ATP Storage Research Articles

Organ donation and organ assessment after primary circulatory death and secondary brain death

The global organ shortage is the biggest obstacle to expand urgently needed liver transplantation activities. In addition to donation after brain death (DBD), donation after primary circulatory death (DCD) has also been introduced in many European countries to increase the number of donated organs. This article summarizes the legal and ethical aspects of DCD, the practical donation process of DCD, the clinical results of DCD liver transplantation with aspecial focus on organ assessment before a planned DCD liver transplantation. In Europe 11countries have active DCD liver transplantation programs and atotal of 1230 DCD liver transplantations were performed in Europe in 2023. The highest proportion of DCD liver transplantations were recorded in Belgium (52.8%), the Netherlands (42.8%) and Switzerland (32.1%). The adequate selection of donors and recipients is crucial in DCD transplantation and the use of DCD livers particularly depends on the preparedness of the healthcare system for routine machine perfusion. The leaders are Belgium, France and Italy which implant around 68-74% of DCD organs. With an adequate organ assessment, the long-term results of DBD and DCD liver transplantations are comparable. To assess mitochondrial damage and thus organ quality, hypothermic oxygenated machine perfusion (HOPE) was introduced and has the secondary benefit of mitochondrial protection through oxygenation. The establishment of aerobic metabolism in mitochondria under hypothermia leads to areduction of toxic metabolites and the restoration of ATP storage, which subsequently leads to areperfusion light during implantation. Expanding the donor pool with DCD donors can counteract the global organ shortage. With adequate patient selection and routine organ assessment short-term and also long-term outcomes of DBD and DCD liver transplantation are comparable.

Rotary FoF1-ATP Synthase-Driven Flasklike Pentosan Colloidal Motors with ATP Synthesis and Storage.

We report the hierarchical assembly of a chloroplast-derived rotary FoF1-ATPase motor-propelled flasklike pentosan colloidal motor (FPCM) with the ability of the synthesis, storage, and triggered release of biological energy currency ATP. These streamlined and submicrometer-sized hollow flasklike pentosan colloidal motors are prepared by combining a soft-template-based hydrothermal polymerization with a vacuum infusion of chloroplast-derived proteoliposomes containing rotary FoF1-ATPase motors. The generation of proton motive force across the proteoliposomes by injecting an acidic buffer solution promotes the rotation of FoF1-ATPase motors to drive the self-propelled motion of FPCMs, accompanying the inner ATP synthesis and storage. These rotary FoF1-ATPase motor-powered FPCMs exhibit a chemotactic behavior by migrating from their neck opening to their round bottom along a proton gradient of the external environment (negative chemotaxis). Such rotary biomolecular motor-driven flasklike pentosan colloidal motors with ATP synthesis and on-demand release make them promising candidates for engineering novel intelligent nanocarriers to actively regulate cellular metabolism.

Using total adenosine triphosphate (tATP) measurements for cyanobacterial bloom monitoring and response assessment during algaecide treatments

Total adenosine triphosphate (tATP) was investigated for its potential as a rapid indicator of cyanobacterial growth and algaecide effectiveness. tATP and other common bloom monitoring parameters were measured over the growth cycles of cyanobacteria and green algae in laboratory cultures and examined at a drinking water source during an active bloom. Strong correlations (R2>0.78) were observed between tATP and chlorophyll-a in cyanobacteria cultures. tATP offered greater sensitivity by increasing two orders of magnitude approximately 7 d before changes in chlorophyll-a or optical density were observed in Lyngbya sp. and Dolichospermum sp. cultures. Increases in tATP per cell coincided with the onset of exponential growth phases in lab cultures and increase in cell abundance in field samples, suggesting that ATP/cell is a sensitive indicator that may be used to identify the development of blooms. Bench-scale trials using samples harvested during a bloom showed that tATP exhibited a clear dose-response during copper sulfate (CuSO4) and hydrogen peroxide (H2O2) treatment compared to chlorophyll-a and cell counts, indicating that cellular production and storage of ATP decreases even when live and dead cells cannot be distinguished. During Copper (Cu) algaecide application at a reservoir used as a drinking water source, tATP and cell counts decreased following initial algaecide application; however, the bloom rebounded within 10 d showing that the Cu algaecide only has limited effectiveness. In this case, tATP was a sensitive indicator to bloom rebounding after algaecide treatments and correlated positively with cell counts (R2=0.7). These results support the use of tATP as a valuable complementary bloom monitoring tool for drinking water utilities to implement during the monitoring and treatment of cyanobacterial blooms.

