NAADP: a new second messenger comes of age

Nicotinic acid adenine dinucleotide phosphate (NAADP) is one of the most potent stimulators of intracellular Ca(2+) mobilization in a variety of cell types. Its role in physiological processes is increasingly demonstrated by NAADP increases following cellular stimulation. As a second messenger NAADP shows unique features such as the ability to mobilize Ca(2+) from stores that are physically distinct from those connected to the Ca(2+) channels located in the endoplasmic reticulum, namely, the inositol-1,4,5-trisphosphate and the cyclic-ADP-ribose/ ryanodine receptors. Furthermore, the NAADP-induced self-inactivation mechanism is suggestive of an irreversible binding of NAADP to its putative receptor.     

Calcium signaling: NAADP ascends as a new messenger

NAADP is now established as a Ca(2+) messenger by recent studies which have shown that it is active in various cells, identified the targeted Ca(2+) stores and demonstrated a stimulus-activated increase in its endogenous levels.     

NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells

Important questions remain concerning how elevated blood glucose levels are coupled to insulin secretion from pancreatic beta cells and how this process is impaired in type 2 diabetes. Glucose uptake and metabolism in beta cells cause the intracellular Ca(2+) concentration ([Ca(2+)](i)) to increase to a degree necessary and sufficient for triggering insulin release. Although both Ca(2+) influx and Ca(2+) release from internal stores are critical, the roles of inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine dinucleotide phosphate ribose (cADPR) in regulating the latter have proven equivocal. Here we show that glucose also increases [Ca(2+)](i) via the novel Ca(2+)-mobilizing agent nicotinic acid adenine dinucleotide phosphate (NAADP) in the insulin-secreting beta-cell line MIN6. NAADP binds to specific, high-affinity membrane binding sites and at low concentrations elicits robust Ca(2+) responses in intact cells. Higher concentrations of NAADP inactivate NAADP receptors and attenuate the glucose-induced Ca(2+) increases. Importantly, glucose stimulation increases endogenous NAADP levels, providing strong evidence for recruitment of this pathway. In conclusion, our results support a model in which NAADP mediates glucose-induced Ca(2+) signaling in pancreatic beta cells and are the first demonstration in mammalian cells of the presence of endogenous NAADP levels that can be regulated by a physiological stimulus.     

NAADP as a second messenger: neither CD38 nor base-exchange reaction are necessary for in vivo generation of NAADP in myometrial cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) has recently been shown to act as a second messenger controlling intracellular Ca²⁺ responses in mammalian cells. Many questions remain regarding this signaling pathway, including the role of the ryanodine receptor (RyR) in NAADP-induced Ca²⁺ transients. Furthermore, the exact metabolic pathway responsible for the synthesis of NAADP in vivo has not been determined. Here, we demonstrate that the NAADP mediated Ca²⁺ release system is present in human myometrial cells. We also demonstrate that human myometrial cells use the NAADP second messenger system to generate intracellular Ca²⁺ transients in response to histamine. It has been proposed in the past that the NAADP system in mammalian cells is dependent on the presence of functional RyRs. Here, we observed that the histamine-induced Ca²⁺ transients are dependent on both the NAADP and inositol 1,4,5-trisphosphate signaling pathways but are independent of RyRs. The enzyme CD38 has been shown to catalyze the synthesis of NAADP in vitro by the base-exchange reaction. Furthermore, it has been proposed that this enzyme is responsible for the intracellular generation of NAADP in vivo. Using CD38 knockout mice, we observed that both the basal and histamine stimulated levels of NAADP are independent of CD38 and the base-exchange reaction. Our group is the first to demonstrate that NAADP is a second messenger for histamine-elicited Ca²⁺ transients in human myometrial cells. Furthermore, the NAADP mediated mechanism in mammalian cells can be independent of RyRs and CD38. Our data provides novel insights into the understanding of the mechanism of action and metabolism of this new second messenger system. cADP ribose; inositol 1,4,5-trisphosphate; endoplasmic reticulum; ryanodine channel; nicotinic acid adenine dinucleotide phosphate; CD38; base-exchange reaction     

cGAMP：一种新的哺乳动物第二信使

在细菌中已发现多种环二核苷酸如c-di-GMP、c-di-AMP和cGAMP等可作第二信使,但在哺乳动物中一直未鉴定成功。最新研究发现cGAMP在哺乳动物天然免疫信号通路中也发挥着第二信使作用。在DNA结合条件下cGAMP可由cGAMP合成酶（cGAS）催化生成,随后结合干扰素基因激活蛋白（STING）而诱导Ⅰ型干扰素依赖的天然免疫。这些研究为天然免疫信号通路提供了新的视野,有益于免疫治疗药物的开发。     

