Histamine induces ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels leading to Ca2+ mobilization and cell proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADPsensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Exposure of Lycopersicon Esculentum to Microcystin-LR: Effects in the Leaf Proteome and Toxin Translocation from Water to Leaves and Fruits

Natural toxins such as those produced by freshwater cyanobacteria have been regarded as an emergent environmental threat. However, the impact of these water contaminants in agriculture is not yet fully understood. The aim of this work was to investigate microcystin-LR (MC-LR) toxicity in Lycopersicon esculentum and the toxin accumulation in this horticultural crop. Adult plants (2 month-old) grown in a greenhouse environment were exposed for 2 weeks to either pure MC-LR (100 μg/L) or Microcystis aeruginosa crude extracts containing 100 μg/L MC-LR. Chlorophyll fluorescence was measured, leaf proteome investigated with two-dimensional gel electrophoresis and Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)/TOF, and toxin bioaccumulation assessed by liquid chromatography-mass spectrometry (LC-MS)/MS. Variations in several protein markers (ATP synthase subunits, Cytochrome b6-f complex iron-sulfur, oxygen-evolving enhancer proteins) highlight the decrease of the capacity of plants to synthesize ATP and to perform photosynthesis, whereas variations in other proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and ribose-5-phosphate isomerase) suggest an increase of carbon fixation and decrease of carbohydrate metabolism reactions in plants exposed to pure MC-LR and cyanobacterial extracts, respectively. MC-LR was found in roots (1635.21 μg/kg fw), green tomatoes (5.15–5.41 μg/kg fw), mature tomatoes (10.52–10.83 μg/kg fw), and leaves (12,298.18 μg/kg fw). The results raise concerns relative to food safety and point to the necessity of monitoring the bioaccumulation of water toxins in agricultural systems affected by cyanotoxin contamination.