Cesare Usai

Abscisic acid is an endogenous cytokine in human granulocytes with cyclic ADP-ribose as second messenger

Abscisic acid (ABA) is a phytohormone involved in fundamental physiological processes of higher plants, such as response to abiotic stress (temperature, light, drought), regulation of seed dormancy and germination, and control of stomatal closure. Here, we provide evidence that ABA stimulates several functional activities [phagocytosis, reactive oxygen species and nitric oxide (NO) production, and chemotaxis] of human granulocytes through a signaling pathway sequentially involving a pertussis toxin (PTX)-sensitive G protein/receptor complex, protein kinase A activation, ADP-ribosyl cyclase phosphorylation, and consequent cyclic-ADP-ribose overproduction, leading to an increase of the intracellular Ca2+ concentration. The increase of free intracellular ABA and its release by activated human granulocytes indicate that ABA should be considered as a new pro-inflammatory cytokine in humans. This discovery is an intriguing example of conservation of a hormone and its signaling pathway from plants to humans and provides insight into the molecular mechanisms of granulocyte activation, possibly leading to the development of new antiinflammatory drugs.

The transmembrane glycoprotein CD38 is a catalytically active transporter responsible for generation and influx of the second messenger cyclic ADP-ribose across membranes

CD38 is a type II transmembrane glycoprotein expressed in many vertebrate cells. It is a bifunctional ectoenzyme that catalyzes both the synthesis of Cyclic ADP‐ribose (cADPR) from NAD+ and the degradation of cADPR to ADP‐ribose by means of its ADP‐ribosyl cyclase and cADPR‐hydrolase activities, respectively. The cyclase also converts NGD+ to cyclic GDP‐ribose (cGDPR), which is refractory to cADPR‐hydrolase. cADPR, but not cGDPR, is a potent calcium mobilizer from intracellular stores. It has been demonstrated to be a new second messenger involved in the regulation of calcium homeostasis in many cell types, from plants to mammals. The number of physiological processes shown to be regulated by cADPR is steadily increasing. A topological paradox exists because ectocellularly generated cADPR acts intracellularly. Here we demonstrate that the catalytic functioning of CD38 is accompanied by a cADPR (cGDPR) ‐transporting activity across natural and artificial membranes. In resealed membranes from CD38+ human erythrocytes, transport of catalytically generated cADPR or cGDPR was saturation dependent and occurred against a concentration gradient. Likewise, CD38‐reconstituted proteoliposomes were active in concentrating NAD+ (NGD+) ‐derived cADPR (cGDPR) inside the vesicle compartment. Moreover, the cADPR‐transporting activity in CD38 proteoliposomes prevented the hydrolase‐catalyzed degradation to ADPR that occurs conversely with detergent‐solubilized CD38, resulting in selective influx of cADPR. In the CD38 proteoliposomes, catalytically active CD38 exhibited monomeric, dimeric, and tetrameric structures. In CD38 sense‐ but not in antisense‐transfected HeLa cells, externally added NAD+ resulted in significant, transient increases in cytosolic calcium. These data suggest that transmembrane juxtaposition of two or four CD38 monomers can generate a catalytically active channel for selective formation and influx of cADPR (cGDPR) to reach cADPR‐responsive intracellular calcium stores.—Franco, L., Guida, L., Bruzzone, S., Zocchi, E., Usai, C., De Flora, A. The transmembrane glycoprotein CD38 is a catalytically active transporter responsible for generation and influx of the second messenger cyclic ADP‐ribose across membranes. FASEB J. 12, 1507–1520 (1998)

Extracellular NAD+ Is an Agonist of the Human P2Y11 Purinergic Receptor in Human Granulocytes

Micromolar concentrations of extracellular β-NAD+ (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document}) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document} generates both inositol 1,4,5-trisphosphate (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document}-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y11 receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y11-transfected 1321N1 astrocytoma cells: micromolar \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document} promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y11 but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y11, and the down-regulation of P2Y11 expression by short interference RNA prevented \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{NAD}_{e}^{+}\) \end{document}-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\beta}\mathrm{-}\mathrm{NAD}_{e}^{+}\) \end{document} is an agonist of the P2Y11 purinoceptor and that P2Y11 is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.

