ATP Breakdown Research Articles

Administration of a soluble ADPase, AZD3366, on top of ticagrelor confers additional cardioprotective benefits to that of ticagrelor alone

Abstract Background Preclinical and pilot human studies suggest platelet-independent cardioprotective effects of ticagrelor most likely due to its ability to block cellular uptake of adenosine. Adenosine is a potent endogenous protective molecule, concentrations of which locally rise as a response to ischemia because of the breakdown of extracellular ATP by an endothelial ADPase (apyrase, CD39) to AMP that is subsequently converted to adenosine by CD73. Purpose In the present study we used cardiac magnetic resonance (CMR) imaging in a pig model of myocardial infarction (MI) to examine whether administration of a recombinant soluble form of ADPase, AZD3366 (APT102), confers additional benefits to that of ticagrelor alone in terms of reduced infarct size and improved heart function. Methods Pigs (n=20) were administered an oral loading dose of ticagrelor (180mg) and 2h later subjected to MI induction (1.5h closed-chest LAD coronary balloon occlusion). Prior to reperfusion, pigs were randomized to intravenously receive 1) vehicle (n=5); 2) 1mg/kg AZD3366 (n=5); or 3) 3mg/kg AZD3366 (n=5). After reperfusion all pigs were administered ticagrelor (90mg/bid) for 42 days. A non-treated control-MI group (n=5) was run for comparative purposes. Serial-CMR imaging was performed at baseline and 3 and 42 days post-MI for global and regional structural and functional assessments. Light transmittance aggregometry (LTA; challenged by 5, 10 and 20μM ADP) and ear bleeding time were monitored throughout the study. Results Ticagrelor significantly reduced edema formation (29.8±1.9 vs. 13.1±0.9%LV) and limited infarct size (17.7±1.5 vs. 8.2±1.2%LV) at 3 days post-MI as compared to control-MI pigs (p<0.05), an effect that persisted up to 42 days. Infusion of 1mg/kg AZD3366 showed a clear signal towards further prevention of myocardial damage that reached significance at doses of 3mg/kg (additional reduction of 35% and 52% in edema and infarct size, respectively, as compared to pigs treated with ticagrelor alone; p<0.05). Left ventricular ejection fraction was higher in all ticagrelor-treated pigs at 3 and 42 days post-MI vs. control (p<0.05). Yet, regional analysis of the jeopardized myocardium revealed that pigs administered 3mg/kg AZD3366 on top of ticagrelor presented minimal dysfunctional segmental contraction as compared to the mild hypokinetic and akinetic disturbances observed in ticagrelor-alone and control-MI pigs (χ2 p<0.05 vs. all). Ticagrelor inhibited ADP-induced platelet aggregation by 30% and addition of AZD3366 acutely abolished (90% inhibition) LTA at all tested ADP doses, an effect remaining significant up to 3 days post-infusion. Ear bleeding time was not affected by AZD3366. Conclusion Infusion of a soluble recombinant ADPase (AZD3366) on top of ticagrelor leads to a greater cardioprotection as compared to ticagrelor alone. Co-administration of both drugs in AMI patients undergoing revascularization deserves to be investigated. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Ministerio de Ciencia, Innovaciόn y Universidades / Instituto de Salud Carlos III

