Search

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with no effective cure. Astrocytes display a toxic phenotype in ALS and contribute to motoneuron (MN) degeneration. Modulating astrocytes’ neurotoxicity can reduce MN death. Our previous studies showed the beneficial effect of mesenchymal stem cell (MSC) administration in SOD1G93A ALS mice, but the mechanisms are still unclear. We postulated that the effects could be mediated by extracellular vesicles (EVs) secreted by MSCs. We investigated, by immunohistochemical, molecular, and in vitro functional analyses, the activity of MSC-derived EVs on the pathological phenotype and neurotoxicity of astrocytes isolated from the spinal cord of symptomatic SOD1G93A mice and human astrocytes (iAstrocytes) differentiated from inducible neural progenitor cells (iNPCs) of ALS patients. In vitro EV exposure rescued mouse and human ALS astrocytes’ neurotoxicity towards MNs. EVs significantly dampened the pathological phenotype and neuroinflammation in SOD1G93A astrocytes. In iAstrocytes, exposure to EVs increased the antioxidant factor Nrf2 and reduced reactive oxygen species. We previously found nine miRNAs upregulated in MSC-derived EVs. Here, the transfection of SOD1G93A astrocytes with single miRNA mimics reduced astrocytes’ activation and the expression of neuroinflammatory factors. Moreover, miR-466q and miR-467f mimics downregulate Mapk11, while miR-466m-5p and miR-466i-3p mimics promote the nuclear translocation of Nrf2. In iAstrocytes, transfection with miR-29b-3p mimic upregulated NQO1 antioxidant activity and reduced neurotoxicity towards MNs. MSC-derived EVs modulate astrocytes’ reactive phenotype and neurotoxicity through anti-inflammatory and antioxidant-shuttled miRNAs, thus representing a therapeutic strategy in ALS.

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are considered a great promise in the repair and regeneration of bone. Considerable efforts have been oriented towards uncovering the best strategy to promote stem cells osteogenic differentiation. In previous studies, hBM-MSCs exposed to physical stimuli such as pulsed electromagnetic fields (PEMFs) or directly seeded on nanostructured titanium surfaces ($TiO_{2}$) were shown to improve their differentiation to osteoblasts in osteogenic condition. In the present study, the effect of a daily PEMF-exposure on osteogenic differentiation of hBM-MSCs seeded onto nanostructured $TiO_{2}$ (with clusters under 100 nm of dimension) was investigated. $TiO_{2}$-seeded cells were exposed to PEMF (magnetic field intensity: 2 mT; intensity of induced electric field: 5 mV; frequency: 75 Hz) and examined in terms of cell physiology modifications and osteogenic differentiation. Results showed that PEMF exposure affected $TiO_{2}$-seeded cells osteogenesis by interfering with selective calcium-related osteogenic pathways, and greatly enhanced hBM-MSCs osteogenic features such as the expression of early/late osteogenic genes and protein production (e.g., ALP, COL-I, osteocalcin and osteopontin) and ALP activity. Finally, PEMF-treated cells resulted to secrete into conditioned media higher amounts of BMP-2, DCN and COL-I than untreated cell cultures. These findings confirm once more the osteoinductive potential of PEMF, suggesting that its combination with $TiO_{2}$ nanostructured surface might be a great option in bone tissue engineering applications.

Glutamate (Glu)-mediated excitotoxicity is a major cause of amyotrophic lateral sclerosis (ALS) and our previous work highlighted that abnormal Glu release may represent a leading mechanism for excessive synaptic Glu. We demonstrated that group I metabotropic Glu receptors (mGluR1, mGluR5) produced abnormal Glu release in SOD1G93A mouse spinal cord at a late disease stage (120 days). Here, we studied this phenomenon in pre-symptomatic (30 and 60 days) and early-symptomatic (90 days) SOD1G93A mice. The mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) concentration dependently stimulated the release of [3H]d-Aspartate ([3H]d-Asp), which was comparable in 30- and 60-day-old wild type mice and SOD1G93A mice. At variance, [3H]d-Asp release was significantly augmented in 90-day-old SOD1G93A mice and both mGluR1 and mGluR5 were involved. The 3,5-DHPG-induced [3H]d-Asp release was exocytotic, being of vesicular origin and mediated by intra-terminal Ca2+ release. mGluR1 and mGluR5 expression was increased in Glu spinal cord axon terminals of 90-day-old SOD1G93A mice, but not in the whole axon terminal population. Interestingly, mGluR1 and mGluR5 were significantly augmented in total spinal cord tissue already at 60 days. Thus, function and expression of group I mGluRs are enhanced in the early-symptomatic SOD1G93A mouse spinal cord, possibly participating in excessive Glu transmission and supporting their implication in ALS. Please define all abbreviations the first time they appear in the abstract, the main text, and the first figure or table caption.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to loss of upper and lower motor neurons (MNs). The mechanisms of neuronal death are largely unknown, thus prejudicing the successful pharmacological treatment. One major cause for MN degeneration in ALS is represented by glutamate(Glu)-mediated excitotoxicity. We have previously reported that activation of Group I metabotropic Glu receptors (mGluR1 and mGluR5) at glutamatergic spinal cord nerve terminals produces abnormal Glu release in the widely studied SOD1