Dietary polyamines promote intestinal adaptation in an experimental model of short bowel syndrome

Intestinal adaptation does not necessarily recover absorptive capacity in short bowel syndrome (SBS), sometimes resulting in intestinal failure-associated liver disease (IFALD). Additionally, its therapeutic options remain limited. Polyamines (spermidine and spermine) are known as one of the autophagy inducers and play important roles in promoting the weaning process; however, their impact on intestinal adaptation is unknown. The aim of this study was to investigate the impact of polyamines ingestion on adaptation and hepatic lipid metabolism in SBS. We performed resection of two-thirds of the small intestine in male Lewis rats as an SBS model. They were allocated into three groups and fed different polyamine content diets (0%, 0.01%, 0.1%) for 30 days. Polyamines were confirmed to distribute to remnant intestine, whole blood, and liver. Villous height and number of Ki-67-positive cells in the crypt area increased with the high polyamine diet. Polyamines increased secretory IgA and mucin content in feces, and enhanced tissue Claudin-3 expression. In contrast, polyamines augmented albumin synthesis, mitochondrial DNA copy number, and ATP storage in the liver. Moreover, polyamines promoted autophagy flux and activated AMP-activated protein kinase with suppression of lipogenic gene expression. Polyamines ingestion may provide a new therapeutic option for SBS with IFALD.

CiAP2/ERF65 and CiAP2/ERF106, a pair of homologous genes in pecan (Carya illinoensis), regulate plant responses during submergence in transgenic Arabidopsis thaliana

When plants are entirely submerged, photosynthesis and respiration are severely restricted, affecting plant growth and potentially even causing plant death. The AP2/ERF superfamily has been widely reported to play a vital role in plant growth, development and resistance to biotic and abiotic stresses. However, no relevant studies exist on flooding stress in pecan. In this investigation, we observed that CiAP2/ERF65 positively modulated the hypoxia response during submergence, whereas CiAP2/ERF106 was sensitive to submergence. The levels of physiological and biochemical indicators, such as POD, CAT and among others, in CiAP2/ERF65-OE lines were significantly higher than those in wild-type Arabidopsis thaliana, indicating that the antioxidant capacity of CiAP2/ERF65-OE lines was enhanced under submergence. The RNA-seq results revealed that the maintenance of the expression levels of the antenna protein gene, different signaling pathways for regulation, as well as the storage and consumption of ATP, might account for the opposite phenotypes of CiAP2/ERF65 and CiAP2/ERF106. Furthermore, the expression of some stress-related genes was altered during submergence and reoxygenation. Overall, these findings enhance our understanding of submergence stress in pecan, providing important candidate genes for the molecular design and breeding of hypoxia resistant in plants.