Functional atropine sensitive purinergic responses in the healthy rat bladder

While acetylcholine is regarded to be the main directly contractile transmitter substance in the urinary bladder, interactions with other transmitters likely occur. Presently, the interplay between purinergic and cholinergic signalling was investigated to unravel the involvement of the urothelium and efferent neurons in the functionally important purinergically evoked release of acetylcholine in vitro. Functional characterization of receptor subtypes involved in this interplay was also performed. In vitro organ bath experiments with electrical field stimulation (EFS) or administration of agonist were performed in the absence and presence of the neurotoxin tetrodotoxin (TTX; 5×10-7M) and/or receptor antagonists, in intact and urothelium-denuded full thickness rat bladder strip preparations. Interestingly, functional contractions to ATP (10-6-10-3M) remained unaffected by TTX, but were significantly lowered in the presence of the muscarinic antagonist atropine (10-6M). However, in urothelium-denuded strip preparations, this latter phenomenon was not present and the ATP response remained unaltered. To rule out purinergic interference caused by break-down of ATP, experiments were performed in which the stable ATP-analogue αβMeATP (10-7-10-5M) gave rise to functional atropine-sensitive contractions. Furthermore, contractions to ATP were not affected by P2Y6 purinoceptor blockade (by MRS2578; 10-7, 10-5M), nor were relaxatory responses to ATP sensitive to atropine, PPADS (3×10-5M) or αβMeATP. Lastly, relaxations to ADP (10-6-10-3M) or NECA (10-8-10-5M) were unaltered by the presence of atropine. To conclude, purinergic functional contractile, but not relaxatory, responses are supported by the cholinergic transmitter system in vitro, through non-neuronal mechanisms in the urothelium. Involved purinoceptors are of the P2X-subtype, most likely P2X1 and/or P2X3.

Compartmentalization of adenosine metabolism in cancer cells and its modulation during acute hypoxia.

Extracellular adenosine mediates diverse anti-inflammatory, angiogenic and vasoactive effects, and has become an important therapeutic target for cancer, which has been translated into clinical trials. This study was designed to comprehensively assess adenosine metabolism in prostate and breast cancer cells. We identified cellular adenosine turnover as a complex cascade, comprising (1) the ectoenzymatic breakdown of ATP via sequential ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1, officially known as ENPP1), ecto-5'-nucleotidase (CD73, also known as NT5E), and adenosine deaminase reactions, and ATP re-synthesis through a counteracting adenylate kinase and members of the nucleoside diphosphate kinase (NDPK, also known as NME/NM23) family; (2) the uptake of nucleotide-derived adenosine via equilibrative nucleoside transporters; and (3) the intracellular adenosine phosphorylation into ATP by adenosine kinase and other nucleotide kinases. The exposure of cancer cells to 1% O2 for 24 h triggered an ∼2-fold upregulation of CD73, without affecting nucleoside transporters, adenosine kinase activity and cellular ATP content. The ability of adenosine to inhibit the tumor-initiating potential of breast cancer cells via a receptor-independent mechanism was confirmed in vivo using a xenograft mouse model. The existence of redundant pathways controlling extracellular and intracellular adenosine provides a sufficient justification for reexamination of the current concepts of cellular purine homeostasis and signaling in cancer.This article has an associated First Person interview with the first author of the paper.

Effects of stunning methods on pre rigor changes in rainbow trout (Oncorhynchus mykiss) reared at two different temperatures

The effects of two stunning methods (carbon monoxide asphyxia, CO, and electroshock, E) on blood plasma parameters, rigor index, fillet pH and shape changes, ATP breakdown and Adenylate Energy Charge (AEC) in muscle immediately post mortem were investigated in rainbow trout (Oncorhynchus mykiss) kept in tanks containing water set at 8 °C or 12 °C. Both the methods here adopted induced a stress-response which, however, was not able to affect the rigor mortis development and fillet pH. The fillets from the E group showed the strongest length contraction and width increase at 48 h post mortem. The CO stunning method exhibited the highest levels of both ATP and AEC in the muscle immediately after death, equal to 2.28 µmol/g and 0.83, respectively, while 1.12 µmol/g and 0.64 values were found in the E group. In addition, we found that the water temperature might interact with the stunning method and minimise the stress response. In the present trial, the most suitable use of the CO stunning method would be coupled with 12 °C of rearing water temperature to better preserve ATP and AEC in muscles. Highlights CO stunning preserves ATP and AEC in trout muscle better than electrical stunning Water temperature might interact with the stunning and minimise the stress response CO stunning is suggested to be coupled with 12 °C of water temperature Electricity should be avoided at 8 °C of water temperature

The role of purinergic P2Y12 and P2Y13 receptors in ADPβS-induced inhibition of the cardioaccelerator sympathetic drive in pithed rats