Vascular remodelling plays a central role in asthma and chronic obstructive pulmonary disease (COPD). Bradykinin (BK) is a vasoactive proinflammatory peptide mediating acute responses in asthma. We investigated the role of angiogenic factors in relation to BK receptors in asthma and COPD.Bronchial biopsies from 33 patients with COPD, 24 old (≥50 years) patients with (≥50 years) asthma, 18 old control smokers, 11 old control non-smokers, 15 young (≤40yrs) patients with (≤40 years) asthma and 10 young control non-smokers were immunostained for CD31, vascular endothelial growth factor-A (VEGF-A), angiogenin and BK receptors (B2R and B1R). Fibroblast and endothelial co-localisation of relevant molecules were performed by immunofluorescence. BK-induced VEGF-A and angiogenin release was studied (ELISA) in bronchial fibroblasts from subjects with asthma and COPD.In bronchial lamina propria of old patients with asthma, CD31 and VEGF-A(+) cell numbers were higher than old control non-smokers (p0.05). Angiogenin(+), B2R(+) and B1R(+) cell numbers in old patients with asthma were higher than in old control non-smokers, control smokers and patients with COPD (p0.01). Angiogenin(+) cell numbers were higher in patients with COPD than both old control groups (p0.05). In all patients with asthma the number of B2R(+) cells was positively related to the numbers of B1R(+) (rs=0.43), angiogenin(+) (rs=0.42) and CD31 cells (rs=0.46) (p0.01). Angiogenin(+) cell numbers were negatively related to forced expiratory volume in 1 s (rs=-0.415, p=0.008). Double immunofluorescence revealed that CD31 cells of capillary vessels coexpressed B2R and that fibroblasts coexpressed B2R, VEGF-A and angiogenin. BK (10(-6)M) induced significant angiogenin release in fibroblasts from asthma and to a lesser extent in COPD.Unlike COPD, this study suggests the involvement of BK receptors in bronchial vascular remodelling in asthma.

Glutamate-mediated excitotoxicity plays a major role in ALS and reduced astrocytic glutamate transport was suggested as a cause. Based on previous work we have proposed that abnormal release may represent another source of excessive glutamate. In this line, here we studied the modulation of glutamate release in ALS by Group I metabotropic glutamate (mGlu) receptors, that comprise mGlu1 and mGlu5 members. Synaptosomes from the lumbar spinal cord of SOD1/G93A mice, a widely used murine model for human ALS, and controls were used in release, confocal or electron microscopy and Western blot experiments. Concentrations of the mGlu1/5 receptor agonist 3,5-DHPG >0.3 μM stimulated the release of [(3)H]d- aspartate, used to label the releasing pools of glutamate, both in control and SOD1/G93A mice. At variance, ≤0.3 μM 3,5-DHPG increased [(3)H]d-aspartate release in SOD1/G93A mice only. Experiments with selective antagonists indicated the involvement of both mGlu1 and mGlu5 receptors, mGlu5 being preferentially involved in the high potency effects of 3,5-DHPG. High 3,5-DHPG concentrations increased IP3 formation in both mouse strains, whereas low 3,5-DHPG did it in SOD1/G93A mice only. Release experiments confirmed that 3,5-DHPG elicited [(3)H]d-aspartate exocytosis involving intra-terminal Ca(2+) release through IP3-sensitive channels. Confocal microscopy indicated the co-existence of both receptors presynaptically in the same glutamatergic nerve terminal in SOD1/G93A mice. To conclude, activation of mGlu1/5 receptors produced abnormal glutamate release in SOD1/G93A mice, suggesting that these receptors are implicated in ALS and that selective antagonists may be predicted for new therapeutic approaches. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the α subunit of a Gi protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation.

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca2+]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.

