteristics (Table 1). ...
ATP determination is of great importance since this compound is involved in a number of vital biological processes. To monitor ATP concentration levels, we have developed a microbiosensor based on cylindrical platinum microelectrode, covered with a layer of poly-m-phenylendiamine (PPD), and layer of co-immobilised glucose oxidase and hexokinase. Conditions for biosensor measurement of ATP (pH, Mg(2+) and substrates concentration) in vitro and microbiosensor characteristics such as sensitivity, selectivity, reproducibility, storage stability were studied and optimized. Under optimal conditions the microbiosensor can measure ATP concentrations down to a 2.5 microM detection limit with response time about 15 s. Interferences by electroactive compounds like biogenic amines and their metabolites, ascorbic acid, uric acid and L-cystein are rejected in general by the PPD layer. The microbiosensor developed is insensitive to ATP analogues (or substances with similar structure), such as ADP, AMP, GTP and UTP, too. It can be used for ATP analysis in vitro in the reactions consuming or producing macroergic triphosphate molecules to study kinetics of the process and in drug design concerning development of inhibitors specific to target kinases and others target enzymes.
... 0.3 g of -exotoxin was mixed with 1 ml of the supernatant of non producing Bt strains grown in LB medium and subsequently quantified. Replacing UV absorption by fluorescence detection has indeed markedly increased the sensitivity and the selectivity of the determination by RP-HPLC of adenosine and ATP and of their metabolites (15,16). ...
Beta-exotoxin is a nucleotide analogue produced by the entomopathogenic bacterium Bacillus thuringiensis. We have defined two new HPLC procedures for quantification of this exotoxin in culture supernatants of B. thuringiensis grown in poor or rich medium. The sample is prepared either by precipitation in solvent or by solid-phase extraction. Solvent precipitation is achieved treating the sample with acetone and acetonitrile. Solid-phase extraction is performed with a C18 and an anion-exchange cartridge. Reversed-phase HPLC with gradient elution of the prepared samples gives a limit of quantitation of 2 microg/ml for samples prepared by solvent precipitation and of 0.3 microg/ml for samples prepared by solid-phase extraction.
... The separations were monitored at 260 nM. The solvent system was based on one previously described (32). The following solvents were prepared: solvent A: 100 mM KH 2 PO 4 with 2 mM TBAB; solvent B: 100 mM KH 2 PO 4 with 2 mM TBAB and 15% CH 3 CN; solvent C: 100 mM KH 2 PO 4 with 2 mM TBAB and 35% CH 3 CN. ...
The propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica catalyzes the first step of propionate catabolism, i.e., the activation of propionate to propionyl-CoA. The PrpE enzyme was purified, and its kinetic properties were determined. Evidence is presented that the conversion of propionate to propionyl-CoA proceeds via a propionyl-AMP intermediate. Kinetic experiments demonstrated that propionate was the preferred acyl substrate (kcat/Km = 1644 mM(-1) x s(-1)). Adenosine 5'-propyl phosphate was a potent inhibitor of the enzyme, and inhibition kinetics identified a Bi Uni Uni Bi Ping Pong mechanism for the reaction catalyzed by the PrpE enzyme. Site-directed mutagenesis was used to change the primary sequence of the wild-type protein at positions G245A, P247A, K248A, K248E, G249A, K592A, and K592E. Mutant PrpE proteins were purified, and the effects of the mutations on enzyme activity were investigated. Both PrpEK592 mutant proteins (K592A and K592E) failed to convert propionate to propionyl-CoA, and plasmids containing these alleles of prpE failed to restore growth on propionate of S. enterica carrying null prpE alleles on their chromosome. Both PrpEK592 mutant proteins converted propionyl-AMP to propionyl-CoA, suggesting residue K592 played no discernible role in thioester bond formation. To the best of our knowledge, these mutant proteins are the first acyl-CoA synthetases reported that are defective in adenylation activity.
... The eluents, 50 mM KH 2 PO 4 , 50 mM K 2 HPO 4 , 4 mM TBAB, and 10% methanol, adjusted to pH 6.0 with H 3 PO 4 , were prepared in the day of use and Wltered through a 0.22-m Wlter (Millipore). The methodology used was modiWed from the original protocol proposed by Kawamoto et al. (1998), for the best separation and reproducibility under ours conditions. The hydrolysis of ATP and generation of ADP and AMP was determinate incubating 2 £ 10 7 cells/ml in a mixture containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 50 mM Hepes-Tris buVer, pH 7.2, and 100 M ATP. ...
The presence of Leishmania amazonensis ecto-nucleoside triphosphate triphosphohydrolase activities was demonstrated using antibodies against different NTPDase members by Western blotting, flow cytometry, and immunoelectron microscopy analysis. Living promastigote cells sequentially hydrolyzed the ATP molecule generating ADP, AMP, and adenosine, indicating that this surface enzyme may play a role in the salvage of purines from the extracellular medium. The L. amazonensis ecto-NTPDase activities were insensitive to Triton X-100, but they were enhanced by divalent cations, such as Mg(2+). In addition, the ecto-NTPDase activities decreased with time for 96 h when promastigotes were grown in vitro. On the other hand, these activities increased considerably when measured in living amastigote forms. Furthermore, the treatment with adenosine, a mediator of several relevant biological phenomena, induced a decrease in the reactivity with anti-CD39 antibody, raised against mammalian E-NTPDase, probably because of down regulation in the L. amazonensis ecto-NTPDase expression. Also, adenosine and anti-NTPDase antibodies induced a significant diminishing in the interaction between promastigotes of L. amazonensis and mouse peritoneal macrophages.