The temperature-signaling cascade in sponges involves a heat-gated cation channel, abscisic acid, and cyclic ADP-ribose.

Sponges (phylum Porifera) are the phylogenetically oldest metazoan animals, their evolution dating back to 600 million years ago. Here we demonstrate that sponges express ADP-ribosyl cyclase activity, which converts NAD+ into cyclic ADP-ribose, a potent and universal intracellular Ca2+ mobilizer. In Axinella polypoides (Demospongiae, Axinellidae), ADP-ribosyl cyclase was activated by temperature increases by means of an abscisic acid-induced, protein kinase A-dependent mechanism. The thermosensor triggering this signaling cascade was a heat-activated cation channel. Elucidation of the complete thermosensing pathway in sponges highlights a number of features conserved in higher organisms: (i) the cation channel thermoreceptor, sensitive to heat, mechanical stress, phosphorylation, and anesthetics, shares all of the functional characteristics of the mammalian heat-activated background K+ channel responsible for central and peripheral thermosensing; (ii) involvement of the phytohormone abscisic acid and cyclic ADP-ribose as its second messenger is reminiscent of the drought stress signaling pathway in plants. These results suggest an ancient evolutionary origin of this stress-signaling cascade in a common precursor of modern Metazoa and Metaphyta.

Ligand-induced internalization of CD38 results in intracellular Ca2+ mobilization: role of NAD+ transport across cell membranes

CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP‐ribose (cADPR). cADPR is a universal second messenger that releases calcium from intracellular stores. Since cADPR is generated by CD38 at the outer surface of many cells, where it acts intracellularly, increasing attention is paid to addressing this topological paradox. Recently, we demonstrated that CD38 is a catalytically active, unidirectional transmembrane transporter of cADPR, which then reaches its receptor‐operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin‐coated endocytotic vesicles upon incubating CD38+ cells with either NAD+ or thiol compounds: these endocytotic vesicles can convert cytosolic NAD into cADPR despite an asymmetric unfavorable orientation that makes the active site of CD38 intravesicular. Here we demonstrate that the cADPR‐generating activity of the endocytotic vesicles results in remarkable and sustained increases of intracellular free calcium concentration in different cells exposed to either NAD+, or GSH, or N‐acetylcysteine. This effect of CD38‐internalizing ligands on intracellular calcium levels was found to involve a two‐step mechanism: 1) influx of cytosolic NAD+ into the endocytotic vesicles, mediated by a hitherto unrecognized dinucleotide transport system that is saturable, bidirectional, inhibitable by 8‐N3‐NAD+, and characterized by poor dinucleotide specificity, low affinity, and high efficiency; 2) intravesicular CD38‐catalyzed conversion of NAD+ to cADPR, followed by out‐pumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigarginsensitive stores. This unknown intracellular trafficking of NAD+ and cADPR based on two distinctive and specific transmembrane carriers for either nucleotide can affect the intracellular calcium homeostasis in CD38+ cells. —Zocchi, E., Usai, C., Guida, L., Franco, L., Bruzzone, S., Passalacqua, M., De Flora, A. Ligand‐induced internalization of CD38 results in intracellular Ca2+ mobilization: role of NAD+ transport across cell membranes. FASEB J. 13, 273–283 (1999)

A Self-restricted CD38-connexin 43 Cross-talk Affects NAD+ and Cyclic ADP-ribose Metabolism and Regulates Intracellular Calcium in 3T3 Fibroblasts

