Endogenous ATP Research Articles

P2X7 and A2A receptor endogenous activation protects against neuronal death caused by CoCl2‐induced photoreceptor toxicity in the zebrafish retina

Injured retinas in mammals do not regenerate and heal with loss of function. The adult retina of zebrafish self-repairs after damage by activating cell-intrinsic mechanisms, which are regulated by extrinsic signal interactions. Among relevant regulatory extrinsic systems, purinergic signaling regulates progenitor proliferation during retinogenesis and regeneration and glia proliferation in proliferative retinopathies. ATP-activated P2X7 (P2RX7) and adenosine (P1R) receptors are involved in the progression of almost all retinopathies leading to blindness. Here, we examined P2RX7 and P1R participation in the retina regenerative response induced by photoreceptor damage caused by a specific dose of CoCl2 . First, we found that treatment of uninjured retinas with a potent agonist of P2RX7 (BzATP) provoked photoreceptor damage and mitotic activation of multipotent progenitors. In CoCl2 -injured retinas, blockade of endogenous extracellular ATP activity on P2RX7 caused further neurodegeneration, Müller cell gliosis, progenitor proliferation, and microglia reactivity. P2RX7 inhibition in injured retinas also increased the expression of lin28a and tnfα genes, which are related to multipotent progenitor proliferation. Levels of hif1α, vegf3r, and vegfaa mRNA were enhanced by blockade of P2RX7 immediately after injury, indicating hypoxic like damage and endothelial cell growth and proliferation. Complete depletion of extracellular nucleotides with an apyrase treatment strongly potentiated cell death and progenitor proliferation induced with CoCl2 . Blockade of adenosine P1 and A2A receptors (A2A R) had deleterious effects and deregulated normal timing for progenitor and precursor cell proliferation following photoreceptor damage. ATP via P2RX7 and adenosine via A2A R are survival extracellular signals key for retina regeneration in zebrafish.

Mitochondrial (ATP Synthase) Permeability Transition Pore

ATP synthase has been suggested to contain a mitochondrial megachannel (MCC) or permeability transition pore (mPTP), which contributes the majority of the current associated with the calcium-induced mitochondrial PT. We reconstituted purified monomeric ATP synthase from porcine heart mitochondria into liposomes for patch clamp recordings. These recordings demonstrated multi-conductance, voltage and Ca2+-induced channels with sensitivity to endogenous and pharmacological mPTP and ATP synthase regulators. Pharmacological inhibitors that act by binding to the ATP synthase F1 lost their activity upon F1 removal, suggesting that regulation of channel activity occurs in response to conformational changes within ATP synthase F1. To determine the source of the channel activity, we purified and reconstituted c-subunit rings for patch recordings. These recordings demonstrated channel activity similar to that of monomeric F1FO ATP synthase except that reagents that normally bind within F1 were ineffective, presumably because of the absence of F1. In contrast, addition of purified F1 to the bath during the recordings inactivated the c-subunit channel. Based on our findings we conclude that the c-subunit forms a pore of the mPTP, the intact ATP synthase F1 binds to the c-subunit to inactivate the channel and finally, the F1 contains regulatory subunits that interact with previously known mPTP pharmacological inhibitors and novel endogenous regulators.

The Influence of Exogenous ATP on Functional Responses of Murine Bone Marrow Granulocytes

Extracellular ATP is a potent inflammatory mediator and regulator of neutrophil activity in an inflammatory site. The role of ATP in the regulation of bone marrow granulocyte functions remains unexplored. The aim of this work was to study effects of ATP on the cytoplasmic Ca2+ concentration ([Ca2+]i), generation of reactive oxygen species (ROS) and adhesion of bone marrow granulocytes. Changes of [Ca2+]i were estimated with Fura-2 AM ratiometric probe. To reveal cells with calcium activity in a population we developed an automated procedure based on the calculation of skewness and kurtosis of the [Ca2+]i value distribution. ROS generation was estimated by luminol-dependent chemiluminescence. Cell adhesion was evaluated by a standard absorbance (492 nm) method. The population of bone marrow granulocytes contained cells that generated calcium spikes in response to 10 μM ATP (35.2 ± 4.0%). The automated scanning allowed us to identify four groups of cells: those responding to ATP (23.4 ± 4.9%) or to the addition of Hanks’ solution (16.9 ± 2.5%) or to both ATP and Hanks’ solution (6.7 ± 1.5%), and cells not responding to either stimulus (53.1 ± 8.2%). ATP increased the adhesion of granulocytes, while apyrase, which hydrolyzes ATP, did not significantly change the adhesion. ATP did not influence the respiratory burst initiated by the bacterial peptide N-formyl-MLF (fMLF). Apyrase significantly enhanced the response to fMLF, and cytochalasin D inhibited this effect. In summary, exogenous ATP enhances adhesion and generation of calcium spikes in the population of bone marrow granulocytes. Endogenous ATP likely reduces respiratory burst through a mechanism involving the cytoskeleton. The studied granulocyte population contains cells with different ATP sensitivity.