A novel fluorescent sensor based on an asymmetric anthracene derivative (SSAPA) was designed and synthesized. Using this molecule, a rapid and sensitive assay for detecting Tb3+ and ATP in aqueous solutions was established. The SSAPA molecule had excellent aggregation-induced emission (AIE) performance and good aqueous dispersion ability. This molecule could coordinate with Tb3+ and the fluorescence quenched linearly with the increase in the concentration of Tb3+ from 0.005 to 1.2 μM. Since both Tb3+ and adenosine triphosphate (ATP) have strong binding ability, ATP can compete with Tb3+ from the SSAPA/Tb3+ complex leading to fluorescence recovery. In this way, a brand-new fluorescent "turn-on" assay for ATP in the range from 0.01 to 0.4 μM was developed using the Tb3+-based complex probe. The detection limits for Tb3+ and ATP both reached single-digit nanomole per millilitre (2.8 nM and 4.5 nM, respectively), which demonstrated that this method has high sensitivity. Besides, Tb3+ and ATP also could be well detected in other complex environments such as real water samples or serum samples. This study provides a feasible assay for detecting trace amounts of Tb3+ and ATP in aqueous solutions.
Hexokinases play a critical role in the cellular uptake and utilization of glucose. As such, they are of fundamental importance to all cells. By catalyzing of glucose to produce glucose‐6‐phosphate, hexokinases control the first irreversible step of glucose metabolism and initiating all major pathways of glucose consumption. Our objective was to develop and validate highly sensitive and selective high performance liquid chromatography with photodiode array detector (HPLC‐PDA) assays allowing the determination of adenosine diphosphate (ADP), which was used for determination of the hexokinase activity. Samples were analyzed by HPLC‐PDA using a C18 analytical column (250 x 4.6 mm) for chromatographic separation. Optimal detection was achieved based on isocratic elution with a mobile phase consisting of mixture sodium phosphate monobasic buffer‐methanol. This method met all requirements of specificity, sensitivity, linearity, precision, accuracy, and stability generally accepted in bioanalytical chemistry and was successfully applied to study hexokinase activity in alloxan‐induced diabetic rat model. Determination of hexokinase activity will permit characterization of cellular metabolic state in many diseases, such as cancer and diabetes.
In addition to their roles in intracellular energy metabolism and nucleic acid synthesis, ATP and other nucleotides play important functions as extracellular signaling molecules (Figure 5.1). It is now more than 30 years since Burnstock1 first proposed that extracellular adenine nucleotides and nucleosides can be used for signal transduction at nerve endings in diverse tissues. Implicit in this notion of purine-based neurotransmission was a requirement for ATP (or other nucleotides) to be released and then degraded in a highly localized fashion at sites of cell-to-cell communication. Given the early emphasis on the role of purines in neuronal signaling, initial studies were focused on nucleotide/nucleoside release at neuron-to-neuron synapses, neuron-to-tissue varicosities, or the immediate vicinity of neuroendocrine cells (e.g., adrenal chromaffin cells).2,3 At approximately the same time, studies from the hematological literature were showing that platelets also release large amounts of ATP and ADP during activation of hemostasis and degranulation of dense granules.4-6 These early investigations demonstrated that neurons, neuroendocrine cells, and platelets could all release ATP via classical mechanisms involving exocytotic release of nucleotides copackaged with biogenic amines or other neurotransmitters within specialized secretory vesicles or granules (Figure 5.1 and Figure 5.2).
Analytical characteristics of a biosensor based on glucose oxidase and hexokinase and intended for ATP determination were studied. Platinum disc electrodes were used as amperometric transducers. Range of working potentials for biosensor functioning was shown. An optimal time of enzymes immobilization was determined. Optimal conditions for biosensor functioning during work with biological fluids were selected. Biosensor work in three buffer solutions (PBS, tris and HEPES) was investigated and it was shown that it was possible to obtain various operational characteristics of the biosensor depending on tasks that are assigned to it by varying the composition of sample. Reproducibility of biosensor responses to ATP and glucose during a day and of biosensor preparation was shown. The proposed biosensor can be further used for analysis of glucose and ATP content in water solutions.
In this study, we have developed a biosensor to detect adenosine triphosphate (ATP), based on fluorescence resonance energy transfer (FRET) and making use of the activities of exonuclease I (EXO I) and exonuclease III (EXO III). In the absence of ATP in the biosensor reaction system, the aptasensor is hydrolyzed by EXO I. When ATP is present, it conjugates with the aptasensor and protects it from hydrolysis by EXO I; the aptasensor can then hybridize with a fluorescent sequence linked to graphene oxide (GO). The dsDNA formed by the interaction between the aptasensor and the fluorescent sequence is then recognized and cleaved by EXO III. The increased distance between the fluorescent particle (FAM, 6-carboxyfluorescein) and the GO significantly hinders the FRET and increases the fluorescence of FAM. By incorporating EXO III into the process, the fluorescence signals of the biosensor are therefore greatly amplified and they were found to displayed a good linear relationship with ATP concentration, in the range from 0 to 3 μM. This system can be widely employed for the detection of other biological molecules.
We present here a novel and highly sensitive ion-pair hydrophilic interaction chromatography-tandem mass spectrometry (IP-HILIC-MS/MS) method for quantitation of highly polar acid metabolites like adenine nucleotides. A mobile phase based on diethylamine (DEA) and hexafluoro-2-isopropanol (HFIP) and an aminopropyl (NH2) column were applied for a novel chromatographic separation for the determination of AMP, ADP and ATP in biological matrices. This novel IP-HILIC mechanism could be hypothesized by the ion-pairing reagent (DEA) in the mobile phase forming neutral and hydrophilic complexes with the analytes of polar organic acids. The IP-HILIC-MS/MS assay for adenine nucleotides was successfully validated with satisfactory linearity, sensitivity, accuracy, reproducibility and matrix effects. The lower limit of quantitation (LLOQ) at 2.00ng/mL obtained for ATP showed a least 10-fold higher sensitivity than previous LC-MS/MS assays except nano-LC-MS/MS assay. In summary, this novel IP-HILIC-MS/MS assay provides a sensitive method for nucleotides bioanalysis and shows great potential to determine a number of organic acids in biological matrices.