Bradykinin (BK) induces fibroblast contraction but the structural changes and intracellular mechanisms involved have not been completely explored. We stimulated HFL-1 fibroblasts with BK to assess: 1) fibroblast contractility; 2) the role of alpha-smooth muscle actin (SMA) in contraction by small interfering RNA (siRNA); 3) alpha-SMA protein expression; 4) alpha-SMA and F-actin structure; 5) intracellular calcium concentration; and 6) phosphorylated myosin light-chain (pMLC) and MLC kinase (MLCK) expression. BK triggered concentration- and time-dependent fibroblast gel contraction in conjunction with alpha-SMA over expression, but not in alpha-SMA-siRNA-treated cells. BK also increased alpha-SMA(+) and F-actin(+) cell number and stress fibre polymerisation (detectable at 5-60 min). These BK-induced changes were associated with an increase in intracellular calcium concentration, which peaked within 15 s, and activation of pMLC, which was detectable at 5-60 min. No MLCK content modification was observed. The different manifestations of the BK-induced fibroblast activation were downregulated at different levels (25-100%) by HOE140, a specific BK B2 receptor (B2R) antagonist and by the Ca(2+) chelator, EGTA. Thus, BK-induced fibroblast contraction, associated with differentiation into alpha-SMA(+) myofibroblasts, is mediated through the activation of the B2R and involves the Ca(2+)/calmodulin pMLC-dependent pathway.

BACKGROUND: Increased numbers of activated neutrophils have been reported in the bronchial mucosa of patients with stable chronic obstructive pulmonary disease (COPD), particularly in severe disease. OBJECTIVES: To investigate the expression of neutrophilic chemokines and adhesion molecules in bronchial biopsies from patients with stable COPD of different severity (GOLD stages I-IV) compared with age-matched control subjects, smokers with normal lung function and never smokers. METHODS: The expression of CCL5, CXCL1, 5, 6, 7 and 8, CXCR1, CXCR2, CD11b and CD44 was measured in the bronchial mucosa using immunohistochemistry, confocal immunofluorescence, real-time quantitative polymerase chain reaction (RT-QPCR) and Western blotting (WB). RESULTS: The numbers of CCL5+ epithelial cells and CCL5+ and CXCL7+ immunostained cells were increased in the bronchial submucosa of patients with stable severe COPD compared with control never smokers and smokers with normal lung function. This was also confirmed at the level of mRNA expression. The numbers of CCL5+ cells in the submucosa of patients with COPD were 2-15 times higher than any other chemokines. There was no correlation between the number of these cells and the number of neutrophils in the bronchial submucosa. Compared with control smokers, the percentage of neutrophils co-expressing CD11b and CD44 receptors was significantly increased in the submucosa of patients with COPD. CONCLUSION: The increased expression of CCL5 and CXCL7 in the bronchial mucosa of patients with stable COPD, together with an increased expression of extracellular matrix-binding receptors on neutrophils, may be involved in the pathogenesis of COPD.

Abscisic acid (ABA) is a phytohormone recently identified as a new endogenous pro-inflammatory hormone in human granulocytes. Here we report the functional activation of human monocytes and vascular smooth muscle cells by ABA. Incubation of monocytes with ABA evokes an intracellular Ca2+ rise through the second messenger cyclic ADP-ribose, leading to NF-kappaB activation and consequent increase of cyclooxygenase-2 expression and prostaglandin E2 production and enhanced release of MCP-1 (monocyte chemoattractant protein-1) and of metalloprotease-9, all events reportedly involved in atherogenesis. Moreover, monocytes release ABA when exposed to thrombin-activated platelets, a condition occurring at the injured vascular endothelium; monocyte-derived ABA behaves as an autocrine and paracrine pro-inflammatory hormone-stimulating monocyte migration and MCP-1 release, as well as vascular smooth muscle cells migration and proliferation. These results, and the presence of ABA in human arterial plaques at a 10-fold higher concentration compared with normal arterial tissue, identify ABA as a new signal molecule involved in the development of atherosclerosis and suggest a possible new target for anti-atherosclerotic therapy.

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 Ca 2+ 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.

Exposure to Pulsed Electromagnetic Field (PEMF) has been shown to affect proliferation and differentiation of human mesenchymal stem cells derived from bone marrow stroma (BM-hMSC). These cells offer considerable promise in the field of regenerative medicine, but their clinical application is hampered by major limitations such as poor availability and the time required to differentiate up to a stage suitable for implantation. For this reason, several research efforts are focusing on identifying strategies to speed up the differentiation process. In this work we investigated the in vitro effect of PEMF on Ca2+-related mechanisms promoting the osteogenic differentiation of BM-hMSC. Cells were daily exposed to PEMF while subjected to osteogenic differentiation and various Ca2+-related mechanisms were monitored using multiple approaches for identifying functional and structural modifications related to this process. The results indicate that PEMF exposure promotes chemically induced osteogenesis by mechanisms that mainly interfere with some of the calcium-related osteogenic pathways, such as permeation and regulation of cytosolic concentration, leaving others, such as extracellular deposition, unaffected. The PEMF effect is primarily associated to early enhancement of intracellular calcium concentration, which is proposed here as a reliable hallmark of the osteogenic developmental stage.

