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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.

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 Ca(2+) mobilizer, and (iv) influx of extracellular Ca(2+). Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar NAD(e)(+) generates both inositol 1,4,5-trisphosphate (IP(3)) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca(2+)](i)): a rapid, IP(3)-mediated Ca(2+) release, followed by a sustained influx of extracellular Ca(2+) mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NAD(e)(+)-induced intracellular increases of IP(3), cAMP, cADPR, and [Ca(2+)](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 [Ca(2+)](i) due to the enhanced intracellular production of IP(3), 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 [Ca(2+)](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 [Ca(2+)](i) increase responsible for their functional activation.

GABAB receptors modulate swimming behavior in Paramecium by inhibiting dihydropyridine-sensitive Ca2+ channels via G-proteins. Prolonged occupancy of GABAB receptors by baclofen results in a decrease in GABAB receptor functions. Since changes in the number of cell-surface GABAA receptors have been postulated to be of importance in modulating inhibitory synaptic transmission in neurons, we have studied the cell-surface expression and maintenance of GABAB receptors in P. primaurelia. In this study, we use immunostaining in electron and confocal microscopy to demonstrate that constitutive internalization of GABAB receptors in P. primaurelia is mediated by clathrin-dependent and -independent endocytosis. Indeed, GABAB receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations. Furthermore, when endocytosis is blocked with hypertonic medium, cytosol acidification, filipin or with a peptide that disrupts the association between amphiphysin and dynamin, the effect of baclofen on swimming is increased. These results suggest that GABAB receptor endocytosis into clathrin-coated and -uncoated vesicles represents an important mechanism in the modulation of swimming behavior in Paramecium.

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 incr

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.

The presence in Paramecium of gamma-aminobutyric acid A-type receptors (GABA(A)) and the capability of the protozoon to synthesize and release the GABA neurotransmitter into the environment have already been demonstrated. This study investigates the involvement of the GABA(A) complex in the swimming control of the ciliated protozoon. The GABA(A) receptors were pharmacologically activated by the selective agonist muscimol and the effect on Paramecium primaurelia swimming behavior was analyzed. Paramecium normally swims forward, but the activation of GABA(A) receptors induced a peculiar response, characterized by alternate periods of whirling and forward swim. This effect was inhibited by the GABA(A) selective antagonists bicuculline and picrotoxin in a dose-dependent manner. Moreover, the application of benzodiazepines did not enhance the agonist action but only reduced it. Response to muscimol was also suppressed by nimodipine, a selective antagonist of dihydropyridine-sensitive calcium channels. This inhibition suggests that an inflow of calcium ions through L-type channels mediates the muscimol-induced swimming behavior.

The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels. 2005 Wiley-Liss, Inc.

A multicentre prospective survey on the use of artificial nutrition (AN) and its complications has been carried out on 1657 hospitalised patients including 7.8% in surgical departments, 7.1% in medical departments and 59.9% in intensive care units. Gastrointestinal diseases and cancer were the most frequent pathologies among patients requiring AN. Parenteral nutrition (PN) alone was employed in 1103 (66.5%) patients. In 267 (16.1%) PN was associated with enteral nutrition (EN). 287 (17.3%) patients were fed by the enteral route alone. The average daily energy intake was 35 kcal/kg for central PN, 26 kcal/kg for peripheral PN and 33 kcal/kg for EN. The mean daily nitrogen intake was 0.19 g/kg for central PN, 0.17 g/kg for peripheral PN and 0.20 g/kg for EN. In 10% of patients PN was electrolyte free and in only 50% all the main electrolytes were added. Trace elements were added in 48% of formulations, mainly as multiple combination. Insulin was added in 61% of PN patients, while albumin was given with PN in 20% of patients. Adverse reactions were observed in 10.9% of PN patients and in 11.5% of EN patients. The most frequent complications occurring in parenterally fed patients were infections of the central venous catheter and metabolic alterations (severe electrolyte abnormalities, liver or renal dysfunction, hypo or persistent hyperglycemia). In enterally fed patients the frequent complications were abdominal distension, cramps and diarrhoea. Adverse reactions were responsible for discontinuation of AN in 2.2% of parenterally supported patients and in 5.7% of enterally fed patients.

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.

