[Show abstract][Hide abstract] ABSTRACT: Objective:
Thirty percent of patients with epilepsy are refractory to medication. The majority of these patients have mesial temporal lobe epilepsy (MTLE). This prompts for new pharmacologic targets, like ATP-mediated signaling pathways, since the extracellular levels of the nucleotide dramatically increase during in vitro epileptic seizures. In this study, we investigated whether sodium-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake by isolated nerve terminals of the human neocortex could be modulated by ATP acting via slow-desensitizing P2X7 receptor (P2X7R).
Modulation of [(3) H]GABA and [(14) C]glutamate uptake by ATP, through activation of P2X7R, was investigated in isolated nerve terminals of the neocortex of cadaveric controls and patients with drug-resistant epilepsy (non-MTLE or MTLE) submitted to surgery. Tissue density and distribution of P2X7R in the human neocortex was assessed by Western blot analysis and immunofluorescence confocal microscopy.
The P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-100 μm) decreased [(3) H]GABA and [(14) C]glutamate uptake by nerve terminals of the neocortex of controls and patients with epilepsy. The inhibitory effect of BzATP (100 μm) was prevented by the selective P2X7R antagonist, A-438079 (3 μm). Down-modulation of [(14) C]glutamate uptake by BzATP (100 μm) was roughly similar in controls and patients with epilepsy, but the P2X7R agonist inhibited more effectively [(3) H]GABA uptake in the epileptic tissue. Neocortical nerve terminals of patients with epilepsy express higher amounts of the P2X7R protein than control samples.
High-frequency cortical activity during epileptic seizures releases huge amounts of ATP, which by acting on low-affinity slowly desensitizing ionotropic P2X7R, leads to down-modulation of neuronal GABA and glutamate uptake. Increased P2X7R expression in neocortical nerve terminals of patients with epilepsy may, under high-frequency firing, endure GABA signaling and increase GABAergic rundown, thereby unbalancing glutamatergic neuroexcitation. This study highlights the relevance of the ATP-sensitive P2X7R as an important negative modulator of GABA and glutamate transport and prompts for novel antiepileptic therapeutic targets.
[Show abstract][Hide abstract] ABSTRACT: This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [3H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [3H]ACh was higher (P < 0.05) in detrusor strips from BPH patients. The extracellular hydrolysis of ATP and, subsequent, adenosine formation was slower (t
1/2 73 vs. 36 min, P < 0.05) in BPH detrusor strips. The A1 receptor-mediated inhibition of evoked [3H]ACh release by adenosine (100 μM), NECA (1 μM), and R-PIA (0.3 μM) was enhanced in BPH bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A1 immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A1 inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL−1) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A1 receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A1 receptor activation might be useful to control bladder overactivity in BPH patients.
Full-text · Article · Nov 2015 · Purinergic Signalling
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jan 2015 · Mediators of Inflammation
[Show abstract][Hide abstract] ABSTRACT: Polymorphisms of the P2X7 receptor have been associated with increased risk of fractures in postmenopausal women. Although both osteoblasts and osteoclasts express P2X7 receptors, their function in osteogenesis remains controversial. Here, we investigated the role of the P2X7 receptor on osteogenic differentiation and mineralization of bone marrow mesenchymal stem cell (BMSC) cultures from postmenopausal women (age 71±3 yr, n=18). We focused on the mechanisms related to intracellular [Ca(2+)]i oscillations and plasma membrane-dynamics. ATP, and the P2X7 agonist BzATP (100 μM), increased [Ca(2+)]i in parallel to the formation of membrane pores permeable to TO-PRO-3 dye uptake. ATP and BzATP elicited reversible membrane blebs (zeiosis) in 38 ± 1 and 70 ± 1% of the cells, respectively. P2X7-induced zeiosis was Ca(2+) independent, but involved phospholipase C, protein kinase C, and Rho-kinase activation. BzATP (100 μM) progressively increased the expression of Runx-2 and Osterix transcription factors by 452 and 226% (at d 21), respectively, alkaline phosphatase activity by 88% (at d 28), and mineralization by 329% (at d 43) of BMSC cultures in a Rho-kinase-dependent manner. In summary, reversible plasma membrane zeiosis involving cytoskeleton rearrangements due to activation of the P2X7-Rho-kinase axis promotes osteogenic differentiation and mineralization of BMSCs, thus providing new therapeutic targets for postmenopausal bone loss.-Noronha-Matos, J. B., Coimbra, J., Sá-e-Sousa, A., Rocha, R., Marinhas, J., Freitas, R., Guerra-Gomes, S., Ferreirinha, F., Costa, M A., Correia-de-Sá, P. P2X7-induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow-derived mesenchymal stem cells.