The impact of synaptic vesicle endo-exocytosis on the trafficking of nerve terminal heterotransporters was studied by monitoring membrane expression and function of the GABA transporter-1 (GAT-1) and of type-1/2 glycine (Gly) transporters (GlyT-1/2) at spinal cord glutamatergic synaptic boutons. Experiments were performed by inducing exocytosis in wild-type (WT) mice, in amphiphysin-I knockout (Amph-I KO) mice, which show impaired endocytosis, or in mice expressing high copy number of mutant human SOD1 with a Gly93Ala substitution (SOD1(G93A)), a model of human amyotrophic lateral sclerosis showing constitutively excessive Glu exocytosis. Exposure of spinal cord synaptosomes from WT mice to a 35mM KCl pulse increased the expression of GAT-1 at glutamatergic synaptosomal membranes and enhanced the GAT-1 heterotransporter-induced [(3)H]d-aspartate ([(3)H]d-Asp) release. Similar results were obtained in the case of GlyT-1/2 heterotransporters. Preventing depolarization-induced exocytosis normalized the excessive GAT-1 and GlyT-1/2 heterotransporter-induced [(3)H]d-Asp release in WT mice. Impaired endocytosis in Amph-I KO mice increased GAT-1 membrane expression and [(3)H]GABA uptake in spinal cord synaptosomes. Also the GAT-1 heterotransporter-evoked release of [(3)H]d-Asp was augmented in Amph-I KO mice. The constitutively excessive Glu exocytosis in SOD1(G93A) mice resulted in augmented GAT-1 expression at glutamatergic synaptosomal membranes and GAT-1 or GlyT-1/2 heterotransporter-mediated [(3)H]d-Asp release. Thus, endo-exocytosis regulates the trafficking of GAT-1 and GlyT-1/2 heterotransporters sited at spinal cord glutamatergic nerve terminals. As a consequence, it can be hypothesized that the excessive GAT-1 and GlyT-1/2 heterotransporter-mediated Glu release, in the spinal cord of SOD1(G93A) mice, is due to the heterotransporter over-expression at the nerve terminal membrane, promoted by the excessive Glu exocytosis.

Fanconi Anemia (FA) is a rare and complex inherited blood disorder associated with bone marrow failure and malignancies. Many alterations in FA physiology appear linked to red-ox unbalance including alterations in the morphology and structure of nuclei, intermediate filaments and mitochondria, defective respiration, reduced ATP production and altered ATP/AMP ratio. These defects are consistently associated with impaired oxygen metabolism indeed treatment with antioxidants N-acetylcysteine (NAC) and resveratrol (RV) does rescue FA physiology. Due to the importance of the intracellular calcium signaling and its key function in the control of intracellular functions we were interested to study calcium homeostasis in FA. We found that FANCA cells display a dramatically low intracellular calcium concentration ([Ca(2+)]i) in resting conditions. This condition affects cellular responses to stress. The flux of Ca(2+) mobilized by H2O2 from internal stores is significantly lower in FANCA cells in comparison to controls. The low basal [Ca(2+)]i in FANCA appears to be an actively maintained process controlled by a finely tuned interplay between different intracellular Ca(2+) stores. The defects associated with the altered Ca(2+) homeostasis appear consistently overlapping those related to the unbalanced oxidative metabolism in FA cells underlining a contiguity between oxidative stress and calcium homeostasis.

Cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, was recently demonstrated to stimulate proliferation of various cell types. The role of cADPR in a specific process of monocyte- and plasma-mediated activation of T-lymphocytes by lipopolysaccharide (LPS) was addressed using human mononuclear cells from peripheral blood (PBMCs). Incubation of PBMCs with 0.1 μg/ml of LPS for 24 h provided a doubling in the intracellular levels of cADPR as compared with unstimulated PBMCs. The cADPR increase was abolished either by prior removal of monocytes or by pre-incubating a whole PBMC population with a monoclonal antibody against the monocyte marker CD14. The increased concentrations of intracellular cADPR elicited by LPS stimulation were paralleled by significant increases in NAD + levels and in the activities of ectocellular and membrane-bound fractions of ADP-ribosyl cyclase/ cADPR hydrolase activities. A cytosolic ADP-ribosyl cyclase was also detectable in PBMCs and its activity was comparably enhanced by LPS stimulation. This soluble cyclase is distinguished from the membrane-bound cyclase by both substrate and inhibitor sensitivities. LPS-stimulated PBMCs showed 2-3-fold increases of intracellular calcium ([Ca2+]), and these changes were prevented completely by the cADPR antagonist 8-Br-cADPR and by ryanodine. Both compounds, and the cyclase inhibitor nicotinamide, significantly inhibited the T-lymphocyte proliferation induced by LPS in PBMCs. These results demonstrate that cADPR plays a role of second messenger in the adaptive immune recognition process of LPS-stimulated proliferation of PBMCs., link_to_subscribed_fulltext