Plastic but repeatable: rapid adjustments of mitochondrial function and density during reproduction in a wild bird species.

Most of the energy fluxes supporting animal performance flow through mitochondria. Hence, inter-individual differences in performance might be rooted in inter-individual variations in mitochondrial function and density. Furthermore, because the energy required by an individual often changes across life stages, mitochondrial function and density are also expected to show within-individual variation (i.e. plasticity). No study so far has repeatedly measured mitochondrial function and density in the same individuals to simultaneously test for within-individual repeatability and plasticity of mitochondrial traits. Here, we repeatedly measured mitochondrial DNA copy number (a proxy of density) and respiration rates from blood cells of female pied flycatchers (Ficedula hypoleuca) at the incubation and chick-rearing stages. Mitochondrial density and respiration rates were all repeatable (R = [0.45; 0.80]), indicating high within-individual consistency in mitochondrial traits across life-history stages. Mitochondrial traits were also plastic, showing a quick (i.e. 10 days) downregulation from incubation to chick-rearing in mitochondrial density, respiratory activity, and cellular regulation by endogenous substrates and/or ATP demand. These downregulations were partially compensated by an increase in mitochondrial efficiency at the chick-rearing stage. Therefore, our study provides clear evidence for both short-term plasticity and high within-individual consistency in mitochondrial function and density during reproduction in a wild bird species.

Histochemical Approach for Simultaneous Detection of Ectonucleotidase and Alkaline Phosphatase Activities in Tissues.

Studies on pathophysiology and the therapeutic potential of extracellular ATP and other purines represent an important and rapidly evolving field. The integral response of the cell is determined by multiple factors, including the release of endogenous ATP, co-expression of different types of nucleotide- and adenosine-selective receptors, as well as the specific makeup of ectoenzymes governing the duration and magnitude of purinergic signaling. Current findings support the presence of an extensive network of purine-converting ectoenzymes that are co-expressed to a variable extent among the mammalian tissues and share similarities in substrate specificity. Here, we describe a histochemical approach for simultaneous detection of ecto-nucleotidase and tissue-nonspecific alkaline phosphatase (TNAP) activities in the same tissue slice. Further employment of this technique for staining human palatine tonsil cryosections revealed selective distribution of the key ectoenzymes within certain tonsillar structures, including germinal centers and connective tissues (ecto-5'-nucleotidase/CD73), as well as interfollicular area (TNAP and NTPDase1/CD39).

P2Y receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

P2Y receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on P2Y Receptors [3, 5]) are activated by the endogenous ligands ATP, ADP, uridine triphosphate, uridine diphosphate and UDP-glucose. The relationship of many of the cloned receptors to endogenously expressed receptors is not yet established and so it might be appropriate to use wording such as 'uridine triphosphate-preferring (or ATP-, etc.) P2Y receptor' or 'P2Y1-like', etc., until further, as yet undefined, corroborative criteria can be applied [46, 109, 187, 375, 388].Clinically used drugs acting on these receptors include the dinucleoside polyphosphate diquafosol, agonist of the P2Y2 receptor subtype, approved in Japan for the management of dry eye disease [236], and the P2Y12 receptor antagonists prasugrel, ticagrelor and cangrelor, all approved as antiplatelet drugs [52, 316].

