Adenosine Triphosphate Transport Research Articles

Effects of Time-Dependent Adenosine Triphosphate Consumption Caused by Neuron Firing on Adenosine Triphosphate Concentrations in Synaptic Boutons Containing and Lacking a Stationary Mitochondrion.

The precise mechanism behind the supply of adenosine triphosphate (ATP) to approximately half of the presynaptic release sites in axons that lack a stationary mitochondrion is not fully understood. This paper presents a mathematical model designed to simulate the transient ATP concentration in presynaptic en passant boutons. The model is utilized to investigate how the ATP concentration responds to increased ATP demand during neuronal firing in boutons with a stationary mitochondrion and those without one. The analysis suggests that neuron firing may cause oscillations in the ATP concentrations, with peak-to-peak amplitudes ranging from 0.06% to 5% of their average values. However, this does not deplete boutons lacking a mitochondrion of ATP; for physiologically relevant values of model parameters, their concentration remains approximately 3.75 times higher than the minimum concentration required for synaptic activity. The variance in average ATP concentrations between boutons containing a stationary mitochondrion and those lacking one ranges from 0.3% to 0.8%, contingent on the distance between the boutons. The model indicates that diffusion-driven ATP transport is rapid enough to adequately supply ATP molecules to boutons lacking a stationary mitochondrion.

Tandem Mass Tag Labeling–Based Analysis to Characterize Muscle-Specific Proteome Changes During Postmortem Aging of Bison Longissimus Lumborum and Psoas Major Muscles

The objective of the study was to examine the variations in sarcoplasmic proteomes of bison longissimus lumborum (LL) and psoas major (PM) muscles during postmortem aging utilizing tandem mass tag isobaric labeling coupled with liquid chromatography–mass spectrometry for the categorization of muscles with muscle-specific inherent color stability. A total of 576 proteins were identified in both bison LL and PM muscles, where 97 proteins were identified as differentially abundant (fold change > 1.5, P < 0.05) from the 3 comparisons between muscles during postmortem aging periods (PM vs. LL at 2 d, 7 d, and 14 d). Between muscles, the most abundant protein groups were based on functions such as electron transport chain or oxidative phosphorylation, tricarboxylic acid cycle, adenosine triphosphate transport, carbohydrate metabolism, fatty acid oxidation, chaperones, oxygen transport, muscle contraction, calcium signaling, and protein synthesis. In PM, most of the proteins from electron transport chain, tricarboxylic acid cycle, fatty acid oxidation, adenosine triphosphate and oxygen transport, and muscle contraction were more abundant or exhibited increased expression during aging compared with LL. On the other hand, the proteins involved in carbohydrate metabolism, chaperone function, and protein synthesis mostly exhibited decreased expression in PM muscles relative to LL. These results clearly demonstrate that the proteins associated with oxidative metabolism showed increased expression in PM muscles. This indicates that oxidative damage and subsequent color deterioration resulted in bison PM muscles being attacked by the reactive oxygen species produced during those metabolic processes. In contrast, proteins involved in glycolysis and chaperone activity exhibited a decrease in expression in bison PM muscles, resulting in a decline in color stability compared with LL. Because glycolytic enzymes generate reducing equivalents and chaperones maintain the native folded protein structure, they are consequently responsible for the color stability in LL muscles compared with PM.

Design of Lipophilic Split Aptamers as Artificial Carriers for Transmembrane Transport of Adenosine Triphosphate

Transmembrane transport plays an important role in many physiological functions, and mimicking this biological process in artificial systems has potential applications in biosensing, drug delivery,...