CD38 is a bifunctional ectoenzyme, predominantly expressed on hematopoietic cells during differentiation, that catalyzes the synthesis (cyclase) and the degradation (hydrolase) of cyclic ADP-ribose (cADPR), a powerful calcium mobilizer from intracellular stores. Due to the well established role of calcium levels in the regulation of apoptosis, proliferation, and differentiation, the CD38/cADPR system seems to be a likely candidate involved in the control of these fundamental processes. The ectocellular localization of the cyclase activity, however, contrasts with the intracellular site of action of cADPR. Here we demonstrate that ectocellular expression of human CD38 in CD38(-) HeLa and 3T3 cells results in intracellular CD38 substrate (NAD+ + NADH) consumption and product (cADPR) accumulation. Furthermore, a causal relationship is established between presence of intracellular cADPR, partial depletion of thapsigargin-sensitive calcium stores, increase in basal free cytoplasmic calcium concentration, and decrease of cell doubling time. The significant shortening of the S phase in CD38(+) HeLa cells, as compared with controls, demonstrates an effect of intracellular cADPR on the mammalian cell cycle.

Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-alpha, IL-4 and IFN-gamma to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as G(T) values; (d) epidermal growth factor receptor (EGFR)-dependent mitogen-activated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62 +/- 2% in control cultures and significantly decreased in the presence of TNF-alpha (47 +/- 3%), IL-4 (43 +/- 1%) and INF-gamma (35 +/- 3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. GT values were, respectively, 1.030 +/- 0.0, 1.300 +/- 0.04, 1.260 +/- 0.020 and 2.220 +/- 0.015 (mS/cm(2)) x 1000 in control cultures and in those exposed to TNF-alpha, IFN-gamma and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were markedly prevented when Calu-3 cells were cultured in the presence of an EGFR inhibitor (AG1478, 1 mu M) or a MAP kinase inhibitor (U0126, 25 mu M). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.

Bradykinin (BK) induces differentiation of lung fibroblasts into myofibroblasts, which play an important role in extracellular matrix remodeling in the airways of asthmatic patients. It is unclear whether this process is affected by antiasthma therapies. Here, we evaluated whether a glucocorticoid, budesonide (BUD), and a long-acting β2-agonist, formoterol (FM), either alone or in combination, modified BK-induced lung fibroblast differentiation, and affected the BK-activated intracellular signaling pathways.Human fetal lung fibroblasts were incubated with BUD (0.001-0.1 μM) and/or FM (0.0001-0.1 μM) before exposure to BK (0.1 or 1 μM). Fibroblast differentiation into α-smooth-muscle-actin-positive (α-SMA⁺) myofibroblasts, BK2 receptor (B2R) expression, extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation (p-ERK1/2), intracellular Ca²⁺ concentration ([Ca²⁺]i), and p65 nuclear factor kappa B translocation were evaluated.BUD (0.1 μM) and FM (0.1 μM), either alone or in combination, completely inhibited BK-induced α-SMA protein expression and decreased the numbers of α-SMA⁺ fibroblasts, with a clear reduction in α-SMA stress fibers organization. BUD also completely inhibited the increase of B2R, whereas FM with or without BUD had no effect. BK-induced increases of [Ca²⁺]i and p-ERK1/2 were significantly reduced to similar levels by BUD and FM, either alone or in combination, whereas p65 translocation was completely inhibited by all treatments.Both BUD and FM, either alone or in combination, effectively inhibited the BK-induced differentiation of fibroblasts into α-SMA⁺ myofibroblasts and the intracellular signaling pathways involved in fibroblast activation. These results suggest that BUD and FM combination therapy has potential to inhibit fibroblast-dependent matrix remodeling in the airways of asthmatic patients.