The Preferential Inhibitory Effect of Olmesartan, a New Angiotensin II Type 1 Antagonist, on Sympathetic Nerve Terminals in Isolated Canine Splenic Artery

Effects of olmesartan (RNH-6270: (5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxy-4-(1-hydroxy-1-methylethyl)-2-propyl{4-[2-(tetrazol-5-yl)-phenyl]phenyl}methylimidazol-5-carboxylase, an active form of olmesartan medoxomil (CS-866)) was investigated in isolated, perfused canine splenic arterial preparations. Neither exogenous noradrenaline- nor ATP-induced vasoconstrictor responses were modified by treatment with the used concentrations of olmesartan (1 – 100 nM). A high concentration of 10 nM angiotensin II caused a potentiation of either noradrenaline- and ATP-induced constrictions, although 1 nM angiotensin II did not induce any potentiating effects for these responses. These potentiations were inhibited by olmesartan in a concentration-related manner. Periarterial nerve electrical stimulation (PNS) readily induced a biphasic constriction consisting of an initial P2X purinoceptor-mediated vasoconstriction followed by a prolonged mainly α1-adrenoceptor-mediated response. PNS-induced 1st and 2nd peaked responses were significantly inhibited by olmesartan in a concentration-related manner. With a low concentration of 1 nM angiotensin II, which did not induce any vascular effects by itself, PNS-induced responses were markedly enhanced. The enhanced responses were inhibited by olmesartan. It is concluded that endogenous angiotensin II exerts its stimulating action on the releases of ATP and noradrenaline from the periarterial sympathetic nerve terminal, and olmesartan has an inhibitory property on angiotensin II-induced potentiation of endogenous ATP- and noradrenaline-induced responses.

Grape Seed Proanthocyanidins Protect N2a Cells against Ischemic Injury via Endoplasmic Reticulum Stress and Mitochondrial-associated Pathways.

Grape seed proanthocyanidins (GSPs) are a group of polyphenolic bioflavonoids, which possess a variety of biological functions and pharmacological properties. We studied the neuroprotective effects of GSP against oxygen-glucose deprivation/reoxygenation (OGD/R) injury and the potential mechanisms in mouse neuroblastoma N2a cells. OGD/R was conducted in N2a cells. Cell viability was evaluated by CCK-8 and LDH release assay. Apoptosis was assessed by TUNEL staining and flow cytometry. Protein levels of cleaved caspase-3, Bax and Bcl-2 were detected by Western blotting. CHOP, GRP78 and caspase-12 mRNA levels were assessed by real-time PCR. JC-1 dying was used to detect mitochondrial membrane potential. ROS levels, activities of endogenous antioxidant enzymes and ATP production were examined to evaluate mitochondrial function. GSP increased cell viability after OGD/R injury in a dose-dependent manner. Furthermore, GSP inhibited cell apoptosis, reduced the mRNA levels of CHOP, GRP78 and caspase-12 (ER stressassociated genes), restored mitochondrial membrane potential and ATP generation, improved activities of endogenous anti-oxidant ability (T-AOC, GXH-Px, and SOD), and decreased ROS level. Our findings suggest that GSP can protect N2a cells from OGD/R insult. The mechanism of anti-apoptotic effects of GSP may involve attenuating ER stress and mitochondrial dysfunction.

An Acidic-Microenvironment-Driven DNA Nanomachine Enables Specific ATP Imaging in the Extracellular Milieu of Tumor.

Extracellular ATP is an emerging target for cancer treatment because it is a key messenger for shaping the tumor microenvironment (TME) and regulating tumor progression. However, it remains a great challenge to design biochemical probes for targeted imaging of extracellular ATP in the TME. A TME-driven DNA nanomachine (Apt-LIP) that permits spatially controlled imaging of ATP in the extracellular milieu of tumors with ultrahigh signal-to-background ratio is reported. It operates in response to the mild acidity in the TME with the pH (low) insertion peptide (pHLIP) module, thus allowing the specific anchoring of the structure-switching signaling aptamer unit to the membrane of tumor cells for "off-on" fluorescence imaging of the extracellular ATP. Apt-LIP allows for acidity driven visualization of different extracellular concentrations of exogenous ATP, as well as the monitoring of endogenous ATP release from cells. Furthermore, it is demonstrated that Apt-LIP represents a promising platform for the specific imaging of the extracellular ATP in both primary and metastatic tumors. Ultimately, since diverse aptamers are obtained through in vitro selection, this design strategy can be further applied for precise detection of various extracellular targets in the TME.

