Extracellular Nicotinamide Adenine Dinucleotide Research Articles

Extracellular NAD+ response to post-hepatectomy liver failure: bridging preclinical and clinical findings

Liver fibrosis progressing to cirrhosis is a major risk factor for liver cancer, impacting surgical treatment and survival. Our study focuses on the role of extracellular nicotinamide adenine dinucleotide (eNAD+) in liver fibrosis, analyzing liver disease patients undergoing surgery. Additionally, we explore NAD+’s therapeutic potential in a mouse model of extended liver resection and in vitro using 3D hepatocyte spheroids. eNAD+ correlated with aspartate transaminase (AST) and bilirubin after liver resection (AST: r = 0.2828, p = 0.0087; Bilirubin: r = 0.2584, p = 0.0176). Concordantly, post-hepatectomy liver failure (PHLF) was associated with higher eNAD+ peaks (n = 10; p = 0.0063). Post-operative eNAD+ levels decreased significantly (p < 0.05), but in advanced stages of liver fibrosis or cirrhosis, this decline not only diminished but actually showed a trend towards an increase. The expression of NAD+ biosynthesis rate-limiting enzymes, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), were upregulated significantly in the liver tissue of patients with higher liver fibrosis stages (p < 0.0001). Finally, the administration of NAD+ in a 3D hepatocyte spheroid model rescued hepatocytes from TNFalpha-induced cell death and improved viability (p < 0.0001). In a mouse model of extended liver resection, NAD+ treatment significantly improved survival (p = 0.0158) and liver regeneration (p = 0.0186). Our findings reveal that eNAD+ was upregulated in PHLF, and rate-limiting enzymes of NAD+ biosynthesis demonstrated higher expressions under liver fibrosis. Further, eNAD+ administration improved survival after extended liver resection in mice and enhanced hepatocyte viability in vitro. These insights may offer a potential target for future therapies.

LBP-015 The effect of precursors on extracellular nicotinamide adenine dinucleotide in plasma

LBP-015 The effect of precursors on extracellular nicotinamide adenine dinucleotide in plasma

Optimized protocol for quantification of extracellular nicotinamide adenine dinucleotide: evaluating clinical parameters and pre-analytical factors for translational research.

Nicotinamide adenine dinucleotide (NAD+), a coenzyme for more than 500 enzymes, plays a central role in energy production, metabolism, cellular signaling, and DNA repair. Until recently, NAD+ was primarily considered to be an intracellular molecule (iNAD+), however, its extracellular species (eNAD+) has recently been discovered and has since been associated with a multitude of pathological conditions. Therefore, accurate quantification of eNAD+ in bodily fluids such as plasma is paramount to answer important research questions. In order to create a clinically meaningful and reliable quantitation method, we analyzed the relationship of cell lysis, routine clinical laboratory parameters, blood collection techniques, and pre-analytical processing steps with measured plasma eNAD+ concentrations. Initially, NAD+ levels were assessed both intracellularly and extracellularly. Intriguingly, the concentration of eNAD+ in plasma was found to be approximately 500 times lower than iNAD+ in peripheral blood mononuclear cells (0.253 ± 0.02 μM vs. 131.8 ± 27.4 μM, p = 0.007, respectively). This stark contrast suggests that cellular damage or cell lysis could potentially affect the levels of eNAD+ in plasma. However, systemic lactate dehydrogenase in patient plasma, a marker of cell damage, did not significantly correlate with eNAD+ (n = 33; r = -0.397; p = 0.102). Furthermore, eNAD+ was negatively correlated with increasing c-reactive protein (CRP, n = 33; r = -0.451; p = 0.020), while eNAD+ was positively correlated with increasing hemoglobin (n = 33; r = 0.482; p = 0.005). Next, variations in blood drawing, sample handling and pre-analytical processes were examined. Sample storage durations at 4°C (0-120 min), temperature (0° to 25°C), cannula sizes for blood collection and tourniquet times (0 - 120 s) had no statistically significant effect on eNAD+ (p > 0.05). On the other hand, prolonged centrifugation (> 5 min) and a faster braking mode of the centrifuge rotor (< 4 min) resulted in a significant decrease in eNAD+ levels (p < 0.05). Taken together, CRP and hemoglobin appeared to be mildly correlated with eNAD+ levels whereas cell damage was not correlated significantly to eNAD+ levels. The blood drawing trial did not show any influence on eNAD+, in contrast, the preanalytical steps need to be standardized for accurate eNAD+ measurement. This work paves the way towards robust eNAD+ measurements, for use in future clinical and translational research, and provides an optimized hands-on protocol for reliable eNAD+ quantification in plasma.