J. Neurochem. (2010) 113, 489–501. Abstract The effect of GABA on glutamate release from astrocytes has been studied in healthy mice and in a murine transgenic model of amyotrophic lateral sclerosis (ALS), using mouse spinal cord gliosomes labeled with [3H]d-aspartate ([3H]d-ASP). GABA concentration-dependently evoked the release of [3H]d-ASP. The effect of GABA was not mimicked by GABAA or GABAB receptor agonists or counteracted by antagonists, excluding receptor involvement. However, it was prevented by the GABA transport inhibitor N-(4,4-phenyl-3-butenyl)-nipecotic acid (SKF 89976A), suggesting participation of GABA transporters type 1 (GAT1) placed on glutamate-releasing astrocyte-derived gliosomes. Accordingly, GAT1 co-expressed with glutamate–aspartate transporter (GLAST) and glutamate transporter type 1 (GLT1) in the majority of glial particles. [3H]d-aspartate release was Ca2+-independent and not blocked by the glutamate uptake inhibitor dl-threo-b-benzyloxyaspartic acid (dl-TBOA); instead, it was abrogated by the anion channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). The GAT1-mediated release of [3H]d-ASP was significantly enhanced in spinal cord gliosomes from the mouse model of ALS. This excessive [3H]d-ASP release was very precocious, largely preceding the onset of the disease symptoms. These data indicate that GAT1, GLAST and GLT1 coexist on the same gliosome in mouse spinal cord and that activation of GAT1 transporters elicits glutamate release by anion channel opening. This phenomenon might have pathological relevance, because [3H]d-ASP release is enhanced in experimental ALS.

ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5',5'"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509-14514). P24, but not P18, proved to increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca(2+) through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca(2+) influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC(50) of approximately 1 mum. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca(2+)](i) has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146-23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca(2+)](i) to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A.

Abscisic acid (ABA) is a hormone involved in pivotal physiological functions in higher plants, such as response to abiotic stress and control of seed dormancy and germination. Recently, ABA was demonstrated to be autocrinally produced by human granulocytes, β pancreatic cells, and mesenchymal stem cells (MSC) and to stimulate cell-specific functions through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). Here we show that ABA expands human uncommitted hemopoietic progenitors (HP) in vitro, through a cADPR-mediated increase of the intracellular calcium concentration ([Ca2+]i). Incubation of CD34+ cells with micromolar ABA also induces transcriptional effects, which include NF-κB nuclear translocation and transcription of genes encoding for several cytokines. Human MSC stimulated with a lymphocyte-conditioned medium produce and release ABA at concentrations sufficient to exert growth-stimulatory effects on co-cultured CD34+ cells, as demonstrated by the inhibition of colony growth in the presence of an anti-ABA monoclonal antibody. These results provide a remarkable example of conservation of a stress hormone and of its second messenger from plants to humans and identify ABA as a new hemopoietic growth factor involved in the cross-talk between HP and MSC.

Background Through a variety of biochemical mechanisms, cigarette smoke may damage airway epithelium, altering its normal structure and function. Injury to epithelium may include changes in tight junction integrity with impairment of epithelial barrier function. Methods and results To study the effect of the exposure to CS condensate on TJ integrity, two human bronchial epithelial cell lines, BEAS-2B and 16HBE14o-, were used. Exposure of the two HBECs to CSC resulted in a time-dependent and concentration-dependent disassembly of TJs, which were already detectable at 24 h at all the CSC concentrations tested, associated with changes in cell shape, suggesting cell damage. However, a significant inhibition of cell growth and an increase in DNA fragmentation were detected only at the highest CSC concentration tested (20%) at 48 and 72 h, respectively. The involvement of epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase (ERK) 1/2 cascade in CSC-induced damage was shown by the observation that exposure to CSC (5%) induced a marked phosphorylation of ERK1/2, already detectable after 5-min incubation and confirmed by the demonstration that not only ERK1/2 phosphorylation but also CSC-induced TJ disassembly and DNA fragmentation were partially inhibited by a mitogen-activated protein kinase kinase inhibitor (U0126) and completely blocked by a EGFR inhibitor (AG1478). Conclusion CSC-induced damage to airway epithelium includes disassembly of TJs, modulated through the EGFR-ERK1/2 signaling pathway.

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.

