Delivery Of ATP Research Articles

Balanced mitochondrial function at low temperature is linked to cold adaptation in Drosophila species.

The ability of ectothermic animals to live in different thermal environments is closely associated with their capacity to maintain physiological homeostasis across diurnal and seasonal temperature fluctuations. For chill-susceptible insects, such as Drosophila, cold tolerance is tightly linked to ion and water homeostasis obtained through a regulated balance of active and passive transport. Active transport at low temperature requires a constant delivery of ATP and we therefore hypothesize that cold-adapted Drosophila are characterized by superior mitochondrial capacity at low temperature relative to cold-sensitive species. To address this, we investigated how experimental temperatures from 1 to 19 °C affected mitochondrial substrate oxidation in flight muscle of seven Drosophila species and compared it to a measure of species cold tolerance (CTmin, the temperature inducing cold coma). Mitochondrial oxygen consumption rates measured using a substrate-uncoupler-inhibitor-titration (SUIT) protocol showed that cooling generally reduced oxygen consumption of the electron transport system across species, as was expected due to thermodynamic effects. Complex I is the primary consumer of oxygen at non-stressful temperatures, but low temperature decreases complex I respiration to a much greater extent in cold-sensitive species than in cold-adapted species. Accordingly, cold-induced reduction of complex I correlates strongly with CTmin. The relative contribution of other substrates (proline, succinate and glycerol-3-phosphate) increased as temperature decreased, particularly in the cold-sensitive species. At present, it is unclear whether the oxidation of alternative substrates can be used to offset the effects of the temperature-sensitive complex I, and the potential functional consequences of such a substrate switch are discussed.

The role of cluster of differentiation 163-positive macrophages in wound healing: a preliminary study and a systematic review.

This is a literature assessment of essential information and current knowledge that pertains to the potential role for cluster of differentiation (CD) 163+ macrophages in different wound healing models, including extremely rapid tissue regeneration for regenerative medicine purposes. We intend to focus on the beneficial strategies that activate macrophage performance in order to advance the CD163+ macrophage-based therapy approaches to accelerate wound healing. We conducted an extensive literature search of peer reviewed articles obtained from the PubMed, Google Scholar, Scopus, Web of Science, and Cochrane databases by using the keywords "wound healing, CD163+ macrophages, diabetes mellitus, and burn." There were no limitations in terms of publication date. Our search resulted in 300 papers from which 17 articles were screened according to the inclusion criteria. We divided the selected articles into four distinct groups: healthy humans (n = 5); healthy animals (n = 7); humans with diabetes (n = 2); and animals with diabetes (n = 3). CD163 is a biomarker of the M2c macrophage subtype in mammals. Functions of M2c macrophages include angiogenesis, matrix maturation, and phagocytosis, and they activate prior to wounding. M2c produces many cytokines and growth factors, and also contains receptors for numerous cytokines and growth factors. Induction of M2c macrophages from tissue-resident macrophages in the wound bed by a suitable agent, such as delivery of intracellular ATP, appears to induce rapid granulation tissue formation without hypertrophic scarring and significantly reduces the lag time of the wound healing process.

Adenosine and ATP\u03b3S protect against bacterial pneumonia-induced acute lung injury

Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, disrupts the alveolar-capillary barrier, triggering pulmonary vascular leak thus inducing acute lung injury (ALI). Extracellular purines, adenosine and ATP, protected against ALI induced by purified LPS. In this study, we investigated whether these purines can impact vascular injury in more clinically-relevant E.coli (non-sterile LPS) murine ALI model. Mice were inoculated with live E. coli intratracheally (i.t.) with or without adenosine or a non-hydrolyzable ATP analog, adenosine 5′-(γ-thio)-triphosphate (ATPγS) added intravenously (i.v.). After 24 h of E. coli treatment, we found that injections of either adenosine or ATPγS 15 min prior or adenosine 3 h after E.coli insult significantly attenuated the E.coli-mediated increase in inflammatory responses. Furthermore, adenosine prevented weight loss, tachycardia, and compromised lung function in E. coli-exposed mice. Accordingly, treatment with adenosine or ATPγS increased oxygen saturation and reduced histopathological signs of lung injury in mice exposed to E. coli. Lastly, lung-targeting gene delivery of adenosine or ATPγS downstream effector, myosin phosphatase, significantly attenuated the E. coli-induced compromise of lung function. Collectively, our study has demonstrated that adenosine or ATPγS mitigates E. coli-induced ALI in mice and may be useful as an adjuvant therapy in future pre-clinical studies.

