Rate Of ATP Release Research Articles

K+ Channel Activation Inhibits Allergen-Stimulated ATP Release by Human Bronchial Epithelial Cells

Allergens derived from the fungus Alternaria alternata stimulate the release of multiple alarmins including ATP, IL-33 and DNA fragments from human bronchial epithelial cells (hBECs). Oxidative stress induced by Alternaria extract activates ATP release by opening voltage-dependent anion channels (VDAC-1) in the apical membrane and by stimulating exocytosis of ATP containing membrane vesicles. The present study shows that pretreatment of hBECs with K2P channel inhibitors prior to Alternaria exposure significantly increases the initial rate of ATP release and inhibition of ATP loading into membrane vesicles with bafilomycin A1 blocks the response. Furthermore, pretreatment with the K+ channel openers, EBIO (KCa3.1 channels), NS11021 (KCa1.1 channels) and pinacidil (KATP channels) each inhibited the bafilomycin A1-dependent component of ATP release, reducing the initial rate of ATP secretion by ~50%. K2P channel blockers caused depolarization whereas K+ channel openers produced hyperpolarization of the cell membrane. Additionally, K2P channel inhibitors also increased Alternaria-stimulated Ca2+ uptake into hBECs while K+ channel openers reduced the Alternaria response by ~50%. Imaging experiments involving visualization of fluorescently labeled MARCKS proteins in the apical membrane showed that treatment with Alternaria caused a significant decrease in fluorescence intensity, however pretreatment with K+ channel openers, but not K2P channel blockers, inhibited the effect. These results are consistent with recent findings showing that MARCKS proteins bind to the plasma membrane through an electrostatic association with PIP2 and that phosphorylation by PKC or membrane hyperpolarization following K+ channel activation disrupts this interaction resulting in MARCKS detachment from the membrane. MARCKS detachment uncouples the membrane from the actin cytoskeleton, facilitating vesicle docking, fusion and release of ATP. This study was supported by NIH grants (AI128729 and AI169530) to HK and SMO and by USDA-AES 0016134 to SMO. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanosensitive ATP Release Through Transient Membrane Rupture in Alveolar Epithelial Cells

Mechanical ventilation (MV) is critical for the survival of patients with respiratory distress, but it can also exacerbate their symptoms: a condition called ventilator‐induced lung injury (VILI). Innate protection to attenuate VILI symptoms includes adenosine (Ado), an anti‐inflammatory mediator known to promote resolution of pulmonary edema. Airways‐localized Ado is a breakdown product of ATP by ectoenzymes, and high level of ATP has been found in the airways at the onset of MV treatment. Since stretch is a potent ATP secretion stimulus, this elevated ATP level has been explained by the intense deformation experienced by lung cells during MV. While ATP‐permeable channels or exocytosis have been frequently proposed as probable mechanisms for mechanosensitive ATP release, the pathway for cell stretch‐induced ATP secretion remains to be firmly identified. Here, we present an investigation on alveolar cells with a quantitative ATP imaging technique.Rat‐derived primary cultures of alveolar epithelial type 2 cells grown in a compliant silicone chamber for 7 days were uniaxially stretched to 30% linear deformation at varying strain rate and hold time. Level of extracellular ATP in medium was quantified with an ATP‐dependent luciferase‐luciferin light‐emitting reaction and wide field of view imaging of the whole stretch chamber (2×18 mm). Pre‐ and post‐stretch uptake of the plasma membrane impermeant DNA intercalator‐type propidium iodide (PI) dye was evaluated by epifluorescence microscopy.We first observed a strong spatial and temporal correlation between density distribution of extracellular ATP and PI uptake in response to stretch, suggesting a conductive pathway for ATP secretion. High rate of ATP release per cell ruled out involvement of ATP‐permeable channels however, and was instead consistent with a membrane rupture process. Quantification of PI fluorescence uptake suggested that almost all of the detected extracellular ATP leaked through those transient cracks. In addition, the fast decline of ATP response as soon as cell deformation ceased (half‐life ~5 seconds), excluded the involvement of slow acting membrane repair processes for the closure of the ATP‐permeable pathways but rather suggested rapid recoil of elastic membrane components. Further proof implicating the viscoelastic plasma membrane came from two prominent characteristics of the stretch‐induced ATP response: relaxation and sensitivity to strain rate, both hallmarks of viscoelasticity.This study gathered evidence to support an alternate mechanism of ATP release by mechanical stretch; one occurring through transient membrane rupture. This lytic mechanism is most likely non‐lethal however, due to the fast resealing of these membrane breaks resulting in minimal trauma on cells. We expect such cellular ATP release mechanism to contribute for the early detection of ATP inside mechanically‐ventilated lungs. This knowledge may help to shape a MV load history optimized to stimulate ATP release, and subsequent Ado formation, in order to minimize VILI‐associated symptoms.Support or Funding InformationSupported by the Canadian Institutes of Health Research (PJT166157 to R.G.)