A new spectrofluorimetric method was developed for determination of adenosine disodium triphosphate (ATP). We studied the interactions between ciprofloxacin (CIP)‐ Tb complex and adenosine disodium triphosphate (ATP) by using UV–Visible absorption and fluorescence spectra. Using CIP‐ Tb as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the CIP‐Tb complex at λ = 545 nm and the enhanced fluorescence intensity of Tb ion is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The linear ranges for ATP are 1.00 × 10∼1.00 × 10 mol/L with detection limits of 4.60 × 10 mol/L. This method is simple, practical, and relatively free of interference from coexisting substances and can be successfully applied to determination of ATP in samples. The mechanism of fluorescence enhancement between CIP‐ Tb complex and ATP was also studied.The authors are grateful to the National Natural Science Foundation of China for its financial support. (Grant No. 20271043).
A simple spectrofluorimetric method was developed for determination of trace amount of adenosine disodium triphosphate (ATP). Under the optimum conditions, we studied the interaction between CIP-Y3+-ATP complex by using absorption and fluorescence spectra. It was observed that ATP remarkably enhanced the fluorescence intensity of the CIP-Y3+ complex at λem=415 nm and the enhanced fluorescence intensity of Y3+ ion remained proportional to the concentration of ATP. The developed method was successfully applied for the determination of ATP in ATP injection sample. A suitable mechanism of fluorescence enhancement between CIP-Y3+ and the CIP-Y3+-ATP system was proposed and discussed.
A new spectrofluorimetric method was developed for determination of trace amount of adenosine disodium triphosphate (ATP). Using norfloxacin (NFLX)–terbium (Tb3+) as a fluorescent probe, in the buffer solution of pH=7.40, ATP can remarkably enhance the fluorescence intensity of the NFLX–Tb3+ complex at λ=545nm and the enhanced fluorescence intensity of Tb3+ ion is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The dynamic range for the determination of ATP is 1.00×10−6–1.60×10−5mol/L with detection limit of 4.13×10−8mol/L. This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of ATP in samples.
SUMMARY • Our recent studies of the tail artery of SHR.Cg-Leprcp/NDmcr (SHR-cp) rats showed dysfunction of presynaptic inhibitory adenosine-receptor on the sympathetic nerve and promotion of adenyl purine release from the endothelium. We examined the influence of hypotensive agents as an initial approach to understanding whether hypertension participates in these two changes in vascular function. • In the tail artery of SHR-cp rats treated with amlodipine (8 mg/kg, p.o.) and moxonidine (4 mg/kg, p.o.) for 10 weeks, 2-chloroadenosine, the adenosine receptor agonist, did not inhibit the contractions evoked by electrical nerve stimulation (1 Hz). In this artery, the release of adenyl purines evoked by noradrenaline was significantly decreased. Treatments with amlodipine and moxonidine increased the amount of plasma adenyl purines and decreased the systolic blood pressure. • Dysfunction of prejunctional adenosine-receptor on the sympathetic nerve terminal of SHR-cp rats’ tail artery seems not to be directly caused by hypertension. The extracellular level of ATP in SHR-cp rats seems to be regulated by blood pressure in a complicated manner.
GCN5-type N-acetyltransferases (GNATs) are enzymes that catalyse the transfer of the acetyl group from acetyl-CoA to a primary amine. GNATs are conserved in all domains of life. Some members of this family of enzymes acetylate the side-chain of specific lysine residues in proteins of diverse function. In bacteria, GNAT-catalysed protein acetylation regulates carbon metabolism, RNA metabolism and transcriptional regulation. Metabolic regulation in Streptomyces species is of interest due to the role of these organisms in natural product synthesis. Here we identify SlPatA, a GNAT in Streptomyces lividans with unique domain organization, and a new acetylation target, namely acetoacetyl-CoA synthetase (SlAacS). The latter has homologues in all domains of life. In vitro and in vivo evidence show that SlAacS is a bona fide acetoacetyl-CoA synthetase. SlPatA acetylates SlAacS more efficiently than it does acetyl-CoA synthetase, an enzyme known to be under acetylation control. SlPatA acetylates SlAacS at the active-site residue Lys617 and acetylation inactivates SlAacS. Acetylated SlAacS was deacetylated by a sirtuin-type protein deacetylase. SlAacS acetylation/deacetylation may represent a conserved mechanism for regulation of acetoacetyl-CoA synthetase activity in all domains of life.
The quenching of the fluorescence of a europium(III)– trifluoro-(2-thenoyl) acetone chelate solubilized in Brij-35 micelles with adenosine triphosphoric acid (ATP) was studied. It was shown that the sensitization of fluorescence with Gd3+ ions lowers the detection limit for ATP. The possibility of the direct determination of ATP in fruit juices with a detection limit of 8.0 10–8 M was demonstrated.
The following methods for the determination of adenosine triphosphate reported in the past 25 years are considered: bioluminescence
methods with the use of the firefly luciferase enzyme (with sensitivity to 10−14 M); chromatographic methods (ion-exchange, thin-layer, and high performance liquid chromatography) for the determination
of adenine nucleotides in mixtures with other nucleotides, nucleosides, and nitrogen bases; and fluorescence, spectrophotometric,
and electrochemical techniques (including those with the use of sensors), which are promising but not commonly used for the
determination of adenine nucleotides. The advantages and disadvantages of these methods are demonstrated.
An amperometric biosensor for adenosine 5 -triphosphate (ATP) was developed applying a com-petitive assay of two glucose converting enzymes: glucose oxidase (GOD) and hexokinase (HEX). Competition between GOD and HEX for glucose in presence of ATP, lead to a decrease in the current coming from the hydrogen peroxide generated by the GOD, and allows ATP detection. The biosensor was realized on commercial screen-printed electrodes modified with carbon nanotubes (CNTs). Confinement of CNTs in a polycarbonate membrane, Chitosan and Nafion polymers was investigated as possible solutions for implantable sensors. Nafion gave the best performances, with a sensitivity of 25 pA/M mm −2 , and a detection limit of 257 M. The sensor resulted able to measure ATP concentrations in the range of hundred of mol/l.