Connexin 43 (Cx43) hexameric hemichannels, recently demonstrated to mediate NAD+ transport, functionally interact in the plasma membrane of several cells with the ectoenzyme CD38 that converts NAD+ to the universal calcium mobilizer cyclic ADP-ribose (cADPR). Here we demonstrate that functional uncoupling between CD38 and Cx43 in CD38-transfected 3T3 murine fibroblasts is paralleled by decreased [Ca2+] i levels as a result of reduced intracellular conversion of NAD+ to cADPR. A sharp inverse correlation emerged between [Ca2+] i levels and NAD+ transport (measured as influx into cells and as efflux therefrom), both in the CD38+ cells (high [Ca2+] i , low transport) and in the CD38− fibroblasts (low [Ca2+] i , high transport). These differences were correlated with distinctive extents of Cx43 phosphorylation in the two cell populations, a lower phosphorylation with high NAD+ transport (CD38− cells) and vice versa (CD38+ cells). Conversion of NAD+-permeable Cx43 to the phosphorylated, NAD+-impermeable form occurs via Ca2+-stimulated protein kinase C (PKC). Thus, a self-regulatory loop emerged in CD38+ fibroblasts whereby high [Ca2+] i restricts further Ca2+mobilization by cADPR via PKC-mediated disruption of the Cx43-CD38 cross-talk. This mechanism may avoid: (i) leakage of NAD+from cells; (ii) depletion of intracellular NAD+ by CD38; (iii) overproduction of intracellular cADPR resulting in potentially cytotoxic [Ca2+] i .

Abscisic Acid Is an Endogenous Stimulator of Insulin Release from Human Pancreatic Islets with Cyclic ADP Ribose as Second Messenger

Abscisic acid (ABA) is a plant stress hormone recently identified as an endogenous pro-inflammatory cytokine in human granulocytes. Because paracrine signaling between pancreatic β cells and inflammatory cells is increasingly recognized as a pathogenetic mechanism in the metabolic syndrome and type II diabetes, we investigated the effect of ABA on insulin secretion. Nanomolar ABA increases glucose-stimulated insulin secretion from RIN-m and INS-1 cells and from murine and human pancreatic islets. The signaling cascade triggered by ABA in insulin-releasing cells sequentially involves a pertussis toxin-sensitive G protein, cAMP overproduction, protein kinase A-mediated activation of the ADP-ribosyl cyclase CD38, and cyclic ADP-ribose overproduction. ABA is rapidly produced and released from human islets, RIN-m, and INS-1 cells stimulated with high glucose concentrations. In conclusion, ABA is an endogenous stimulator of insulin secretion in human and murine pancreatic β cells. Autocrine release of ABA by glucose-stimulated pancreatic β cells, and the paracrine production of the hormone by activated granulocytes and monocytes suggest that ABA may be involved in the physiology of insulin release as well as in its dysregulation under conditions of inflammation.

Glia re-sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate

Glial subcellular re‐sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia‐specific proteins glial fibrillary acidic protein (GFAP) and S‐100, but not the neuronal proteins 95‐kDa postsynaptic density protein (PSD‐95), microtubule‐associated protein 2 (MAP‐2) and β‐tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin‐αM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1–5 µm) efficiently stimulated the release of tritium from gliosomes pre‐labelled with [3H]d‐aspartate and of endogenous glutamate in a Ca2+‐dependent and bafilomycin A1‐sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca2+‐dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d‐aspartate release in a concentration‐ (0.1–3 mm) and Ca2+‐dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin‐1, vesicular‐associated membrane protein type 2 (VAMP‐2), 23‐kDa synaptosome‐associated protein (SNAP‐23) and 25‐kDa synaptosome‐associated protein (SNAP‐25)] co‐existing with GFAP immunoreactivity. Moreover, GFAP or VAMP‐2 co‐expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several ∼30‐nm non‐clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate‐accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.

Multi-photon excitation microscopy

Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments.

Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors

Cyclic ADP‐ribose (cADPR) is a universal second messenger that regulates many calcium‐related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD+ (CD38 and BST‐1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 μM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3‐deaza‐cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells.—Podestà, M., Zocchi, E., Pitto, A., Usai, C., Franco, L., Bruzzone, S., Guida, L., Bacigalupo, A., Scadden, D. T., Walseth, T. F., De Flora, A., Daga, A. Extracellular cyclic ADP‐ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors. FASEB J. 14, 680–690 (2000)