Regulation of miR-1-Mediated Connexin 43 Expression and Cell Proliferation in Dental Epithelial Cells.

Many genes encoding growth factors, receptors, and transcription factors are induced by the epithelial-mesenchymal interaction during tooth development. Recently, numerous functions of microRNAs (miRNAs) are reportedly involved in organogenesis and disease. miRNAs regulate gene expression by inhibiting translation and destabilizing mRNAs. However, the expression and function of miRNAs in tooth development remain poorly understood. This study aimed to analyze the expression of miRNAs produced during tooth development using a microarray system to clarify the role of miRNAs in dental development. miR-1 showed a unique expression pattern in the developing tooth. miR-1 expression in the tooth germ peaked on embryonic day 16.5, decreasing gradually on postnatal days 1 and 3. An in situ hybridization assay revealed that miR-1 is expressed at the cervical loop of the dental epithelium. The expression of miR-1 and connexin (Cx) 43, a target of miR-1, were inversely correlated both in vitro and in vivo. Knockdown of miR-1 induced the expression of Cx43 in dental epithelial cells. Interestingly, cells with miR-1 downregulation proliferated slower than the control cells. Immunocytochemistry revealed that Cx43 in cells with miR-1 knockdown formed both cell-cell gap junctions and hemichannels at the plasma membrane. Furthermore, the rate of ATP release was higher in cells with miR-1 knockdown than in control cells. Furthermore, Cx43 downregulation in developing molars was observed in Epiprofin-knockout mice, along with the induction of miR-1 expression. These results suggest that the expression pattern of Cx43 is modulated by miR-1 to control cell proliferation activity during dental epithelial cell differentiation.

Critical role of ATP-induced ATP release for Ca2+ signaling in nonsensory cell networks of the developing cochlea

Spatially and temporally coordinated variations of the cytosolic free calcium concentration ([Ca2+]c) play a crucial role in a variety of tissues. In the developing sensory epithelium of the mammalian cochlea, elevation of extracellular adenosine trisphosphate concentration ([ATP]e) triggers [Ca2+]c oscillations and propagation of intercellular inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ waves. What remains uncertain is the relative contribution of gap junction channels and connexin hemichannels to these fundamental mechanisms, defects in which impair hearing acquisition. Another related open question is whether [Ca2+]c oscillations require oscillations of the cytosolic IP3 concentration ([IP3]c) in this system. To address these issues, we performed Ca2+ imaging experiments in the lesser epithelial ridge of the mouse cochlea around postnatal day 5 and constructed a computational model in quantitative adherence to experimental data. Our results indicate that [Ca2+]c oscillations are governed by Hopf-type bifurcations within the experimental range of [ATP]e and do not require [IP3]c oscillations. The model replicates accurately the spatial extent and propagation speed of intercellular Ca2+ waves and predicts that ATP-induced ATP release is the primary mechanism underlying intercellular propagation of Ca2+ signals. The model also uncovers a discontinuous transition from propagating regimes (intercellular Ca2+ wave speed > 11 μm⋅s-1) to propagation failure (speed = 0), which occurs upon lowering the maximal ATP release rate below a minimal threshold value. The approach presented here overcomes major limitations due to lack of specific connexin channel inhibitors and can be extended to other coupled cellular systems.