The effect of nicorandil, an ATP-sensitive K+ channel opener, on the level of intracellular Ca2+ ([Ca2+]i) and on ATP release in endothelial cells of the rat caudal artery was examined using a fluorescent confocal microscopic imaging system and high-performance liquid chromatography (HPLC) with fluorescent detection, respectively. Nicorandil significantly increased [Ca2+]i and the overflow of ATP and its metabolites. The former reaction was abolished in the absence of extracellular Ca2+, but it did not change in the presence of thapsigargin or cyclopiazonic acid. The increase in the overflow of ATP and [Ca2+]i induced by nicorandil was markedly suppressed by glibenclamide, an ATP-sensitive K+ channel blocker. The increase of [Ca2+]i induced by nicorandil was significantly and inversely correlated with the level of intracellular ATP in the endothelial cells, suggesting that activation of ATP-sensitive K+ channels by nicorandil increases Ca2+ influx in endothelial cells. The increase of [Ca2+]i might be associated with ATP release.
Ischemia/reperfusion injury in the intestine: Important roles for PKC, MAPK, and adenosine
Cancer cells must detach from the primary focus to initiate the process of metastasis. Previously, we demonstrated that intracellular Ca ²⁺ levels are increased in endothelial cells in the presence of cancer cells and that ATP derived from these cells causes this increase. The present study clarifies the mechanism of ATP release from cancer cells by investigating the effects of Cl ⁻ channel inhibitors and other drugs on ATP release from human fibrosarcoma cells (HT‐1080 cells).
Levels of extracellular ATP and its metabolites were measured using high‐performance liquid chromatography (HPLC) with fluorescent detection.
Significantly more extracellular ATP was released by suspended than by adherent HT‐1080 cells. The Cl ⁻ channel inhibitors 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (100 µmol/L), gadolinium (100 µmol/L) and niflumic acid (100 µmol/L) all significantly inhibited ATP release from HT‐1080 cells (1 × 10 ³ /mL) to 39.7 ± 6.5, 28.5 ± 2.5 and 82.5 ± 4.1% of control, respectively.
Neither of the p‐glycoprotein inhibitors (i.e. 50 µmol/L quinidine and 90 µmol/L verapamil) had any effect on ATP release from HT‐1080 cells. The gap junction hemichannel inhibitor Gap26 (300 µmol/L) slightly, but significantly, decreased ATP release by approximately 20%. The gap junction inhibitor 18‐α‐glycyrrhetinic acid (10 µmol/L) tended to inhibit ATP release from HT‐1080 cells, but the difference did not reach statistical significance.
These findings indicate that Cl ⁻ channels play the most important role in ATP release from detached cancer cells and that gap junction hemichannels are also associated with ATP release.
Protein glycation has been implicated in the development of diabetic complications and other health disorders, which mainly arise from accumulation of advanced glycation endproducts (AGEs) in vivo. Methylglyoxal (MGO), a typical reactive intermediate carbonyl formed in early glycation process, can react non-enzymatically with N-terminal amino groups on proteins, leading to their inactivation and generation of detrimental AGEs. Recently, it was reported that activity of creatine kinase (CK, EC 2.7.3.2) could be reduced or even eliminated completely after incubation with MGO in vitro. CK activity is usually determined by conventional colorimetric assays. However, these methods are not appropriate for monitoring the influence of MGO on CK activity since MGO can also directly react with creatine, a substrate of CK. In this study, an efficient and much more accurate HPLC approach was established to investigate the effect of MGO on CK activity. Aminoguanidine was utilized to eliminate interference from the undesirable reaction between residual MGO and creatine. It was found that higher concentrations of MGO and longer incubation time for CK and MGO caused more pronounced reduction in CK activity. This HPLC method greatly facilitates acquisition of kinetic data about CK reaction and through further improvement it may be adopted to rapidly screen potential inhibitors of MGO-induced glycation.
To investigate the intracellular concentrations of adenosine phosphates in Escherichia coli, especially during bioreactor cultivations, a method that enables reproducible determination of adenosine phosphates in culture solutions containing at least 0.25 g dry cell weight/L has been developed. The detection limits of AMP, ADP, and ATP were found to be as low as 1 pmol. The method involves fast sampling, instantaneous inactivation of cell metabolism, extraction of nucleotides, and quantitative analysis by ion-pair reversed-phase HPLC.
1. The role of ATP in the regulatory volume decrease (RVD) after hypotonic cell swelling was examined in cultured endothelial cells isolated from the rat caudal artery.
2. Hypotonic stress increased [Ca2+]i in addition to increasing the overflow of ATP and cell volume. The hypotonicity induced increase in [Ca2+]i was prevented by pyridoxalphosphate-6-azophenyl-1-2′,4′-disulphonic acid (PPADS; a P2 purinoceptor antagonist), U-73122 (a phospholipase C inhibitor) and thapsigargin (a Ca2+ pump inhibitor). However, the hypotonicity induced increase in cell volume was potentiated by PPADS, U-73122 and thapsigargin.
3. Similar changes were observed in cells treated with 2-methylthioATP, a P2Y purinoceptor agonist, but not by α,β-methylene ATP, a P2X purinoceptor agonist. Thus, it appears that the responses observed following hypotonic stress are mediated by activation of P2Y purinoceptors.
4. On the basis of these findings, it is suggested that ATP, which is released by hypotonicity, may participate in the RVD as a substantial regulator or initiator via P2 purinoceptor-induced increases in [Ca2+]i.
A newly available chromatography column packing material that employs hybrid particle technology was used to improve the analysis of adenosine compounds. Using a TBAS buffer/acetonitrile gradient this material permits separation of etheno-adenosine compounds in less than 4 min with excellent resolution and sensitivity (50 fmol). Variability of compound quantification is small (coefficients of variation 0.23+/-0.14% for 50 pmol and 1.70+/-0.53% for 0.5 pmol). The new method is well suited for the analysis of adenosine compounds in small biological samples and permits a high sample throughput in autosampler setups with high precision and reproducibility.