No preview · Article · Aug 2014 · The FASEB Journal
[Show abstract][Hide abstract] ABSTRACT: It has been suggested that there is a link between epinephrine synthesis and the development of β2-adrenoceptor-mediated effects, but it remains to be determined if this development is triggered by epinephrine. The aim of this study was to characterize the β-adrenoceptor-mediated relaxation and facilitation of norepinephrine release in the aorta of phenylethanolamine-N-methyltransferase-knockout (Pnmt-KO) mice. Catecholamines were quantified by reverse-phase HPLC-ED. Aorta rings were mounted in a myograph to determine concentration-response curves to selective β1- or β2-adrenoceptor agonists in the absence or presence of selective β1- or β2-adrenoceptor antagonists. Aortic rings were also preincubated with (3)H-norepinephrine to measure tritium overflow elicited by electrical stimulation in the presence of increasing concentrations of non-selective β or selective β2-adrenoceptor agonists. β2-adrenoceptor protein density was evaluated by western-blotting and β2-adrenoceptor localization by immunohistochemistry. Epinephrine is absent in Pnmt-KO mice. The potency and the maximal effect of β2-adrenoceptor agonist terbutaline were lower in Pnmt-KO than in WT mice. The selective β2-adrenoceptor antagonist ICI-118551 antagonized the relaxation caused by terbutaline in wild type (WT) but not in Pnmt-KO mice. Isoproterenol and terbutaline induced concentration-dependent increase in tritium overflow in WT mice only. β2-Adrenoceptor protein density was decreased in membrane aorta homogenates of Pnmt-KO mice and this finding was supported by immunofluorescence confocal microscopy. In conclusion, epinephrine is crucial for β2-adrenoceptor-mediated vasodilation and facilitation of norepinephrine release. In the absence of epinephrine, β2-adrenoceptor protein density was decreased in aorta cell membranes, thus potentially hindering its functional activity.
Full-text · Article · Aug 2014 · Journal of Pharmacology and Experimental Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Background
Increased sympathetic activity has been implicated in hypertension. Adenosine has been shown to play a role in blood flow regulation. In the present study, the endogenous adenosine neuromodulatory role, in mesenteric arteries from normotensive and spontaneously hypertensive rats, was investigated.
Methods and Results
The role of endogenous adenosine in sympathetic neurotransmission was studied using electrically-evoked [3H]-noradrenaline release experiments. Purine content was determined by HPLC with fluorescence detection. Localization of adenosine A1 or A2A receptors in adventitia of mesenteric arteries was investigated by Laser Scanning Confocal Microscopy. Results indicate a higher electrically-evoked noradrenaline release from hypertensive mesenteric arteries. The tonic inhibitory modulation of noradrenaline release is mediated by adenosine A1 receptors and is lacking in arteries from hypertensive animals, despite their purine levels being higher comparatively to those determined in normotensive ones. Tonic facilitatory adenosine A2A receptor-mediated effects were absent in arteries from both strains. Immunohistochemistry revealed an adenosine A1 receptors redistribution from sympathetic fibers to Schwann cells, in adventitia of hypertensive mesenteric arteries which can explain, at least in part, the absence of effects observed for these receptors.
Data highlight the role of purines in hypertension revealing that an increase in sympathetic activity in hypertensive arteries is occurring due to a higher noradrenaline/ATP release from sympathetic nerves and the loss of endogenous adenosine inhibitory tonus. The observed nerve-to-glial redistribution of inhibitory adenosine A1 receptors in hypertensive arteries may explain the latter effect.
[Show abstract][Hide abstract] ABSTRACT: 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
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.
Full-text · Article · Aug 2014 · Mediators of Inflammation
[Show abstract][Hide abstract] ABSTRACT: Purpose: Adenosine fine-tuning regulates cardiovascular function. The negative chronotropic effect of adenosine depends on the activation of the most abundant adenosine receptor in the heart, the A1AR, leading to GIRK/Kir3.1/3.4 channels opening in the SA node. The A1AR-mediated responses in the atrial muscle also depend on KCa2/SK channel function (unpublished observations). Since these channels are involved in atrial repolarization and in human rhythm disturbances, we aimed at studying the interplay between A1AR and SK channels both in situ and single-cell preparations of rat atria.