A “noisy” electrical stimulation protocol favors muscle regeneration in vitro through release of endogenous ATP

An in vitro system of electrical stimulation was used to explore whether an innovative “noisy” stimulation protocol derived from human electromyographic recordings (EMGstim) could promote muscle regeneration. EMGstim was delivered to cultured mouse myofibers isolated from Flexor Digitorum Brevis, preserving their satellite cells. In response to EMGstim, immunostaining for the myogenic regulatory factor myogenin, revealed an increased percentage of elongated myogenin-positive cells surrounding the myofibers. Conditioned medium collected from EMGstim-treated cell cultures, promoted satellite cells differentiation in unstimulated myofiber cell cultures, suggesting that extracellular soluble factors could mediate the process. Interestingly, the myogenic effect of EMGstim was mimicked by exogenously applied ATP (0.1 μM), reduced by the ATP diphosphohydrolase apyrase and prevented by blocking endogenous ATP release with carbenoxolone.In conclusion, our results show that “noisy” electrical stimulations favor muscle progenitor cell differentiation most likely via the release of endogenous ATP from contracting myofibres. Our data also suggest that “noisy” stimulation protocols could be potentially more efficient than regular stimulations to promote in vivo muscle regeneration after traumatic injury or in neuropathological diseases.

DNA Logic Operations in Living Cells Utilizing Lysosome-Recognizing Framework Nucleic Acid Nanodevices for Subcellular Imaging.

DNA logic nanodevices that in situ operate within living cells have attracted increasing interest and shown great promise for gene regulation and target recognition. A challenge remains how to control their activation inside specific cellular compartments. Toward this goal, here we report a lysosome-recognizing framework nucleic acid (FNA) nanodevice using an i-motif and an ATP-binding aptamer (ABA) incorporated into a DNA triangular prism (DTP) as the logic-controlling units. Once entering the lysosomal compartments, the FNA device responds to lysosomal pH and ATP via the folding of i-motif and ABA, which triggers a structural change of FNA and the release of a reporter structure for subcellular imaging. With endogenous proton and ATP as two inputs, an AND logic gate is built and in situ operated within living lysosomes by pH and ATP modulation with external drug stimuli. Given the abnormal levels of pH and ATP within some cancer cells or dysfunctional lysosomal cells, in this context our designed FNA logic device may find extended applications in controllable drug release and disease treatment.

Involvement of P2X7 Receptors in the Osteogenic Differentiation of Mesenchymal Stromal/Stem Cells Derived from Human Subcutaneous Adipose Tissue.

The ionotropic P2X7 receptor (P2X7R) is involved in bone homeostasis but its role in osteogenesis is controversial. Thus, we investigated the expression of P2X7R and the effects exerted by its modulation in mesenchymal stromal cells from human subcutaneous adipose tissue (S-ASCs), which have potential therapeutic application in bone regenerative medicine. We found that undifferentiated S-ASCs expressed P2X7R and its functional splice variants P2X7AR and P2X7BR. Cell stimulation by P2X7R agonist BzATP (100μM) neither modified proliferation nor caused membrane pore opening while increasing intracellular Ca2+ levels and migration. The P2X7R antagonist A438079 reversed these effects. However, 25-100μM BzATP, administered to S-ASCs undergoing osteogenic differentiation, dose-dependently decreased extracellular matrix mineralization and expression of osteogenic transcription factors Runx2, alkaline phosphatase and osteopontin. These effects were not coupled to cell proliferation reduction or to cell death increase, but were associated to decrease in P2X7AR and P2X7BR expression. In contrast, expression of P2X7R, especially P2X7BR isoform, significantly increased during the osteogenic process. Noteworthy, the antagonist A438079, administered alone, at first restrained cell differentiation, enhancing it later. Accordingly, A438079 reversed BzATP effects only in the second phase of S-ASCs osteogenic differentiation. Apyrase, a diphosphohydrolase converting ATP/ADP into AMP, showed a similar behavior. Altogether, findings related to A438079 or apyrase effects suggest an earlier and prevailing pro-osteogenic activity by endogenous ATP and a later one by adenosine derived from endogenous ATP metabolism. Conversely, P2X7R pharmacological stimulation by BzATP, mimicking the effects of high ATP levels occurring during tissue injuries, depressed receptor expression/activity impairing MSC osteogenic differentiation.