To characterize the influence of nitric oxide (NO) donors on the intestinal absorption of macromolecules, the relationship between the release rate of NO from NO donors and their absorption-enhancing effects and the effects of several scavengers and generators on the absorption-enhancing effects of NO donor were investigated. The t1/2 values of the NO release rate from 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-1-propanamine (NOC5), 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC7) and N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine (NOC12) are 25, 5 and 100min, respectively. The absorption-enhancing effects of NO donors on the absorption of fluorescein isothiocyanate dextrans with an average molecular weight of 4400 (FD-4) are NOC5 > NOC7 > NOC12 in the colon. The lowest enhancing effect of NOC12 may be due to the slow rate of NO release. The enhancing effect of NOC7 rapidly disappeared compared with the effect of NOC5. The results raise the possibility that the difference between NOC5 and NOC7 on enhancing effect is related to the t1/2 of the NO release. The NOC7-induced enhancing effect was prevented by the co-administration of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide sodium salt (C-PTIO), an NO scavenger; tiron, an O2(-) scavenger; mannitol, an OH* scavenger, and deferoxamine, peroxynitrate scavenger. Pyrogallol, an O2(-) generator, potentiated the NOC7-induced enhancing effect. These results support a role for peroxynitrate, and possibly OH*, in the NO donor-induced intestinal enhancing effect.
This review is aimed at providing readers with a comprehensive reference article about the distribution and function of P2 receptors in all the organs, tissues, and cells in the body. Each section provides an account of the early history of purinergic signaling in the organ?cell up to 1994, then summarizes subsequent evidence for the presence of P2X and P2Y receptor subtype mRNA and proteins as well as functional data, all fully referenced. A section is included describing the plasticity of expression of P2 receptors during development and aging as well as in various pathophysiological conditions. Finally, there is some discussion of possible future developments in the purinergic signaling field.
To obtain further information on time course and mechanisms of cell death after poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, we used HeLa cells exposed for 1 h to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. This treatment activated PARP-1 and caused a rapid drop of cellular NAD(H) and ATP contents, culminating 8-12 h later in cell death. PARP-1 antagonists fully prevented nucleotide depletion and death. Interestingly, in the early 60 min after challenge with N-methyl-N'-nitro-N-nitrosoguanidine, mitochondrial membrane potential and superoxide production significantly increased, whereas cellular ADP contents decreased. Again, these events were prevented by PARP-1 inhibitors, suggesting that PARP-1 hyperactivity leads to mitochondrial state 4 respiration. Mitochondrial membrane potential collapsed at later time points (3 h), when mitochondria released apoptosis-inducing factor and cytochrome c. Using immunocytochemistry and targeted luciferase transfection, we found that, despite an exclusive localization of PARP-1 and poly(ADP-ribose) in the nucleus, ATP levels first decreased in mitochondria and then in the cytoplasm of cells undergoing PARP-1 activation. PARP-1 inhibitors rescued ATP (but not NAD(H) levels) in cells undergoing hyper-poly(ADP-ribosyl)ation. Glycolysis played a central role in the energy recovery, whereas mitochondria consumed ATP in the early recovery phase and produced ATP in the late phase after PARP-1 inhibition, further indicating that nuclear poly(ADP-ribosyl)ation rapidly modulates mitochondrial functioning. Together, our data provide evidence for rapid nucleus-mitochondria cross-talk during hyper-poly(ADP-ribosyl)ation-dependent cell death.
A new spectrofluorimetric method has been developed for the determination of adenosine disodium triphosphate (ATP). We studied the interactions between the doxycycline (DC)-Eu3+ complex and adenosine disodium triphosphate (ATP) by using UV-visible absorption and fluorescence spectra. Using doxycycline (DC)-Eu3+ as a fluorescence probe, under the optimum conditions, ATP could remarkably enhance the fluorescence intensity of the DC-Eu3+ complex at lambda = 612 nm. The enhanced fluorescence intensity of the Eu3+ ion was in proportion to the concentration of ATP. The optimum conditions for the determination of ATP were also investigated. The linear ranges for ATP were 1.00 x 10(-7) - 2.00 x 10(-6) mol L(-1) with detection limits of 4.07 x 10(-8) mol L(-1). This method is simple, practical and relatively free of interference from coexisting substances, and can be successfully applied to the determination of ATP in samples. The mechanism of fluorescence enhancement between the doxycycline (DC)-Eu3+ complex and ATP was also studied.
We reported previously that IGF-I inhibits burn-induced muscle proteolysis. Recent studies suggest that activation of the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway with downstream phosphorylation of Forkhead box O transcription factors is an important mechanism of IGF-I-induced anabolic effects in skeletal muscle. The potential roles of other mechanisms in the anabolic effects of IGF-I are less well understood. In this study we tested the roles of mammalian target of rapamycin and glycogen synthase kinase-3beta (GSK-3beta) phosphorylation as well as MAPK- and calcineurin-dependent signaling pathways in the anticatabolic effects of IGF-I by incubating extensor digitorum longus muscles from burned rats in the presence of IGF-I and specific signaling pathway inhibitors. Surprisingly, the PI3K inhibitors LY294002 and wortmannin reduced basal protein breakdown. No additional inhibition by IGF-I was noticed in the presence of LY294002 or wortmannin. Inhibition of proteolysis by IGF-I was associated with phosphorylation (inactivation) of GSK-3beta. In addition, the GSK-3beta inhibitors, lithium chloride and thiadiazolidinone-8, reduced protein breakdown in a similar fashion as IGF-I. Lithium chloride, but not thiadiazolidinone-8, increased the levels of phosphorylated Foxo 1 in incubated muscles from burned rats. Inhibitors of mammalian target of rapamycin, MAPK, and calcineurin did not prevent the IGF-I-induced inhibition of muscle proteolysis. Our results suggest that IGF-I inhibits protein breakdown at least in part through a PI3K/Akt/GSK3beta-dependent mechanism. Additional experiments showed that similar mechanisms were responsible for the effect of IGF-I in muscle from nonburned rats. Taken together with recent reports in the literature, the present results suggest that IGF-I inhibits protein breakdown in skeletal muscle by multiple mechanisms, including PI3K/Akt-mediated inactivation of GSK-3beta and Foxo transcription factors.