Numerical simulation of nucleotide transport in the human thoracic aorta

Extracellular adenine nucleotides ATP and ADP on vascular endothelial cells may play a role in the localization of atherogenesis by regulating the release of nitric oxide from endothelial cells and modulating intracellular calcium levels. To quantitatively investigate the concentration distribution of nucleotides on the luminal surface of the human thoracic aorta, we numerically simulated the transport of nucleotides using an aorta model constructed based on MRI images and analyzed the effects of different factors on nucleotide transport, such as ATP release rate (SATP), pulsatile flow and the absence of ATP in the main blood stream. The numerical results revealed that the combined concentration of ATP and ADP (cw−ATP+ADP) on the aortic surface varied from place to place, being relatively low in disturbed flow regions. In addition, cw−ATP+ADP was significantly affected by SATP. For relatively slow SATP, such as the moderate sigmoidal release model, cw−ATP+ADP was very low in certain flow regions with low wall shear stress. However, for very rapid SATP, such as the rapid linear release model, cw−ATP+ADP was relatively high in these same regions. The results also demonstrated that for relatively slow SATP, pulsatile blood flow enhanced cw−ATP+ADP. However, for very rapid SATP, pulsatile blood flow would reduce cw−ATP+ADP. Moreover, the absence of ATP within the main blood stream would not influence the distribution of cw−ATP+ADP. In conclusion, the concentration distribution of nucleotides along the aortic wall was quite uneven and determined by both the ATP release rate and the blood flow pattern in the aorta.

Zebrafish: A model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics

Mercury, anthropogenic release of uranium (U), and nanoparticles constitute hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. The impact of such pollutants on living organisms has been up to now approached by classical toxicology in which huge doses of toxic compounds, environmentally irrelevant, are displayed through routes that never occur in the lifespan of organisms (for instance injecting a bolus of mercury to an animal although the main route is through prey and fish eating). We wanted to address the effect of such pollutants on the muscle and brain mitochondrial bioenergetics under realistic conditions, at unprecedented low doses, using an aquatic model animal, the zebrafish Danio rerio.We developed an original method to measure brain mitochondrial respiration: a single brain was put in 1.5mL conical tube containing a respiratory buffer. Brains were gently homogenized by 13 strokes with a conical plastic pestle, and the homogenates were immediately used for respiration measurements. Skinned muscle fibers were prepared by saponin permeabilization.Zebrafish were contaminated with food containing 13μg of methylmercury (MeHg)/g, an environmentally relevant dose. In permeabilized muscle fibers, we observed a strong inhibition of both state 3 mitochondrial respiration and cytochrome c oxidase activity after 49 days of MeHg exposure. We measured a dramatic decrease in the rate of ATP release by skinned muscle fibers. Contrarily to muscles, brain mitochondrial respiration was not modified by MeHg exposure although brain accumulated twice as much MeHg than muscles.When zebrafish were exposed to 30μg/L of waterborne U, the basal mitochondrial respiratory control ratio was decreased in muscles after 28 days of exposure. This was due to an increase of the inner mitochondrial membrane permeability.The impact of a daily ration of food containing gold nanoparticles of two sizes (12 and 50nm) was investigated at a very low dose for 60 days (40ng gold/fish/day). Mitochondrial dysfunctions appeared in brain and muscle for both tested sizes.In conclusion, at low environmental doses, dietary or waterborne heavy metals impinged on zebrafish tissue mitochondrial respiration. Due to its incredible simplicity avoiding tedious and time-consuming mitochondria isolation, our one-pot method allowing brain respiratory analysis should give colleagues the incentive to use zebrafish brain as a model in bioenergetics.This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Sustained release of ATP encapsulated in chitosan oligosaccharide nanoparticles

The chemical cross-linked chitosan oligosaccharide (CSO) nanoparticles containing ATP/CSO ionic complex nano-components were prepared using combination techniques of W/O miniemulsion, chemical cross-linking and ionic complexation. The resulted nanoparticles had about 110 nm diameter and 20 mV surface zeta potential. The ATP loading efficiencies in nanoparticles could reach up to 40.6–69.5%. It was found that the ATP loading efficiency increased with increasing the amount and the molecular weight of chitosan oligosaccharide, and decreased with increasing molar ratio of glutaraldehyde to chitosan oligosaccharide. In vitro ATP release from chemical cross-linked CSO nanoparticles could continue for 24 h, and could also be adjusted by the amount and molecular weight of CSO, and the molar ratio of glutaraldehyde to CSO. The higher molecular weight and smaller amount of CSO, and the lower molar ratio of glutaraldehyde to CSO led the slower ATP release rate. Furthermore, it was also found that the CSO nanoparticles could be uptaken by HepG-2 tumor cells, and could be applied for intracellular drug delivery.