Large 10 × 10 single cell grid networks of human hNT astrocytes on raised parylene-C/SiO2 substrates

Objective. The ‘Astrocytic Network’ is an emerging research field for researchers in cell biology. Culturing astrocytes in organised networks is a novel method for permitting controlled studies and investigations into the calcium transients of such networks. Recent research has photolithographically patterned hNT astrocytes on parylene-C inlayed SiO2 trench grid networks. However, it was observed that the trench networks could not specifically immobilise the astrocyte cell bodies to the nodes of the networks. Approach. In this study, for the first time, we demonstrate how it is possible to establish grid networks of human hNT astrocytes on raised parylene-C structures where the cell bodies are specifically organised down to the single-cell level on nodes of the grid and connected throughout. Main results. Here, we report these to be the largest patterned single-cell grid network of astrocytes of their kind consisting of 100 cells in a 10 × 10 grid arrangement to an 80% efficiency. We quantify the level of patterning through six cell patterning assessment indices: the parylene adhesion index (PAI); SiO2 attraction index (SAI); node index (NI) and connectivity interval (χI), number of components (k) and fielder value (λss) and report that the best connected network is obtained with 65 µm node size, 90 µm node spacing, and 5 µm interconnecting track width (PAI = 0.77 ± 0.040, SAI = 0.12 ± 0.049, NI = 0.81 ± 0.066, χI = 0.25 ± 0.064, k = 2.33 ± 1.528, λss = 0.0249 ± 0.0018). We finally demonstrate, through delivery of ATP, that the networks are functional on the raised parylene-C grid structures. Significance. The significance of this study is that it determines the optimal dimensions to obtain highly organised, large, interlinked, single-cell networks which provide an effective platform to investigate calcium communication within astrocytic networks in an accurate, controlled and repeatable manner.

Significant upregulation of U1 and U4 spliceosomal snRNAs by ATP nanoliposomes explains acceleration of wound healing, due to increased pre-mRNA processing to functional mRNA

Delayed wound healing is one of the hallmarks of diabetic complications and certain autoimmune inflammatory diseases. Extensive wound healing studies in rabbits have indicated that the delivery of ATP encapsulated in unilamellar nanoliposomes causes rapid cell proliferation and fast tracks the wound healing process. In the current study, we explored the possible molecular mechanism underlying this response by comparing gene expression in cultured rabbit kidney cells treated with either ATP nanoliposomes (containing 1 mg Mg-ATP/ml formulation) or control nanoliposomes (containing 1 mg/ml unmetabolisable gamma-thio-ATP/ml formulation). High-quality total RNA was isolated 24 h from the cells and subjected to RNA seq technology, which revealed significant overexpression of specific noncoding RNAs. The U1 spliceosomal RNA, U1 snRNA, was upregulated more than 250-fold following treatment with ATP nanoliposomes. This multifunctional U1 spliceosomal RNA may function in transcription by speeding up the critical splicing step, thereby facilitating faster processing of pre-mRNA to translatable mRNA.

KO of 5-InsP7 kinase activity transforms the HCT116 colon cancer cell line into a hypermetabolic, growth-inhibited phenotype

The inositol pyrophosphates 5-InsP7 (diphosphoinositol pentakisphosphate) and 1,5-InsP8 (bis-diphosphoinositol tetrakisphosphate) are highly energetic cellular signals interconverted by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks). Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP8 and raises 5-InsP7 levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP7] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP7] provides direct support for an energy-sensing attribute (i.e., 1 mM Km for ATP) of the 5-InsP7-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP7 levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in PPIP5K-/- HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP8 and 5-InsP7 as drivers of the hypermetabolic and p53-elevated phenotypes, we used IP6K2 RNAi and the pan-IP6K inhibitor, N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine (TNP), to return 5-InsP7 levels in PPIP5K-/- cells to those of WT cells without rescuing 1,5-InsP8 levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP7 regulates p53 expression, whereas 1,5-InsP8 regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP8 to bioenergetic homeostasis.