N-hydroxypipecolic acid triggers systemic acquired resistance through extracellular NAD(P)

Systemic acquired resistance (SAR) is a long-lasting broad-spectrum plant defense mechanism induced in distal systemic tissues by mobile signals generated at the primary infection site. Despite the discoveries of multiple potential mobile signals, how these signals cooperate to trigger downstream SAR signaling is unknown. Here, we show that endogenous extracellular nicotinamide adenine dinucleotide (phosphate) [eNAD(P)] accumulates systemically upon pathogen infection and that both eNAD(P) and the lectin receptor kinase (LecRK), LecRK-VI.2, are required in systemic tissues for the establishment of SAR. Moreover, putative mobile signals, e.g., N-hydroxypipecolic acid (NHP), trigger de novo systemic eNAD(P) accumulation largely through the respiratory burst oxidase homolog RBOHF-produced reactive oxygen species (ROS). Importantly, NHP-induced systemic immunity mainly depends on ROS, eNAD(P), LecRK-VI.2, and BAK1, indicating that NHP induces SAR primarily through the ROS-eNAD(P)-LecRK-VI.2/BAK1 signaling pathway. Our results suggest that mobile signals converge on eNAD(P) in systemic tissues to trigger SAR through LecRK-VI.2.

Damage-induced NAD release activates intestinal CD4+ and CD8+ T cell via P2X7R signaling

BackgroundPostoperative ileus (POI) is characterized by the activation of inflammation triggered by tissue damage. Damage-associated molecular patterns (DAMPs) reportedly induce local inflammation after injury. However, the impact of DAMPs on intestinal resident lymphocytes during POI remains poorly elucidated. MethodsPOI in mice was induced via intestinal manipulation (IM). The concentration of nicotinamide adenine dinucleotide (NAD) was detected after IM. The gastrointestinal motility of the mice was assessed after IM or NAD injection. Cytokine production and calcium influx in T cells were investigated after NAD stimulation using flow cytometry. ResultsThe concentration of extracellular NAD significantly increased after IM administration, and NAD directly impaired gastrointestinal motility. Intraperitoneal injection of NAD promoted the expression of TNF-α in intestinal CD8+ and CD4+ T cells, but only IFN-γ production by CD8+ T cells was significantly promoted by NAD injection. Granzyme B production in CD8+ and CD4+ T cells decreased after administration. Concordantly, the same results were observed in NAD stimulation of intestinal CD3+ T cells in vitro. Blocking the P2X7R-related membrane enzyme ART2.2 significantly diminished the pro-inflammatory effect of NAD. ConclusionIM includes the release of NAD derived from damaged tissues, consequently promoting pro-inflammatory cytokine production in intestinal CD4+ and CD8+ T lymphocytes. NAD-induced intestinal T cells activation may be associated with POI progression in the mouse.

SAT162 - Regulation of extracellular Nicotinamide adenine dinucleotide in patients with liver fibrosis: new insights into conventional hepatobiliary parameters

SAT162 - Regulation of extracellular Nicotinamide adenine dinucleotide in patients with liver fibrosis: new insights into conventional hepatobiliary parameters

Identification of the Mouse T Cell ADP-Ribosylome Uncovers ARTC2.2 Mediated Regulation of CD73 by ADP-Ribosylation.