Partial Reactions of the ATP Synthesis Reaction in the E. Coli βD380C Mutant F1Fo-ATP Synthase

The initial stage of ATP synthesis by the E. coli wild type (WT) and mutant βD380C F1Fo-ATPase was compared at saturating substrate concentrations and a proton-motive force (pmf) of ∼315 mV. The enzymes catalyze both ATP hydrolysis (Baylis Scanlon et al., 2008, J. Biol. Chem. 283, 26228-26240) and ATP synthesis with similar steady-state parameters. ATP synthesis by the WT enzyme proceeded with neither burst nor lag-phase while ATP synthesis by βD380C (+DTT) showed a burst phase with a stoichiometry of ATP/F1Fo equal to 1. The burst was simulated using a kinetic model in which the βD380C mutant has a less than 10-fold increase in Pi binding rate in combination with a less than 10-fold decrease in ATP release rate compared to WT. Resolution of the burst allows us to distinguish the partial reactions of the ATP synthesis pathway.Comparison of the ATP release rates in ATP hydrolysis (Baylis Scanlon et al., 2007, Biochemistry 46, 8785-8797) and ATP synthesis suggest differences in the cooperative behavior in the two directions of the reaction. In ATP hydrolysis, positive cooperativity in catalysis is induced by nucleotide (ATP) only, while in ATP synthesis it is induced by both nucleotide (ADP) and the pmf. Fast steady-state ATP hydrolysis proceeds through a trisite mechanism, while ATP synthesis uses a bisite mechanism.We further analyzed the effects of the mutation by forming a stator-rotor disulfide cross-link, βD380C-γC87 induced by DTNB. The cross-linked enzyme catalyzed absolutely no ATP synthesis in the millisecond time domain after inducing a pmf. This result suggests that γ subunit mobility is required for the ATP synthetic reaction to occur and is consistent with the model that high affinity Pi binding cannot be achieved without ΔμH+-dependent γ-subunit rotation.

At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish (Danio rerio)

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for human health through fish eating. To study the impact of MeHg on mitochondrial structure and function, we contaminated the model fish species Danio rerio with food containing 13μg of MeHg per gram, an environmentally relevant dose. Mitochondria from contaminated zebrafish muscles presented structural abnormalities under electron microscopy observation. In permeabilized muscle fibers, we observed, a strong inhibition of both state 3 mitochondrial respiration and functionally isolated maximal cytochrome c oxidase (COX) activity after 49 days of MeHg exposure. However, the state 4 respiratory rate remained essentially unchanged. This suggested a defect at the level of ATP synthesis. Accordingly, we measured a dramatic decrease in the rate of ATP release by skinned muscle fibers using either pyruvate and malate or succinate as respiratory substrates. However, the amount and the assembly of the ATP synthase were identical in both control and contaminated muscle mitochondrial fractions. This suggests that MeHg induced a decoupling of mitochondrial oxidative phosphorylation in the skeletal muscle of zebrafish. Western blot analysis showed a 30% decrease of COX subunit IV levels, a 50% increase of ATP synthase subunit α, and a 40% increase of the succinate dehydrogenase Fe/S protein subunit in the contaminated muscles. This was confirmed by the analysis of gene expression levels, using RT-PCR. Our study provides a basis for further analysis of the deleterious effect of MeHg on fish health via mitochondrial impairment.

Acute increase of intraocular pressure releases ATP into the anterior chamber

Throughout the body, mechanical perturbations are transduced into neurochemical signals by the release of ATP from non-neuronal cells. As an increased intraocular pressure (IOP) can initiate mechanical changes, we hypothesized that extracellular levels of ATP might be increased in the anterior chamber of human patients with primary acute angle closure glaucoma (PAACG). ATP levels in aqueous humor samples were determined using the luciferin–luciferase assay. Samples were obtained from 18 controls scheduled for cataract extraction and from 14 subjects with angle closure glaucoma during paracentesis. All subjects with angle closure glaucoma had remarkably elevated levels of ATP in their aqueous humor. The mean ATP concentration was ninefold higher for patients with angle closure glaucoma than for control. The concentration of ATP was correlated with IOP levels, further supporting a causal relationship. ATP levels were not influenced by the duration of the PAACG attack, suggesting the rate of ATP release was sustained. We conclude that increased IOP leads to increased levels of extracellular ATP in the anterior chamber. This elevated ATP may contribute to the extreme pain experienced by subjects with angle closure glaucoma, and may serve as a source of elevated adenosine in the anterior chamber.

