Cátia Vieira

Localization and function of adenosine receptor subtypes at the longitudinal muscle--myenteric plexus of the rat ileum.

Adenosine plays a dual role on acetylcholine (ACh) release from myenteric motoneurons via the activation of high-affinity inhibitory A₁ and facilitatory A(2A) receptors. The therapeutic potential of adenosine-related compounds for controlling intestinal motility and inflammation, prompted us to investigate further the role of low-affinity adenosine receptors, A(2B) and A₃, on electrically-evoked (5 Hz, 200 pulses) [³H]ACh release from myenteric neurons. Immunolocalization studies showed that A(2B) receptors exhibit a pattern of distribution similar to the glial cell marker, GFAP. Regarding A₁ and A₃ receptors, they are mainly distributed to cell bodies of ganglionic myenteric neurons, whereas A(2A) receptors are localized predominantly on cholinergic nerve terminals. Using selective antagonists (DPCPX, ZM241385 and MRS1191), data indicate that modulation of evoked [³H]ACh release is balanced through tonic activation of inhibitory (A₁) and facilitatory (A(2A) and A₃) receptors by endogenous adenosine. The selective A(2B) receptor antagonist, PSB603, alone was devoid of effect and failed to modify the inhibitory effect of NECA. The A₃ receptor agonist, 2-Cl-IB MECA (1-10 nM), concentration-dependently increased the release of [³H]ACh. The effect of 2-Cl-IB MECA was attenuated by MRS1191 and by ZM241385, which selectively block respectively A₃ and A(2A) receptors. In contrast to 2-Cl-IB MECA, activation of A(2A) receptors with CGS21680C attenuated nicotinic facilitation of ACh release induced by focal depolarization of myenteric nerve terminals in the presence of tetrodotoxin. Tandem localization of excitatory A₃ and A(2A) receptors along myenteric neurons explains why stimulation of A₃ receptors (with 2-Cl-IB MECA) on nerve cell bodies acts cooperatively with prejunctional facilitatory A(2A) receptors to up-regulate acetylcholine release. The results presented herein consolidate and expand the current understanding of adenosine receptor distribution and function in the myenteric plexus of the rat ileum, and should be taken into consideration for data interpretation regarding the pathophysiological implications of adenosine on intestinal motility disorders.

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

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.

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.

Muscarinic M3 facilitation of acetylcholine release from rat myenteric neurons depends on adenosine outflow leading to activation of excitatory A2A receptors

Abstract  Acetylcholine (ACh) is a major excitatory neurotransmitter in the myenteric plexus, and it regulates its own release acting via muscarinic autoreceptors. Adenosine released from stimulated myenteric neurons modulates ACh release preferentially via facilitatory A2A receptors. In this study, we investigated how muscarinic and adenosine receptors interplay to regulate ACh from the longitudinal muscle–myenteric plexus of the rat ileum. Blockade of the muscarinic M2 receptor with 11‐[[2‐1[(diethylamino) methyl‐1‐piperidinyl]‐ acetyl]]‐5,11‐dihydro‐6H‐pyrido [2,3‐b][1,4] benzodiazepine‐6‐one (AF‐DX 116), 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP) and atropine facilitated [3H]ACh release evoked by short stimulation trains (5 Hz, 200 pulses). Prolonging stimulus train length (>750 pulses) shifted muscarinic autoinhibition towards facilitatory M3 receptors activation, as predicted by blockade with J104129 (a selective M3 antagonist), 4‐DAMP and atropine, whereas the selective M2 antagonist, AF‐DX 116, was without of effect. Blockade of A2A receptors with ZM 241385, inhibition of adenosine transport with dipyridamole, and inhibition of ecto‐5′‐nucleotidase with concanavalin A, all attenuated release inhibition caused by 4‐DAMP. J104129 and 4‐DAMP, but not AF‐DX 116, decreased (∼60%) evoked adenosine outflow (5 Hz, 3000 pulses). Oxotremorine (300 μmol L−1) facilitated the release of [3H]ACh (34 ± 4%, n = 5) and adenosine (57 ± 3%, n = 6) from stimulated myenteric neurons. 4‐DAMP, dipyridamole and concanavalin A prevented oxotremorine‐induced facilitation. ZM 241385 blocked oxotremorine facilitation of [3H]ACh release, but kept adenosine outflow unchanged. Thus, ACh modulates its own release from myenteric neurons by activating inhibitory M2 and facilitatory M3 autoreceptors. While the M2 inhibition is prevalent during brief stimulation periods, muscarinic M3 facilitation is highlighted during sustained nerve activity as it depends on extracellular adenosine accumulation leading to activation of facilitatory A2A receptors.

Post-inflammatory Ileitis Induces Non-neuronal Purinergic Signaling Adjustments of Cholinergic Neurotransmission in the Myenteric Plexus

Uncoupling between ATP overflow and extracellular adenosine formation changes purinergic signaling in post-inflammatory ileitis. Adenosine neuromodulation deficits were ascribed to feed-forward inhibition of ecto-5′-nucleotidase/CD73 by high extracellular adenine nucleotides in the inflamed ileum. Here, we hypothesized that inflammation-induced changes in cellular density may also account to unbalance the release of purines and their influence on [3H]acetylcholine release from longitudinal muscle-myenteric plexus preparations of the ileum of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-treated rats. The population of S100β-positive glial cells increase, whereas Ano-1-positive interstitial cells of Cajal (ICCs) diminished, in the ileum 7-days after the inflammatory insult. In the absence of changes in the density of VAChT-positive cholinergic nerves detected by immunofluorescence confocal microscopy, the inflamed myenteric plexus released smaller amounts of [3H]acetylcholine which also became less sensitive to neuronal blockade by tetrodotoxin (1 μM). Instead, [3H]acetylcholine release was attenuated by sodium fluoroacetate (5 mM), carbenoxolone (10 μM) and A438079 (3 μM), which prevent activation of glial cells, pannexin-1 hemichannels and P2X7 receptors, respectively. Sodium fluoroacetate also decreased ATP overflow without significantly affecting the extracellular adenosine levels, thus indicating that surplus ATP release parallels reactive gliosis in post-inflammatory ileitis. Conversely, loss of ICCs may explain the lower amounts of adenosine detected in TNBS-treated preparations, since blockade of Cav3 (T-type) channels existing in ICCs with mibefradil (3 μM) or inhibition of the equilibrative nucleoside transporter 1 with dipyridamole (0.5 μM), both decreased extracellular adenosine. Data indicate that post-inflammatory ileitis operates a shift on purinergic neuromodulation reflecting the upregulation of ATP-releasing enteric glial cells and the depletion of ICCs accounting for decreased adenosine overflow via equilibrative nucleoside transporters.