ATP is a cotransmitter released with other neurotransmitters from sympathetic nerves, where it stimulates purinergic receptors. Purinergic adenosine P1 receptors (coupled to Gi/o proteins) produce sympatho-inhibition in several autonomic effectors by prejunctional inhibition of neurotransmitter release. Similarly, signalling through P2Y12 and P2Y13 receptors coupled to Gi/o proteins is initiated by the ATP breakdown product ADP. Hence, this study has pharmacologically investigated a possible role of ADP-induced inhibition of the cardioaccelerator sympathetic drive in pithed rats, using a stable ADP analogue (ADPβS) and selective antagonists for the purinergic P2Y1, P2Y12 and P2Y13 receptors. Accordingly, male Wistar rats were pithed and: (i) pretreated i.v. with gallamine (25mg/kg) and desipramine (50μg/kg) for preganglionic spinal (C7-T1) stimulation of the cardioaccelerator sympathetic drive (n = 78); or (ii) prepared for receiving i.v. injections of exogenous noradrenaline (n = 12). The i.v. continuous infusions of ADPβS (10 and 30μg/kg/min) dose-dependently inhibited the tachycardic responses to electrical sympathetic stimulation, but not those to exogenous noradrenaline. The cardiac sympatho-inhibition produced by 30μg/kg/min ADPβS was (after i.v. administration of compounds) (i) unchanged by 1-ml/kg bidistilled water or 300-μg/kg MRS 2500 (P2Y1 receptor antagonist), (ii) abolished by 300-μg/kg PSB 0739 (P2Y12 receptor antagonist) and (iii) partially blocked by 3000-μg/kg MRS 2211 (P2Y13 receptor antagonist). Our results suggest that ADPβS induces a cardiac sympatho-inhibition that mainly involves the P2Y12 receptor subtype and, probably to a lesser extent, the P2Y13 receptor subtype. These receptors may represent therapeutic targets for treating cardiovascular pathologies, including stroke and myocardial infarctions.

Characterisation of nerve-mediated ATP release from bladder detrusor muscle and its pathological implications.

This study aims to characterise the molecular mechanisms that determine variability of atropine resistance of nerve-mediated contractions in human and guinea pig detrusor smooth muscle. Atropine resistance of nerve-mediated contractions and the role of P2X1 receptors, were assessed in isolated preparations from guinea pigs and also humans with or without overactive bladder syndrome, from which the mucosa was removed. Nerve-mediated ATP release was measured directly with amperometric ATP-sensitive electrodes. Ecto-ATPase activity of guinea pig and human detrusor samples was measured in vitro by measuring the concentration-dependent rate of ATP breakdown. The transcription of ecto-ATPase subtypes in human samples was measured by qPCR. Atropine resistance was greatest in guinea pig detrusor, absent in human tissue from normally functioning bladders, and intermediate in human overactive bladder. Greater atropine resistance correlated with reduction of contractions by the ATP-diphosphohydrolase apyrase, directly implicating ATP in their generation. E-NTPDase-1 was the most abundantly transcribed ecto-ATPase of those tested, and transcription was reduced in tissue from human overactive, compared to normal, bladders. E-NTPDase-1 enzymic activity was inversely related to the magnitude of atropine resistance. Nerve-mediated ATP release was continually measured and varied with stimulation frequency over the range of 1-16Hz. Atropine resistance in nerve-mediated detrusor contractions is due to ATP release and its magnitude is inversely related to E-NTPDase-1 activity. ATP is released under different stimulation conditions compared with ACh, implying different routes for their release.

The Ionotropic P2X4 Receptor has Unique Properties in the Heart by Mediating the Negative Chronotropic Effect of ATP While Increasing the Ventricular Inotropy.

