ATP Amplification Research Articles

Inhibition of urothelial P2X3 receptors prevents desensitization of purinergic detrusor contractions in the rat bladder.

To evaluate whether P2X3 receptors (P2X3R) are expressed in the bladder urothelium and to determine their possible function in modulating purinergic detrusor contractions in the rat urinary bladder. The expression of urothelial receptors was determined using conventional immunohistochemistry in bladders from normal Sprague-Dawley rats. The urothelial layer was removed by incubation with protamine, and disruption of the urothelium was confirmed using haematoxylin and eosin staining on bladder sections. Open cystometry was used to determine the effects of both urothelial removal as well as intravesical application of a specific P2X3R antagonist on bladder properties from intact and protamine-treated rats. Isometric contractile responses to potassium chloride (KCl) depolarization, electrical field stimulation (EFS) or chemical P2X activation were determined in normal and urothelium-denuded bladder strips, with and without application of the P2X3R antagonist. Immunohistochemical staining showed high expression of P2X3R in the medial and basal layers of the urothelium. Removal of the urothelial layer disturbed normal bladder performance in vivo and eliminated the effects of the P2X3R antagonist on increasing the contractile interval and reducing the amplitude of voiding contractions. Removal of the urothelium did not affect bladder strip contractile responses to KCl depolarization or EFS. Pharmacological inhibition of P2X3R prevented desensitization to P2X-mediated detrusor muscle contractions during EFS only in the strips with an intact urothelium. A concentration-dependent, specific inhibition of P2X3R also prevented desensitization of purinergic contractile responses in intact bladder strips. In the rat bladder, medial and basal urothelial cells express P2X3R, and specific inhibition of the receptor leads to a more hyporeflexive bladder condition. This pathway may involve P2X3R driving a paracrine amplification of ATP released from umbrella cells to increase afferent transmission in the sub-urothelial sensory plexus and desensitization of P2X1-mediated purinergic detrusor contractions.

Sensitive Electrochemical ATP Assay Combined with Enzymatic ATP Amplification Reaction

Sensitive Electrochemical ATP Assay Combined with Enzymatic ATP Amplification Reaction

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria

Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 104 colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PPi and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PPi-recycling loop was completed using ATP sulfurylase and adenosine 5′ phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water.

Reciprocating-flow ATP amplification system for increasing the number of amplification cycles

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76N. The backpressure on the columns was 0.03MPa in 1–10 ATP amplification cycles, and no increases in backpressure were observed.

Continuous-flow ATP amplification system on a chip

In this study, we constructed a novel microfluidic device for continuous-flow ATP amplification, using the SU-8:PDMS method. Sepharose beads immobilized with adenylate kinase and pyruvate kinase was packed into a microfluidic chamber to form lamination layer. Dry film type SU-8 was suitable to form a very thick mold for beads column reactor and its dam structure. A good correlation between amplified luminescence and initial ATP concentration was observed in this system. The gradient of amplification when performing six cycles of continuous-flow ATP amplification was 1.72 N .

Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87 N. Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

The LiMA Technology: Measurement of ATP on a Nucleic Acid Testing Platform

Detection of bacterial growth is used in the diagnosis and treatment of infectious disease, blood screening, food safety, product quality assurance, and life science research. The measurement of intracellular ATP content has long been the standard for rapid bacterial growth and viability measurement (1)(2)(3). The current methods used to detect ATP include luminescence generated by the enzyme system firefly luciferase/luciferin. The chemistry involved in this process is simple and can be used with a wide range of luminescence equipment, from handheld devices to sophisticated, laboratory-based instruments for high-throughput applications(4). We have developed an alternative, quantitative assay method for ATP based on a nucleic acid testing (NAT) format. During the past decade NAT has become the method of choice for bacterial identification (5). NAT hardware, such as thermal cyclers, isothermal instruments, and real-time/kinetic product detection systems, are now commonplace laboratory equipment. The new method, LiMA (Ligase Mediated ATP Amplification Assay)(6), uses DNA ligase, an ATP-requiring enzyme(7), to join 2 oligonucleotides in a nicked-DNA substrate and create a template that can be amplified in a DNA amplification reaction (Fig. 1⇓ ). Before the LiMA process, the ligase is treated with pyrophosphate to ensure that all the enzyme molecules are in the deadenylated form. Thus the ligase is inactive until it binds a molecule of ATP, which leads to the loss of the pyrophosphate moiety from ATP and the formation of a covalent enzyme—AMP intermediate linked to a lysine side-chain in the enzyme. In this reaction the enzyme becomes charged by an ATP molecule; essentially, a sample is scavenged for its ATP content. In the next step of the reaction the AMP nucleotide is transferred to the 5′ phosphate of the nicked-DNA strand, followed by attack on the AMP-DNA bond by the 3′-OH of …

