M.T. Magalhães-Cardoso

Histamine Induces ATP Release from Human Subcutaneous Fibroblasts, via Pannexin-1 Hemichannels, Leading to Ca2+ Mobilization and Cell Proliferation

Background: Chronic pain may involve connective tissue remodeling due to inflammatory mediators. Results: Histamine H1 receptor activation causes ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels. Conclusion: Responses of skin fibroblasts to histamine are amplified by autocrine ATP release and P2Y1 purinoceptor activation. Significance: Amplification of histamine-mediated Ca2+ mobilization and growth of human fibroblasts by purines may be a novel therapeutic target for painful fibrotic diseases. 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 ADP-sensitive 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.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

BackgroundChronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts.ResultsBradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2-octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor.ConclusionsBradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

Role of ecto-NTPDases on UDP-sensitive P2Y6 receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women

This study aimed at investigating the expression and function of uracil nucleotide‐sensitive receptors (P2Y2, P2Y4, and P2Y6) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration‐dependently increased [Ca2+]i in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y6 receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y6 receptors with MRS 2578 prevented [Ca2+]i rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y2, P2Y4, and P2Y6 receptors. While the expression of P2Y6 receptors remained fairly constant (7∼21 days), P2Y2 and P2Y4 became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, ‐2, and ‐3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP‐sensitive P2Y6 receptors coupled to increases in [Ca2+]i. Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation. J. Cell. Physiol. 227: 2694–2709, 2012.

Relative contribution of ecto‐ATPase and ecto‐ATPDase pathways to the biphasic effect of ATP on acetylcholine release from myenteric motoneurons

Background and purpose:  The relative contribution of distinct ecto‐nucleotidases to the modulation of purinergic signalling may depend on differential tissue distribution and substrate preference.

Tetanic failure due to decreased endogenous adenosine A2A tonus operating neuronal Cav1 (L-type) influx in Myasthenia gravis

J. Neurochem. (2011) 117, 797–811.

Deficits in Endogenous Adenosine Formation by Ecto-5′-Nucleotidase/CD73 Impair Neuromuscular Transmission and Immune Competence in Experimental Autoimmune Myasthenia Gravis

AMP dephosphorylation via ecto-5′-nucleotidase/CD73 is the rate limiting step to generate extracellular adenosine (ADO) from released adenine nucleotides. ADO, via A2A receptors (A2ARs), is a potent modulator of neuromuscular and immunological responses. The pivotal role of ecto-5′-nucleotidase/CD73, in controlling extracellular ADO formation, prompted us to investigate its role in a rat model of experimental autoimmune myasthenia gravis (EAMG). Results show that CD4+CD25+FoxP3+ regulatory T cells express lower amounts of ecto-5′-nucleotidase/CD73 as compared to controls. Reduction of endogenous ADO formation might explain why proliferation of CD4+ T cells failed upon blocking A2A receptors activation with ZM241385 or adenosine deaminase in EAMG animals. Deficits in ADO also contribute to neuromuscular transmission failure in EAMG rats. Rehabilitation of A2AR-mediated immune suppression and facilitation of transmitter release were observed by incubating the cells with the nucleoside precursor, AMP. These findings, together with the characteristic increase in serum adenosine deaminase activity of MG patients, strengthen our hypothesis that the adenosinergic pathway may be dysfunctional in EAMG. Given that endogenous ADO formation is balanced by ecto-5′-nucleotidase/CD73 activity and that A2ARs exert a dual role to restore use-dependent neurocompetence and immune suppression in myasthenics, we hypothesize that stimulation of the two mechanisms may have therapeutic potential in MG.

L‐Citrulline inhibits [3H]acetylcholine release from rat motor nerve terminals by increasing adenosine outflow and activation of A1 receptors

Nitric oxide (NO) production and depression of neuromuscular transmission are closely related, but little is known about the role of L‐citrulline, a co‐product of NO biosynthesis, on neurotransmitter release.

Tuning adenosine A1 and A2A receptors activation mediates l-citrulline-induced inhibition of [3H]-acetylcholine release depending on nerve stimulation pattern

