6PGD Upregulation is Associated with Chemo- and Immuno-Resistance of Renal Cell Carcinoma via AMPK Signaling-Dependent NADPH-Mediated Metabolic Reprograming.

BACKGROUND: The essential role of 6-phosphogluconate dehydrogenase (6PGD), the enzyme catalyzing the oxidative pentose phosphate pathway, in tumor growth and metabolism has garnered attention in recent years. In this work, we are the first to demonstrate that aberrant activation of 6PGD is a feature in renal cell carcinoma (RCC) and is critically involved in renal carcinogenesis and chemo- and immuno-resistance. MATERIALS AND METHODS: 6PGD expression and activity were systematically analyzed in normal and malignant renal cells and tissues. The roles of 6PGD and its downstream mechanism were investigated using gain-of-function and loss-of-function approaches. RESULTS: 6PGD expression and enzyme activity were increased in RCC cells and patients' samples. Activation of 6PGD via gain-of-function approach promoted growth of normal kidney but not RCC cells, and alleviated the efficacy of chemotherapeutic (e.g., 5-FU) and immunotherapeutic (e.g., IFN-α) agents. In contrast, 6PGD inhibition using siRNA knockdown and pharmacological inhibitor physcion augmented the inhibitory effects of 5-FU and IFN-α in RCC. Mechanistic studies demonstrated that 6PGD inhibition activated AMPK signaling, leading to ACC1 enzyme inhibition and reduction of lipid synthesis. In addition, 6PGD inhibition disrupted NADPH and NADH homeostasis in RCC cells as shown by the decreased level of NADPH and NADH, and suppressed SIRT-1 activity. AMPK inhibition by siRNA knockdown reversed the inhibitory effects of physcion, demonstrating that the effect of 6PGD inhibition is AMPK activation dependent. CONCLUSIONS: Our work provides preclinical evidence that 6PGD inhibition may represent a potential therapeutic strategy to augment the efficacy of RCC standard of care drugs.

NAD(P)-Driven Redox Status Contributes to Desiccation Tolerance in Acer seeds.

Desiccation tolerance is a developmental program enabling seed survival in a dry state and is common in seeds categorized as orthodox. We focused on NAD and its phosphorylated form (NADP) because their continual switching between reduced (NAD(P)H) and oxidized (NAD(P)+) forms is involved in the modulation of redox signaling and the determination of the reducing power and further antioxidant responses. Norway maple and sycamore seeds representing the orthodox and recalcitrant categories, respectively, were used as models in a comparison of responses to water loss. The process of desiccation up to 10% water content (WC) was monitored in Norway maple seeds, while dehydration up to 30% WC was monitored in desiccation-sensitive sycamore seeds. Norway maple and sycamore seeds, particularly their embryonic axes, exhibited a distinct redox status during dehydration and desiccation. High NADPH levels, NAD+ accumulation, low and stable NAD(P)H/NAD(P)+ ratios expressed as reducing power and high NADPH-dependent enzyme activity were reported in Norway maple seeds and were considered attributes of orthodox-type seeds. The contrasting results of sycamore seeds contributed to their low antioxidant capacity and high sensitivity to desiccation. NADPH deficiency, low NADPH-dependent enzyme activity and lack of NAD+ accumulation were primary features of sycamore seeds, with implications for their NAD(P)H/NAD(P)+ ratios and reducing power and with effects on many seed traits. Thus, we propose that the distinct levels of pyridine nucleotides and their redox status contribute to orthodox and recalcitrant phenotype differentiation in seeds by affecting cellular redox signaling, metabolism and the antioxidant system.

Pyridine nucleotide regulation of hepatic endoplasmic reticulum calcium uptake.