Different behavior of agmatine in liver mitochondria: Inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 μM or 100 μM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H 2O 2 generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H 2O 2 and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron–sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca 2+. Instead, 1 mM agmatine generates larger amounts of H 2O 2 than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca 2+-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell.

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.

Excitable Spatio-Temporal Chaos in a Model of Glycolysis

We study the spatio-temporal dynamics of a qualitative model of the glycolytic reaction catalyzed by phosphofructokinase. Adding the nonlinear regulation by fructose-2,6-bisphosphate, the model is found to exhibit complex oscillations and two types of chaos. In particular, a region of excitable chaos is found in parameter space. In a spatially extended system, this excitable chaos gives rise to spontaneous irregular spiking as well as self-organized formation of spots of high ATP concentration with “center-surround” characteristics. The deterministic spiking has noise-like temporal properties but shows a pronounced “memory”, which we attribute to the transient aparition of a Turing-pattern that exists nearby in parameter space. The model thus provides an example of an excitable medium that prohibits (rather than permits) the propagation of concentration waves. This could be exploited by the living cell to self-organize pronounced local amplification of ATP.

Utilization of spectral absorption for measurement of adenylate cyclase activity

The purpose of this study was to improve our previously described enzymatic fluorometric assay of the adenylate cyclase activity. Using physicochemical characteristics of NADPH, of which a 0.1 mmol/L solution would have an optical density of 0.627, we measured the adenylate cyclase activity by the spectral absorption of NADPH. The assay consists of two parts: pharmacological modulation of adenylate cyclase and measurement of newly synthesized cyclic AMP. The latter part involves four steps: enzymatic destruction of noncyclic adenine nucleotides and phosphorylated metabolites, conversion of cyclic AMP to ATP, amplification of ATP by enzymatic cycling, and measurement of NADPH with spectral absorption, which is generated in proportion to initial cyclic AMP levels. This new assay was tested in membrane preparations made from rat hearts in comparison with the previously described fluorometric assay. We obtained identical results by spectrophotometry and fluorometry with high reproducibility. Because the fluorometric assay possesses a high sensitivity while the spectrophotometric method is advantageous because of its wide analytical range of cyclic AMP measurement, combination of fluorometric and spectrophotometric methods may offer a convenient way to measure the adenylate cyclase activities in various samples. J. Clin. Lab. Anal. 14:115–119, 2000. © 2000 Wiley-Liss, Inc.

Utilization of spectral absorption for measurement of adenylate cyclase activity

The purpose of this study was to improve our previously described enzymatic fluorometric assay of the adenylate cyclase activity. Using physicochemical characteristics of NADPH, of which a 0.1 mmol/L solution would have an optical density of 0.627, we measured the adenylate cyclase activity by the spectral absorption of NADPH. The assay consists of two parts: pharmacological modulation of adenylate cyclase and measurement of newly synthesized cyclic AMP. The latter part involves four steps: enzymatic destruction of noncyclic adenine nucleotides and phosphorylated metabolites, conversion of cyclic AMP to ATP, amplification of ATP by enzymatic cycling, and measurement of NADPH with spectral absorption, which is generated in proportion to initial cyclic AMP levels. This new assay was tested in membrane preparations made from rat hearts in comparison with the previously described fluorometric assay. We obtained identical results by spectrophotometry and fluorometry with high reproducibility. Because the fluorometric assay possesses a high sensitivity while the spectrophotometric method is advantageous because of its wide analytical range of cyclic AMP measurement, combination of fluorometric and spectrophotometric methods may offer a convenient way to measure the adenylate cyclase activities in various samples.