Conducted dilatation to ATP and K+ in rat skeletal muscle arterioles.

AimDuring exercise in humans, circulating levels of ATP and K+ increase at a time when blood flow increases to satisfy metabolic demand. Both molecules can activate arteriolar K+ channels to stimulate vasodilatation; here, it is established whether conducted dilatation is observed in a skeletal muscle bed.MethodsIsolated and cannulated rat cremaster arterioles were used to assess both local and conducted responses. Agents were either added to the bath, focally pulse‐ejected to the downstream end of arterioles, or in triple‐cannulated arterioles, luminally perfused into the downstream branches to assess both local and conducted responses.ResultsThe endothelium‐dependent agonist ACh and the KATP channel opener levcromakalim each stimulated both local and conducted vasodilatation. Focal, bolus delivery of ATP (10 μ m) or KCl (33 mm) to the outside of arterioles stimulated a biphasic vasomotor response: rapid vasoconstriction followed by dilatation as each washed away. At lower concentrations of KCl (19 mm), constriction was avoided, and instead, Ba2+‐sensitive local dilatation and conducted dilatation were both observed. Luminal perfusion of ATP avoided constriction and activated P2Y1 receptors stimulating vasodilatation secondary to opening of KC a channels. In triple‐cannulated arterioles, either ATP (10 μ m) or K+ (15 mm) luminally perfused into daughter branches of a bifurcation stimulated local dilatation which conducted into the parent arteriole.ConclusionThe recognized physiological autocrine and paracrine mediators ATP and K+ each act to evoke both local and conducted vasodilatation in rat cremaster arterioles. Therefore, in situations when circulating levels are raised, such as during exercise, these agents can act as important regulators of blood flow.

Abstract 1701: PENAO, a novel mitochondria-targeted agent, has shown potent antitumor effect on glioblastoma in vitro and in vivo.

Abstract Despite advances in multimodal therapy, the prognosis in the great majority of patients with glioblastoma (GBM) is still dismal as almost all tumors relapse and result in disease-related death. New therapeutic strategies are urgently needed. PENAO, (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid) is a novel mitochondria-targeted agent that inactivates adenine nucleotide translocase (ANT) located on the inner-membrane of mitochondria. PENAO blocks ANT delivery of ATP to mitochondrial-bound hexokinase II. This blockage inhibits glucose metabolism and triggers the mitochondrial permeability transition pore to leak which results in proliferation arrest and apoptotic cell death. In vitro, we tested the efficacy of PENAO on a panel of glioblastoma cell lines including immortalized and primary glioblastoma neural stem (GNS) cell lines. PENAO demonstrated potent anti-proliferative activity in all cells. The half-maximal inhibitory concentration (IC50) values for PENAO were in the range of 0.3-6 μM. Blocking ANT with PENAO resulted in inhibition of glioblastoma cell migration at concentration of 1.4 μM. PENAO (0.3-5 μM) increased caspase 3 and 9 activity levels indicative of the intrinsic apoptotic pathway. Proper functioning of ANT is also of importance for maintaining mitochondrial integrity. Low micromolar concentrations of PENAO inhibited oxygen utilization, increased the cytosolic levels of superoxide and depolarized the mitochondrial trans-membrane potential, which are responsible for the activation of mitochondria-mediated apoptosis in glioblastoma cells. In vivo, PENAO was found to cross the intact blood-brain-barrier in mice and continuous administration of 1 mg/kg/day PENAO to 10 mice bearing subcutaneous glioblastoma tumors for 28 days resulted in 8 partial and 2 complete tumor remissions. No signs or symptoms of treatment toxicity were noticed. To further inhibit glucose metabolism of glioblastoma, we tested the effectiveness of PENAO in combination with 2-deoxy-D-glucose, a glucose analogue that inhibits glycolysis via competitive inhibition of hexokinase. This dual-targeting of glucose metabolism by inhibiting both mitochondrial respiration and glycolysis led to synergistic effects and improved efficacy when measuring proliferation arrest in vitro. By targeting the glioblastoma aberrant metabolism of glucose better treatments for this cancer may arise. Citation Format: Han Shen, Peter P. Luk, Sylvia A. Chung, Stephanie Decollogne, Pierre J. Dilda, Philip J. Hogg, Kerrie L. McDonald. PENAO, a novel mitochondria-targeted agent, has shown potent antitumor effect on glioblastoma in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1701. doi:10.1158/1538-7445.AM2013-1701