Ceramide: a novel second messenger

Sphingomyelin used to be considered only as a structural molecule of the outer leaflet of the plasma membrane of mammalian cells. However, in the last five years it has become clear that sphingomyelin is rapidly degraded and resynthesized in a pathway that could function in signal transduction. Indeed, it is now known that this pathway plays a role in signal transduction for at least one cytokine, tumour necrosis factor alpha. It seems possible that other cell surface receptors will also utilize this pathway for signalling.     

The Ca2+-releasing messenger NAADP, a new player in the nervous system.

Many physiological processes are controlled by a great diversity of Ca2+ signals. Within cell, Ca2+ signals depend upon Ca2+ entry and/or Ca2+ release from internal Ca2+ stores. The control of Ca2+-store mobilization is ensured by a family of messengers comprising inositol 1,4,5 trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). From recent works, new concepts have emerged where activation of the cells by outside stimuli, acting at the plasma membrane, results in the synthesis of multiple Ca2+-releasing messengers which may interact and shape complex Ca2+ signals in the cytosol as well as in the nucleus. This contribution will cover the most recent advances on NAADP signalling with some emphasis on neurons.     

NAADP as an intracellular messenger regulating lysosomal calcium-release channels

Recent studies into the mechanisms of action of the Ca(2+)-mobilizing messenger NAADP (nicotinic acid-adenine dinucleotide phosphate) have demonstrated that a novel family of intracellular Ca(2+)-release channels termed TPCs (two-pore channels) are components of the NAADP receptor. TPCs appear to be exclusively localized to the endolysosomal system. These findings confirm previous pharmacological and biochemical studies suggesting that NAADP targets acidic Ca(2+) stores rather than the endoplasmic reticulum, the major site of action of the other two principal Ca(2+)-mobilizing messengers, InsP(3) and cADPR (cADP-ribose). Studies of the messenger roles of NAADP and the function of TPCs highlight the novel role of lysosomes and other organelles of the endocytic pathway as messenger-regulated Ca(2+) stores which also affects the regulation of the endolysosomal system.     

Ca2+ signalling: a new route to NAADP

The LKB1 serine/threonine kinase is a tumour suppressor responsible for the inherited familial cancer disorder Peutz-Jeghers syndrome and is inactivated in a large percentage of human lung cancers. LKB1 acts a master kinase, directly phosphorylating and activating a family of 14 AMPK (AMP-activated protein kinase)-related kinases which control cell metabolism, cell growth and cell polarity. In this issue of the Biochemical Journal, Hardie and colleagues discover an alternative splice form of LKB1 that alters the C-terminus of the protein containing a few known sites of post-translational regulation. Although widely expressed, the short isoform (LKB1(s)) is the sole splice isoform expressed in testes, and its expression peaks at the time of spermatid maturation. Male mice lacking the LKB1(s) isoform have dramatic defects in spermatozoa, resulting in sterility.     

The nuclear phosphoinositide 3-kinase/AKT pathway: a new second messenger system

Lipid second messengers, particularly those derived from the polyphosphoinositide cycle, play a pivotal role in several cell signaling networks. Phosphoinositide 3-kinases (PI3Ks) generate specific inositol lipids that have been implicated in a plethora of cell functions. One of the best-characterized targets of PI3K lipid products is the serine/threonine protein kinase Akt. Recent findings have implicated Akt in cancer progression because it stimulates cell proliferation and suppresses apoptosis. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid molecules are important components of signaling pathways operating within the nucleus. PI3Ks, their lipid products, and Akt have also been identified at the nuclear level. In this review, we shall summarize the most updated findings about these molecules in relationship with the nuclear compartment and provide an overview of the possible mechanisms by which they regulate important cell functions.     