Allergic sensitization increases the amount of extracellular ATP hydrolyzed by guinea pig leukocytes.

Increased levels of ATP have been found in the bronchoalveolar lavage of patients with asthma, and subjects with this disease, but not healthy subjects, develop bronchospasm after nebulization with ATP. Because the main mechanism for controlling the noxious effects of extracellular ATP is its enzymatic hydrolysis, we hypothesized that allergic sensitization is accompanied by a decreased functioning of such hydrolysis. In the present study, peripheral blood leukocytes from sensitized and non-sensitized guinea pigs were used for determining the extracellular metabolism (as assessed by inorganic phosphate production) of ATP, ADP, AMP, or adenosine, and for detecting possible changes in the expression (qPCR and Western blot) of major ectonucleotidases (NTPDase1, NTPDase3, and NPP1) and purinoceptors (P2X1, P2X7, P2Y4, and P2Y6). Contrary to our hypothesis, we found that leukocytes from allergic animals produced higher amounts of inorganic phosphate after stimulation with ATP and ADP, as compared with leukocytes from non-sensitized animals. Although at first glance, this result suggested that sensitization caused higher efficiency of ectonucleotidases, their mRNA and protein expressions were unaffected. On the other hand, after sensitization, we found a significant increase in the protein expression of P2X7 and P2Y4, two purinoceptors known to be responsible for ATP release after activation. We concluded that allergic sensitization increased the amount of ATP hydrolyzed by ectonucleotidases, the latter probably not due to the enhanced efficiency of its enzymatic breakdown, but rather due to an increased release of endogenous ATP or other nucleotides, partly mediated by enhanced expression or P2X7 and P2Y4 receptors.

Reconfigurable Bioinspired Framework Nucleic Acid Nanoplatform Dynamically Manipulated in Living Cells for Subcellular Imaging.

In nature, the formation of spider silk fibers begins with dimerizing the pH-sensitive N-terminal domains of silk proteins (spidroins) upon lowering pH, and provides a natural masterpiece for programmable assembly. Inspired by the similarity of pH-dependent dimerization behaviors, introduced here is an i-motif-guided model to mimic the initial step of spidroin assembly at the subcellular level. A framework nucleic acid (FNA) nanoplatform is designed using two tetrahedral DNA nanostructures (TDNs) with different branched vertexes carrying a bimolecular i-motif and a split ATP aptamer. Once TDNs enter acidic lysosomes within living cells, they assemble into a heterodimeric architecture, thereby enabling the formation of a larger-size framework and meanwhile subcellular imaging in response to endogenous ATP, which can be dynamically manipulated by adjusting intracellular pH and ATP levels with external drug stimuli.

BLOOD PLATELETS AS ACTIVATORS AND REGULATORS OF INFLAMMATORY AND IMMUNE REACTIONS. PART 1. BASIC CHARACTERISTICS OF PLATELETS AS INFLAMMATORY CELLS