Xanthine oxidase inhibitors withdrawal syndrome

Abstract Background In humans, uric acid is the final metabolite of adenosine triphosphate, an energy source. Xanthine oxidase (XO) contributes to urate metabolism, and XO inhibitors (XOI) suppress the production of uric acid and could store ATP. However, recent studies showed that discontinuation of XOI may lead to ATP depletion and increased death as a withdrawal syndrome. We examined whether the XOI withdrawal syndrome actually occurs. Methods This study retrospectively analyzed the Japanese Registry Of All cardiac and vascular Diseases (JROAD) database, which included 8,927,858 patients out of 9,825,635 data from April 2014 to March 2020, excluding 330,847 patients younger than 20 years and 566,930 patients older than 90 years. Of these, 356,405 were hospitalized patients with cardiovascular disease who were receiving uric acid-lowering drugs, and 315,388 were on XOI (41,017 were on other uric acid-lowering medications)(Fig). The XOI adherents were divided into the following four groups, and the number of deaths and mortality rates for each were identified as follows. Results (1) Those who were on the drug at admission and continued the drug at discharge: 219,020 cases, of which 59 deaths (0.03%). (2) 89,035 patients were discharged from the hospital on the medication they were on at the time of admission, of which 17,663 died (19.84%). (3) 7,241 patients who were not on any medication at admission (medication was given during hospitalization) and continued on medication at discharge, including 11 deaths (0.15%). (4) No medication at admission (medication was given during hospitalization) and stopped at discharge: 92 cases, of which 11 deaths (11.96%). Continuous medication group: 226,261 cases, including (1) and (3), of which 70 deaths, (0.03%). Withdrawal group: 89,127 cases, including (2) and (4), of which 17,674 deaths (19.8%). The number of deaths in the withdrawal group was 620 times higher than that in the continuation group (p<0.001). Conclusion XOI withdrawal syndrome is detected in our study. However, some of the deaths in the XOI discontinuation group were due to the fact that other medications were also discontinued, which indicate impossible to determine a direct causal relationship between XOI withdrawal and death. Further studies are needed to check the relationship between XOI withdrawal and death in other algorithms.Fig. Flow-chart of the study

Intrathecal administration of botulinum toxin type a antagonizes neuropathic pain by countering increased vesicular nucleotide transporter expression in the spinal cord of chronic constriction injury of the sciatic nerve rats

Botulinum toxin type A (BoNT/A) induces direct analgesic effects in neuropathic pain by inhibiting the release of substance P, calcitonin gene-related peptide (CGRP) and glutamate. Vesicular nucleotide transporter (VNUT) was responsible for the storage and release of ATP in vivo, and one of the mechanisms underlying neuropathic pain is VNUT-dependent release of extracellular ATP from dorsal horn neurons. However, the analgesic effect of BoNT/A by affecting the expression of VNUT remained largely unknown. Thus, in this study, we aimed to elucidate the antinociceptive potency and analgesic mechanism of BoNT/A in chronic constriction injury of the sciatic nerve (CCI) induced neuropathic pain. Our results showed that a single intrathecal injection of 0.1 U BoNT/A seven days after CCI surgery produced significant analgesic activity and decreased the expression of VNUT in the spinal cord of CCI rats. Similarly, BoNT/A inhibited the CCI-induced increase in ATP content in the rat spinal cord. Overexpression of VNUT in the spinal cord of CCI-induced rats markedly reversed the antinociceptive effect of BoNT/A. Furthermore, 33 U/mL BoNT/A dramatically reduced the expression of VNUT in pheochromocytoma (PC12) cells but overexpressing SNAP-25 increased VNUT expression in PC12 cells. Our current study is the first to demonstrate that BoNT/A is involved in neuropathic pain by regulating the expression of VNUT in the spinal cord in rats.

Is the vesicular nucleotide transporter a molecular target of eicosapentaenoic acid?

Vesicular nucleotide transporter (VNUT), an active transporter for nucleotides in secretory vesicles, is responsible for the vesicular storage of ATP and plays an essential role in purinergic chemical transmission. Inhibition of VNUT decreases the concentration of ATP in the luminal space of secretory vesicles, followed by decreased vesicular ATP release, resulting in the blockade of purinergic chemical transmission. Very recently, Miyaji and colleagues reported that eicosapentaenoic acid (EPA) is a potent VNUT inhibitor and effective in treating neuropathic and inflammatory pain and insulin resistance through inhibition of vesicular storage and release of ATP. However, our validation study indicated that, in bovine adrenal chromaffin granule membrane vesicles, EPA inhibited the formation of an electrochemical gradient of protons across the membrane with the concentration of 50% inhibition (IC50) being 1.0μM without affecting concanamycin B-sensitive ATPase activity. Essentially, similar results were obtained with proteoliposomes containing purified vacuolar H+-ATPase. Consistent with these observations, EPA inhibited the ATP-dependent uptakes of ATP and dopamine by chromaffin granule membrane vesicles, with ID50 being 1.2 and 1.0μM, respectively. Furthermore, EPA inhibited ATP-dependent uptake of L-glutamate by mouse brain synaptic vesicles with ID50 being 0.35μM. These results indicate that EPA at sub-μM acts as a proton conductor and increases proton permeability across the membrane, regardless of the presence or absence of VNUT, thereby inhibiting non-specifically the vesicular storage of neurotransmitters. Thus, EPA may affect a broader range of chemical transmission than proposed.