Sustained ATP‐mediated vasodilation of cerebral arteries requires Ca2+ influx via TRPC3 channels to produce endothelial SKCa channel activation and hyperpolarization

Extracellular ATP acts through endothelial cell (EC) purinoceptors to produce vasodilation of cerebral arteries (CA). IKCa channels have been established in this mechanism; however, the role of SKCa channels is not clear. We hypothesize that sustained ATP‐mediated hyperpolarization and vasodilation requires Ca2+ influx via EC TRPC3 channels to produce activation of SKCa channels.MethodsVascular diameter was measured following luminal delivery of ATP in mouse pressurized superior cerebellar arteries (SCA). Changes in EC Ca2+ were measured by biosensor in SCA isolated from Cx40BAC‐GCaMP2 transgenic mice. Membrane currents and voltage were measured in freshly isolated EC from CA of WT and TRPC3 knock out (KO) mice using perforated patch whole cell recording in voltage or current clamp mode.ResultsApplication of a selective TRPC3 blocker (Pyr3) significantly attenuated the duration of endothelial Ca2+ increase and vasodilation to ATP in pressurized SCA and abbreviated ATP‐mediated hyperpolarization in freshly isolated ECs. Responses were replicated with arteries and EC isolated from TRPC3 KO mice. Selective SKCa channel inhibition (UCL1684) mirrored the abbreviated time course of EC hyperpolarization, suggesting a common pathway.ConclusionWe propose that Ca2+ influx via TRPC3 promotes SKCa activation to produce the sustained component of cerebral vasorelaxation to ATP. Supported by R01HL088435 to SPM

Expression and localization of creatine kinase in the preimplantation embryo

Creatine Kinase (CK) catalyses the "creatine shuttle," the reversible conversion of creatine phosphate to creatine with the liberation of ATP. This article examines the potential role of the creatine shuttle in the provision of ATP during mouse preimplantation embryo development. Using quantitative PCR, transcripts of four subunit isoforms of CK--CKM, CKB, CKMT1, and CKMT2--were detectable at all developmental stages, from the presumptive zygote to late blastocyst, but there was no obvious pattern in gene expression. By contrast, total CK biochemical activity, measured by a novel method, was relatively constant from the 2- to 8-cell stage, before exhibiting a significant decrease in activity at the blastocyst stage. Immunocytochemical studies revealed a marked association of CKB with the mitotic spindle in 2- and 4-cell mouse embryos, consistent with the proposition that the creatine shuttle plays a key role in local delivery of ATP during cytokinesis. Endogenous creatine was detected in the blastocyst at a level of 0.53 pmol/embryo. In conclusion, we believe that creatine phosphate can now be added to the list of potential sources of ATP during preimplantation development.

ATP release and purinergic signaling: a common pathway for particle-mediated inflammasome activation

Deposition of uric acid crystals in joints causes the acute and chronic inflammatory disease known as gout and prolonged airway exposure to silica crystals leads to the development of silicosis, an irreversible fibrotic pulmonary disease. Aluminum salt (Alum) crystals are frequently used as vaccine adjuvant. The mechanisms by which crystals activate innate immunity through the Nlrp3 inflammasome are not well understood. Here, we show that uric acid, silica and Alum crystals trigger the extracellular delivery of endogenous ATP, which just precedes the secretion of mature interleukin-1β (IL-1β) by macrophages, both events depending on purinergic receptors and connexin/pannexin channels. Interestingly, not only ATP but also ADP and UTP are involved in IL-1β production upon these Nlrp3 inflammasome activators through multiple purinergic receptor signaling. These findings support a pivotal role for nucleotides as danger signals and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome.