Mouse T cells express the ecto-ADP-ribosyltransferase ARTC2.2, which can transfer the ADP-ribose group of extracellular nicotinamide adenine dinucleotide (NAD+) to arginine residues of various cell surface proteins thereby influencing their function. Several targets of ARTC2.2, such as P2X7, CD8a and CD25 have been identified, however a comprehensive mouse T cell surface ADP-ribosylome analysis is currently missing. Using the Af1521 macrodomain-based enrichment of ADP-ribosylated peptides and mass spectrometry, we identified 93 ADP-ribsoylated peptides corresponding to 67 distinct T cell proteins, including known targets such as CD8a and CD25 but also previously unknown targets such as CD73. We evaluated the impact of ADP-ribosylation on the capability of CD73 to generate adenosine from adenosine monophosphate. Our results show that extracellular NAD+ reduces the enzymatic activity of CD73 HEK cells co-transfected with CD73/ARTC2.2. Importantly, NAD+ significantly reduced CD73 activity on WT CD8 T cells compared to ARTC2ko CD8 T cells or WT CD8 T cells treated with an ARTC2.2-blocking nanobody. Our study provides a comprehensive list of T cell membrane proteins that serve as targets for ADP-ribosylation by ARTC2.2 and whose function may be therefore affected by ADP-ribosylation.

The Extracellular NADome Modulates Immune Responses.

The term NADome refers to the intricate network of intracellular and extracellular enzymes that regulate the synthesis or degradation of nicotinamide adenine dinucleotide (NAD) and to the receptors that engage it. Traditionally, NAD was linked to intracellular energy production through shuffling electrons between oxidized and reduced forms. However, recent data indicate that NAD, along with its biosynthetic and degrading enzymes, has a life outside of cells, possibly linked to immuno-modulating non-enzymatic activities. Extracellular NAD can engage puriginergic receptors triggering an inflammatory response, similar - to a certain extent – to what described for adenosine triphosphate (ATP). Likewise, NAD biosynthetic and degrading enzymes have been amply reported in the extracellular space, where they possess both enzymatic and non-enzymatic functions. Modulation of these enzymes has been described in several acute and chronic conditions, including obesity, cancer, inflammatory bowel diseases and sepsis. In this review, the role of the extracellular NADome will be discussed, focusing on its proposed role in immunomodulation, together with the different strategies for its targeting and their potential therapeutic impact.

Plasma-Functionalized Isotactic Polypropylene Assembled with Conducting Polymers for Bacterial Quantification by NADH Sensing.

Rapid detection of bacterial presence on implantable medical devices is essential to prevent biofilm formation, which consists of densely packed bacteria colonies able to withstand antibiotic-mediated killing. In this work, a smart approach is presented to integrate electrochemical sensors for detecting bacterial infections in biomedical implants made of isotactic polypropylene (i-PP) using chemical assembly. The electrochemical detection is based on the capacity of conducting polymers (CPs) to detect extracellular nicotinamide adenine dinucleotide (NADH) released from cellular respiration of bacteria, which allows distinguishing prokaryotic from eukaryotic cells. Oxygen plasma-functionalized free-standing i-PP, coated with a layer (≈1.1µm in thickness) of CP nanoparticles obtained by oxidative polymerization, is used as working electrode for the anodic polymerization of a second CP layer (≈8.2µm in thickness), which provides very high electrochemical activity and stability. The resulting layered material, i-PPf /CP2 , detects the electro-oxidation of NADH in physiological media with a sensitivity 417 µA cm-2 and a detection limit up to 0.14× 10-3 m, which is below the concentration of extracellular NADH found for bacterial cultures of biofilm-positive and biofilm-negative strains.

Extracellular Hydrolysis of NAD+and its Secondary Metabolites by a new Aspergillus sp

In some organisms, the metabolism of extracellular nicotinamide adenine dinucleotide (NAD+) plays an important role in purine salvage and uptake. Since filamentous fungi are used to degrade NAD and produce beneficial secondary metabolites, various enzymes, fermented foods and organic acids. Many strains belonging to filamentous fungi were screened for the enzymes production that catalyzed hydrolytic dephosphorylation and deamination of NAD and its secondary metabolites. A fungal isolate which was found to be the most prominent strain was identified genetically as Aspergillus fumigatous. Extracts of this new strain catalyzes the hydrolysis the ester bond between the two nucleotides of NAD by pyrophosphatase activity, which catalyzes NAD to nicotinamide mononucleotide (NMN) and adenosine monophosphate (AMP), at that point NMN and AMP are hydrolyzed to nicotinamide riboside (NR) and adenosine separately by 5’–nucleotidase activity This research reports also existence of deaminase activity hydrolyzed adenosine to inosine. The study demonstrated major enzymatic pathways involved in NAD degradation and identification of intermediates formed by Aspergillus fumigatous. Separation and partial purification of these enzymes and some kinetic properties of them were achieved