NLRP3 Inflammasome Activation Through Heart-Brain Interaction Initiates Cardiac Inflammation and Hypertrophy During Pressure Overload.

Mechanical stress on the heart, such as high blood pressure, initiates inflammation and causes hypertrophic heart disease. However, the regulatory mechanism of inflammation and its role in the stressed heart remain unclear. IL-1β (interleukin-1β) is a proinflammatory cytokine that causes cardiac hypertrophy and heart failure. Here, we show that neural signals activate the NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3) inflammasome for IL-1β production to induce adaptive hypertrophy in the stressed heart. C57BL/6 mice, knockout mouse strains for NLRP3 and P2RX7 (P2X purinoceptor 7), and adrenergic neuron-specific knockout mice for SLC17A9, a secretory vesicle protein responsible for the storage and release of ATP, were used for analysis. Pressure overload was induced by transverse aortic constriction. Various animal models were used, including pharmacological treatment with apyrase, lipopolysaccharide, 2'(3')-O-(4-benzoylbenzoyl)-ATP, MCC950, anti-IL-1β antibodies, clonidine, pseudoephedrine, isoproterenol, and bisoprolol, left stellate ganglionectomy, and ablation of cardiac afferent nerves with capsaicin. Cardiac function and morphology, gene expression, myocardial IL-1β and caspase-1 activity, and extracellular ATP level were assessed. In vitro experiments were performed using primary cardiomyocytes and fibroblasts from rat neonates and human microvascular endothelial cell line. Cell surface area and proliferation were assessed. Genetic disruption of NLRP3 resulted in significant loss of IL-1β production, cardiac hypertrophy, and contractile function during pressure overload. A bone marrow transplantation experiment revealed an essential role of NLRP3 in cardiac nonimmune cells in myocardial IL-1β production and cardiac phenotype. Pharmacological depletion of extracellular ATP or genetic disruption of the P2X7 receptor suppressed myocardial NLRP3 inflammasome activity during pressure overload, indicating an important role of ATP/P2X7 axis in cardiac inflammation and hypertrophy. Extracellular ATP induced hypertrophic changes of cardiac cells in an NLRP3- and IL-1β-dependent manner in vitro. Manipulation of the sympathetic nervous system suggested sympathetic efferent nerves as the main source of extracellular ATP. Depletion of ATP release from sympathetic efferent nerves, ablation of cardiac afferent nerves, or a lipophilic β-blocker reduced cardiac extracellular ATP level, and inhibited NLRP3 inflammasome activation, IL-1β production, and adaptive cardiac hypertrophy during pressure overload. Cardiac inflammation and hypertrophy are regulated by heart-brain interaction. Controlling neural signals might be important for the treatment of hypertensive heart disease.

Mechanisms and Drug Intervention for Doxorubicin-Induced Cardiotoxicity Based on Mitochondrial Bioenergetics.

Doxorubicin (DOX) is an anthracycline chemotherapy drug, which is indispensable in antitumor therapy. However, its subsequent induction of cardiovascular disease (CVD) has become the primary cause of mortality in cancer survivors. Accumulating evidence has demonstrated that cardiac mitochondrial bioenergetics changes have become a significant marker for doxorubicin-induced cardiotoxicity (DIC). Here, we mainly summarize the related mechanisms of DOX-induced cardiac mitochondrial bioenergetics disorders reported in recent years, including mitochondrial substrate metabolism, the mitochondrial respiratory chain, myocardial ATP storage and utilization, and other mechanisms affecting mitochondrial bioenergetics. In addition, intervention for DOX-induced cardiac mitochondrial bioenergetics disorders using chemical drugs and traditional herbal medicine is also summarized, which will provide a comprehensive process to study and develop more appropriate therapeutic strategies for DIC.