Highlights From The Literature

Highlights From The Literature

The role of Tyro 3 subfamily receptors in the regulation of hemostasis and megakaryocytopoiesis

The molecular mechanisms regulating megakaryocytopoiesis and hemostasis remain largely unknown. The Tyro 3 subfamily of receptor tyrosine kinases (RTK), which is composed of three members (Tyro 3, Axl and Mer), plays important roles in various tissues, such as those in the nervous, immune and reproductive systems. Here, we investigate the roles of the Tyro 3 RTK subfamily in regulating megakaryocytopoiesis and hemostasis. Single, double, and triple knock-out mice for the three Tyro 3 RTK were used in the study. Bleeding time, platelet count, megakaryocyte count, megakaryocyte ploidy, rate of proplatelet formation, platelet aggregation and ATP release were used as criteria to evaluate hemostasis, megakaryocytopoiesis and platelet function. Mice lacking all three receptors had impaired hemostasis and mild thrombocytopenia, which may be due to platelet dysfunction and defective megakaryocytopoiesis. Mice lacking different combinations of two receptors of the Tyro 3 RTK subfamily had normal platelet counts in peripheral blood, but exhibited impaired hemostasis and platelet function. Although knock-out mice for any single receptor had normal hemostasis and megakaryocytopoiesis, they exhibited a mild platelet dysfunction. The Tyro 3 RTK subfamily plays important roles in regulating hemostasis, megakaryocytopoiesis and platelet function.

Differential effects of cyclic and constant stress on ATP release and mucociliary transport by human airway epithelia

In the lungs, the first line of defence against bacterial infection is the thin layer of airway surface liquid (ASL) lining the airway surface. The superficial airway epithelium exhibits complex regulatory pathways that blend ion transport to adjust ASL volume to maintain proper mucociliary clearance (MCC). We hypothesized that stresses generated by airflow and transmural pressures during breathing govern ASL volume by regulating the rate of epithelial ATP release. Luminal ATP, via interactions with apical membrane P2-purinoceptors, regulates the balance of active ion secretion versus absorption to maintain ASL volume at optimal levels for MCC. In this study we tested the hypothesis that cyclic compressive stress (CCS), mimicking normal tidal breathing, regulates ASL volume in airway epithelia. Polarized tracheobronchial epithelial cultures from normal and cystic fibrosis (CF) subjects responded to a range of CCS by increasing the rate of ATP release. In normal airway epithelia, the CCS-induced increase in ASL ATP concentration was sufficient to induce purinoceptor-mediated increases in ASL height and MCC, via inhibition of epithelial Na(+)-channel-mediated Na(+) absorption and stimulation of Cl(-) secretion through CFTR and the Ca(2+)-activated chloride channels. In contrast, static, non-oscillatory stress did not stimulate ATP release, ion transport or MCC, emphasizing the importance of rhythmic mechanical stress for airway defence. In CF airway cultures, which exhibit basal ASL depletion, CCS was partially effective, producing less ASL volume secretion than in normal cultures, but a level sufficient to restore MCC. The present data suggest that CCS may (1) regulate ASL volume in the normal lung and (2) improve clearance in the lungs of CF patients, potentially explaining the beneficial role of exercise in lung defence.

Physiological Regulation of ATP Release at the Apical Surface of Human Airway Epithelia

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin ( approximately 7 microm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of approximately 250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached approximately 1 microm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.