Background: Mounting evidence indicate that reducing the sinoatrial node (SAN) activity may be a useful therapeutic strategy to control of heart failure. Purines, like ATP and its metabolite adenosine, consistently reduce the SAN spontaneous activity leading to negative cardiac chronotropy, with variable effects on the force of myocardial contraction (inotropy). Apart from adenosine A1 receptors, the human SAN expresses high levels of ATP-sensitive ionotropic P2X4 receptors (P2X4R), yet their cardiac role is unexplored. Methods: Here, we investigated the activity of P2 purinoceptors on isolated spontaneously beating atria (chronotropy) and on 2 Hz-paced right ventricular (RV, inotropy) strips from Wistar rats. Results: ATP (pEC 50 = 4.05) and its stable analogue ATPγS (pEC 50 = 4.69) concentration-dependently reduced atrial chronotropy. Inhibition of ATP breakdown into adenosine by NTPDases with POM-1 failed to modify ATP-induced negative chronotropy. The effect of ATP on atrial rate was attenuated by a broad-spectrum P2 antagonist, PPADS, as well as by 5-BDBD, which selectively blocks the P2X4R subtype; however, no effect was observed upon blocking the A1 receptor with DPCPX. The P2X4R positive allosteric modulator, ivermectin, increased the negative chronotropic response of ATP. Likewise, CTP, a P2X agonist that does not generate adenosine, replicated the P2X4R-mediated negative chronotropism of ATP. Inhibition of the Na+/Ca2+ exchanger (NCX) with KB-R7943 and ORM-10103, but not blockage of the HCN channel with ZD7288, mimicked the effect of the P2X4R blocker, 5-BDBD. In paced RV strips, ATP caused a mild negative inotropic effect, which magnitude was 2 to 3-fold increased by 5-BDBD and KB-R7943. Immunofluorescence confocal microscopy studies confirm that cardiomyocytes of the rat SAN and RV co-express P2X4R and NCX1 proteins. Conclusions: Data suggest that activation of ATP-sensitive P2X4R slows down heart rate by reducing the SAN activity while increasing the magnitude of ventricular contractions. The mechanism underlying the dual effect of ATP in the heart may involve inhibition of intracellular Ca2+-extrusion by bolstering NCX function in the reverse mode. Thus, targeting the P2X4R activation may create novel well-tolerated heart-rate lowering drugs with potential benefits in patients with deteriorated ventricular function.

Inhibition of vascular smooth muscle cell calcification by ATP analogues.

Arterial medial calcification (AMC) has been associated with phenotypic changes in vascular smooth muscle cells (VSMCs) that reportedly makes them more osteoblast-like. Previous work has shown that ATP/UTP can inhibit AMC directly via P2 receptors and indirectly by NPP1-mediated hydrolysis to produce the mineralisation inhibitor, pyrophosphate (PPi). This study investigated the role of P2X receptors in the inhibitory effects of extracellular nucleotides on VSMC calcification. We found that Bz-ATP, α,β-meATP and β,γ-meATP inhibited calcification by up to 100%. Culture in a high-phosphate medium (2mM) was associated with increased VSMC death and apoptosis; treatment with Bz-ATP, α,β-meATP and β,γ-meATP reduced apoptosis to levels seen in non-calcifying cells. Calcification was also associated with alterations in the protein levels of VSMC (e.g. SM22α and SMA) and osteoblast-associated (e.g. Runx2 and osteopontin) markers; Bz-ATP, α,β-meATP and β,γ-meATP attenuated these changes in protein expression. Long-term culture with Bz-ATP, α,β-meATP and β,γ-meATP resulted in lower extracellular ATP levels and an increased rate of ATP breakdown. P2X receptor antagonists failed to prevent the inhibitory effects of these analogues suggesting that they act via P2X receptor-independent mechanisms. In agreement, the breakdown products of α,β-meATP and β,γ-meATP (α,β-meADP and methylene diphosphonate, respectively) also dose-dependently inhibited VSMC calcification. Furthermore, the actions of Bz-ATP, α,β-meATP and β,γ-meATP were unchanged in VSMCs isolated from NPP1-knockout mice, suggesting that the functional effects of these compounds do not involve NPP1-mediated generation of PPi. Together, these results indicate that the inhibitory effects of ATP analogues on VSMC calcification and apoptosis in vitro may be mediated, at least in part, by mechanisms that are independent of purinergic signalling and PPi.