Efficacy and safety of Mildronate in treatment of postinfectious asthenic syndrome (clinical examples)

Asthenic syndrome is a non‑specific syndrome that is often found in general medical practice in patients with various somatic pathologies both during an exacerbation of a chronic disease, and after an acute process. The leading symptoms in the clinic of asthenia are pathological weakness and fatigue. According to various researchers, they are found in 15–45 % of people. To date, there is no universally accepted definition and clear classification of this syndrome. The main pathogenetic concepts are the psychosocial, infectious‑immune theory of asthenia. The development of asthenic syndrome in patients with somatic pathology may be associated with the depletion of the functional capabilities of the nervous system due to disturbances in the blood supply to the brain, auto‑toxicity or exposure to exogenous toxic factors in bacterial, viral infections. There is no specific treatment for this condition. This article presents two clinical cases of the development of post‑infection asthenic syndrome in a 47‑year‑old patient against community‑acquired pneumonia and in a 36‑year‑old patient after an acute respiratory‑viral infection of moderate severity. In both cases, drug treatment with Mildronate was carried out with a positive trend in the form of regression of asthenia symptoms, side effects during treatment. Mildronate belongs to the class of cytoprotectors – antihypoxants, activates glycolysis and prevents impaired transport of adenosine triphosphate, providing a sufficient level of energy synthesis, which allows cells to maintain homeostasis and morphological integrity. The immunoadjuvant properties of Mildronate are very important in post‑infectious asthenic syndrome, since a violation of the immune response plays a central role in its pathogenesis. The immunoadjuvant properties of Mildronate have been discovered in a number of studies.

Free-standing flexible and biomimetic hybrid membranes for ions and ATP transport

The transport of metabolites across robust, flexible and free-standing biomimetic membranes made of three perforated poly (lactic acid) (pPLA) layers, separated by two anodically polymerized conducting layers of poly (3,4-ethylenedioxythiophene-co-3-dodecylthiophene), and functionalized on the external pPLA layers with a voltage dependent anion channel (VDAC) protein, has been demonstrated. The three pPLA layers offer robustness and flexibility to the bioactive platform and the possibility of obtaining conducing polymer layers by in situ anodic polymerization. The incorporation of dodecylthiophene units, which bear a 12 carbon atoms long linear alkyl chain, to the conducting layers allows mimicking the amphiphilic environment offered by lipids in cells, increasing 32% the efficiency of the functionalization. Electrochemical impedance measurements in NaCl and adenosine triphosphate (ATP) solutions prove that the integration of the VDAC porin inside the PLA perforations considerably increases the membrane conductivity and is crucial for the electrolyte diffusion. Such results open the door for the development of advanced sensing devices for a broad panel of biomedical applications.

A Little AXER ABC: ATP, BiP, and Calcium Form a Triumvirate Orchestrating Energy Homeostasis of the Endoplasmic Reticulum

Pioneering work in the 1990s started to address an interesting question. How is the main cellular energy source, adenosine triphosphate (ATP), imported into the mammalian endoplasmic reticulum (ER)? Despite its high-energy demand, large volume, and structural as well as functional complexity, the ER harbors no intricate system for ATP synthesis or regeneration. Although the original biochemical reconstitution approaches established hallmarks of the ATP transport into the ER including nucleotide selectivity, affinity, and antiport mode, the more recent live-cell imaging methods employing sensitive, localized molecular probes identified the elusive ATP/adenosine diphosphate (ADP) exchanger. According to its selectivity and localization, the identified SLC35B1 protein was rebranded AXER. Here, we discuss the identification and regulation of AXER plus the cytosolic partners (AMP-activated protein kinase, AMPK) and subcellular structures (mitochondrial–ER contact sites, MERCs) acting in concert with it to orchestrate energy homeostasis of the mammalian ER. Furthermore, we combine the two seemingly controversial regulatory mechanisms (lowER and CaATiER) in a unifying hypothesis.

P3356Antiarrhythmic therapy with amiodarone decreases the uptake of 82-Rubidium-PET: studies in a rodent model