Killer or helper? The mechanism underlying the role of adenylate activated kinase in sound conditioning.

ObjectiveTo investigate whether sound conditioning influences auditory system protection by activating adenylate activated kinase (AMPK), and if such adaption protects ribbon synapses from high-intensity noise exposure.Materials and methodsCBA mice (12 weeks old) were randomly divided into four groups (n = 24 mice per group): control, sound conditioning (SC), sound conditioning plus noise exposure (SC+NE), and noise exposure (NE). Hearing thresholds were assessed before testing, after sound conditioning, and 0, 3, 7, and 14 days after 110 dB noise exposure. Amplitudes and latencies of wave I at 90 dB intensity were assessed before test, after conditioning, and at 0 and 14 days after 110 dB noise exposure. One cochlea from each mouse was subjected to immunofluorescence staining to assess synapse numbers and AMPK activation, while the other cochlea was analyzed for phosphorylated adenylate activated kinase (p-AMPK) protein expression by western blot.ResultsThere was no significant difference in auditory brainstem response (ABR) threshold between SC and control mice. The degree of hearing loss of animals in the two SC groups was significantly reduced compared to the NE group after 110 dB noise exposure. Animals in the SC group showed faster recovery to normal thresholds, and 65 dB SPL sound conditioning had a stronger auditory protection effect. After sound conditioning, the amplitude of ABR I wave in the SC group was higher than that in the control group. Immediately after noise exposure (D0), the amplitudes of ABR I wave decreased significantly in all groups; the most significant decrease was in the NE group, with amplitude in 65SC+NE group significantly higher than that in the 85SC+NE group. Wave I latency in the SC group was significantly shorter than that in the control group. At D0, latency was prolonged in the NE group compared with the control group. In contrast, there was no significant difference in latency between the 65SC+NE and 85SC+NE groups. Further, at D14, there was no significant difference between the NE and control groups, while latency remained significantly shorter in the 65SC+NE and 85SC+NE groups compared with controls. Number of ribbon synapses in SC mice did not differ significantly from that in controls. After 110 dB noise exposure, there were significantly more ribbon synapses in the SC+NE group than the NE group. Ribbon synapses of all groups were recovered 14 days after the noise exposure, while the SC group had a shorter recovery time than the non-SC groups (p < 0.05). AMPK was highly activated in the SC group, and p-AMPK expression was detected; however, after 110 dB noise exposure, the strongest protein expression was detected in the NE group, followed by the SC+NE groups, and the lowest protein expression was detected in the control group.ConclusionSound conditioning animals were more noise resistant and recovered hearing faster than non-SC animals. Further, 65 dB SPL SC offered better hearing protection than 85 dB SPL SC. Early AMPK activation may protect hearing by increasing ATP storage and reducing the release of large quantities of p-AMPK, which could help to inhibit synapse damage.

Quinacrine is not a vital fluorescent probe for vesicular ATP storage.

Quinacrine, a fluorescent amphipathic amine, has been used as a vital fluorescent probe to visualize vesicular storage of ATP in the field of purinergic signaling. However, the mechanism(s) by which quinacrine represents vesicular ATP storage remains to be clarified. The present study investigated the validity of the use of quinacrine as a vial fluorescent probe for ATP-storing organelles. Vesicular nucleotide transporter (VNUT), an essential component for vesicular storage and ATP release, is present in very low density lipoprotein (VLDL)-containing secretory vesicles in hepatocytes. VNUT gene knockout (Vnut-/-) or clodronate treatment, a VNUT inhibitor, disappeared vesicular ATP release (Tatsushima et al., Biochim Biophys Acta Molecular Basis of Disease 2021, e166013). Upon incubation of mice's primary hepatocytes, quinacrine accumulates in a granular pattern into the cytoplasm, sensitive to 0.1-μM bafilomycin A1, a vacuolar ATPase (V-ATPase) inhibitor. Neither Vnut-/- nor treatment of clodronate affected quinacrine granular accumulation. In vitro, quinacrine is accumulated into liposomes upon imposing inside acidic transmembranous pH gradient (∆pH) irrespective of the presence or absence of ATP. Neither ATP binding on VNUT nor VNUT-mediated uptake of ATP was affected by quinacrine. Consistently, VNUT-mediated uptake of quinacrine was negligible or under the detection limit. From these results, it is concluded that vesicular quinacrine accumulation is not due to a consequence of its interaction with ATP but due to ∆pH-driven concentration across the membranes as an amphipathic amine. Thus, quinacrine is not a vital fluorescent probe for vesicular ATP storage.