A new spectrofluorimetric method was developed for determination of adenosine disodium triphosphate (ATP). We studied the interactions between oxytetracycline (OTC)-Eu3+ complex and adenosine disodium triphosphate (ATP) by using UV-vis absorption and fluorescence spectra. Using oxytetracycline (OTC)-Eu3+ as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the OTC-Eu3+ complex at lambda = 612 nm and the enhanced fluorescence intensity of Eu3+ ion is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The linear ranges for ATP are 8.00 x 10(-8)-1.50 x 10(-6) mol L(-1) with detection limits of 2.67 x 10(-9) mol L(-1). This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of ATP in samples. The mechanism of fluorescence enhancement between oxytetracycline (OTC)-Eu3+ complex and ATP was also studied.
Both insulin and metformin have been shown to attenuate hyperglycemia and reduce net muscle protein catabolism following burn injury. The purpose of this study was to compare the peripheral metabolic effects of insulin and metformin in severe burn patients.
Six adult patients with burns greater than 40% of their body surface underwent metabolic evaluation utilizing isotopic dilution of phenylalanine, femoral arterial and venous blood sampling, and sequential biopsies of leg muscle. Following baseline measurements, insulin was infused into the femoral artery at 0.45 mIU/min 100 mL leg volume. Patients were then given metformin (850 mg every 8 hours) for seven days with repeat metabolic evaluation before and during intra-arterial infusion of insulin.
Intra-arterial administration of insulin significantly increased insulin concentrations within the femoral vein, creating hyperinsulinemia localized to the extremity. Metformin had no significant effect on either peripheral glucose clearance or the rate of glucose oxidation. Furthermore, the availability of ATP and energy charge within muscle was not overtly affected by either insulin or metformin. Metformin did significantly increase the fractional synthetic rate of muscle protein which increased even further with insulin administration. Both metformin and insulin separately increased the rate of muscle protein synthesis as calculated using three compartment modeling.
This study demonstrates a significant anabolic effect on muscle protein with metformin and a modest response with insulin. Findings also suggest that metformin and insulin may work synergistically to further improve muscle protein kinetics.
We demonstrated previously that stimulation of the P2Y receptor enhanced the macromolecular permeability of cultured endothelial cell monolayers via the paracellular pathway. To determine whether the P2Y receptor participates in the regulation of permeability in intact microvessels, we have examined the effects of exogenous and endogenous ATP on the permeation of the surface tissue of perfused rat tail caudal artery using a fluorescein isothiocyanate-dextran (FD-4; MW 4400; 1.0 mg mL(-1)). The permeation of FD-4 was assessed by a confocal fluorescence imaging system. We found that 2-methylthioadenosine 5'-triphosphate, a P2Y receptor agonist, enhanced the fluorescence intensity of FD-4 in the surface of the rat caudal artery tissue and that it was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, a P2 receptor antagonist. Also, noradrenaline, a sympathetic neurotransmitter, and bradykinin, an inflammatory autacoid, enhanced the fluorescence intensity of FD-4 in the surface tissue of the rat caudal artery. The enhancement by noradrenaline was significantly inhibited by the P2 receptor antagonist. In addition, noradrenaline and bradykinin caused the release of ATP, ADP, AMP and adenosine from the endothelium of the rat caudal artery. These results indicated that the exogenous and endogenous ATP increased the macromolecular permeability of blood capillaries via the P2Y receptor. Such purinergic regulation of endothelial permeability may function in physiological and pathophysiological conditions.
Noradrenaline‐induced release of endogenous adenine nucleotides (ATP, ADP, AMP) and adenosine from both rat caudal artery and thoracic aorta was characterized, using high‐performance liquid chromatography with fluorescence detection.
Noradrenaline, in a concentration‐dependent manner, increased the overflow of ATP and its metabolites from the caudal artery. The noradrenaline‐induced release of adenine nucleotides and adenosine from the caudal artery was abolished by bunazosin, an α‐adrenoceptor antagonist, but not by idazoxan, an α 2 ‐adrenoceptor antagonist. Clonidine, α 2 ‐adrenoceptor agonist, contracted caudal artery smooth muscle but did not induce release of adenine nucleotides or adenosine.
Noradrenaline also significantly increased the overflow of ATP and its metabolites from the thoracic aorta in the rat; however, the amount of adenine nucleotides and adenosine released from the aorta was considerably less than that released from the caudal artery.
Noradrenaline significantly increased the overflow of ATP and its metabolites from cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the aorta was also much less than that from cultured endothelial cells from the caudal artery. In cultured smooth muscle cells from the caudal artery, a significant release of ATP or its metabolites was not observed.
These results suggest that there are vascular endothelial cells that are able to release ATP by an α 1 ‐adrenoceptor‐mediated mechanism, but that these cells are not homogeneously distributed in the vasculature.