Measurement of adenylate cyclase activity in the right ventricular endomyocardial biopsy samples from patients with chronic congestive heart failure.

A highly sensitive fluorometric assay technique was adopted in order to examine the adenylate cyclase activity in the minute right ventricular endomyocardial biopsy samples from patients with chronic congestive heart failure (n = 10). Norepinephrine (10(-4) M) and adenosine (10(-3) M) were incubated for 30 min with 10 microl of membrane preparation (1-2 mg protein/mg) to analyze the extent of the receptor-coupled adenylate cyclase activity. Forskolin (10(-4) M) stimulation was used to estimate the maximum adenylate cyclase activity (pmol/mg protein/min, mean +/- SE). The new microanalytical cyclic AMP assay involves four steps: enzymatic destruction of noncyclic adenine nucleotides and phosphorylated metabolites, conversion of cyclic AMP to ATP, amplification of ATP by enzymatic cycling, and fluorometric measurement of NADPH, which is generated in proportion to initial cyclic AMP levels. Basal and forskolin-stimulated maximum adenylate cyclase activities were 75 +/- 8 and 123 +/- 15, respectively. Norepinephrine increased the adenylate cyclase activity to 107 +/- 14, while adenosine tended to decrease it to 65 +/- 7. In addition, elimination of adenosine by adenosine deaminase (10 U/ml) slightly increased the adenylate cyclase activity to 82 +/- 9. These results indicate that the adenylate cyclase activity can be measured in minute endomyocardial biopsy samples. Use of this new approach shows promise of becoming a new and potentially important way to predict the efficacy of pharmacological treatment.

Kinetic Aspects of ATP Amplification Reactions

The accurate determination of very low concentrations of ATP is important in many areas of pure and applied biochemistry, particularly hygiene monitoring in the food industry. Two mathematical models have been used to show that a luciferase-linked ATP recycling enzyme assay can determine sample ATP concentration from the time taken for observed bioluminescence to reach half its maximum value. Such a time-based assay has the potential to detect very low ATP concentrations in less than 10 min without the need for a sensitive photometer.

Enzymatic fluorometric assay for tissue cAMP.

cAMP is commonly measured using either immunoassay or high-performance liquid chromatography. The current methods are sensitive but may lack versatility and be expensive; also, radioactivity is potentially harmful to the operator and environment. Given these concerns, we developed a highly sensitive enzymatic fluorometric assay for cAMP. The method consists of five steps: (1) destruction of interfering compounds with apyrase, 5' nucleotidase, adenosine deaminase, and alkaline phosphatase; (2) conversion of cAMP to AMP; (3) conversion of AMP to ATP; (4) amplification of ATP by ATP-ADP cycling; and (5) fluorometric measurement of resultant NADPH. cAMP was measured in male Sprague Dawley rats anesthetized with pentobarbital. Stimulated rats (n = 4) received isoproterenol (16 micrograms/kg, s.q.) and aminophylline (20 mg/kg, s.q.), whereas controls (n = 4) received no additional drug. With the enzymatic fluorometric assay, cAMP content in heart, liver, and kidney (pmol/mg wet wt, mean +/- SEM) was 0.34 +/- 0.03, 0.33 +/- 0.03, and 0.92 +/- 0.11 in the control group and 0.77 +/- 0.10, 0.66 +/- 0.04, and 1.53 +/- 0.12 in the stimulated group, respectively. The total assay duration including sample reading procedure varied at 4.5-9.5 hr, depending on its sensitivity. cAMP from the same samples was measured using a commercially available enzyme immunoassay kit and was found to be very similar to the enzymatic fluorometric assay. We conclude that this new assay is sensitive, safe, versatile, and inexpensive and can be used to measure cAMP in multiple types of tissue, including biopsy samples weighing < 200 micrograms.