Kinetics of urothelial ATP release

Recent reports have proposed that the urothelium can sense mechanical stretch and communicate this information to sensory afferent neurons by the release of ATP into the vicinity of P2X-containing neurons. This report investigates the bidirectional release of ATP by in vitro rabbit urothelium. ATP was measured using the luciferin-luciferase assay. Immediately after washing of both sides of the epithelium, there was a linear increase in ATP content in the mucosal compartment with a rate of 23 +/- 6.5 fmol x min(-1) x cm(-2) (n = 18). Serosal ATP content increased as a saturating exponential function, suggesting a constant rate of release and degradation of ATP by ectonucleotidases/exonucleotidases. The presence of a serosal ectonucleotidase/exonucleotidases was demonstrated by the time-dependent decrease in exogenously added ATP. The maximum rate of hydrolysis was 11 pmol x min(-1) x cm(-2) with a K(m) of 0.49 microM. The time course of serosal ATP release was modeled as a constant rate of release (d: mol x min(-1) x cm(-2)) and rate constant of hydrolysis (k(h): min(-)). In control conditions d was 18 fmol x min(-1) x cm(-2) and k(h) of 0.056 +/- 0.01 min(-) (n = 18). Steady-state serosal chamber content is 370 +/- 90 fmol/cm(2), and concentration is 50 +/- 1.2 x 10(-12) M. Stretching the tissue resulted in a transient fivefold increase in the rate of mucosal ATP release and a transient sixfold increase in serosal ATP release. Half-osmotic strength solutions increased mucosal release by 10-fold and serosal release by 5-fold. Tissue damage resulted in a step-increase in mucosal chamber ATP content by 6.6 +/- 1 pmol/cm(2) and serosal chamber ATP by 0.1 +/- 0.06 pmol/cm(2) (n = 5).

Photoreaction of caged ATP studied by the time-resolved transient grating method.

The photoreaction of caged ATP, P3-[1-(2-nitrophenyl)ethyl]adenosine-5'-triphosphate, has been investigated using the time-resolved transient grating (TG) method. We found that a feature of the TG signal time profile depends sensitively on the grating wavenumber (q) after the photoexcitation of caged ATP. This q-dependent feature of the TG signal was interpreted based on a model where the ATP release rate is comparable to the molecular diffusion process. We found that the TG signals at various q can be consistently analyzed based on this model and the ATP release rate determined. The enthalpy and volume changes of the reaction have been determined by quantitative measurement of the grating and photoacoustic signals.

Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6).

In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin-luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing-type chamber. We developed a LL pulse protocol to determine the rate of ATP release (R(ATP)) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure R(ATP) as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 microl of A6 cells released ATP at a rate of 66 +/- 8 fmol min(-1). A sudden reduction of the basolateral osmolality from 260 to 140 mosmol (kg H(2)O)(-1) elevated R(ATP) rapidly to a peak value of 1.89 +/- 0.11 pmol min(-1) (R(ATP)(peak)) followed by a plateau phase reaching 0.51 +/- 0.07 pmol min(-1) (R(ATP)(plat)). Both R(ATP)(peak) and R(ATP)(plat) values increased with the degree of dilution. The magnitude of R(ATP)(plat) remained constant as long as the hyposmolality was maintained. Similarly, a steady ATP release of 0.78 +/- 0.08 pmol min(-1) was recorded after gradual dilution of the basolateral osmolality to 140 mosmol (kg H(2)O)(-1). This R(ATP) value, induced in the absence of cell swelling, is comparable to R(ATP)(plat). Therefore, the steady ATP release is unrelated to membrane stretching, but possibly caused by the reduction of intracellular ionic strength during cell volume regulation. Independent determinations of dose-response curves for peak [Ca(2+)](i) increase in response to exogenous ATP and basolateral hyposmolality demonstrated that the exogenous ATP concentration, required to mimic the osmotic reduction, was linearly correlated with R(ATP)(peak). The link between the ATP release and the fast [Ca(2+)](i) transient was also demonstrated by the depression of both phenomena by Cl(-) removal from the basolateral perfusate. The data are consistent with the notion that during hypotonicity, basolateral ATP release activates purinergic receptors, which underlies the suramin-sensitive rise of [Ca(2+)](i) during the hyposmotic shock.

Constitutive Release of ATP and Evidence for Major Contribution of Ecto-nucleotide Pyrophosphatase and Nucleoside Diphosphokinase to Extracellular Nucleotide Concentrations