The essential schistosome tegumental ectoenzyme SmNPP5 can block NAD-induced T cell apoptosis

ABSTRACT Infection with intravascular platyhelminths of the genus Schistosoma can result in the debilitating disease schistosomiasis. Schistosomes (blood flukes) can survive in the host for many years. We hypothesize that proteins on their host-interactive surface modify the worm’s external environment to help insure worm survival. Previously, we have shown that a surface ectoenzyme of Schistosoma mansoni, SmNPP5 – a nucleotide pyrophosphatase/phosphodiesterase – can cleave ADP and block platelet aggregation in vitro. In this work, we show that both adult schistosomes and recombinant SmNPP5 can cleave the exogenous purinergic signaling molecule nicotinamide adenine dinucleotide (NAD). In doing so, worms and rSmNPP5 can prevent NAD-induced apoptosis of T cells in vitro. Since regulatory T cells (Tregs) are especially prone to such NAD-induced cell death (NICD), we hypothesize that schistosome cleavage of NAD promotes Treg survival which creates a more immunologically hospitable environment for the worms in vivo. In addition to SmNPP5, schistosomes express another host-interactive NAD-degrading enzyme, SmNACE. We successfully suppressed the expression of SmNPP5 and SmNACE (singly or together) using RNAi. Only SmNPP5-suppressed worms, and not SmNACE-suppressed worms, were significantly impaired in their ability to cleave exogenous NAD compared to controls. Therefore, we contend that ectoenzyme SmNPP5 on the surface of the worm is primarily responsible for extracellular NAD cleavage and that this helps modulate the host immune environment by preventing Treg cell death.

Astrocytes and Microglia Are Resistant to NAD+-Mediated Cell Death Along the ARTC2/P2X7 Axis

ADP-ribosylation of the P2X7k splice variant on mouse T cells by Ecto-ADP-ribosyltransferase ARTC2.2 in response to its substrate extracellular nicotinamide adenine dinucleotide (NAD+) triggers cell death. Since NAD+ is released as a danger signal during tissue damage, this NAD+-induced cell death (NICD) may impact the survival of other cell populations co-expressing P2X7 and of one of the ARTC2 isoforms (ARTC2.1, ARTC2.2). NICD of brain-resident, non-T cell populations has only been rudimentarily investigated. In this study, we evaluated the susceptibility of two glia cell populations, astrocytes and microglia, towards NICD. We found that astrocytes and microglia strongly upregulate cell surface levels of ARTC2.1 and ADP-ribosylation of cell surface proteins in response to treatment with lipopolysaccharide (LPS) and the mitogen-activated protein kinase kinase (MEK) 1 and 2 inhibitor U0126, but do not respond to extracellular NAD+ with P2X7 activation and induction of cell death. Furthermore, we found that astrocytes and microglia preferentially express the ADP-ribosylation-insensitive P2X7a splice variant, likely accounting for the resistance of these cells to NICD.

Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells.

Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD+ and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD+ intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.

The nanomolar sensing of nicotinamide adenine dinucleotide in human plasma using a cycling assay in albumin modified simulated body fluids

Nicotinamide adenine dinucleotide (NAD), a prominent member of the pyridine nucleotide family, plays a pivotal role in cell-oxidation protection, DNA repair, cell signalling and central metabolic pathways, such as beta oxidation, glycolysis and the citric acid cycle. In particular, extracellular NAD+ has recently been demonstrated to moderate pathogenesis of multiple systemic diseases as well as aging. Herein we present an assaying method, that serves to quantify extracellular NAD+ in human heparinised plasma and exhibits a sensitivity ranging from the low micromolar into the low nanomolar domain. The assay achieves the quantification of extracellular NAD+ by means of a two-step enzymatic cycling reaction, based on alcohol dehydrogenase. An albumin modified revised simulated body fluid was employed as standard matrix in order to optimise enzymatic activity and enhance the linear behaviour and sensitivity of the method. In addition, we evaluated assay linearity, reproducibility and confirmed long-term storage stability of extracellular NAD+ in frozen human heparinised plasma. In summary, our findings pose a novel standardised method suitable for high throughput screenings of extracellular NAD+ levels in human heparinised plasma, paving the way for new clinical discovery studies.