Pyridine nucleotides serve an array of intracellular metabolic functions such as, to name a few, shuttling electrons in enzymatic reactions, safeguarding the redox state against reactive oxygen species, cytochrome P450 (CYP) enzyme detoxification pathways and, relevant to this study, the regulation of ion fluxes. In particular, the maintenance of a steep calcium gradient between the cytosol and endoplasmic reticulum (ER), without which apoptosis ensues, is achieved by an elaborate combination of energy-requiring ER membrane pumps and efflux channels. In liver microsomes, net calcium uptake was inhibited by physiological concentrations of NADP. In the presence of 1 mmol/L NADP, calcium uptake was attenuated by nearly 80%, additionally, this inhibitory effect was blunted by concomitant addition of NADPH. No other nicotinamide containing compounds -save a slight inhibition by NAADP-hindered calcium uptake; thus, only oxidized pyridine nucleotides, or related compounds with a phosphate moiety, had an imposing effect. Moreover, the NADP inhibition was evident even after selectively blocking ER calcium efflux channels. Given the fundamental role of endoplasmic calcium homeostasis, it is plausible that changes in cytosolic NADP concentration, for example, during anabolic processes, could regulate net ER calcium uptake.

Ned-19 inhibition of parasite growth and multiplication suggests a role for NAADP mediated signalling in the asexual development of Plasmodium falciparum.

BACKGROUND: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. RESULTS: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca2+ levels. CONCLUSIONS: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca2+ oscillations suggests a potential role of NAADP in regulating Ca2+ signalling of P. falciparum.

Two-pore channels (TPCs): Novel voltage-gated ion channels with pleiotropic functions.

Two-pore channels (TPC1-3) comprise a subfamily of the eukaryotic voltage-gated ion channels (VGICs) superfamily that are mainly expressed in acidic stores in plants and animals. TPCS are widespread across the animal kingdom, with primates, mice and rats lacking TPC3, and mainly act as Ca+ and Na+ channels, although it was also suggested that they could be permeable to other ions. Nowadays, TPCs have been related to the development of different diseases, including Parkinson´s disease, obesity or myocardial ischemia. Due to this, their study has raised the interest of the scientific community to try to understand their mechanism of action in order to be able to develop an efficient drug that could regulate TPCs activity. In this review, we will provide an updated view regarding TPCs structure, function and activation, as well as their role in different pathophysiological processes.

Polyamine- and NADPH-dependent generation of ROS during Helicobacter pylori infection: A blessing in disguise.

Helicobacter pylori is a Gram-negative bacterium that specifically colonizes the gastric ecological niche. During the infectious process, which results in diseases ranging from chronic gastritis to gastric cancer, the host response is characterized by the activation of the innate immunity of gastric epithelial cells and macrophages. These cells thus produce effector molecules such as reactive oxygen species (ROS) to counteract the infection. The generation of ROS in response to H. pylori involves two canonical pathways: 1) the NADPH-dependent reduction of molecular oxygen to generate O2•-, which can dismute to generate ROS; and 2) the back-conversion of the polyamine spermine into spermidine through the enzyme spermine oxidase, leading to H2O2 production. Although these products have the potential to affect the survival of bacteria, H. pylori has acquired numerous strategies to counteract their deleterious effects. Nonetheless, ROS-mediated oxidative DNA damage and mutations may participate in the adaptation of H. pylori to its ecological niche. Lastly, ROS have been shown to play a major role in the development of the inflammation and carcinogenesis. It is the purpose of this review to summarize the literature about the production of ROS during H. pylori infection and their role in this infectious gastric disease.

Glucose 6-phosphate dehydrogenase and the kidney.

PURPOSE OF REVIEW: Glucose 6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway. G6PD is the main source of the essential cellular reductant, NADPH. The purpose of this review is to describe the biochemistry of G6PD and NADPH, cellular factors that regulate G6PD, normal physiologic roles of G6PD, and the pathogenic role altered G6PD/NADPH plays in kidney disease. RECENT FINDINGS: NADPH is required for many essential cellular processes such as the antioxidant system, nitric oxide synthase, cytochrome p450 enzymes, and NADPH oxidase. Decreased G6PD activity and, as a result, decreased NADPH level have been associated with diabetic kidney disease, altered nitric oxide production, aldosterone-mediated endothelial dysfunction, and dialysis-associated anemia. Increased G6PD activity is associated with all cancers including kidney cancer. Inherited G6PD deficiency is the most common mutation in the world that is thought to be a relatively mild disorder primarily associated with anemia. Yet, intriguing studies have shown an increased prevalence of diabetes mellitus in G6PD-deficient people. It is not known if G6PD-deficient people are at more risk for other diseases. SUMMARY: Much more research needs to be done to determine the role of altered G6PD activity (inherited or acquired) in the pathogenesis of kidney disease.