Abstract Introduction Myocardial perfusion imaging using positron emission tomography (PET) with 82Rubidium (82Rb) is used as a standard clinical examination. 82Rb-PET provides important information for the correct diagnosis and aids in prognostic risk assessment in patients with ischemic heart disease. 82Rb enters the myocardial cells by active transport of the sodium/potassium adenosine triphosphate transporter. Some antiarrhythmic drugs inhibits sodium/potassium adenosine triphosphate transporter. Purpose To determent if the uptake of 82Rb-PET using standard uptake value (SUV) is affected by administration of antiarrhythmic drugs. Methods Twenty-one Sprague-Dawley rats were baseline PET/CT scanned with 82Rb, using a Siemens preclinical PET/CT Inveon MM system and a Cardiogen-82 Rb generator. After baseline scan, the rats were divided into three groups. One control group without any treatment, and two received treatment with either orally administered Metoprolol 50mg/kg/24h or orally administered Amiodarone 120mg/kg/24h for seven days. All three groups were again PET/CT scanned with 82Rb-PET after the last administration on day seven. Data are presented as mean ±SEM. Results In the control group, there was no difference in mean SUV in the heart from day one to day seven (SUVmean 3.47±0.11 vs 3.45±0.35 p=0.97). In the group treated with metoprolol, likewise there was no difference in mean SUV in the heart from day one to day seven (SUVmean 3.87±0.13 vs 4.4±0.21 p=0.096). In contrast, in the group treated with amiodarone there was a significant difference in mean SUV from day one to day seven (SUVmean 4.14±0.15 vs 3.38±0.18 p=0.001). In all three groups there was no difference in mean SUV from day one to day seven in the liver and kidney. Conclusions The antiarrhythmic drug amiodarone had a clear impact on SUVmean in the heart. To the best of our knowledge, this is the first time it has been shown, that amiodarone lowers the SUV in 82Rb-PET in the heart. Amiodarone therefore seems to change to uptake of 82Rb in the myocyte. This could potentially influence the calculation to estimate absolute flow through the coronary arteries and lead to wrong conclusions regarding myocardial perfusion in patients treated with amiodarone. Further studies are needed to characterize the mechanism and to confirm the effect clinically.

Compartmentalization of Bioenergetic Substrate Delivery in Intact Cells

The intracellular production and transport of energetic substrate adenosine triphosphate (ATP) produced by mitochondria is dependent on multiple factors. These include local metabolic demand, mitochondrial motility and intracellular location, mitochondrial intermembrane potential, bioenergy substrate diffusion within the cell cytosol, and energy transport to the cell nucleus, which itself does not contain any mitochondria. Herein, we demonstrate via cell-based experiment and scaling argument that intracellular bioenergy transport is readily compartmentalized into perinuclear and peripheral regions of the cell. We draw on direct fluorescence-based measurement of quantum dot tracking, high-resolution respirometry, mitochondrial dynamics, and intermembrane potential to assess intracellular quantum dot diffusion to define the intracellular milieu for small molecule transport, and chemical perturbations which challenge cells by altering bioenergetics states. We identify a heterogeneous environment for intracellular bioenergy transport, with a dominant feature being present: the intracellular bioenergy distribution in response to pharmacologically induced cell challenge is determined to be preservation of perinuclear mitochondrial ATP-linked respiration in order to preserve, maintain, or otherwise support bioenergy delivery to meet the metabolic requirements of the cell nucleus whereas there is a decrement in bioenergetic capacity in the cell periphery. This dynamic effect of motile intracellular bioenergy production yields efficient transport of ATP in the maintenance of cellular health.

Etidronate attenuates tactile allodynia by spinal ATP release inhibition in mice with partial sciatic nerve ligation.

Etidronate is widely used as a therapeutic agent for osteoporosis. We have recently shown that intrathecal administration of etidronate into mice produces an analgesic effect against the capsaicin-induced nociceptive behavior. However, the effect of etidronate on neuropathic pain at the spinal level remains unknown. Therefore, we examined whether etidronate attenuates pain after partial sciatic nerve ligation (PSNL). We evaluated tactile allodynia 7days after PSNL by measuring paw withdrawal with the von Frey filament test. The mRNA and protein levels of SLC17A9 in the ipsilateral lumbar dorsal spinal cord of PSNL-operated mice were determined using real-time PCR and western blotting, respectively. PSNL-induced tactile allodynia was attenuated by oral and intrathecal administration of etidronate, with maximum efficiency at 90 and 60min after injection, respectively. The anti-allodynic effect of intrathecally administered etidronate was completely inhibited by an intrathecal administration of adenosine triphosphate (ATP). The solute carrier family, SLC17, mediates the transport of pain transmitters, like ATP and glutamate. Indeed, we detected several members of the SLC17 family in the mouse dorsal lumbar spinal cord. Among the detected mRNAs, only Slc17a9, encoding for neuronal vesicular ATP transporter, was significantly increased upon PSNL. SLC17A9 protein levels were also significantly increased. In mice subjected to PSNL, SLC17A9 was present in neurons and microglia, but not in astrocytes of the lumbar superficial dorsal horn. Collectively, our results suggest that etidronate produces its anti-allodynic effects by inhibiting SLC17A9-dependent exocytotic ATP release from the dorsal horn in mice subjected to PSNL.