Exogenous Nicotinamide Adenine Dinucleotide Induces Resistance to Citrus Canker in Citrus.

Nicotinamide adenine dinucleotide (NAD) is a universal electron carrier that participates in important intracellular metabolic reactions and signaling events. Interestingly, emerging evidence in animals indicates that cellular NAD can be actively or passively released into the extracellular space, where it is processed or perceived by ectoenzymes or cell-surface receptors. We have recently shown in Arabidopsis thaliana that exogenous NAD induces defense responses, that pathogen infection leads to release of NAD into the extracellular space at concentrations sufficient for defense activation, and that depletion of extracellular NAD (eNAD) by transgenic expression of the human NAD-hydrolyzing ectoenzyme CD38 inhibits plant immunity. We therefore hypothesize that, during plant–microbe interactions, NAD is released from dead or dying cells into the extracellular space where it interacts with adjacent naïve cells’ surface receptors, which in turn activate downstream immune signaling. However, it is currently unknown whether eNAD signaling is unique to Arabidopsis or the Brassicaceae family. In this study, we treated citrus plants with exogenous NAD+ and tested NAD+-induced transcriptional changes and disease resistance. Our results show that NAD+ induces profound transcriptome changes and strong resistance to citrus canker, a serious citrus disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). Furthermore, NAD+-induced resistance persists in new flushes emerging after removal of the tissues previously treated with NAD+. Finally, NAD+ treatment primes citrus tissues, resulting in a faster and stronger induction of multiple salicylic acid pathway genes upon subsequent Xcc infection. Taken together, these results indicate that exogenous NAD+ is able to induce immune responses in citrus and suggest that eNAD may also be an elicitor in this woody plant species.

Ecto-ADP-ribosyltransferase ARTC2.1 functionally modulates Fc\u03b3R1 and Fc\u03b3R2B on murine microglia

Mammalian ecto-ADP-ribosyltransferases (ecto-ARTs or also ARTCs) catalyze the ADP-ribosylation of cell surface proteins using extracellular nicotinamide adenine dinucleotide (NAD+) as substrate. By this post-translational protein modification, ecto-ARTs modulate the function of various target proteins. A functional role of ARTC2 has been demonstrated for peripheral immune cells such as T cells and macrophages. Yet, little is known about the role of ecto-ARTs in the central nervous system and on microglia. Here, we identified ARTC2.1 as the major ecto-ART expressed on murine microglia. ARTC2.1 expression was strongly upregulated on microglia upon co-stimulation with LPS and an ERK1/2 inhibitor or upon IFNβ stimulation. We identified several target proteins modified by ARTC2.1 on microglia with a recently developed mass spectrometry approach, including two receptors for immunoglobulin G (IgG), FcγR1 and FcγR2B. Both proteins were verified as targets of ARTC2.1 in vitro using a radiolabeling assay with 32P-NAD+ as substrate. Moreover, ADP-ribosylation of both targets strongly inhibited their capacity to bind IgG. In concordance, ARTC2.1 induction in WT microglia and subsequent cell surface ADP-ribosylation significantly reduced the phagocytosis of IgG-coated latex beads, which was unimpaired in NAD+/DTT treated microglia from ARTC2.1−/− mice. Hence, induction of ARTC2.1 expression under inflammatory conditions, and subsequent ADP-ribosylation of cell surface target proteins could represent a hitherto unnoticed mechanism to regulate the immune response of murine microglia.

A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana.