Involvement of A3 Adenosine Receptor in Neuroblastoma Progression via Modulation of the Hypoxic/Angiogenic Pathway.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. The clinical course may range from spontaneous regression towards ganglioneuroblastoma/ganglioneuroma or maturation to a very aggressive form characterized by an extensive hypoxic area. In solid tumors, extracellular microenvironment hypoxia induces the transcription of hypoxia-inducible factors (HIFs) leading to synthesis of pro-angiogenic factor, VEGF; also, it increases extracellular adenosine production from ATP breakdown. To date, the role of this nucleoside in the hypoxic/angiogenic pathway characterizing the core of cancer mass has not been investigated yet. Therefore, the aim of the present study was to analyze the adenosine effect on modulation of the HIF-1α/2α/VEGF pathway mediated through A3 AR binding. To this end, we have used a selective A3 AR agonist IB-MECA or antagonist VUF 5574 in an in vitro model of malignant undifferentiated and all-trans retinoic acid (RA)-differentiated SH-SY5Y cells, representing the benign form of NB. Our results have shown that specific A3 AR stimulation induces HIF and VEGF expression through the activation of mitogen-activated protein kinase/Erk kinase signaling cascade. In conclusion, the data suggest that A3 AR may represent a marker of NB malignancy as well as a drug target for treatment of this solid tumor. Graphical Abstract.

ATP Synthase: Structure, Function and Inhibition.

Oxidative phosphorylation is carried out by five complexes, which are the sites for electron transport and ATP synthesis. Among those, Complex V (also known as the F1F0 ATP Synthase or ATPase) is responsible for the generation of ATP through phosphorylation of ADP by using electrochemical energy generated by proton gradient across the inner membrane of mitochondria. A multi subunit structure that works like a pump functions along the proton gradient across the membranes which not only results in ATP synthesis and breakdown, but also facilitates electron transport. Since ATP is the major energy currency in all living cells, its synthesis and function have widely been studied over the last few decades uncovering several aspects of ATP synthase. This review intends to summarize the structure, function and inhibition of the ATP synthase.

Analysis of Lactate Dehydrogenase & ATPase activity in fish, Gambusia affinis at different period of exposureto chlorpyrifos

The acute toxicity of an organophosphate insecticide chlorpyrifos, on biochemical parameters of fish Gambusia affinis was evaluated under static condition. The sub lethal concentration of the insecticide chlorpyrifos was found to be 0.284 ppm indicating the high toxicity of the insecticide. Fishes were exposed to 1/10th of sub lethal (0.0284 ppm) and the alteration of enzyme activity in liver and kidney of fish Gambusia affinis were studied at 15, 30 and 45 days of exposure. A significant increase in LDH activity in both tissue Liver and Kidney and the inhibition of ATPase leads to decreased ATP breakdown and reduced the availability of free energy was observed.

Quality of fresh saithe (Pollachius virens) in modified atmosphere packages as affected by the gas composition

Abstract The experimental design was set up to study the effect of different modified atmospheres (CO2 (67 or 33%) balanced with either O2 or N2) on autolytic- and microbiological deterioration of chilled saithe (Pollachius virens). As controls, vacuum packaged saithe was used. The results showed a positive effect of gas mixtures containing O2 on physiochemical and microbial product quality. Discriminating factors were; lower psychrotrophic count, slower breakdown of ATP, lower contents of certain biogenic amines (e.g. cadaverine) and reduced drip loss during storage. A high CO2 concentration (67%) in the packaging atmosphere was moreover found to inhibit microbial proliferation. Vacuum-packaged saithe stand out negatively with highest DL and reduced sensory shelf life and physiochemical and microbial quality. It was moreover found that increased drip loss, and higher contents of hypoxanthine and cadaverine in the muscle tissue was related to the microbiological ecology (i.e. increased growth of Shewanella spp. and Photobacterium spp.).

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

PL - 032 Physiological triggers involved in reduced slow myosin expression in disused postural muscle

Purinergic Profiling of Regulatory T-cells in Patients With Episodic Migraine.