Involvement of VNUT-exocytosis in transient receptor potential vanilloid 4-dependent ATP release from gastrointestinal epithelium.

Adenosine triphosphate (ATP) modulates mechanosensitive vagal afferent nerves in the gastrointestinal tract. ATP is stored in secretory vesicles via the ATP transporter VNUT. Recently, the bisphosphate clodronate was reported to inhibit VNUT and was suggested to be a safe potent therapeutic option for chronic pain. Transient receptor potential vanilloid 4 (TRPV4) is activated by mechanical stimuli and some epoxyeicosatrienoic acids and becomes sensitized under inflammatory conditions. We have previously reported that TRPV4 and VNUT are expressed in mouse esophageal keratinocytes and that TRPV4 activation induces ATP release in gastric epithelial cells. Here we show the expression of TRPV4 and VNUT in normal human gastrointestinal cell derived cell lines (GES-1 and CCD 841) and in tissues from normal and VNUT-KO mice. TRPV4 agonists (GSK101 or 8,9-EET) induced an increase in cytosolic Ca2+ and/or current responses in mouse primary colonic epithelial cells and CCD 841 cells, but not in cells isolated from TRPV4-KO mice. TRPV4 agonists (GSK101 or 5.6-EET) also induced ATP release in GES-1 and CCD 841 cells, which could be blocked by the VNUT inhibitor, clodronate. Thus, VNUT inhibition with clodronate could represent a novel therapeutic option for visceral pain.

New insight into foregut functions of xenobiotic detoxification in the cockroach Periplaneta americana.

The physiological functions of insect foregut, especially in xenobiotic detoxification, are scarcely reported because of unimportance in appearance and insufficient molecular information. The cockroach Periplaneta americana, an entomological model organism, provides perfect material to study physiological functions of foregut tissue due to its architectural feature. Through Illumina sequencing of foregut tissue from P. americana individuals (control) or insects treated with cycloxaprid, as a novel neonicotinoid insecticide, 54 193166 clean reads were obtained and further assembled into 53853 unigenes with an average length of 366 bp. Furthermore, the number of unigenes involved in xenobiotic detoxification was analyzed, mainly including 70 cytochrome P450s, 12 glutathione S-transferases (GSTs), seven carboxylesterases (CarEs) and seven adenosine triphosphate-binding cassette (ABC) transporters. Compared to control, the expression of 22 xenobiotic detoxification unigenes was up-regulated after cycloxaprid application, mainly containing 18 P450s, one GST, two CarEs and one ABC adenosine triphosphate transporter, indicating that the oxidation-reduction was the major reactive process to cycloxaprid application. Through quantitative real-time polymerase chain reaction analysis, the expression of selected unigenes (six P450s, one GST and one CarE) was up-regulated at least two-fold following cycloxaprid treatment, and was generally in agreement with transcriptome data. Compared to the previous midgut transcriptome of P. americana, it looks like the expressive abundance of the xenobiotic detoxification unigenes might be important factors to the detoxifying functional differences between foregut and midgut. In conclusion, insect foregut would also play important roles in the physiological processes related to xenobiotic detoxification.

