Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes

UV-B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV-B of human granulocytes and keratinocytes, the cells involved in UV-triggered skin inflammation. The intracellular ABA concentration increased in UV-B-exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV-B-induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE(2)), and tumor necrosis factor-α (TNF-α) by UV-B irradiated keratinocytes. Lanthionine synthetase C-like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV-B-triggered release of PGE(2), TNF-α, and NO and ROS production. These results indicate that UV-B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation.     

The h-Current in the Substantia Nigra pars Compacta Neurons: A Re-examination

The properties of the hyperpolarization-activated cation current (I_h) were investigated in rat substantia nigra - pars compacta (SNc) principal neurons using patch-clamp recordings in thin slices. A reliable identification of single dopaminergic neurons was made possible by the use of a transgenic line of mice expressing eGFP under the tyrosine hydroxylase promoter. The effects of temperature and different protocols on the I_h kinetics showed that, at 37°C and minimizing the disturbance of the intracellular milieu with perforated patch, this current actually activates at potentials more positive than what is generally indicated, with a half-activation potential of −77.05 mV and with a significant level of opening already at rest, thereby substantially contributing to the control of membrane potential, and ultimately playing a relevant function in the regulation of the cell excitability. The implications of the known influence of intracellular cAMP levels on I_h amplitude and kinetics were examined. The direct application of neurotransmitters (DA, 5-HT and noradrenaline) physiologically released onto SNc neurons and known to act on metabotropic receptors coupled to the cAMP pathway modify the I_h amplitude. Here, we show that direct activation of dopaminergic and of 5-HT receptors results in I_h inhibition of SNc DA cells, whereas noradrenaline has the opposite effect. Together, these data suggest that the modulation of I_h by endogenously released neurotransmitters acting on metabotropic receptors –mainly but not exclusively linked to the cAMP pathway- could contribute significantly to the control of SNc neuron excitability.     

The effect of deleting p110delta on the phenotype and function of PTEN-deficient B cells

Control of the intracellular levels of phosphatidylinositol-(3, 4, 5)-trisphosphate by PI3K and phosphatase and tensin homolog (PTEN) is essential for B cell development and differentiation. Deletion of the PI3K catalytic subunit p110delta leads to a severe reduction in B1 and marginal zone (MZ) B cells, whereas deletion of PTEN results in their expansion. We have examined the relationship between these two molecules by generating mice with a B cell-specific deletion of PTEN (PTENB) and a concurrent germline deletion of p110delta. The expanded B1 cell population of PTENB mice was reduced to normal levels in PTENB/p110delta mutant mice, indicating a critical role for the p110delta isoform in the expansion of B1 cells. However, numbers of MZ B cells in the PTENB/p110delta mutants was intermediate between wild-type and PTENB-deficient mice, suggesting an additional role for other PI3K catalytic isoforms in MZ differentiation. Furthermore, the defective class switch recombination in PTENB B cells was only partially reversed in PTENB/p110delta double mutant B cells. These results demonstrate an epistatic relationship between p110delta and PTEN. In addition, they also suggest that additional PI3K catalytic subunits contribute to B cell development and function.     

NAADP+ is an agonist of the human P2Y11 purinergic receptor

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 pathways of oxalate formation from phenylalanine,tyrosine, tryptophan and ascorbic acid in the rat

The metabolic pathway by which L-[¹⁴C₁]phenylalanine, L-[¹⁴C₁]tyrosine, L-[¹⁴C₁]tryptophan, and L-[¹⁴C₁]ascorbic acid are converted to [¹⁴C]oxalate have been investigated in the male rate. Only [¹⁴C]oxalate was detected in the urine of rats injected with L-[¹⁴C₁]ascorbic acid, but [¹⁴C]-labeled oxalate, glycolate, glyoxylate, glycolaldehyde, glycine, and serine were recovered from the [¹⁴C₁]-labeled aromatic amino acids. DL-Phenyllactate, an inhibitor of glycolic acid oxidase and glycolic acid dehydrogenase, reduced the amount of [¹⁴C]oxalate recovered in the urine of rats given the [¹⁴C₁]-labeled aromatic amino acids, but increased the amount of [¹⁴C]glycolate formed from L-[¹⁴C₁]-phenylalanine and L-[¹⁴C₁]tyrosine and the amount of [¹⁴C]glycolate produced from [¹⁴C₁]tryptophan. Based on the [¹⁴C]labeled intermediates identified and the relative distribution of the radioactivity, it is postulated that phenylalanine and tyrosine are converted to oxalate via glycolate which is oxidized directly to oxalate by glycolic acid dehydrogenase. Tryptophan is metabolized via glyxylate which is oxidized directly to oxalate by glycolic acid oxidase. Neither glycolate, glyoxylate, glycolic acid oxidase or glycolic acid dehydrogenase are involved in the formation of oxalate from ascorbic acid.     

Protein oxidative damage and redox imbalance induced by ionising radiation in CHO cells

Reactive oxygen species (ROS) are important mediators of the cytotoxicity induced by the direct reaction of ionising radiation (IR) with all critical cellular components, such as proteins, lipids, and nucleic acids. The derived oxidative damage may propagate in exposed tissues in a dose- and spatiotemporal dependent manner to other cell compartments, affecting intracellular signalling, and cell fate. To understand how cell damage is induced, we studied the oxidative events occurring immediately after cell irradiation by analysing the fate of IR-derived ROS, the intracellular oxidative damage, and the modification of redox environment accumulating in Chinese hamster ovary (CHO) within 1?h after cell irradiation (dose range 0–10?Gy). By using the immuno-spin trapping technique (IST), spectrophotometric methods, and electron paramagnetic resonance (EPR) spectroscopy, we showed that IR-derived ROS (i) induced an IST-detectable, antioxidant-inhibitable one-electron oxidation of specific intracellular proteins; (ii) altered the glutathione (GSH) content (which was found to increase below 2?Gy, and decrease at higher doses, leading to a redox imbalance); (iii) decreased glutathione peroxidase and glutaredoxin activity; (iv) modified neither glutathione reductase nor thioredoxin reductase activity; (v) were detected by spin trapping technique, but adduct intensity decreased due to cell competition for ROS; and (vi) induced no EPR-detectable radicals assignable to oxidised cellular components. In conclusion, our results showed that IR generated an early high oxidising potential (protein radical intermediates, redox imbalance, modified redox enzyme activity) in irradiated cells potentially able to propagate the damage and induce oxidative modification of secondary targets.     

Pathogenetic determinants in Kawasaki disease: the haematological point of view

Kawasaki disease is a multisystemic vasculitis that can result in coronary artery lesions. It predominantly affects young children and is characterized by prolonged fever, diffuse mucosal inflammation, indurative oedema of the hands and feet, a polymorphous skin rash and non‐suppurative lymphadenopathy. Coronary artery involvement is the most important complication of Kawasaki disease and may cause significant coronary stenosis resulting in ischemic heart disease. The introduction of intravenous immunoglobulin decreases the incidence of coronary artery lesions to less than 5%. The etiopathogenesis of this disease remains unclear. Several lines of evidence suggest that an interplay between a microbial infection and a genetic predisposition could take place in the development of the disease. In this review, we summarize the state of the art of pathogenetic mechanisms of Kawasaki disease underscoring the relevance of haematological features as a novel field of investigation.