Abstract P386: Supplementation Of Tyrode With Fatty Acids And 3-hydroxybutyrate Improves Adult Cardiomyocytes Contractility Of Failing Human Hearts.

Due to its high energy consumption and limited ability to store ATP, the heart is highly dependent of exogenous metabolic substrates. Prior in vivo studies have reported that the development of heart failure is accompanied by a transition from the normal preferential metabolism of free fatty acids (FFA) to increases in glucose utilization and even ketone bodies, which normally provide a modest contribution to energy balance. However, the functional significance of the upregulated ketone metabolism in the failing heart is poorly understood. Recognizing that nearly all prior studies examining isolated cardiomyocyte physiology have used glucose as the sole metabolic substrate, we initiated studies to examine the impact of alternative metabolic substrates on contractility in isolated human cardiomyocytes. To understand the role of substrate alteration cardiomyocyte functionalities, we employed freshly isolated adult human left ventricular cardiomyocytes from 11 non-failing hearts (NF) obtained from organ donors and 13 failing hearts (HF) obtained from transplant recipients. Cardiomyocytes were resuspended in a conventional 5mM Glucose Tyrode solution with alternative substrates (Glucose, FFA, R-3-OHB or Mix (Glucose + FFA + 3-OHB)). Myocytes were field stimulated at 1 Hz and sarcomere length, fractional shortening, contraction velocity and relaxation velocity were measured using a video-based sarcomere length detection system (IonOptix Corp). Studies using isolated cardiac myocyte contractility as readout confirm that myocytes from NF human hearts are omnivorous: high levels of myocyte fractional shortening (FS) can be achieved under unstressed conditions (1 Hz, unloaded) with any substrate (FS Glucose : 0.1315±0.012; FS FFA : 0.1428±0.0127; FS 3OHB : 0.1343±0.014; FS MIX : 0.15467±0.02). In the failing heart, glucose alone is insufficient to produce normal unstressed myocyte fractional shortening (FS Glucose : 0.088±0.009***, p&lt;0.001 compare to NF). However, in failing myocytes, supplementation of physiological levels of glucose with FFA or ketones each enhances myocyte contractility and rates of shortening and re-lengthening (FS FFA : 0.109±0.0127; FS 3OHB : 0.107±0.012; FS MIX : 0.112±0.016). These results suggest that future comparisons of NF vs. HF human myocyte contractility should include conditions with a physiological mix of metabolic substrates.

Airway Epithelial Nucleotide Release Contributes to Mucociliary Clearance.

Mucociliary clearance (MCC) is a dominant component of pulmonary host defense. In health, the periciliary layer (PCL) is optimally hydrated, thus acting as an efficient lubricant layer over which the mucus layer moves by ciliary force. Airway surface dehydration and production of hyperconcentrated mucus is a common feature of chronic obstructive lung diseases such as cystic fibrosis (CF) and chronic bronchitis (CB). Mucus hydration is driven by electrolyte transport activities, which in turn are regulated by airway epithelial purinergic receptors. The activity of these receptors is controlled by the extracellular concentrations of ATP and its metabolite adenosine. Vesicular and conducted pathways contribute to ATP release from airway epithelial cells. In this study, we review the evidence leading to the identification of major components of these pathways: (a) the vesicular nucleotide transporter VNUT (the product of the SLC17A9 gene), the ATP transporter mediating ATP storage in (and release from) mucin granules and secretory vesicles; and (b) the ATP conduit pannexin 1 expressed in non-mucous airway epithelial cells. We further illustrate that ablation of pannexin 1 reduces, at least in part, airway surface liquid (ASL) volume production, ciliary beating, and MCC rates.