Función del sistema NADPH oxidasa en la formación de trampas extracelulares de los neutrófilos (NETs) / Role of nadph oxidase system in the neutrophil extracelular Traps (NETs) formation

Las trampas extracelulares de los neutrófilos son estructuras fundamentalmente compuestas de cromatina y proteínas granulares, que una vez liberadas constituyen un mecanismo de defensa que tiene la capacidad de atrapar y destruir microorganismos patógenos. El proceso que libera estas estructuras es conocido como NETosis y en el caso que provoque muerte celular, esta es diferente a la apoptosis y a la necrosis. Si bien no se conocen todos los eventos moleculares involucrados en la formación de las NETs, se sabe que dependiendo del estímulo, las especies reactivas del oxígeno son esenciales para que ocurra la descondensación de la cromatina y se lleve a cabo el proceso de NETosis(AU)

Neutrophil extracellular traps (NETs) are structures mainly composed of chromatin and granule proteins that once released constitute a defense mechanism due to their ability to trap and destroy pathogen microorganisms. The process by which these structures are released is known as NETosis and in case this may lead to cell death is different to apoptosis and necrosis. Although all the molecular events involved in the formation of NETs are poorly understood, it is known that depending on the stimulus, reactive oxygen species (ROS) are essential to the chromatin decondensation and subsequent NETs formation(AU)

Exploring the biophysical evidence that mammalian two-pore channels are NAADP-activated calcium-permeable channels.

Nicotinic acid adenine dinucleotide phosphate (NAADP) potently releases Ca(2+) from acidic intracellular endolysosomal Ca(2+) stores. It is widely accepted that two types of two-pore channels, termed TPC1 and TPC2, are responsible for the NAADP-mediated Ca(2+) release but the underlying mechanisms regulating their gating appear to be different. For example, although both TPC1 and TPC2 are activated by NAADP, TPC1 appears to be additionally regulated by cytosolic Ca(2+) . Ion conduction and permeability also differ markedly. TPC1 and TPC2 are permeable to a range of cations although biophysical experiments suggest that TPC2 is slightly more selective for Ca(2+) over K(+) than TPC1 and hence capable of releasing greater quantities of Ca(2+) from acidic stores. TPC1 is also permeable to H(+) and therefore may play a role in regulating lysosomal and cytosolic pH, possibly creating localised acidic domains. The significantly different gating and ion conducting properties of TPC1 and TPC2 suggest that these two ion channels may play complementary physiological roles as Ca(2+) -release channels of the endolysosomal system.

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic ß-Adrenoceptor Signaling in the Heart.

Ca(2+)-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca(2+) responses in cardiac myocytes from Tpcn2(-/-) mice, unlike myocytes from wild-type (WT) mice. Ca(2+)/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca(2+) transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of ß-adrenoreceptor stimulation were reduced in myocytes from Tpcn2(-/-) mice. Increases in amplitude of L-type Ca(2+) currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2(-/-) mice showing no loss of ß-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2(-/-) mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute ß-adrenoreceptor stimulation. Hearts from Tpcn2(-/-) mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼ 25 nm). We propose that Ca(2+)-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in ß-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of ß-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of ß-adrenoceptors to hypertrophy and associated arrhythmias.

Activated mTORC1 promotes long-term cone survival in retinitis pigmentosa mice.