"PowerUp Your Microscope" is a software package designed and realized for the optimization of 3D optical microscopy image quality using the Internet and inverse problems computational approaches. The package is mainly devoted to 3D microscopy users, being operative for wide-field, confocal, and multiphoton microscopy. It provides the microscopy community with an extremely easy and comparatively powerful access to advanced image restoration methods. The core of the computational section is the optical system modeling and inverse deconvolution implementation, which is strongly linked to Web-based software and technology. This project constitutes a real and effective migration to the Web, extending computational approaches to image restoration to the whole microscopy user community, regardless of their background.

The presence of opioid, beta-adrenergic and cholinergic receptors has been demonstrated in ciliated protozoa, but little is known about gamma-aminobutyric acid (GABA) receptors. In this study we have analyzed the distribution of GABA(A)-type receptor subunits in Paramecium. Confocal laser microscopy using antibodies specific for alpha(1)-, alpha(2)-, alpha(3)-, alpha(6)-, beta(2/3)-, gamma(2)-, epsilon-, lambda-, and theta-subunits showed that most receptors are aggregated in clusters and are distributed both on cell surface and in the cytoplasm. The intensity of labelling of the alpha(6)-, beta(2/3)- and gamma(2)-subunits was more intense than the alpha(1)-, epsilon-, and theta-subunits, suggesting that the former are present in higher concentrations than the latter.

Recently, the thermosensing pathway in sponges (Porifera) was elucidated. The thermosensor triggering this cascade is a heat-activated cation channel, with the phytohormone abscisic acid (ABA), cyclic ADP-ribose (cADPR) and calcium acting as intracellular messengers, similarly to the drought-stress signaling cascade in higher plants. Here, we investigated the functional effects downstream of the temperature-signaling pathway in Axinella polypoides (Porifera, Demonspongiae). Short-term stimulation followed by long-term depression of amino acid incorporation, oxygen consumption and water filtration were observed after exposure of the sponge to a brief heat stress or to micromolar ABA. These effects could be prevented by the targeted interruption of the signaling pathway either at the level of the cation channel thermosensor or at the level of the cADPR-induced intracellular calcium increase. Moreover, release of cyclase activity into the sea water and generation of extracellular cADPR were observed following brief heat stress. Intact sponge cells were sensitive to extracellular cADPR and addition of purified cyclase increased sponge respiration similarly to heat stress. This is the first observation of functional effects exerted on Metazoa by the phytohormone ABA: conservation of the ABA/cADPR stress-signaling cascade points to its early evolution in a common precursor of modern Metazoa and Metaphyta. The functional effects induced by extracellular cyclase/cADPR suggest an evolutionary origin of cADPR as an ancient stress hormone in Porifera.

In Paramecium, internal Ca(2+) concentration increase coupled to membrane depolarization induces a reversal in the direction of ciliary beating and, consequently, a reversal in swimming direction. The ciliary reversal (CR) duration is correlated to Ca(2+) influx, and the addition of drugs that block the Ca(2+) current leads to a reduction in the backward swimming duration. In this study we have examined the possible function of GABA(B) receptors in P. primaurelia swimming control. The presence of GABA(B) immunoanalogue in Paramecium was evidenced using SDS-PAGE, Western blotting, and confocal laser scanning microscopy. By applying the specific GABA(B) receptor agonist baclofen, a dose-dependent inhibition of the membrane depolarization-induced CR duration was observed. This inhibition was antagonized by phaclofen, persisted when K(+) channel blockers were applied, and disappeared after treatment with nifedipine and verapamil. Moreover, the action of baclofen on depolarization-induced CR was suppressed by treatment with pertussis toxin. Therefore, these experiments suggest that baclofen modulates CR by a G protein (G(0) or G(1)) mediated inhibition of dihydropyridine-sensible calcium channels. Finally, synthesis and release of GABA in the environment by Paramecium have been demonstrated by HPLC. Possible correlations between GABA(B) receptor activation and the regulation of intracellular Ca(2+) levels are discussed.

Sponges are the most ancient known metazoans. Their cells are specialised but not organised into tissues or organs. Recordings of action potential-like propagating electrical impulses suggested that electrical signalling may occur between sponge cells, but the characterization of ionic channels in these cells is still at the beginning. Actually, sponge cell surfaces are covered by a complex glycocalyx and long-chain fatty acids are present in the lipid core of their membranes. In these experimental conditions, a low percentage of tight seals (3%) was obtained applying the patch-clamp technique to cells isolated from the Mediterranean Demospongia Axinella polypoides. This paper shows in detail how difficulties can be overcome making use of trivalent cations in the extracellular solution and how electrophysiological measurements can be performed on sponge cell membranes. A potassium selective conductance is shown as an example. We suggest that the presented methodology could also be applied to other cell types.