Methods: Isometric contraction experiments were performed on isolated spontaneously beating atria from Wistar rats. For whole-cell voltage-clamp recordings, atrial myocytes were enzymatically isolated by retrograde perfusion. Immunolocalization studies (A1AR, GIRK1, KCa2.2 and KCa2.3) were evaluated by confocal microscopy. The SA node was identified as a connexin-43 low / neurofilament-160 high immunoreactive region.
Results: The selective A1AR agonist R-PIA (0.001-1μM) decreased atrial chronotropism and inotropism in concentration-dependent manner. However, the negative chronotropic effect of R-PIA (IC50~0.03 μM) was evidenced at much lower (~30 times) concentrations than the negative inotropic action (IC50~1 μM). Selective blockade of A1AR and of GIRK/Kir3.1/3.4 channels, respectively with DPCPX (100 nM) and tertiapin Q (300 nM), significantly attenuated the effects R-PIA. Blockade of KCa2/SK channels with apamin (30 nM) potentiated the negative inotropic response of R-PIA, without affecting atrial rate. Apamin-induced atrial sensitization to R-PIA was not observed with other cardiodepressant agents, namely oxotremorine (0.01-3 μM). Voltage-clamp experiments demonstrated that adenosine, via A1AR, plays a dual role in atrial cardiomyocytes by activating inwardly rectifying GIRK/Kir3.1/3.4 and inactivating outward Ca2+-activated KCa2/SK currents upon cell depolarization. Immunolabelling of A1AR, KIR3.1, KCa2.2 and KCa2.3 was observed throughout the right atria.
Conclusion: Data demonstrate for the first time that A1AR has opposing effects on distinct K+ currents operating atrial repolarization. While A1AR activation favors the opening of inwardly rectifying GIRK/KIR3.1/3.4 channels modulating atrial automatism, it may also inactivate outward KCa2/SK currents. Taking into consideration that KCa2/SK channel dysfunction has pro-arrhythmic effects drugs targeting the A1AR for supraventricular tachycardia must be used with caution as they might precipitate atrial arrhythmias.
Full-text · Article · Jul 2014 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: Background and purpose:
Despite the abundant expression of the UDP-sensitive P2Y6 receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood.
We compared the effects of UDP and of the selective P2Y6 receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [(3) H]-ACh overflow experiments.
Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y6 receptor antagonist, MRS2578. Effects mediated by urothelial P2Y6 receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y1 receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [(3) H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y1 receptor antagonist, MRS2179. The P2Y6 receptor agonist increased threefold ATP levels in the voided fluid.
Conclusions and implications:
Activation of P2Y6 receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y1 receptors.
Full-text · Article · Apr 2014 · British Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: In contrast to the well-known signaling role of urothelial ATP to control bladder function, the hypothesis that uracil nucleotides (UTP and/or UDP) also exert autocrine/paracrine actions only recently gained experimental support. Urothelial cells express UDP-sensitive P2Y6 receptors, yet their role in the control of bladder activity has been mostly neglected. This study was designed to investigate the ability of PSB0474, a stable UDP analogue which exhibits selectivity for P2Y6 receptors, to modulate urodynamic responses in the anaesthetized rat in vivo. Instillation of PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions. PSB0474-induced bladder overactivity was prevented by the selective P2Y6 antagonist, MRS2578. The increase in the VF produced by PSB0474 was also blocked by inhibitors of pannexin-1 hemichannels, (10)Panx or carbenoxolone, when these drugs were applied inside the bladder lumen but not when they were administered intravenously. Reduction of hemichannels pore permeability with H1152 also prevented PSB0474-induced bladder overactivity, but the exocytosis inhibitor, Exo-1, was inactive. PSB0474 increased by 3-fold the urinary ATP content. Implication of hemichannels permeability on PSB0474-induced ATP release was demonstrated by real-time fluorescence videomicroscopy measuring the uptake of propidium iodide by intact urothelial cells in the absence and in the presence of MRS2578 or carbenoxolone. Confocal microscopy studies confirmed the co-localization of pannexin-1 and P2Y6 receptors in the rat urothelium. Data indicate that activation of P2Y6 receptors causes bladder overactivity in the anaesthetized rat indirectly by releasing ATP from the urothelium via pannexin-1 hemichannels.
No preview · Article · Nov 2013 · Biochemical pharmacology