Retinitis pigmentosa (RP) is an inherited photoreceptor degenerative disorder that results in blindness. The disease is often caused by mutations in genes that are specific to rod photoreceptors; however, blindness results from the secondary loss of cones by a still unknown mechanism. Here, we demonstrated that the mammalian target of rapamycin complex 1 (mTORC1) is required to slow the progression of cone death during disease and that constitutive activation of mTORC1 in cones is sufficient to maintain cone function and promote long-term cone survival. Activation of mTORC1 in cones enhanced glucose uptake, retention, and utilization, leading to increased levels of the key metabolite NADPH. Moreover, cone death was delayed in the absence of the NADPH-sensitive cell death protease caspase 2, supporting the contribution of reduced NADPH in promoting cone death. Constitutive activation of mTORC1 preserved cones in 2 mouse models of RP, suggesting that the secondary loss of cones is caused mainly by metabolic deficits and is independent of a specific rod-associated mutation. Together, the results of this study address a longstanding question in the field and suggest that activating mTORC1 in cones has therapeutic potential to prolong vision in RP.

Teaching the Z-Scheme of electron transport in photosynthesis: a perspective.

This paper deals with how Govindjee taught the Z-Scheme of electron transport in oxygenic photosynthesis at Ravenshaw University, Cuttack, Odisha, India, in 2014, in a unique and highly effective fashion-using students to act as molecules, representing the entire electron transport chain from water to nicotinamide adenine dinucleotide phosphate (NADP(+)). It culminated in a show by B.Sc. students in the garden of the Department of Botany, Ravenshaw University. The first author (PKM) personally acted as Ferredoxin NADP Reductase (FNR) catalyzing the reduction of NADP(+) to NADPH, taking electrons from reduced ferredoxin at the end of Photosystem I. On the other hand, the Q-cycle was played by M.Sc. students, who acted as molecules running this ingenious cycle that produces extra protons. An interesting event was when a student, acting as a herbicide, who was dressed like a devil (fierce looking, in black clothes with a sword; "Yamaraj: The God of Death", as he called himself), stopped all reactions by throwing out QB, the second plastoquinone molecule of Photosystem II, and that too aggressively, taking its position instead. The second author was the major organizer of the Z-scheme show. We provide here a basic background on the process, a bit on Govindjee's teaching, and some selected pictures from the drama played in March, 2014 at Ravenshaw University. Here, we also recognize the teacher Govindjee for his ingenious and fun-filled teaching methods that touched the hearts and the souls of the students as well as the teachers of Ravenshaw University. He was rated as one of the most-admired teachers of plant biology at our university.

Calcium mobilizing second messengers derived from NAD.

Nicotinamide adenine dinucleotide (NAD) has been known since a long period of time as co-factor of oxidoreductases. However, in the past couple of decades further roles have been assigned to NAD. Here, metabolism of NAD to the Ca²âº mobilizing second messengers cyclic adenosine diphosphoribose, nicotinic acid adenine dinucleotide phosphate and adenosine diphosphoribose is reviewed. Moreover, the mechanisms of Ca²âº mobilization by these adenine nucleotides and their putative target Ca²âº channels, ryanodine receptors and transient receptor potential channels are discussed. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Exposure of Madin-Darby canine kidney (MDCK) cells to oxalate and calcium oxalate crystals activates nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase.

OBJECTIVE: To investigate nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity in Madin-Darby canine kidney (MDCK) cells and the production of reactive oxygen species on exposure to oxalate (Ox) or calcium oxalate (CaOx) crystals. METHODS: Monolayers of confluent Madin-Darby canine kidney cells were exposed to 100, 300, 500 µmol, 1 mmol Ox or 33, 66, 132 µg/cm(2) CaOx crystals for 15 minutes, 30 minutes, 1 hour, 2 hours, or 3 hours. After specified periods of exposure to Ox and CaOx crystals, lactate dehydrogenase release, trypan blue exclusion, activation of NADPH oxidase, and superoxide production were determined using standard procedures. The production of Nox4, a membrane associated subunit of the NADPH oxidase enzyme, was determined by western blot analysis. RESULTS: Exposure to Ox and CaOx crystals leads to time- and concentration-dependent activation of NADPH oxidase. Western blot analysis showed an increase in the production of Nox4. The production of superoxide also changed in a time- and concentration-dependent manner, with maximum increases after 30-minute exposure to the highest concentrations of Ox and CaOx crystals. Longer exposures did not change the results or resulted in decreased activities. Exposure to higher concentrations also caused increased lactate dehydrogenase release and trypan blue exclusion indicating cell damage. CONCLUSION: Results indicate that cells of the distal tubular origin are equipped with NADPH oxidase that is activated by exposures to Ox and CaOx crystals. Higher concentrations of both lead to cell injury, most probably through the increased reactive oxygen species production by the exposed cells.