VNUT Is a Therapeutic Target for Type 2 Diabetes and NASH

ATP, used in cells as an energy currency, also acts as an extracellular signaling molecule. Studies of purinergic receptor subtypes have revealed that purinergic chemical transmission plays important roles in various cell types. The vesicular nucleotide transporter (VNUT), the ninth transporter in the SLC17 organic anion transporter family, is essential for vesicular ATP storage and its subsequent release. The VNUT is localized on the membrane of secretory vesicles and actively transports ATP into vesicles using an electrochemical gradient of protons supplied by vacuolar proton ATPase (V-ATPase) as a driving force. ATP acts as a damage-associated molecular pattern (DAMPs), contributing to the persistence of chronic inflammation. Chronic inflammation induces systemic insulin resistance, which is the underlying pathology of type 2 diabetes and non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). We previously demonstrated that ATP transported in insulin granules via the VNUT negatively regulates insulin secretion. We also found that hepatocytes release ATP in a VNUT-dependent manner, which elicits hepatic insulin resistance and inflammation. VNUT-knockout mice exhibited improved glucose tolerance and were resistant to the development of high fat diet-induced NAFLD. In this article, we summarize recent advances in our understanding of the mechanism of the VNUT, the development of inhibitors, and its pathological involvement in type 2 diabetes and NAFLD. The pharmacological inhibition of the VNUT may represent a potential therapeutic approach for the treatment of metabolic diseases.

Clodronate, an inhibitor of the vesicular nucleotide transporter, ameliorates steatohepatitis and acute liver injury

The vesicular nucleotide transporter (VNUT) is responsible for the vesicular storage and release of ATP from various ATP-secreting cells, and it plays an essential role in purinergic signaling. Although extracellular ATP and its degradation products are known to mediate various inflammatory responses via purinoceptors, whether vesicular ATP release affects steatohepatitis and acute liver injury is far less understood. In the present study, we investigated the effects of clodronate, a potent and selective VNUT inhibitor, on acute and chronic liver inflammation in mice. In a model of methionine/choline-deficient diet-induced non-alcoholic steatohepatitis (NASH), the administration of clodronate reduced hepatic inflammation, fibrosis, and triglyceride accumulation. Clodronate also protected mice against high-fat/high-cholesterol diet-induced steatohepatitis. Moreover, prophylactic administration of clodronate prevented d-galactosamine and lipopolysaccharide-induced acute liver injury by reducing inflammatory cytokines and hepatocellular apoptosis. In vitro, clodronate inhibited glucose-induced vesicular ATP release mediated by VNUT and reduced the intracellular level and secretion of triglycerides in isolated hepatocytes. These results suggest that VNUT-dependent vesicular ATP release plays a crucial role in the recruitment of immune cells, cytokine production, and the aggravation of steatosis in the liver. Pharmacological inhibition of VNUT may provide therapeutic benefits in liver inflammatory disorders, including NASH and acute toxin-induced injury.

Selective and stoichiometric incorporation of ATP by self-assembling amyloid fibrils.

ATP acts as a biological hydrotrope preventing protein aggregation. Here, we report a novel chimeric peptide, ACC1-13K8, with an unusual capacity to bind and incorporate ATP while self-assembling into amyloid fibrils. The amino acid sequence combines a highly amyloidogenic segment of insulin's A-chain (ACC1-13) and octalysine (K8). Fibrillization requires binding 2 ATP molecules per ACC1-13K8 monomer and is not triggered by adenosine di- and monophosphates (ADP, AMP). Infrared and CD spectra and AFM-based morphological analysis reveal tight and orderly entrapment of ATP within superstructural hybrid peptide-ATP fibrils. The incorporation of ATP is an emergent property of ACC1-13K8 not observed for ACC1-13 and K8 segments separately. We demonstrate how new functionalities (e.g. ATP storage) emerge from synergistic coupling of amyloidogenic segments with non-amyloidogenic peptide ligands, and suggest that ATP's role in protein misfolding is more nuanced than previously assumed.