Platelets are the smallest blood cells, and yet their total volume and surface area exceed those of all types of leukocytes combined. Platelets are produced by the bone marrow megakaryocytes and megakaryocytes in the lung microvessels. Approximately 50% of all platelets are produced in the lungs, which makes it possible to characterize them as the main site for the production of platelets. In small circuit of blood circulation, there are approximately 30% more platelets than in large circuit. This “excess” of platelets is necessary for the stabilization of the endothelial barrier of the lung vessels regulated by the platelet mediator sphingosine-1-phosphate, a regulator of tight junctions of endothelial cells. The circulating platelets have an amazing ability to “bud” new pro- and pre-platelets, giving rise to new platelets. The removal of platelets from circulation proceeds via their phagocytosis by spleen macrophages (if platelets are covered with IgG or are bound to immune complexes), or Kupffer liver cells and hepatocytes (if platelets have incomplete glycans or desialated proteins). In homeostatic conditions, most of the platelets are removed in liver. Platelet clearance in bacterial infections and sepsis is accelerated because of the activity of bacterial sialidases. Recognition of desialized platelet structures is carried out by the liver cells through the Asgr receptor. Despite DNA absence, the platelets are able to synthesize proteins at mRNAs that are present in majority of platelets. Activation of platelets leads to aggregation and exocytosis of the granule contents, and production of immunomodulating molecules. However, activation of platelets may be incomplete and has various consequences. In a non-classical activation model, platelets can release microparticles that contain about 600 different proteins. About 75% of microparticles in the blood of healthy donors are derived from platelets. Like as immune system cells, platelets are activated by numerous endogenous ligands (alarms), including ADP and ATP, which bind to purinergic receptors P2Y1, P2Y12 andP2X1. Platelets accumulate and retain 99% of the serotonin stored in the body. The platelets contribute to induction of inflammation by releasing proinflammatory cytokines, chemokines, and lipid mediators. In addition, platelets are the source of enzymes that accomplish the capacities of neutrophils and endothelium for production of anti-inflammatory lipid mediators that contribute to tissue repair following acute phase of inflammation.

DNA Polymer Nanoparticles Programmed via Supersandwich Hybridization for Imaging and Therapy of Cancer Cells.

Spherical nucleic acid (SNA) constructs are promising new single entity materials, which possess significant advantages in biological applications. Current SNA-based drug delivery system typically employed single-layered ss- or ds-DNA as the drug carriers, resulting in limited drug payload capacity and disease treatment. To advance corresponding applications, we developed a novel DNA-programmed polymeric SNA, a long concatamer DNA polymer that is uniformly distributed on gold nanoparticles (AuNPs), by self-assembling from two short alternating DNA building blocks upon initiation of immobilized capture probes on AuNPs, through a supersandwich hybridization reaction. The long DNA concatamer of polymeric SNA enables to allow high-capacity loading of bioimaging and therapeutics agents. We demonstrated that both of the fluorescence signals and therapeutic efficacy were effectively inhibited in resultant polymeric SNA. By further modifying with the nucleolin-targeting aptamer AS1411, this polymeric SNA could be specifically internalized into the tumor cells through nucleolin-mediated endocytosis and then interact with endogenous ATP to cause the release of therapeutics agents from long DNA concatamer via a structure switching, leading to the activation of the fluorescence and selective synergistic chemotherapy and photodynamic therapy. This nanostructure can afford a promising targeted drug transport platform for activatable cancer theranostics.

Inflammatory Cytokines Facilitate the Sensitivity of P2X7 Receptors Toward Extracellular ATP at Neural Progenitor Cells of the Rodent Hippocampal Subgranular Zone

Organotypic hippocampal slice cultures were used to model the effects of neuroinflammatory conditions following an epileptic state on functional P2X7 receptors (Rs) of subgranular zone (SGZ) neural progenitor cells (NPCs). The compound, 4-aminopyridine (4-AP), is known to cause pathological firing of neurons, consequently facilitating the release of various transmitter substances including ATP. Lipopolysaccharide (LPS) and interleukin-1β (IL-1β) both potentiated the dibenzoyl-ATP (Bz-ATP)-induced current amplitudes in NPCs, although via different mechanisms. Whereas LPS acted via promoting ATP release, IL-1β acted via its own receptor to directly influence P2X7Rs. Thus, the effect of LPS was inhibited by the ecto-ATPase inhibitor, apyrase, but not by the IL-1β antagonist, interleukin-1RA (IL-1RA); by contrast, the effect of IL-1β was inhibited by IL-1RA, but not by apyrase. Eventually, incubation with 4-AP upregulated the number of nestin/glial fibrillary acidic protein/P2X7R immunoreactive cells and their appropriate staining intensity, suggesting increased synthesis of P2X7Rs at NPCs. In conclusion, inflammatory cytokines accumulating after epilepsy-like neuronal firing may facilitate the effect of endogenous ATP at P2X7Rs of NPCs, thereby probably promoting necrosis/apoptosis and subsequent cell death.

Purinergic receptor types in the hypothalamic-neurohypophysial system.