Meldonium

Meldonium

A Computational Model of a Microfluidic Device to Measure the Dynamics of Oxygen-Dependent ATP Release from Erythrocytes

Erythrocytes are proposed to be involved in blood flow regulation through both shear- and oxygen-dependent mechanisms for the release of adenosine triphosphate (ATP), a potent vasodilator. In a recent study, the dynamics of shear-dependent ATP release from erythrocytes was measured using a microfluidic device with a constriction in the channel to increase shear stress. The brief period of increased shear stress resulted in ATP release within 25 to 75 milliseconds downstream of the constriction. The long-term goal of our research is to apply a similar approach to determine the dynamics of oxygen-dependent ATP release. In the place of the constriction, an oxygen permeable membrane would be used to decrease the hemoglobin oxygen saturation of erythrocytes flowing through the channel. This paper describes the first stage in achieving that goal, the development of a computational model of the proposed experimental system to determine the feasibility of altering oxygen saturation rapidly enough to measure ATP release dynamics. The computational model was constructed based on hemodynamics, molecular transport of oxygen and ATP, kinetics of luciferin/luciferase reaction for reporting ATP concentrations, light absorption by hemoglobin, and sensor characteristics. A linear model of oxygen saturation-dependent ATP release with variable time delay was used in this study. The computational results demonstrate that a microfluidic device with a 100 µm deep channel will cause a rapid decrease in oxygen saturation over the oxygen permeable membrane that yields a measurable light intensity profile for a change in rate of ATP release from erythrocytes on a timescale as short as 25 milliseconds. The simulation also demonstrates that the complex dynamics of ATP release from erythrocytes combined with the consumption by luciferin/luciferase in a flowing system results in light intensity values that do not simply correlate with ATP concentrations. A computational model is required for proper interpretation of experimental data.

Computational Analysis of a Microfluidic Device for Measuring Oxygen‐Dependent ATP Release from Erythrocytes

Release of adenosine triphosphate (ATP) from erythrocytes is believed to be a key component of the system for regulating microvascular blood flow. Recently, shear rate‐dependant ATP release from erythrocytes was measured using a microfluidic device (Wan et al, PNAS, 2008). These authors found ATP release occurred within 25–75ms following increased shear. The present work seeks to apply a similar approach to oxygen‐dependant ATP release, the dynamics of which are thought to be important in regulation of microvascular O2 delivery. Our computational model allows for the analysis of such a system to: i. design the optimal device to measure ATP release and ii. quantitatively interpret the resulting output signal. A computational model was constructed based on hemodynamics, convective‐diffusive transport of O2 and ATP, ATP/luciferin‐luciferase reaction kinetics, and optics. The computational model shows erythrocyte O2 levels can be altered rapidly enough to measure a delay in ATP release of 25ms. We show that this computational model is appropriate for analyzing properties associated with erythrocyte ATP release including the relationship between hemoglobin O2 saturation and magnitude of ATP release.

English

Endothelial cells under physiological conditions can alter energy balance by alteration in synthesis, metabolism and transport of adenosine triphosphate (ATP), which failed during endothelial cell dysfunction. ATP depleted endothelial cells are unable to perform their physiological functions as energy dependent protein secretion. Isolated human umbilical vein endothelial cells (HUVEC) from fresh umbilical cords were treated with 10 mM 2-deoxyglucose and 0.1 pg/ml of oligomycin for 6 h to induce ATP depletion. Nitric oxide (NO), von Willebrand factor (vWF), lactate dehydrogenase (LDH) release and trypan-blue exclusion were compared between treated and untreated cells. We observed a slight decrease in nitric oxide levels (P = 0.09) and vWF (P = 0.395) in the setting of 49.36% ATP depletion. There was no significant change in LDH release and cell viability between treated and untreated cells (P > 0.05). Since vWF exocytosis is an energy consuming process, decreased secretion of vWF in the isolated near-half percent of ATP depletion is not seemingly at odds. The application of vWF exocytosis fades as a candidate marker for ATP depletion induced injury, in cultured endothelial cells. Nitric oxide level and vWF secretion are not candidate markers of endothelial cell dysfunction in isolated partial ATP- depleted HUVECs. Measures such as arachidonic acid synthesis may be better alternatives.   Key words: Endothelial cell dysfunction, adenosine triphosphate (ATP) depletion, nitric oxide, von Willebrand factor (vWF).