Abscisic acid (ABA) is a phytohormone involved in fundamental processes in higher plants. Endogenous ABA biosynthesis occurs also in lower Metazoa, in which ABA regulates several physiological functions by activating ADP-ribosyl cyclase (ADPRC) and causing overproduction of the Ca2+-mobilizing second messenger cyclic ADP-ribose (cADPR), thereby enhancing intracellular Ca2+ concentration ([Ca2+]i). Recently, production and release of ABA have been demonstrated to take place also in human granulocytes, where ABA behaves as a proinflammatory hormone through the same cADPR/[Ca2+]i signaling pathway described in plants and in lower Metazoa. On the basis of the fact that human mesenchymal stem cells (MSC) express ADPRC activity, we investigated the effects of ABA and of its second messenger, cADPR, on purified human MSC. Both ABA and cADPR stimulate the in vitro expansion of MSC without affecting differentiation. The underlying mechanism involves a signaling cascade triggered by ABA binding to a plasma membrane receptor and consequent cyclic AMP-mediated activation of ADPRC and of the cADPR/[Ca2+]i system. Moreover, ABA stimulates the following functional activities of MSC: cyclooxygenase 2-catalyzed production of prostaglandin E2 (PGE2), release of several cytokines known to mediate the trophic and immunomodulatory properties of MSC, and chemokinesis. Remarkably, ABA proved to be produced and released by MSC stimulated by specific growth factors (e.g., bone morphogenetic protein-7), by inflammatory cytokines, and by lymphocyte-conditioned medium. These data demonstrate that ABA is an autocrine stimulator of MSC function and suggest that it may participate in the paracrine signaling among MSC, inflammatory/immune cells, and hemopoietic progenitors. Disclosure of potential conflicts of interest is found at the end of this article.

Abscisic acid (ABA) is a plant stress hormone recently identified as an endogenous pro-inflammatory cytokine in human granulocytes. Because paracrine signaling between pancreatic beta 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 beta cells. Autocrine release of ABA by glucose-stimulated pancreatic beta 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.

Nicotinic acid adenine dinucleotide phosphate (NAADP+) is an intracellular second messenger releasing Ca2+ from intracellular stores in different cell types. In addition, it is also active in triggering [Ca2+](i) increase when applied extracellularly and various underlying mechanisms have been proposed. Here, we used hP2Y(11)-transfected 1321N1 astrocytoma cells to unequivocally establish whether extracellular NAADP+ is an agonist of the P2Y(11) receptor, as previously reported for beta-NAD+ [I. Moreschi, S. Bruzzone, R.A. Nicholas, et al., Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes, J. Biol. Chem. 281 (2006) 31419-31429]. Extracellular NAADP+ triggered a concentration-dependent two-step elevation of [Ca2+](i) in 1321N1-hP2Y(11) cells, but not in wild-type 1321N1 cells, secondary to the intracellular production of IP(3), cAMP and cyclic ADP-ribose (cADPR). Specifically, the transient [Ca2+](i) rise proved to be related to IP(3) overproduction and to consequent Ca2+ mobilization, while the sustained [Ca2+](i) elevation was caused by the cAMP/ADP-ribosyl cyclase (ADPRC)/cADPR signalling cascade and by influx of extracellular Ca2+. In human granulocytes, endogenous P2Y(11) proved to be responsible for the NAADP+-induced cell activation (as demonstrated by the use of NF157, a selective and potent inhibitor of P2Y(11)), unveiling a role of NAADP+ as a pro-inflammatory cytokine. In conclusion, we provide unequivocal evidence for the activation of a member of the P2Y receptor subfamily by NAADP+.

The multifunctional protein high-mobility group box 1 (HMGB1) is expressed in restricted areas of adult brain where it can act as a proinflammatory cytokine. We report here that HMGB1 affects CNS transmission by inducing glutamatergic release from glial (gliosomes) but not neuronal (synaptosomes) resealed subcellular particles isolated from mouse cerebellum and hippocampus. Confocal microscopy showed that gliosomes are enriched with glia-specific proteins such as GFAP and S-100, but not with neuronal proteins such as PSD-95, MAP-2, and beta-tubulin III. Furthermore, gliosomes exhibit labeling neither for integrin-alphaM nor for myelin basic protein, specific for microglia and oligodendrocytes, respectively. The gliosomal fraction contains proteins of the exocytotic machinery coexisting with GFAP. Consistent with ultrastructural analysis, several approximately 30-nm nonclustered vesicles are present in the gliosome cytoplasm. Finally, gliosomes represent functional organelles that actively export glutamate when subjected to releasing stimuli, such as ionomycin or ATP, by mechanisms involving extracellular Ca(2+) and Ca(2+) release from intracellular stores. HMGB1-induced release of the stable glutamate analogue [(3)H]d-aspartate and endogenous glutamate form gliosomes, whereas nerve terminals were insensitive to the protein. The HMGB1-evoked release of glutamate was independent on modifications of cytosolic Ca(2+) concentration, but it was blocked by dl-threo-beta-benzyloxyaspartate, suggesting the involvement of transporter-mediated release mechanisms. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 does not block the HMGB1 effect, indicating a role for the glial glutamate-aspartate transporter (GLAST) subtype in this response. HMGB1 bind to gliosomes but not to synaptosomes and can physically interact with GLAST and receptor for advanced glycation end products (RAGE). Taken together, these results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammalian brain.