Many different types of purinergic receptors are present in the Hypothalamic-Neurohypophysial System (HNS), which synthesizes and releases vasopressin and oxytocin. The specific location of purinergic receptor subtypes has important functional repercussions for neuronal activity and synaptic output. Yet, until the advent of receptor KOs, this had been hindered by the low selectivity of the available pharmacological tools. The HNS offers an excellent opportunity to differentiate the functional properties of these purinergic receptors in cell bodies vs. terminals of the same physiological system. P2X2, P2X3, P2X4 and P2X7 receptors are present in vasopressin terminals while oxytocin terminals exclusively express the P2X7 subtype. The latter is not functional in the cell bodies of the HNS. These purinergic receptor subtypes are permeable to sodium vs. calcium in varying amounts and this could play an important role in the release of vasopressin vs. oxytocin during bursting activity. Endogenous ATP and its metabolite, adenosine, have autocrine and paracrine modulatory effects on the release of these neuropeptides during physiological stimulation. Finally, we hypothesize that during such action potential bursts, ATP potentiates the release of vasopressin but not of oxytocin, and that adenosine, via A1 receptors, inhibits the release of both neuropeptides. This article is protected by copyright. All rights reserved.

Enhanced squalene biosynthesis in Yarrowia lipolytica based on metabolically engineered acetyl-CoA metabolism

As a bioactive triterpenoid, squalene is widely used in the food industry, cosmetics, and pharmacology. Squalene’s major commercial sources are the liver oil of deep-sea sharks and plant oils. In this study, we focused on the enhancement of squalene biosynthesis in Yarrowia lipolytica, with particular attention to the engineering of acetyl-CoA metabolism based on genome-scale metabolic reaction network analysis. Although the overexpression of the rate-limiting endogenous ylHMG1 (3-hydroxy-3-methylglutaryl-CoA reductase gene) could improve squalene synthesis by 3.2-fold over that by the control strain, the availability of the key intracellular precursor, acetyl-CoA, was found to play a more significant role in elevating squalene production. Analysis of metabolic networks with the newly constructed genome-scale metabolic model of Y. lipolytica iYL_2.0 showed that the acetyl-CoA pool size could be increased by redirecting carbon flux of pyruvate dehydrogenation towards the ligation of acetate and CoA or the cleavage of citrate to form oxaloacetate and acetyl-CoA. The overexpression of either acetyl-CoA synthetase gene from Salmonella enterica (acs*) or the endogenous ATP citrate lyase gene (ylACL1) resulted in a more than 50% increase in the cytosolic acetyl-CoA level. Moreover, iterative chromosomal integration of the ylHMG1, asc*, and ylACL1 genes resulted in a significant improvement in squalene production (16.4-fold increase in squalene content over that in the control strain). We also found that supplementation with 10 mM citrate in a flask culture further enhanced squalene production to 10 mg/g DCW. The information obtained in this study demonstrates that rationally engineering acetyl-CoA metabolism to ensure the supply of this key metabolic precursor is an efficient strategy for the enhancement of squalene biosynthesis.

Macrophage P2X4 receptors augment bacterial killing and protect against sepsis.

The macrophage is a major phagocytic cell type, and its impaired function is a primary cause of immune paralysis, organ injury, and death in sepsis. An incomplete understanding of the endogenous molecules that regulate macrophage bactericidal activity is a major barrier for developing effective therapies for sepsis. Using an in vitro killing assay, we report here that the endogenous purine ATP augments the killing of sepsis-causing bacteria by macrophages through P2X4 receptors (P2X4Rs). Using newly developed transgenic mice expressing a bioluminescent ATP probe on the cell surface, we found that extracellular ATP levels increase during sepsis, indicating that ATP may contribute to bacterial killing in vivo. Studies with P2X4R-deficient mice subjected to sepsis confirm the role of extracellular ATP acting on P2X4Rs in killing bacteria and protecting against organ injury and death. Results with adoptive transfer of macrophages, myeloid-specific P2X4R-deficient mice, and P2rx4 tdTomato reporter mice indicate that macrophages are essential for the antibacterial, antiinflammatory, and organ protective effects of P2X4Rs in sepsis. Pharmacological targeting of P2X4Rs with the allosteric activator ivermectin protects against bacterial dissemination and mortality in sepsis. We propose that P2X4Rs represent a promising target for drug development to control bacterial growth in sepsis and other infections.