Intracellular ATP Levels Are a Pivotal Determinant of Chemoresistance in Colon Cancer Cells

Altered metabolism in cancer cells is suspected to contribute to chemoresistance, but the precise mechanisms are unclear. Here, we show that intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds. Drug-resistant cells were characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of intracellular ATP, and enhanced HIF-1α-mediated signaling. Interestingly, direct delivery of ATP into cross-chemoresistant cells destabilized HIF-1α and inhibited glycolysis. Thus, drug-resistant cells exhibit a greater "ATP debt" defined as the extra amount of ATP needed to maintain homeostasis of survival pathways under genotoxic stress. Direct delivery of ATP was sufficient to render drug-sensitive cells drug resistant. Conversely, depleting ATP by cell treatment with an inhibitor of glycolysis, 3-bromopyruvate, was sufficient to sensitize cells cross-resistant to multiple chemotherapeutic drugs. In revealing that intracellular ATP levels are a core determinant of chemoresistance in colon cancer cells, our findings may offer a foundation for new improvements to colon cancer treatment.

Abstract 995: The role of intracellular ATP in regulating glycolysis, HIF1α and drug resistance of cancer cells

Abstract Cancer is also considered to be a metabolic disease due to a shift of glucose metabolism from oxidative phosphorylation to anaerobic glycolysis. This study is to understand the effect of intracellular ATP of cancer cells on various metastatsis related phenotypes, including glycolysis, hypoxia stability and drug resistance. In this study, we delivered ATP directly into cancer cells by embedding ATP into liposomes. Normal cells and its corresponding tumor cells were measured for their whole cell ATP content. Glycolysis was evaluated by measurement of glucose consumption and lactate accumulation of cell culture media. Cell cycle was analyzed using flow cytometry after drug treatment and delivery of ATP. HIF-1α expression level was measured by western blot after delivery of ATP. In summary, normal cells have overall higher ATP content than tumor cells. After delivery of ATP, cells consumed less glucose and produced less lactate acid, which indicated a decrease in glycolysis. ATP liposomes prevented MDA-MB-231 cells from G2 arrest by adriamycin. HIF1a was down regulated after delivery of ATP. Taken together, ATP content is lower in cancer cells than normal cells, and intracellular ATP plays a role in regulating glycolysis, HIF-1α stability and drug resistance of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 995. doi:10.1158/1538-7445.AM2011-995

Abstract 2824: PENAO: A small molecule tumor metabolism inhibitor

Abstract Tumors reorganize the metabolic steps used by normal tissues for glucose utilization and ATP production. Normal tissues rely heavily on oxidative phosphorylation for ATP, while tumor cells employ a route that includes a much greater dependency on glycolysis. This aberrant metabolism allows for tumor survival when oxygen is limiting while providing a mechanism to poison the extracellular environment with acid, which facilitates invasion and metastasis. In particular, tumors harness a crucial enzyme, hexokinase II (HKII), to entrap and channel glucose toward glycolysis. HKII is bound to mitochondria via interaction with the voltage dependent anion channel (VDAC). This affords HKII preferential access to the mitochondrial ATP via inner-membrane adenine nucleotide translocase (ANT) to rapidly phosphorylate and trap the incoming glucose, effectively initiating and regulating the glycolytic flux across the tumor cytosol. We have developed a novel covalent inhibitor of ANT called PENAO, (4-(N-(S-penicillaminylacetyl)amino)phenylarsonous acid) (1). This molecule blocks ANT delivery of ATP to VDAC-bound HKII, thus inhibiting tumor cell metabolism, and triggers the mitochondrial permeability transition pore, resulting in proliferation arrest and apoptotic cell death. PENAO rapidly enters the cell via an organic ion transporter and reacts with Cys160 and Cys257 on the matrix face of ANT, which leads to arrest of proliferation of the cell and then apoptosis. PENAO targets both proliferating endothelial and tumor cells. It is very well tolerated in mice and has potent anti-tumor activity in murine tumor models when administered either subcutaneously or intravenously. PENAO is the most potent anti-tumor compound of its type so far reported and will enter a Phase I/IIa clinical trial in adults with solid tumors refractory to standard therapy in 2011. 1. Dilda PJ, Decollogne S, Weerakoon L, et al. Optimization of the antitumor efficacy of a synthetic mitochondrial toxin by increasing the residence time in the cytosol. J Med Chem 2009;52(20):6209-16. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2824. doi:10.1158/1538-7445.AM2011-2824