Nicotinamide adenine dinucleotide (NAD+) participates in intracellular and extracellular signaling events unrelated to metabolism. In animals, purinergic receptors are required for extracellular NAD+ (eNAD+) to evoke biological responses, indicating that eNAD+ may be sensed by cell-surface receptors. However, the identity of eNAD+-binding receptors still remains elusive. Here, we identify a lectin receptor kinase (LecRK), LecRK-I.8, as a potential eNAD+ receptor in Arabidopsis. The extracellular lectin domain of LecRK-I.8 binds NAD+ with a dissociation constant of 436.5 ± 104.8 nM, although much higher concentrations are needed to trigger in vivo responses. Mutations in LecRK-I.8 inhibit NAD+-induced immune responses, whereas overexpression of LecRK-I.8 enhances the Arabidopsis response to NAD+. Furthermore, LecRK-I.8 is required for basal resistance against bacterial pathogens, substantiating a role for eNAD+ in plant immunity. Our results demonstrate that lectin receptors can potentially function as eNAD+-binding receptors and provide direct evidence for eNAD+ being an endogenous signaling molecule in plants.

Effect of Exogenous Extracellular Nicotinamide Adenine Dinucleotide (NAD⁺) on Bioelectric Activity of the Pacemaker and Conduction System of the Heart.

In rat sinoatrial node, NAD(+) (10 μM) reduced the rate of spontaneous action potentials, duration of action potentials, and the velocity of slow diastolic depolarization, but the rate of action potential front propagation increases. In passed rabbit Purkinje fibers, NAD(+) (10 μM) reduced the duration of action potentials. Under conditions of spontaneous activity of Purkinje fibers, NAD(+) reduced the fi ring rate and the rate of slow diastolic depolarization. The effects of extracellular NAD(+) on bioelectric activity of the pacemaker (sinoatrial node) and conduction system of the heart (Purkinje fibers) are probably related to activation of P1 and P2 purinoceptors.

Identification of UshA as a major enzyme for NAD degradation in Escherichia coli

Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH are essential cofactors for many redox biocatalysts. Because these cofactors are consumed in stoichiometric amounts, whole-cell biocatalysts have been routinely employed in order to reduce the costs. To further improve the efficacy of redox biocatalysts, it is essential to maintain the stability of nicotinamide cofactors, for which it is attractive to block degradation pathways for NAD(H). While the biosynthesis of NAD(H) has been well studied, it is less understood how NAD(H) are degraded. Here we demonstrated that UshA was a major periplasmic enzyme for NAD degradation in Escherichia coli. Purified recombinant UshA showed high pyrophosphatase activity with the catalytic efficiencies for hydrolysis of NAD and NADH at 3.7μM−1s−1 and 1.4μM−1s−1, respectively. Deletion of the ushA gene from the chromosome led to faster cell growth and improved extracellular NAD stability by 3-fold under conditions similar to whole-cell biocatalysis. These results significantly enriched our understanding on NAD metabolism, and should facilitate many applications including designing more robust redox biocatalysts.

Scant Extracellular NAD Cleaving Activity of Human Neutrophils is Down-Regulated by fMLP via FPRL1.

Extracellular nicotinamide adenine dinucleotide (NAD) cleaving activity of a particular cell type determines the rate of the degradation of extracellular NAD with formation of metabolites in the vicinity of the plasma membrane, which has important physiological consequences. It is yet to be elucidated whether intact human neutrophils have any extracellular NAD cleaving activity. In this study, with a simple fluorometric assay utilizing 1,N6-ethenoadenine dinucleotide (etheno-NAD) as the substrate, we have shown that intact peripheral human neutrophils have scant extracellular etheno-NAD cleaving activity, which is much less than that of mouse bone marrow neutrophils, mouse peripheral neutrophils, human monocytes and lymphocytes. With high performance liquid chromatography (HPLC), we have identified that ADP-ribose (ADPR) is the major extracellular metabolite of NAD degradation by intact human neutrophils. The scant extracellular etheno-NAD cleaving activity is decreased further by N-formyl-methionine-leucine-phenylalanine (fMLP), a chemoattractant for neutrophils. The fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist. These findings show that a much less extracellular etheno-NAD cleaving activity of intact human neutrophils compared to other immune cell types is down-regulated by fMLP via a low affinity fMLP receptor FPRL1.