The influence of climate change on human cardiovascular function

Climate change is considered to have great impact on human health. The heat waves have been associated with excess morbidity and mortality of cardiovascular diseases (CVD) across various populations and geographic locations. Important role in the heat-induced cardiovascular damage has endothelial dysfunction. It has been noticed that hot weather can impair tone and structure of the blood vessels via interfering with variety of biological factors such as nitric oxide synthesize, cytokine production and systemic inflammation. Also, due to dehydration and increased blood viscosity, by promoting thrombogenesis, heat has important impact on patients with atherosclerosis. During chronic exposure to the cold or hot weather cardiovascular function can be decreased, leading to a higher risk of developing heart attack, malignant cardiac arrhythmias, thromboembolic diseases and heat-induced sepsis like shock. It has been shown that changes in the ambient temperature through increasing blood pressure, blood viscosity, and heart rate, contribute to the cardiovascular mortality. The majority of deaths due to heat waves especially affect individuals with preexisting chronic CVD. This population can experience a decline in the health status, since extreme ambient temperature affects pharmacokinetic parameters of many cardiovascular drugs. Increased mortality from ischemic or hemorrhagic stroke can also be related to extreme temperature variations. On a cellular level, higher ambient temperature can limit storage of ATP and O2 increase amount of free radicals and toxic substances and induce neuronal apoptotic signal transduction, which all can lead to a stroke. Preserving cardiovascular function in context of extreme climate changing tends to be particularly challenging.

In Memoriam Geoffrey Burnstock: Creator of Purinergic Signaling

Geoff Burnstock (1929–2020) discovered purinergic signaling in a fastidious research that started in early 1960 and culminated in a concept of purinergic nerves in 1972. Subsequently, Geoff developed the concept of purinergic transmission and demonstrated ATP storage, release, and degradation in the context of cotransmission, which was another fundamental concept developed by him. Purinergic transmission contributes to the most fundamental physiological functions such as sensory transduction, regulation of heart rate, smooth muscle contraction, bile secretion, endocrine regulation, immune responses, as well as to various pathophysiological conditions, including inflammation, cancer, neuropathic pain, diabetes, and kidney failure.

Evans Blue Reduces Neuropathic Pain Behavior by Inhibiting Spinal ATP Release.

Upon peripheral nerve injury, vesicular ATP is released from damaged primary afferent neurons. This extracellular ATP subsequently activates purinergic receptors of the spinal cord, which play a critical role in neuropathic pain. As an inhibitor of the vesicular nucleotide transporter (VNUT), Evans blue (EB) inhibits the vesicular storage and release of ATP in neurons. Thus, we tested whether EB could attenuate neuropathic pain behavior induced by spinal nerve ligation (SNL) in rats by targeting VNUT. An intrathecal injection of EB efficiently attenuated mechanical allodynia for five days in a dose-dependent manner and enhanced locomotive activity in an SNL rat model. Immunohistochemical analysis showed that EB was found in VNUT immunoreactivity on neurons in the dorsal root ganglion and the spinal dorsal horn. The level of ATP in cerebrospinal fluid in rats with SNL-induced neuropathic pain decreased upon administration of EB. Interestingly, EB blocked ATP release from neurons, but not glial cells in vitro. Eventually, the loss of ATP decreased microglial activity in the ipsilateral dorsal horn of the spinal cord, followed by a reduction in reactive oxygen species and proinflammatory mediators, such as interleukin (IL)-1β and IL-6. Finally, a similar analgesic effect of EB was demonstrated in rats with monoiodoacetate-induced osteoarthritis (OA) pain. Taken together, these data demonstrate that EB prevents ATP release in the spinal dorsal horn and reduces the ATP/purinergic receptor-induced activation of spinal microglia followed by a decline in algogenic substances, thereby relieving neuropathic pain in rats with SNL.