Micromolar concentrations of extracellular beta-NAD+ (NAD(e)+) 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 NAD(e)+ 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 NAD(e)+-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 P2Y(11) receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y(11)-transfected 1321N1 astrocytoma cells: micromolar NAD(e)+ promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y(11) but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y(11), and the down-regulation of P2Y(11) expression by short interference RNA prevented NAD(e)+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that beta-NAD(e)+ is an agonist of the P2Y(11) purinoceptor and that P2Y(11) is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.

Co-localization of transporters able to recapture the released or endogenously synthesized transmitter (homotransporters) and of transporters that can selectively take up transmitters/modulators originating from neighbouring structures (heterotransporters) has been demonstrated to occur within the same axon terminal of several neuronal phenotypes. Activation of terminal heterotransporters invariably leads to the release of the transmitter specific to the terminal. Heterotransporters are also increasingly reported to exist on neuronal soma/dendrites and nerve terminals, on the basis of morphological experiments. The functions of somatodendritic heterotransporters has been investigated only in a very limited number of cases. Release-regulating GABA heterotransporters of the GAT-1 type exist on Glu nerve terminals in different rodent brain regions including spinal cord. Activation of GABA heterotransporters provokes release of Glu, which takes place by reversal of the Glu homotransporter and by anion channel opening. Interestingly, the release of Glu induced by GABA in spinal cord is dramatically enhanced in a transgenic mouse model of amyotrophic lateral sclerosis and this effect seems to represent the most precocious mechanism that increases extracellular Glu concentration, reported to occur in the pathomechanism.

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.

Beta-NAD+e (extracellular beta-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 alpha and the beta 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 microM NAD+e. Thus extracellular beta-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation.

Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD(+) by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38(+) feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1(+) stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1(+) stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1(+) human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1(+) feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine- and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1(+) feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.

Native human HL-60 cells do not express CD38, a multifunctional ectoenzyme, which generates cyclic ADP-ribose (cADPR), a potent calcium mobilizer. However, when HL-60 cells are induced to differentiate to granulocytes by treatment with retinoic acid (RA), they express CD38 and accumulate cADPR. Both processes play a causal role in RA-induced differentiation. Other granulocyte differentiation-inducers, including dimethyl sulfoxide (Me(2)SO), fail to induce CD38 expression. We investigated whether treatment of HL-60 cells with Me(2)SO involves any changes in the cADPR/intracellular calcium ([Ca(2+)](i)) signaling system and, specifically, whether Me(2)SO affects those nucleoside transporters (NT) (both equilibrative (ENT) and concentrative (CNT)) that mediate influx of extracellular cADPR. Semiquantitative polymerase chain reaction analysis of transcripts, binding of [(3)H]nitrobenzylthioinosine (NBMPR) to intact cells, and influx experiments of extracellular cADPR (with selective inhibitors of NT as NBMPR or in specific conditions) were performed in native and Me(2)SO-differentiated HL-60 cells. The native cells showed uptake of cADPR across ENT2, whereas influx of cADPR into the Me(2)SO-differentiated cells occurred mostly by concentrative processes mediated by CNT3 and by an NBMPR-inhibitable concentrative NT designated cs-csg. Me(2)SO-differentiated, but not native HL-60 cells, accumulated cADPR and showed increased [Ca(2+)](i) levels when grown in a transwell co-culture setting over CD38-transfected 3T3 fibroblasts where nanomolar cADPR concentrations are present in the medium. NBMPR inhibited both responses of Me(2)SO-induced cells. Thus, concentrative influx of extracellular cADPR across CNT3 and cs-csg NT could substitute in the absence of CD38 in eliciting cADPR-dependent [Ca(2+)](i) increases in granulocyte-differentiated HL-60 cells, as well as in other CD38(-) cells.

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 outpumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigargin-sensitive 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.

This paper reports a method of acquiring and codifying branching profiles of axonal arborizations. Using a microcomputer interfaced with a motor driven microscope scanning stage, an acquisition strategy was developed. This strategy allows large profiles (covering up to 1250 mm2) to be measured with an accuracy of a few microns. Each histological section is associated with a specific coordinate system. This system was found to locate the profiles with high precision and reliability in the x-y microscope stage. The overall tracing accuracy of the system is fully discussed.

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.