Microsomal oxidative stress induced by NADPH is inhibited by nitrofurantoin redox biotranformation.

Nitrofurantoin is used in the antibacterial therapy of the urinary tract. This therapy is associated with various adverse effects whose mechanisms remain unclear. Diverse studies show that the nitro reductive metabolism of nitrofurantoin leads to ROS generation. This reaction can be catalyzed by several reductases, including the cytochrome P450 (CYP450) reductase. Oxidative stress arising from this nitro reductive metabolism has been proposed as the mechanism underlying the adverse effects associated with nitrofurantoin. There is, however, an apparent paradox between these findings and the ability of nitrofurantoin to inhibit lipid peroxidation provoked by NADPH in rat liver microsomes. This work was aimed to show the potential contribution of different enzymatic systems to the metabolism of this drug in rat liver microsomes. Our results show that microsomal lipid peroxidation promoted by NADPH is inhibited by nitrofurantoin in a concentration-dependent manner. This suggests that the consumption of NADPH in microsomes can be competitively promoted by lipid peroxidation and nitrofurantoin metabolism. The incubation of microsomes with NADPH and nitrofurantoin generated 1-aminohidantoin. In addition, the biotransformation of a classical substrate of CYP450 oxidative system was competitively inhibited by nitrofurantoin. These results suggest that nitrofurantoin is metabolized through CYP450 system. Data are discussed in terms of the in vitro redox metabolism of nitrofurantoin.

Effects of NAD(P)H and its derivatives on the DNA-binding activity of NPAS2, a mammalian circadian transcription factor.

NPAS2 is a transcription factor that regulates mammalian circadian rhythms. It has been suggested that NPAS2 DNA-binding activity is regulated by the intracellular redox state of NAD(P)H, although the mechanism remains unclear. To investigate the NAD(P)H interaction site of murine NPAS2, we performed electrophoretic mobility shift assays using several truncation mutants of the NPAS2 bHLH domain. Among the mutants, NPAS2 containing the N-terminal 61 residues formed a heterodimer with BMAL1 to bind DNA, and NAD(P)H enhanced the binding activity, while NAD(P)H inhibited the DNA-binding activity of the BMAL1 homodimer in a dose-dependent manner. NAD(P)H derivatives such as 2',5'-ADP, nicotinamide, nicotinic acid and nicotinic acid adenine dinucleotide (NAAD) did not affect the DNA-binding activity. Interestingly, NAD(P)(+), previously reported as an inhibitor, did not affect NPAS2 binding activity in the presence or absence of NAD(P)H in our system. These results suggest that NPAS2 DNA-binding activity is specifically enhanced by NAD(P)H independently of NAD(P)(+) and that the N-terminal 1-61 amino acids of NPAS2 are sufficient to sense NAD(P)H.

Biopharmaceutical characterization, metabolism, and brain penetration of the triple reuptake inhibitor amitifadine.