The influence of nerve stimulation pattern on transmitter release inhibition by L-citrulline, the co-product of NO biosynthesis by nitric oxide synthase (NOS), was studied in the rat phrenic nerve-hemidiaphragm. We also investigated the putative interactions between NOS pathway and the adenosine system. L-citrulline (10-470 microM), the NOS substrate L-arginine (10-470 microM) and the NO donor 3-morpholinylsydnoneimine (SIN-1, 1-10 microM), concentration-dependently inhibited [(3)H]-acetylcholine ([(3)H]-ACh) release from rat motor nerve endings. Increasing stimulus frequency from 5 Hz-trains to 50 Hz-bursts enhanced [(3)H]-ACh release inhibition by l-arginine (47 microM) and L-citrulline (470 microM), whereas the effect of SIN-1 (10 microM) remained unchanged. NOS inhibition with N(omega)-nitro-L-arginine (100 microM) prevented the effect of L-arginine, but not that of L-citrulline. Adenosine deaminase (2.5 U/ml) and the adenosine transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine (10 microM), attenuated release inhibition by L-arginine and L-citrulline. With 5 Hz-trains, blockade of A(1) receptors with 1,3-dipropyl-8-cyclopentyl xanthine (2.5 nM), but not of A(2A) receptors with ZM241385 (10nM), reduced the inhibitory action of l-arginine and L-citrulline; the opposite was verified with 50 Hz-bursts. Blockade of muscarinic M(2) autoreceptors with AF-DX116 (10 nM) also attenuated the effects of L-arginine and L-citrulline with 50 Hz-bursts. L-citrulline (470 microM) increased basal adenosine outflow via the equilibrative nucleoside transport system sensitive to NBTI (10 microM), without significantly (P>0.05) changing the nucleoside release subsequent to nerve stimulation. Data indicate that NOS-derived L-citrulline negatively modulates [(3)H]-ACh release by increasing adenosine outflow channelling to A(1) and A(2A) receptors activation depending on the stimulus paradigm. While adenosine acts predominantly at inhibitory A(1) receptors during 5 Hz-trains, inhibition of ACh release by L-citrulline at 50 Hz-bursts depends on the interplay between adenosine A(2A) and muscarinic M(2) receptors.

Feed-Forward Inhibition of CD73 and Upregulation of Adenosine Deaminase Contribute to the Loss of Adenosine Neuromodulation in Postinflammatory Ileitis

Purinergic signalling is remarkably plastic during gastrointestinal inflammation. Thus, selective drugs targeting the “purinome” may be helpful for inflammatory gastrointestinal diseases. The myenteric neuromuscular transmission of healthy individuals is fine-tuned and controlled by adenosine acting on A2A excitatory receptors. Here, we investigated the neuromodulatory role of adenosine in TNBS-inflamed longitudinal muscle-myenteric plexus of the rat ileum. Seven-day postinflammation ileitis lacks adenosine neuromodulation, which may contribute to acceleration of gastrointestinal transit. The loss of adenosine neuromodulation results from deficient accumulation of the nucleoside at the myenteric synapse despite the fact that the increases in ATP release were observed. Disparity between ATP outflow and adenosine deficit in postinflammatory ileitis is ascribed to feed-forward inhibition of ecto-5′-nucleotidase/CD73 by high extracellular ATP and/or ADP. Redistribution of NTPDase2, but not of NTPDase3, from ganglion cell bodies to myenteric nerve terminals leads to preferential ADP accumulation from released ATP, thus contributing to the prolonged inhibition of muscle-bound ecto-5′-nucleotidase/CD73 and to the delay of adenosine formation at the inflamed neuromuscular synapse. On the other hand, depression of endogenous adenosine accumulation may also occur due to enhancement of adenosine deaminase activity. Both membrane-bound and soluble forms of ecto-5′-nucleotidase/CD73 and adenosine deaminase were detected in the inflamed myenteric plexus. These findings provide novel therapeutic targets for inflammatory gut motility disorders.

Inhibition of cholinergic neurotransmission by β3-adrenoceptors depends on adenosine release and A1-receptor activation in human and rat urinary bladders

The direct detrusor relaxant effect of β3-adrenoceptor agonists as a primary mechanism to improve overactive bladder symptoms has been questioned. Among other targets, activation of β3-adrenoceptors downmodulate nerve-evoked acetylcholine (ACh) release, but there is insufficient evidence for the presence of these receptors on bladder cholinergic nerve terminals. Our hypothesis is that adenosine formed from the catabolism of cyclic AMP in the detrusor may act as a retrograde messenger via prejunctional A1 receptors to explain inhibition of cholinergic activity by β3-adrenoceptors. Isoprenaline (1 µM) decreased [3H]ACh release from stimulated (10 Hz, 200 pulses) human (-47 ± 5%) and rat (-38 ± 1%) detrusor strips. Mirabegron (0.1 µM, -53 ± 8%) and CL316,243 (1 µM, -37 ± 7%) mimicked isoprenaline (1 µM) inhibition, and their effects were prevented by blocking β3-adrenoceptors with L748,337 (30 nM) and SR59230A (100 nM), respectively, in human and rat detrusor. Mirabegron and isoprenaline increased extracellular adenosine in the detrusor. Blockage of A1 receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 nM) or the equilibrative nucleoside transporters (ENT) with dipyridamole (0.5 µM) prevented mirabegron and isoprenaline inhibitory effects. Dipyridamole prevented isoprenaline-induced adenosine outflow from the rat detrusor, and this effect was mimicked by the ENT1 inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 30 µM). Cystometry recordings in anesthetized rats demonstrated that SR59230A, DPCPX, dipyridamole, and NBTI reversed the decrease in the voiding frequency caused by isoprenaline (0.1-1,000 nM). Data suggest that inhibition of cholinergic neurotransmission by β3-adrenoceptors results from adenosine release via equilibrative nucleoside transporters and prejunctional A1-receptor stimulation in human and rat urinary bladder.