Expression and Roles of Pannexins in ATP Release in the Pituitary Gland

Pannexins are a newly discovered three-member family of proteins expressed in the brain and peripheral tissues that belong to the superfamily of gap junction proteins. However, in mammals pannexins do not form gap junctions, and their expression and function in the pituitary gland have not been studied. Here we show that the rat pituitary gland expresses mRNA and protein transcripts of pannexins 1 and 2 but not pannexin 3. Pannexin 1 was more abundantly expressed in the anterior lobe, whereas pannexin 2 was more abundantly expressed in the intermediate and posterior pituitary. Pannexin 1 was identified in corticotrophs and a fraction of somatotrophs, the S100-positive pituicytes of the posterior pituitary and AtT-20 (mouse pituitary adrenocorticotropin-secreting cells) and rat immortalized pituitary cells secreting prolactin, whereas pannexin 2 was detected in the S100-positive folliculostellate cells of the anterior pituitary, melanotrophs of the intermediate lobe, and vasopressin-containing axons and nerve endings in the posterior lobe. Overexpression of pannexins 1 and 2 in AtT-20 pituitary cells enhanced the release of ATP in the extracellular medium, which was blocked by the gap junction inhibitor carbenoxolone. Basal ATP release in At-T20 cells was also suppressed by down-regulating the expression of endogenous pannexin 1 but not pannexin 2 with their short interfering RNAs. These results indicate that pannexins may provide a pathway for delivery of ATP, which is a native agonist for numerous P2X cationic channels and G protein-coupled P2Y receptors endogenously expressed in the pituitary gland.

Motility of alveolar mitochondria

Mitochondria are the major source of cellular ATP. As studied best in axons, mitochondrial motility regulates spatial ATP distribution within the cell. However little is known regarding regulation of mitochondrial motility in non‐neuronal cells. Here, we addressed this issue in intact alveoli of mouse lungs. Isolated, perfused lungs were held at constant pulmonary artery, left atrial and alveolar pressures of 10, 2 and 5 cm H2O, respectively. We visualized alveoli by confocal microscopy. To record mitochondrial density, we microinfused alveoli with the mitochondria‐specific dye, MitoTracker Green (MTG). Spontaneous, time‐dependent fluctuations of mitochondrial density were evident at several alveolar locations, suggesting that mitochondria were motile. To quantify the motility, we photobleached MTG‐labeled regions (3–6 μm) to 23±3% of the initial fluorescence. Then we determined time dependent fluorescence recovery after photobleaching (FRAP). Fluorescence recovery was 43±4 % of the initial in 3 min (P<0.05; n=3), indicating repopulation of the photobleached region by fluorescent mitochondria from adjoining regions. Our findings are the first evidence that alveolar epithelial mitochondria are motile. The motility might enable equitable delivery of ATP to different alveolar regions. (Swiss National Foundation, HL57556, HL36024)

Intracellular delivery of adenosine triphosphate enhanced healing process in full-thickness skin wounds in diabetic rabbits

Background Small unilamellar lipid vesicles were used to encapsulate adenosine triphosphate (ATP-vesicles) for intracellular energy delivery and were tested for diabetic skin wounds in rabbits. Methods Diabetes was induced by alloxan. The mean peak blood glucose concentration was 505 mg/dL. One ear was made ischemic and 80 full-thickness wounds were created in 10 animals. ATP-vesicles or saline was used and healing was compared. Results On the non-ischemic ears, mean closure time for ATP-vesicles–treated wounds was 13.7 days versus 16.4 days for saline-treated wounds ( P < .05). On the ischemic ears, mean closure time for ATP-vesicles–treated wounds was 15.3 days versus 19.3 days for saline-treated wounds ( P < .01). Histological study indicated better healing and re-epithelialization in the ATP-vesicles–treated wounds. Conclusions Intracellular delivery of ATP accelerated the healing process of diabetic skin wounds on ischemic and non-ischemic rabbit ears. The mechanisms deserve further study but may be related to improved cellular energy supplies.