Objectives: Immune responses in migraine are poorly characterized, yet implicated in the disease pathogenesis. This study was carried out to characterize purinergic profiles of T-cells in patients with episodic migraine without aura (MWoA) to provide mechanistic evidence for ATP and adenosine involvement in modulation of immune regulation in migraine.Methods: Peripheral blood samples were obtained from patients with migraine (n = 16) and age-matched control subjects (n = 21). Subsets of T-cells were identified by flow cytometry based on specific membrane markers.Results: Migraine patients showed reduced total T-cell counts in the peripheral blood. Whereas the total number of CD3+CD4+, CD3+CD8+, or regulatory T lymphocytes (Treg) was not changed, the proportion of Treg CD45R0+CD62L– and CD45R0–CD62L– cells was increased. Interestingly, in migraine, less Treg cells expressed CD39 and CD73 suggesting disrupted ATP breakdown to adenosine. The negative correlations were observed between the duration of migraine and the relative number of CD73+CD39– Tregs and total number of CD73-positive CD45R0+CD62L+ Tregs.Conclusion: Obtained data indicate that T-cell populations are altered in episodic migraine and suggest the involvement of Tregs in the pathophysiology of this disorder. Reduced expression of CD39 and CD73 suggests promotion of ATP-dependent pro-inflammatory and reduction of adenosine-mediated anti-inflammatory mechanisms in migraine.

Mechanism of luminal ATP activated chloride secretion in a polarized epithelium.

There are both secretory and absorptive pathways working in tandem to support ionic movement driving fluid secretion across epithelia. The mechanisms exerting control of fluid secretion in the oviduct is yet to be fully determined. This study explored the role of apical or luminal extracellular ATP (ATPe)-stimulated ion transport in an oviduct epithelium model, using the Ussing chamber short-circuit current (Isc) technique. Basal Isc in oviduct epithelium in response to apical ATPe comprises both chloride secretion and sodium absorption and has distinct temporal phases. A rapid transient peak followed by a sustained small increase above baseline. Both phases of the apical ATPe Isc response are sensitive to anion (HCO3-, Cl-) and cation (Na+) replacement. Additionally, the role of apical chloride channels, basolateral potassium channels and intracellular calcium in supporting the peak Isc current was confirmed. The role of ATP breakdown to adenosine resulting in the activation of P2 receptors was supported by examining the effects of non-hydrolyzable forms of ATP. A P2YR2 potency profile of ATP = UTP > ADP was generated for the apical membrane, suggesting the involvement of the P2YR2 subtype of purinoceptor. A P2X potency profile of ATP = 2MeSATP > alpha,beta-meATP > BzATP was also generated for the apical membrane. In conclusion, these results provide strong evidence that purinergic activation of apical P2YR2 promotes chloride secretion and is thus an important factor in fluid formation by the oviduct.