Delayed Preconditioning by Sevoflurane Elicits Changes in the Mitochondrial Proteome in Ischemia-Reperfused Rat Hearts

Delayed myocardial preconditioning by volatile anesthetics involves changes in DNA transcription and translation. Mitochondria play a central role in myocardial ischemia/reperfusion (I/R) injury and in ischemic or pharmacologic preconditioning. In this study, we investigated whether there are alterations in myocardial mitochondrial protein expression after volatile anesthetic preconditioning (APC) to examine the underlying mechanisms of delayed cardioprotection. Thirty-six Sprague-Dawley rats were randomly assigned to 1 of 3 groups (n = 12 for each group). Rats in the delayed APC group were exposed to sevoflurane (2.5% for 60 minutes) 24 hours before myocardial ischemia was induced. Myocardial ischemia in the I/R and APC groups was induced by left coronary artery occlusion for 30 minutes, followed by 120 minutes of reperfusion. The control group received no treatment. The mitochondria fractions were prepared by differential centrifugation with density gradient isolation for proteomic analysis. Two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry was used to identify differences in the protein expression from mitochondria of the rat hearts. Fifteen differentially expressed mitochondrial proteins between the APC group and I/R group were identified and the expression patterns of 2 of the proteins were confirmed by Western blot analysis. These proteins were associated with mitochondrial substrate metabolism, respiration, and adenosine triphosphate (ATP)/adenosine diphosphate transport. The modifications of the mitochondrial proteome suggest an enhanced capacity of mitochondria to maintain myocardial ATP levels after I/R injury. Delayed sevoflurane myocardial preconditioning induces mitochondrial proteome remodeling, which mainly involves proteins that are related to ATP generation and transport. Therefore, proteomic changes related to bioenergetic balance may be the mechanistic basis of delayed anesthetic myocardial preconditioning.

Endothelial Nitric Oxide Synthase and Calcium Production in Arterial Geometries: An Integrated Fluid Mechanics/Cell Model

It is well known that atherosclerosis occurs at very specific locations throughout the human vasculature, such as arterial bifurcations and bends, all of which are subjected to low wall shear stress. A key player in the pathology of atherosclerosis is the endothelium, controlling the passage of material to and from the artery wall. Endothelial dysfunction refers to the condition where the normal regulation of processes by the endothelium is diminished. In this paper, the blood flow and transport of the low diffusion coefficient species adenosine triphosphate (ATP) are investigated in a variety of arterial geometries: a bifurcation with varying inner angle, and an artery bend. A mathematical model of endothelial calcium and endothelial nitric oxide synthase cellular dynamics is used to investigate spatial variations in the physiology of the endothelium. This model allows assessment of regions of the artery wall deficient in nitric oxide (NO). The models here aim to determine whether 3D flow fields are important in determining ATP concentration and endothelial function. For ATP transport, the effects of a coronary and carotid wave form on mass transport is investigated for low Womersley number. For the carotid, the Womersley number is then increased to determine whether this is an important factor. The results show that regions of low wall shear stress correspond with regions of impaired endothetial nitric oxide synthase signaling, therefore reduced availability of NO. However, experimental work is required to determine if this level is significant. The results also suggest that bifurcation angle is an important factor and acute angle bifurcations are more susceptible to disease than large angle bifurcations. It has been evidenced that complex 3D flow fields play an important role in determining signaling within endothelial cells. Furthermore, the distribution of ATP in blood is highly dependent on secondary flow features. The models here use ATP concentration simulated under steady conditions. This has been evidenced to reproduce essential features of time-averaged ATP concentration over a cardiac cycle for small Womersley numbers. However, when the Womersley number is increased, some differences are observed. Transient variations are overall insignificant, suggesting that spatial variation is more important than temporal. It has been determined that acute angle bifurcations are potentially more susceptible to atherogenesis and steady-state ATP transport reproduces essential features of time-averaged pulsatile transport for small Womersley number. Larger Womersley numbers appear to be an important factor in time-dependent mass transfer.

Reversed activity of mitochondrial adenine nucleotide translocator in ischemia-reperfusion.