Adenine compounds can be measured in picomole amounts using liquid chromatography of the fluorescent 1, N-etheno derivatives. The limit of detection for the etheno derivatives in tissue extracts, however, is tissue-dependent due to interference by nucleotides and fluorescent components which are normally present. Prior to derivatization nucleotides were partially removed from extracts of lymphocytes and erythrocytes by treatment with Dowex AG1-X2 anion exchange resin. Samples were analyzed using either a Partisil PXS 10/25 SCX column eluted with 100 mM NH4H2PO4, pH 4.5, at a flow rate of 2 ml/min; or using two μBondapak/C18 reversed-phase columns eluted with 5 mM KH2PO,4:25% methanol (V/V) pH 7.5, at a flow rate of 1 ml/min. Adenosine was found to be 0.07 nmole/ml in normal adult human plasma. The urine of a child with severe combined immunodeficiency disease associated with absence of adenosine deaminase contained a normal amount of adenosine (5–6 nmole/ml), but contained a high level (∼60 nmole/ml) of deoxyadenosine. Deoxyadenosine was not detected (
A previous method of determination of adenine compounds by high-performance liquid chromatography, using bromoacetaldehyde as a fluorescent reagent and a column of Hitachi gel No. 3012-N, was improved and extended to biological materials, especially to measure enzyme activities. A column packed with finer beads, Hitachi gel No. 3013-N, was found to be better than that of No. 3012-N, judging from the analysis time and resolution. ADP, from the hydrolysis of ATP by Na, K-ATPase, was determined quantitatively, and the enzyme activity was inhibited with ouabain. cAMP obtained from ATP by reaction with adenylate cyclase was also determined in the presence of various concentrations of L-epinephrine or sodium fluoride. The ATP levels in human blood were determined, and the cellular levels of ATP and ADP in neuroblastoma NIE 115 were examined as a function of cell growth.
A sensitive and specific assay for measurement of adenine nucleotides and adenosine by paired-ion high-performance liquid chromatography is described. The 1,N6-ethenoderivatives of ATP (epsilon-ATP), ADP (epsilon-ADP), AMP (epsilon-AMP), and adenosine (epsilon-Ado), formed by reaction with chloroacetaldehyde at 37 degrees C, were separated under isocratic conditions in 20 min. These compounds are strongly fluorescent at an emission wavelength of 280 nm, rendering a lowest detection limit of 2-5 pmol per injection. The detector responded linearly over the measured ranges (5-100 pmol for epsilon-Ado and 5-4000 pmol for nucleotides). Specificity was confirmed enzymatically. alpha, beta-Methyleneadenosine 5'-diphosphate could be used as an internal standard for measurement of the nucleotides. Significant amounts of NADH appeared as a separate peak in hypoxic tissue. Recoveries from snap-frozen kidney were 88, 92, 76, and 63% for AMP, ADP, ATP, and adenosine, with SD for recovery of 1.0, 10.5, 8.3, and 5.6%, respectively. This method was successfully used to measure adenine nucleotides and adenosine in oxygenated and hypoxic perfused rat kidneys.
To avoid some of the disadvantages associated with using radiolabeling to investigate adenyl purine content and release from excitable tissues, a reverse-phase high-pressure liquid chromatographic method utilizing fluorescence detection for the measurement of picomole amounts of endogenous ATP and its 6-amino purine analogs has been developed. This procedure has been used to determine the content of adenyl purines in the guinea pig vas deferens and that released from the tissue following stimulation of adrenergic nerves. The total tissue content was measured to be 1.6, 0.76, and 0.10 mumol/g of ATP, ADP, and AMP, respectively. However, adenosine could not be detected. Hypoxia caused a significant decrease in ATP content concomitant with an increase in adenosine content to 0.04 mumol/g. Following transmural electrical stimulation of the guniea pig vas deferens, the release of the following purine compounds was detected: ATP (0.106 nmol/g), ADP (0.242 nmol/g), AMP (0.035 nmol/g), and adenosine (0.454 nmol/g).
An isocratic high-performance liquid chromatographic system for the quantitation of AMP, ADP and ATP is presented. The separations were achieved at room temperature by reversed-phase chromatography (Supelcosil LC-18). The standard solvent was 220 mM potassium phosphate, pH 6.9, 1% (v/v) methanol and 0.3 mM tetrabutylammonium hydrogen sulphate. A selective retention of the adenine nucleotides as a group relative to the mono-, di- and triphosphates of guanosine, uridine and cytidine was observed under these experimental conditions. The adopted procedure was applied to the separation of adenine nucleotides in biological extracts, i.e., human myocard. The adenine nucleotides in an extract of myocard were quantitated in less than 20 min. Only 5-10 mg (wet weight) of myocard were needed in order to determine the energy charge of a myocardial sample. Also inosine was easily quantitated in this liquid chromatographic system.
The developed method of ion-pair reverse-phase high-pressure liquid chromatography (hplc) enables one to separate adenine nucleotides in cardiac tissue extract within 10 min with recovery of about 98% and lowest detection limit of 10−6m. Separation of creatine and phosphocreatine is performed by ion-pair reverse-phase hplc (recovery about 98%) using detector wavelengths of 210 nm; the lowest detection limit is in the range of 3 × 10−6m.
1. Noradrenaline-induced release of endogenous adenine nucleotides (ATP, ADP, AMP) and adenosine from both rat caudal artery and thoracic aorta was characterized, using high-performance liquid chromatography with fluorescence detection. 2. Noradrenaline, in a concentration-dependent manner, increased the overflow of ATP and its metabolites from the caudal artery. The noradrenaline-induced release of adenine nucleotides and adenosine from the caudal artery was abolished by bunazosin, an alpha 1-adrenoceptor antagonist, but not by idazoxan, an alpha 2-adrenoceptor antagonist. Clonidine, an alpha 2-adrenoceptor agonist, contracted caudal artery smooth muscle but did not induce release of adenine nucleotides or adenosine. 3. Noradrenaline also significantly increased the overflow of ATP and its metabolites from the thoracic aorta in the rat; however, the amount of adenine nucleotides and adenosine released from the aorta was considerably less than that released from the caudal artery. 4. Noradrenaline significantly increased the overflow of ATP and its metabolites from cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the aorta was also much less than that from cultured endothelial cells from the caudal artery. In cultured smooth muscle cells from the caudal artery, a significant release of ATP or its metabolites was not observed. 5. These results suggest that there are vascular endothelial cells that are able to release ATP by an alpha 1-adrenoceptor-mediated mechanism, but that these cells are not homogeneously distributed in the vasculature.