MP88-13 TUMOR SUPPRESSOR TSC1 IS A NEW HSP90 COCHAPERONE THAT FACILITATES FOLDING OF KINASE AND NON-KINASE CLIENTS

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2018MP88-13 TUMOR SUPPRESSOR TSC1 IS A NEW HSP90 COCHAPERONE THAT FACILITATES FOLDING OF KINASE AND NON-KINASE CLIENTS Mark Woodford, Rebecca Sager, Adam Blanden, Stewart Loh, David Gutmann, Oleg Shapiro, Dimitra Bourboulia, Michael Wong, Gennady Bratslavsky, and Mehdi Mollapour Mark WoodfordMark Woodford More articles by this author , Rebecca SagerRebecca Sager More articles by this author , Adam BlandenAdam Blanden More articles by this author , Stewart LohStewart Loh More articles by this author , David GutmannDavid Gutmann More articles by this author , Oleg ShapiroOleg Shapiro More articles by this author , Dimitra BourbouliaDimitra Bourboulia More articles by this author , Michael WongMichael Wong More articles by this author , Gennady BratslavskyGennady Bratslavsky More articles by this author , and Mehdi MollapourMehdi Mollapour More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2933AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mutation of either TSC1 or TSC2 causes tuberous sclerosis, a multisystem genetic syndrome that can lead to formation of benign tumors in the kidneys, lungs, and other organs and has also been linked to epilepsy and autism. The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however the precise mechanism remains elusive. Molecular chaperones such as heat shock protein-90 (Hsp90) are essential for the stability and activation of numerous signaling proteins, including tumor suppressors. The objective of this work was to determine the role of Hsp90 in the stability and activity of TSC. We hypothesize that Hsp90 mediates TSC formation and thus is essential for the stability of Tsc2. METHODS Tsc1-FLAG and Tsc2-FLAG were transiently expressed and isolated from HEK293 cells. Interacting proteins were identified by co-immunoprecipitation. Hsp90 chaperone function is coupled to its ATPase activity. We have developed an assay to measure this activity by determining the rate of ATP breakdown and inorganic phosphate production. Hsp90 was isolated from WT and TSC1-/- mice and assayed for ATPase activity in vitro. RESULTS Tsc2 interacts with the molecular chaperone Hsp90 both in vitro and in vivo. Treatment of HEK293 cells with Hsp90 inhibitors such as ganetespib or SNX2112 caused ubiquitination and proteasomal degradation of Tsc2; therefore Tsc2 is a new client of Hsp90. However, Tsc1 stability does not depend on Hsp90. Tsc1 potently decelerates Hsp90 ATPase activity and facilitates the interaction of Tsc2 and other clients with Hsp90. We also showed that Tsc1 competes with the activating co-chaperone Aha1 for binding to Hsp90, providing a reciprocal regulatory mechanism for chaperoning of kinase and non-kinase clients. CONCLUSIONS Tsc1 interacts with Tsc2 and protects Tsc2 from degradation. Many pathogenic mutations of Tsc2 destabilize the protein. Our data suggest that lack of interaction of pathogenic Tsc2 mutants with Tsc1 and consequently Hsp90 lead to their degradation. Further, these newly elucidated roles of Tsc1 and Tsc2 suggest a potential mechanism for the observed differences between TSC1 and TSC2 mutant disease. In the absence of Tsc2, Tsc1 will still perform its critical role as a co-chaperone of Hsp90. However when Tsc1 is lost, the stability of both Tsc2 and many other Hsp90 clients is dramatically reduced, leading to a different phenotypic manifestation. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1202 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Mark Woodford More articles by this author Rebecca Sager More articles by this author Adam Blanden More articles by this author Stewart Loh More articles by this author David Gutmann More articles by this author Oleg Shapiro More articles by this author Dimitra Bourboulia More articles by this author Michael Wong More articles by this author Gennady Bratslavsky More articles by this author Mehdi Mollapour More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of high-pressure processing on taste-related ATP breakdown compounds and aroma volatiles in grass-fed beef during vacuum aging.

ObjectiveThis study aimed to observe whether high-pressure processing (HPP) affected aroma development and the degradation rate of umami taste-related ATP breakdown products, specifically inosinic acid in grass-fed beef during vacuum aging.MethodsStrip loin (longissimus lumborum) cuts obtained from six grass-fed Friesian Holstein steers (32 months old) on day 4 post slaughter were vacuum-packed and subjected to pressurization at 300 and 500 MPa for 180 s at 15°C±2°C. The samples were then stored for 4 weeks at 5°C±0.5°C under vacuum and compared with the control (0.1 MPa).ResultsHPP increased the shear force value, promoted moisture loss and lipid oxidation, induced surface paleness, stabilized pH during aging, and reduced bacterial load and growth. The shear force value of 500 MPa-treated samples remained higher than the control after aging, while no significant differences were found between the control and 300 MPa-treated samples. Degradation of inosinic acid and inosine occurred during pressurization, resulting in an increase in hypoxanthine content. However, the degradation rate in HPP-treated samples during aging was slower; therefore, inosinic acid and inosine content remained higher than in control samples. No significant differences were found in hypoxanthine content at the end of aging. HPP intensified the levels of hexanal, octanal, 2-methylbutanal, 3-methylbutanal, benzaldehyde, and 2,5-dimethylpyrazine in cooked-aged beef samples.ConclusionHPP induced aroma development and delayed the degradation of inosinic acid. However, it also reduced the postmortem tenderization rate.

The Inside Story of Adenosine.