Graft dysfunction as a result of preservation injury remains a major clinical problem in liver transplantation. This is related in part to accumulation of mitochondrial calcium. In an attempt to sustain cell and mitochondrial integrity during ischemia, intramitochondrial F(0)F(1) adenosine triphosphate (ATP) synthase reverses its activity and hydrolyzes ATP to maintain the mitochondrial transmembrane potential (mdeltapsi). It is not known how cytoplasmic ATP becomes available for hydrolysis by this enzyme. The authors hypothesized that mitochondrial adenine nucleotide translocator (ANT) reverses its activity during ischemia, making cytoplasmic ATP available for hydrolysis by F(0)F(1) ATP synthase. Rat livers were perfused with cold University of Wisconsin solution at 4 degrees C (39.2 degrees F)through the portal vein and processed immediately or after 24 hr of cold storage. Mitochondria were separated by differential centrifugation. ATP-dependent mitochondrial calcium-45 (45Ca)2+ uptake was determined after incubation with ATP (5 mM) or adenosine diphosphate (ADP) (5 mM) with or without 15 microM of bongkrekic acid (BA), an ANT blocker; the nonhydrolyzable analog of ATP (adenosine 5'-beta,gamma-imidotriphosphate [AMP-PNP]) served as the negative control. All measurements were performed in triplicate. Student t test, P<0.05 was taken as significant. Inhibition of ANT by BA prevents mitochondrial Ca2+ accumulation in the presence of ATP and high 45Ca2+ concentrations, and increased extramitochondrial 45Ca2+ stimulated mitochondrial 45Ca2+ uptake in the presence of ATP but not ADP, AMP-PNP, or BA. These data demonstrate that ANT plays an important role in mitochondrial Ca2+ uptake under ischemic conditions by reversing its activity and allowing transport of extramitochondrial ATP into the matrix for hydrolysis by reversed F(0)F(1) ATP synthase.

Characterization of the ATP transporter in the reconstituted rough endoplasmic reticulum proteoliposomes

Adenosine triphosphate (ATP) transporter from rat liver rough endoplasmic reticulum (RER) was solubilized and reconstituted into phosphatidylcholine liposomes. The RER proteoliposomes, resulting from optimizing some reconstitution parameters, had an apparent K m value of 1.5 μM and a V max of 286 pmol min −1 (mg protein) −1 and showed higher affinity for ATP and a lower V max value than intact RER ( K m of 6.5 μM and V max of 1 nmol). ATP transport was time- and temperature-dependent, inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, which is known as an inhibitor of anion transporters including ATP transporter, but was not affected by atractyloside, a specific inhibitor of mitochondrial ADP/ATP carrier. The internal and external effects of various nucleotides on the ATP transport were examined. ATP transport was cis-inhibited strongly by ADP and weakly by AMP. ADP-preloaded RER proteoliposomes showed a specific increase of ATP transport activity while AMP-preloaded RER proteoliposomes did not show the enhanced overshoot peak in the ATP uptake plot. These results demonstrate the ADP/ATP antiport mechanism of ATP transport in rat liver RER.

The effects of temperature, pH, and magnesium on the diffusion coefficient of ATP in solutions of physiological ionic strength

Intracellular diffusive transport of adenosine triphosphate (ATP) is critical to cellular metabolism. Physical models predict that diffusion coefficients ( D) of small molecules are functions of temperature and viscosity of the diffusive environment. Therefore, changes in body temperature, commonly experienced by poikilotherms, are expected to result in changes in the rate of intracellular ATP transport. However, it has been postulated that changes in the electrical charge of ATP may influence the interaction between ATP and the cytosol and that the temperature sensitivity of D ATP may deviate from the predicted relationship. To investigate the effects of changes in electrical charge on the temperature sensitivity of D ATP, we measured D ATP under various conditions of temperature, pH, and pMg 2+. Changes in pH and pMg 2+ were used to alter the net charge of ATP, and D ATP was measured in solutions of physiological ionic strength. Results showed a positive correlation between D ATP and temperature; D ATP = 1.75 ± 0.09, 3.68 ± 0.14, and 4.64 ± 0.13 (mean ± S.E.M.) × 10 −6 cm 2/s at 5°C, 25°C, and 40°C, respectively. Changes in pH and pMg 2+ did not significantly influence D ATP, and the change in D ATP with respect to temperature was similar to that predicted on the basis of changes in temperature and viscosity of the aqueous medium.