The components of the ectonucleotidase pathway at the immunoaffinity-purified striatal cholinergic synapse have been studied. The ecto-ATPase (EC 3.6.1.15) had a Km of 131 microM, whereas the ecto-ADPase (EC 3.6.1.6) had a Km of 58 microM, was Ca(2+)-dependent, and was inhibited by the ATP analogue 5'-adenylylimidodiphosphate (AMPPNP). The ecto-5'-nucleotidase (EC 3.1.3.5) had a Km of 21 microM, was inhibited by AMPPNP and alpha,beta-methylene ADP, and by a specific antiserum. The Vmax values of the ATPase, ADPase, and 5'-nucleotidase enzymes present at this synapse were in a ratio of 30:14:1. Very little ecto-adenylate kinase activity was detected on these purified synapses. The intraterminal 5'-nucleotidase enzyme, which amounted to 40% of the total 5'-nucleotidase activity, was inhibited by AMPPNP, alpha,beta-methylene ADP, and the antiserum, and also had the same kinetic properties as the ectoenzyme. The time course of ATP degradation to adenosine outside the nerve terminals showed a delay, followed by a period of sustained adenosine production. The delay in adenosine production was proportional to the initial ATP concentration, was a consequence of feedforward inhibition of the ADPase and 5'-nucleotidase, and was inversely proportional to the ecto-5'-nucleotidase activity. The function and characteristics of this pathway and the central role of 5'-nucleotidase in the regulation of extraterminal adenosine concentrations are discussed.
Electrical field stimulation elicits the release of catecholamines, adenine nucleotides, and adenosine from the rabbit pulmonary artery in a frequency dependent manner. To enhance our ability to investigate the release of endogenous adenine nucleotides and adenosine from this and other biological preparations, a new analytical procedure has been developed. This procedure involves the use of an internal standard, 9-beta-D-arabinofuranosyladenine (IS), the derivatization of ATP, ADP, AMP, adenosine (Ado), and IS with chloroacetaldehyde, the isocratic high-performance liquid chromatographic separation of these ethenopurine derivatives on an Ultron N-phenyl HPLC column, and their detection and quantitation by fluorescence spectroscopy. This procedure has enhanced sensitivity and reliability over existing procedures due to the stability of the chromatographic baseline and the use of an internal standard. When this analytical procedure was utilized to measure the adenine nucleotides and Ado that are released from the rabbit pulmonary artery in response to electrical field stimulation, it was observed that the release of endogenous ATP, ADP, AMP, and Ado exceeded that of endogenous norepinephrine. A molar ratio (6-amino purines:catecholamines) of approximately 2000:1 was obtained at a stimulation frequency of 16 Hz. This observation suggests an important extracellular role for adenine nucleotides and nucleosides in the physiology of vascular tissues.
ATP and noradrenaline are co-stored in synaptic vesicles in sympathetic nerves and when co-released act postjunctionally to evoke contraction of visceral and vascular smooth muscle. In the original purinergic nerve hypothesis it was proposed that ATP would then be sequentially broken down to ADP, AMP and adenosine. Although such breakdown can be measured, it is not clear how the time-scale of breakdown compares with the time-course of the postjunctional actions of ATP. We have investigated the role of ectoATPase in modulating purinergic neurotransmission in the guinea-pig vas deferens using ARL67156 (formerly FPL67516), a recently developed inhibitor of ectoATPase. ARL67156 (1-100 microM) potentiated neurogenic contractions in a concentration-dependent manner. Onset of potentiation was rapid and the effect reversed rapidly on washout of the drug. The effect was also frequency dependent, being greater at lower frequencies. The purinergic component of the neurogenic contraction was isolated using the alpha 1 antagonist prazosin (100 nM) and ARL67156 caused a similar potentiation. ARL67156 also potentiated contractions evoked by exogenous ATP (100 microM), but had no effect on those of the stable analogue alpha, beta-methylene ATP (500 nM). In the presence of the P2 purinoceptor antagonist PPADS (100 microM), ARL67156 also had no effect on contractions evoked by noradrenaline (10 microM) or KCI (40 mM). These results are consistent with an inhibitory action of ARL67156 on ectoATPase and suggest that ectoATPase modulates purinergic transmission in the guinea-pig vas deferens. When released from sympathetic nerves, ATP acts at the P2X purinoceptor, a ligand-gated cation channel, to evoke depolarization and contraction. In single acutely dissociated smooth muscle cells of the rat tail artery, studied under voltage-clamp conditions, ATP and its analogues evoke an inward current, with a rank order potency of 2-methylthioATP = ATP > alpha, beta-methylene ATP. This is very different from the order of potency for evoking contraction in whole vessel rings, which is alpha, beta-methylene ATP > > 2-methylthioATP > or = ATP. This discrepancy can be explained by a previously unrecognized attenuation of the action of ATP and 2-methylthioATP, but not alpha, beta-methylene ATP, by ectoATPase in whole tissues.
ATP and other nucleotides can be released from cells through regulated pathways, or following the loss of plasma membrane integrity. Once outside the cell, these compounds take on new roles as intercellular signaling molecules that elicit a broad spectrum of physiological responses through the activation of numerous cell surface receptor subtypes. This review summarizes recent advances in the molecular characterization of ATP receptors and discusses roles for cloned receptors in established and novel physiological processes.
Efficient control of synaptic transmission requires a rapid mechanism for terminating the actions of neurotransmitters. For amino acids and monoamines, this is achieved by their uptake into the cell by specific high-affinity transporters; acetylcholine is first broken down in the extracellular space and then choline is taken up by the cell. Because ATP is hydrolysed to adenosine by membrane-bound enzymes (ectonucleotidases) that are present in most tissues, it has been assumed that these enzymes terminate the neurotransmitter actions of ATP in the brain and in the periphery. We show here, however, that stimulation of sympathetic nerves innervating the guinea-pig vas deferens releases not only neuronal ATP, but also soluble nucleotidases that break down this ATP to adenosine, indicating that inactivation of ATP is increased by nerve activity. This release of specific nucleotidases together with ATP represents a new mechanism for terminating the actions of a neurotransmitter.