Nucleotides are important extracellular signaling molecules. At least five mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP, or UDP. Although the existence of ectoenzymes that metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. Therefore, we have studied the kinetics of accumulation and metabolism of endogenous ATP in the extracellular medium of four different cell lines. ATP concentrations reached a maximum immediately after change of medium and decreased thereafter with a single exponential decay (t(1/2);1 approximately;230-40 min). ATP levels did not fall to zero but attained a base-line concentration that was independent of the medium volume and of the initial ATP concentration. Although the base-line concentration of ATP remained stable for up to 12 h, [gamma-(32)P]ATP added to resting cells as a radiotracer was completely degraded within 120 min, indicating that steady state reflected a basal rate of ATP release balanced by ATP hydrolysis (20-200 fmol x min(-)(1) x cell(-)(6)). High performance liquid chromatography analysis revealed that the gamma-phosphate of ATP was rapidly, although transiently, transferred during steady state to species subsequently identified as UTP and GTP, indicating the existence of both ecto-nucleoside diphosphokinase activity and the accumulation of endogenous UDP and GDP. Conversely, addition of [gamma-(32)P]UTP to resting cells resulted in transient formation of [gamma-(32)P]ATP, indicating phosphorylation of endogenous ADP by nucleoside diphosphokinase. The final (32)P-products of [gamma-(32)P]ATP metabolism were [(32)P]orthophosphoric acid and a (32)P-labeled species that was further purified and identified as [(32)P]inorganic pyrophosphate. In C6 cells, the formation of [(32)P]pyrophosphate from [gamma-(32)P]ATP at steady state exceeded by 3-fold that of [(32)P]orthophosphate. These results illustrate for the first time a constitutive release of ATP and other nucleotides and reveal the existence of a complex extracellular metabolic pathway for released nucleotides. In addition to the existence of an ecto-ATPase activity, our results suggest a major scavenger role of ecto-ATP pyrophosphatase and a transphosphorylating activity of nucleoside diphosphokinase.

Quantitative analysis of the killer activity of some oenological yeasts. Effect of some additives

<p style="text-align: justify;">The Killer factor was discovered in 1963. Since this time it has been widely studied and nowadays a lot is known about genetics of the factor, the biochemistry of the toxin and also about the way of action of the toxin on sensitive yeasts. The yeast strains are classified in three groups : killer strains, sensitive strains and neutral strains. The killer strain is able to kill sensitive strains while neutral strains are unable to kill any strain and remain unaffected by the toxin. Certainly this classification depends on the couple of strains (killer and sensitive) taken as references. It has been clearly established that the toxin acts on the sensitive cells by inducing important losses of ATP : due to holes created by the toxin in the cell membrane, ATP leaves the cell.</p><p style="text-align: justify;">In the field of Enology, it’s generally accepted that a killer yeast has more probabilities to have a good implantation in a non sterile medium (as the must is) than a neutral or a sensitive strain. Nevertheless, it is difficult to have a precise idea of the sensitivity of a strain as well as of its toxicity, as most of the methods are only able to give a qualitative information.</p><p style="text-align: justify;">In this paper, a new method of evaluation of the killer effect is presented. Also some results dealing with its application to the classification of some enological yeast strains are given and discussed.</p><p style="text-align: justify;">This method is based on the measurement of the ATP released by the sensitive strain under the action of the killer toxin. The criterion that we define is the initial rate of ATP release, that means the quantity of ATP lost in the two first hours by the sensitive cells in contact with the toxin. In a first step, it is shown that this method is reliable and also that it is in a good agreement with the method using the flow cytometry. ATP leak could be correlated with the amount of affected cells (dead and damaged cells). So, using this method, it becomes possible to classify easily yeast strains with respect to their sensitivity or toxicity. Several killer yeast strains were tested against a sensitive strain and different sensitive strains were submitted to the action of a killer toxin produced by a killer strain. It was shown that the losses of ATP were quite different according to the sensitivity of the strain. The initial rate of ATP release was found to be in a range of 0.00 to 0.12 micromole/L/h. The second part of the work deals with the study of the possible effect of some products used for winemaking on the efficiency of the killer effect. The products we have studied were bentonite, tannic acid and enological tannins. It is shown that these products may interact with the killer effect. So, bentonite, for example is able to inhibit completely the efficiency of the killer toxin, as soon as its concentration is about 10 grams/hL.</p>

Structural Changes of the Sarcoplasmic Reticulum Ca 2+-ATPase upon Nucleotide Binding Studied by Fourier Transform Infrared Spectroscopy

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca 2+-ATPase upon nucleotide binding were recorded in H 2O and 2H 2O at −7°C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P 3-1-(2-nitrophenyl) adenosine 5′-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca 2+-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release ( k app ≅ 3 s −1 in H 2O and k app ≅ 7 s −1 in 2H 2O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca 2+-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of α-helical and β-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the γ-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the γ-phosphate group in the interactions between the Ca 2+-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716 cm −1 (H 2O) and 1706 cm −1 ( 2H 2O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm −1 (H 2O) and 1514 cm −1 ( 2H 2O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21.