Intracellular Adenosine Triphosphate Delivery Enhanced Skin Wound Healing in Rabbits

Small unilamellar lipid vesicles were used to encapsulate adenosine triphosphate (ATP-vesicles) for intracellular energy delivery. This technique was tested in full-thickness skin wounds in 16 adult rabbits. One ear was rendered ischemic by using a minimally invasive surgery. The other ear served as a normal control. Four circular full-thickness wounds were created on the ventral side of each ear. ATP-vesicles or saline was used and the wounds were covered with Tegaderm (3M, St. Paul, MN). Dressing was changed and digital photos were taken daily until all the wounds were healed. The mean healing times of ATP-vesicles-treated wounds were significantly shorter than that of saline-treated wounds on ischemic and nonischemic ears. Histologic study indicated better-developed granular tissue and reepithelialization in the ATP-vesicles-treated wounds. The wounds treated by ATP-vesicles exhibited extremely fast granular tissue growth. More CD31 positive cells were seen in the ATP-vesicles-treated wounds. This preliminary study shows that direct intracellular delivery of ATP can accelerate the healing process of skin wounds on ischemic and nonischemic rabbit ears. The extremely fast granular tissue growth was something never seen or reported in the past.

ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear.

Extracellular ATP controls various signaling systems including propagation of intercellular Ca(2+) signals (ICS). Connexin hemichannels, P2x7 receptors (P2x7Rs), pannexin channels, anion channels, vesicles, and transporters are putative conduits for ATP release, but their involvement in ICS remains controversial. We investigated ICS in cochlear organotypic cultures, in which ATP acts as an IP(3)-generating agonist and evokes Ca(2+) responses that have been linked to noise-induced hearing loss and development of hair cell-afferent synapses. Focal delivery of ATP or photostimulation with caged IP(3) elicited Ca(2+) responses that spread radially to several orders of unstimulated cells. Furthermore, we recorded robust Ca(2+) signals from an ATP biosensor apposed to supporting cells outside the photostimulated area in WT cultures. ICS propagated normally in cultures lacking either P2x7R or pannexin-1 (Px1), as well as in WT cultures exposed to blockers of anion channels. By contrast, Ca(2+) responses failed to propagate in cultures with defective expression of connexin 26 (Cx26) or Cx30. A companion paper demonstrates that, if expression of either Cx26 or Cx30 is blocked, expression of the other is markedly down-regulated in the outer sulcus. Lanthanum, a connexin hemichannel blocker that does not affect gap junction (GJ) channels when applied extracellularly, limited the propagation of Ca(2+) responses to cells adjacent to the photostimulated area. Our results demonstrate that these connexins play a dual crucial role in inner ear Ca(2+) signaling: as hemichannels, they promote ATP release, sustaining long-range ICS propagation; as GJ channels, they allow diffusion of Ca(2+)-mobilizing second messengers across coupled cells.

Ischemic skeletal muscle protection by direct delivery of ATP – the role of purinergic and adenosine receptors

Fusogenic Lipid vesicles loaded with ATP (FLVs‐ATP) have been used to conserve critical levels of intracellular ATP to minimize tissue damage in ischemia/reperfusion injury. Although beneficial its precise mechanism was unclear. The purpose of this study was to determine the role of purinergic and adenosine receptors during ATP delivery to ischemic skeletal muscles. We hypothesized that the ATP from FLVs‐ATP activates purinergic and adenosine receptors on cells, and has an ATP sparing effect. We used phosphocreatine (PCr) half life as an index of internal depletion of ATP in order to distinguish between intracellular and extracellular ATP (FLVs‐ATP). Extensor Digitorum Longus muscles (n=10/group) were perfused: Heparinized Lactated Ringer's (HLR); Free ATP; ATPgamma; Adenosine; FLVs; FLVs‐ATP; FLVs‐ATP + purinergic blocker; FLVs‐ATP + adenosine blockers. 31 Phosphate Nuclear Magnetic Resonance was used to measure PCr levels and calculate its half life. We found that FLVs‐ATP with purinergic receptor blockers was beneficial illustrating that purinergic receptors may have played a minor/no role in prolonging the PCr half life. In contrast FLVs‐ATP + adenosine receptors blockers showed significant difference compared to purinergic blockers in the PCr half life (p&lt;0.05) but not significant to HLR indicating that the beneficial effects of exogenous ATP are being mediated by adenosine receptors.