Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic beta cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38(+)/LANCL2(+) HeLa transfected with a chimeric G-protein (G alpha(q/i)) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G(i). Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.

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.

Astrocytes possess different, efficient ways to generate complex changes in intracellular calcium concentrations, which allow them to communicate with each other and to interact with adjacent neuronal cells. Here we show that cultured hippocampal astrocytes coexpress the ectoenzyme CD38, directly involved in the metabolism of the calcium mobilizer cyclic ADP‐ribose, and the NAD⁺ transporter connexin 43. We also demonstrate that hippocampal astrocytes can release NAD⁺ and respond to extracellular NAD⁺ or cyclic ADP‐ribose with intracellular calcium increases, suggesting the existence of an autocrine cyclic ADP‐ribose‐mediated signalling. Cyclic ADP‐ribose‐induced calcium changes are in turn responsible for an increased glutamate and GABA release, this effect being completely inhibited by the cyclic ADP‐ribose specific antagonist 8‐NH₂‐cADPR. Furthermore, addition of NAD⁺ to astrocyte‐neuron co‐cultures results in a delayed intracellular calcium transient in neuronal cells, which is strongly but not completely inhibited by glutamate receptor blockers. These data indicate that an astrocyte‐to‐neuron calcium signalling can be triggered by the CD38/cADPR system, which, through the activation of intracellular calcium responses in astrocytes, is in turn responsible for the increased release of neuromodulators from glial cells.

β-NAD+e (extracellular β-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the α and the β extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1–10 μM NAD+e. Thus extracellular β-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation.

In several cell types, a regulated efflux of NAD⁺ across Connexin 43 hemichannels (Cx43 HC) can occur, and extracellular NAD⁺ (NAD⁺_e) affects cell-specific functions. We studied the capability of bone marrow-derived human mesenchymal stem cells (MSC) to release intracellular NAD⁺ through Cx43 HC. NAD⁺ efflux, quantified by a sensitive enzymatic cycling assay, was significantly upregulated by low extracellular Ca²⁺ (5–6-fold), by shear stress (13-fold), and by inflammatory conditions (3.1- and 2.5-fold in cells incubated with lipopolysaccharide (LPS) or at 39°C, respectively), as compared with untreated cells, whereas it was downregulated in Cx43-siRNA-transfected MSC (by 53%) and by cell-to-cell contact (by 45%). Further, we show that NAD⁺_e activates the purinergic receptor P2Y₁₁ and a cyclic adenosin monophosphate (cAMP)/cyclic ADP-ribose/[Ca²⁺]_i signaling cascade, involving the opening, unique to MSC, of L-type Ca²⁺ channels. Extracellular NAD⁺ enhanced nuclear translocation of cAMP/Ca²⁺-dependent transcription factors. Moreover, NAD⁺, either extracellularly added or autocrinally released, resulted in stimulation of MSC functions, including proliferation, migration, release of prostaglandin E₂ and cytokines, and downregulation of T lymphocyte proliferation compared with controls. No detectable modifications of MSC markers and of adipocyte or osteocyte differentiation were induced by NAD⁺_e. Controls included Cx43-siRNA transfected and/or NAD⁺-glycohydrolase-treated MSC (autocrine effects), and NAD⁺-untreated or P2Y₁₁-siRNA-transfected MSC (exogenous NAD⁺). These findings suggest a potential beneficial role of NAD⁺_e in modulating MSC functions relevant to MSC-based cell therapies.