Amitifadine (EB-1010, formerly DOV 21,947) is a serotonin-preferring triple reuptake inhibitor that is a drug candidate for major depressive disorder. We investigated several relevant biopharmaceutic and drug-like characteristics of amitifadine using in vitro methodology and additionally determined the in vivo brain to plasma ratio of the drug in rats. Amitifadine was highly plasma protein bound with over 99% of drug bound to human plasma proteins. Using Caco-2 cell lines, amitifadine was bidirectionally highly permeable and showed no evidence of active secretion. Amitifadine was metabolized slowly by human hepatocytes and the major metabolite was the lactam EB-10101. In vitro studies using human liver microsomes demonstrated that EB-10101 was formed by monoamine oxidase A (MAO-A) and a NADPHdependent enzyme, possibly a cytochrome P450 (CYP) isoform. Amitifadine was a moderate inhibitor of the human isoforms of the major drug metabolizing enzymes CYP2D6, CYP3A4, CYP2C9, and CYP2C19 (IC50 = 9 - 100 µM), but was a potent inhibitor of human CYP2B6 (IC50 = 1.8 µM). The brain to plasma ratio for amitifadine varied from 3.7 - 6.5 at various time points, indicating preferential partitioning into rat brain versus plasma. The low affinity for the major drug metabolizing CYP enzymes and metabolism by multiple pathways may reduce pharmacokinetic drug-drug interactions and effects of enzyme polymorphisms. Overall, these studies suggest that amitifadine has drug-like characteristics favorable for drug development.

N-terminal tagging of two-pore channels interferes with NAADP action.

NAADP (nicotinic acid-adenine dinucleotide phosphate) is the most potent Ca2+-releasing second messenger known to date. Since its discovery in 1995 identifying the NAADP receptor protein/Ca2+ channel has been a major persuit of the Ca2+ signalling community. In their paper 'The N-terminal region of two-pore channel 1 regulates trafficking and activation by NAADP' published in this issue of the Biochemical Journal Patel and colleagues describe that the N-terminus of one of the NAADP receptor protein/Ca2+ channel candidates, TPC1 (two-pore channel 1), is crucial for protein targeting and for sensitivity to NAADP.

The N-terminal region of two-pore channel 1 regulates trafficking and activation by NAADP.

TPCs (two-pore channels) are NAADP (nicotinic acid-adenine dinucleotide phosphate)-sensitive Ca2+-permeable ion channels expressed on acidic organelles. In the present study we show that deletion of the N-terminal region redirects TPC1 to the ER (endoplasmic reticulum). The introduction of fluorophores at the N-terminus of TPC1 does not affect its subcellular location, but does reversibly abolish NAADP sensitivity. Our results reveal a dual role for the N-terminus in localization and function of TPC1.

Extracellular NAADP affords cardioprotection against ischemia and reperfusion injury and involves the P2Y11-like receptor.

BACKGROUND AND PURPOSE: Extracellular nucleotides may play important regulatory roles within the cardiovascular system and notably in cardioprotection. We aimed to look for a possible pharmacological preconditioning effect of extracellular NAADP ([NAADP]e) against ischemia/reperfusion injury. [NAADP]e has been recently reported to be a full agonist of the P2Y11 receptor. Therefore, we characterized the involvement of the P2Y11-like receptor in mediating ischemic/reperfusion tolerance induced by [NAADP]e. EXPERIMENTAL APPROACH: The cardioprotective effects of [NAADP]e were evaluated in a model of ischemia/reperfusion carried out on Langendorff perfused rat hearts. This model was also instrumented with a microdialysis probe. Furthermore, using isolated cardiomyocytes, we assessed cAMP, inositol phosphate accumulation and prosurvival protein kinases activation induced by [NAADP]e pretreatement. RESULTS: Pretreatment with 1µM [NAADP]e induced cardioprotective effects with regards to functional recovery, necrosis and arrhythmogenesis (p<0.05). These effects were completely suppressed with NF157, an antagonist of the P2Y11 receptor. Moreover, global ischemia induced a time-dependent increase in interstitial concentration of adenosine, NAADP and UTP. In cardiomyocyte cultures, NF157 suppressed cAMP and inositol phosphate accumulation induced by [NAADP]e. [NAADP]e induced phosphorylation of ERK 1/2, AKT and its downstream target GSK-3ß (p<0.05). These activations were also suppressed by NF157. CONCLUSIONS: Evidence suggests that NAADP signalling at the P2Y11-like receptor affords significant cardioprotection against ischemia/reperfusion injury. Besides adenosine and UTP, microdialysis study supports a potential endogenous role of [NAADP]e.