Several physiological functions of adenosine (Ado) appear to be mediated by four G protein-coupled Ado receptors. Ado is produced extracellularly from the catabolism of the excreted ATP, or intracellularly from AMP, and then released through its transporter. High level of intracellular Ado occurs only at low energy charge, as an intermediate of ATP breakdown, leading to hypoxanthine production. AMP, the direct precursor of Ado, is now considered as an important stress signal inside cell triggering metabolic regulation through activation of a specific AMP-dependent protein kinase. Intracellular Ado produced from AMP by allosterically regulated nucleotidases can be regarded as a stress signal as well. To study the receptor-independent effects of Ado, several experimental approaches have been proposed, such as inhibition or silencing of key enzymes of Ado metabolism, knockdown of Ado receptors in animals, the use of antagonists, or cell treatment with deoxyadenosine, which is substrate of the enzymes acting on Ado, but is unable to interact with Ado receptors. In this way, it was demonstrated that, among other functions, intracellular Ado modulates angiogenesis by regulating promoter methylation, induces hypothermia, promotes apoptosis in sympathetic neurons, and, in the case of oxygen and glucose deprivation, exerts a cytoprotective effect by replenishing the ATP pool.

Endothelial cells cope with hypoxia-induced depletion of ATP via activation of cellular purine turnover and phosphotransfer networks

Intravascular ATP and adenosine have emerged as important regulators of endothelial barrier function, vascular remodeling and neovascularization at various pathological states, including hypoxia, inflammation and oxidative stress. By using human umbilical vein endothelial cells (HUVEC) and bovine vasa vasorum endothelial cells (VVEC) as representatives of macro- and microvessel phenotypes, this study was undertaken to evaluate cellular mechanisms contributing to physiological adaptation of vascular endothelium to hypoxia, with a particular emphasis on ectoenzymatic purine-converting activities and their link to intracellular ATP homeostasis and signaling pathways. Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), ecto-5′-nucleotidase/CD73 and ecto-adenylate kinase activities were determined by thin-layer chromatography (TLC) with 3H-labelled nucleotide substrates. Exposure of HUVEC and VVEC to 1% O2 for 4–24 h triggered rather moderate activation of ATP breakdown into adenosine via the CD39-CD73 axis. Additional TLC analysis of salvage pathways revealed the enhanced ability of hypoxic HUVEC to convert cell-incorporated [3H]adenosine into [3H]ADP/ATP. Furthermore, following a period of hypoxia, HUVEC underwent concurrent changes in intracellular signaling manifested in the depletion of putative ATP stores and targeted up-regulation of phospho-p53, p70S6K/mTOR and other tyrosine kinases. The revealed complex implication of both extrinsic and intrinsic mechanisms into a tuned hypoxia-induced control of purine homeostasis and signaling may open up further research for the development of pharmacological treatments to improve endothelial cell function under disease conditions associated with a loss of cellular ATP during oxygen deprivation.

Impact of Diets Containing Plant Raw Materials as Fish Meal and Fish Oil Replacement on Rainbow Trout (Oncorhynchus mykiss), Gilthead Sea Bream (Sparus aurata), and Common Carp (Cyprinus carpio) Freshness

The present study aimed to evaluate whether the total or high substitution of fish meal (FM) and fish oil (FO) by sustainable plant raw materials (plant meal and oils) in long-term feeding for rainbow trout, gilthead sea bream, and common carp can result in spoilage alterations during ice storage. These three species were fed throughout their whole rearing cycle with plant-based diets and compared to counterparts that received FM/FO-based diets or commercial-like diets. Sensory QIM schemes adopted for these species and ATP breakdown products (K-value and components) were used to evaluate the freshness. Sensory acceptability of 14, 15, and 12 days was found for rainbow trout, gilthead sea bream, and common carp, respectively. This corresponded to K-values of approximately 80%, 35%, and 65% for rainbow trout, gilthead sea bream, and common carp, respectively. No major effect of dietary history on postmortem shelf life was shown for gilthead sea bream and common carp; neither sensory-perceived nor chemical freshness showed diet-related differences. Rainbow trout fed with the plant-based diet exhibited slightly worse sensory freshness than fish fed with FM/FO-based diets, at the end of shelf life. These findings imply that FM and FO can be successfully substituted without major impacts on shelf life of fish.