Chemical synthesis of the novel Ca2+ messenger NAADP

The first total chemical synthesis of nicotinamide adenine dinucleotide phosphate (beta-NADP, 2) as a single isomer was achieved. This was subsequently converted into the important second messenger nicotinic acid adenine dinucleotide phosphate (p-NAADP) 1 and the identity of this material confirmed by biological evaluation. This flexible synthetic route offers new opportunities for the generation of NAADP 1 analogues that cannot be generated directly from NADP 2 or mainly enzymatic methods.     

Cyclic di-GMP as a second messenger

In many bacteria bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling determines the timing and amplitude of complex biological processes from biofilm formation and virulence to photosynthesis. Thereby, the tightly regulated temporal and spatial activity patterns of GGDEF and EAL domain proteins, which synthesize and degrade c-di-GMP, respectively, are currently being resolved. Although details of the mechanisms of c-di-GMP signaling are not yet determined, the recent presentation of PilZ as a candidate c-di-GMP binding-domain opens the field for experimental investigations. Besides its role as an intracellular signaling molecule in bacteria, c-di-GMP also acts as an intercellular signaling molecule between prokaryotes and also has effects in eukaryotes that could provide a perspective in cancer treatment.     

Lactosylceramide: a lipid second messenger in neuroinflammatory disease

Inflammatory disease plays a critical role in the pathogenesis of many neurological disorders. Astrogliosis and induction of pro-inflammatory mediators such as chemokines, cytokines and inducible nitric oxide synthase (iNOS) are the 'hallmarks' of inflammatory disease. Increased activity of lactosylceramide (LacCer) synthase and increased synthesis of LacCer during glial proliferation, and induction of pro-inflammatory cytokines and iNOS suggests a role for LacCer in these cellular signaling pathways. Studies using complementary techniques of inhibitors and antisense reported that inhibition of LacCer synthesis inhibits glial proliferation, as well as the induction of pro-inflammatory mediators (cytokines and iNOS). This inhibition was bypassed by exogenous LacCer, but not by other related lipids (e.g. glucosylceramide, galactocerebroside, GD1, GM1), indicating a role for LacCer in inflammatory signaling pathways. Furthermore, inhibition of glial proliferation and induction of inflammatory mediators by antisense to Ras GTPase, PI3Kinase and inhibitors of mitogen-activated protein kinase indicate the participation of the phosphoinositide 3-kinase (PI3Kinas)/Ras/mitogen-activated protein kinase/nuclear factor-kappaB (NF-kappaB) signaling pathways in LacCer-mediated inflammatory events thus exposing additional targets for therapeutics for inflammatory disease conditions.     

Phosphatidylinositol-specific phospholipase D: a pathway for generation of a second messenger

We have found a phospholipase D activity in the postnuclear fraction of human neutrophils which is stimulated by incubation of cells with the calcium ionophore A23187. The phospholipase D activity was assessed by both phosphatidate formation and free inositol release from phosphatidylinositol substrate. The phospholipase D activity shows an optimum pH of 7.5 and hydrolyzes specifically phosphatidylinositol. These results suggest that this phosphatidylinositol-specific phospholipase D can play a role in cell activating process.     

CD38: a NAADP degrading enzyme

The role of the multifunctional enzyme CD38 in formation of the Ca(2+)-mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was investigated. Gene silencing of CD38 did neither inhibit NAADP synthesis in intact Jurkat T cells nor in thymus or spleen obtained from CD38 knock out mice. In vitro, both NAADP formation by base-exchange and degradation to 2-phospho adenosine diphosphoribose were efficiently decreased. Thus in vivo CD38 appears to be a NAADP degrading rather than a NAADP forming enzyme, perhaps avoiding desensitizing NAADP levels in intact cells.     

Ceramide as a second messenger: sticky solutions to sticky problems

Much discussion has recently centred around the biochemical mechanisms by which ceramide is produced in signalling pathways. Since ceramide is virtually insoluble in aqueous solutions, the biological effects of ceramide should be considered in the context of its generation within the membrane lipid bilayer. To this end, we now summarize recent data describing some biophysical properties of ceramide that are of relevance for understanding the mode of ceramide action as a second messenger, and, as a consequence, how the site(s) of ceramide generation might impact upon its role in signalling.