Paulo Correia-de-Sá

Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides

1 In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory A1 and excitatory A2a‐adenosine receptors, the rat hippocampal Schaffer fibres/CA1 pyramid synapses and the rat innervated hemidiaphragm. 2 Endogenous adenosine tonically inhibited synaptic transmission, since 0.5‐2 u ml−1 of adenosine deaminase increased both the population spike amplitude (30 ± 4%) and field excitatory post‐synaptic potential (f.e.p.s.p.) slope (27 ± 4%) recorded from hippocampal slices and the evoked [3H]‐acetylcholine ([3H]‐ACh) release from the motor nerve terminals (25 ± 2%). 3 a, b̊‐Methylene adenosine diphosphate (AOPCP) in concentrations (100–200 μm) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39 ± 5% and f.e.p.s.p. slope by 32 ± 3% in hippocampal slices and [3H]‐ACh release from motor nerve terminals by 27 ± 3%. 4 Addition of exogenous 5′‐nucleotidase (5 u Ml−1) prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43–57%, whereas the P2 antagonist, suramin (100 μm), did not modify the effect of AOPCP. 5 In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5‐2 u ml−1). The inhibitory effect of AOPCP was still evident when A1 receptors were blocked by 1,3‐dipropyl‐8‐cyclopentylxanthine (2.5‐5 nM), but was abolished by the A2 antagonist, 3,7‐dimethyl‐1‐propargylxanthine (10 μm). 6 These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CA1 pyramid synapses and in phrenic motor nerve endings.

Inhibitory and excitatory effects of adenosine receptor agonists on evoked transmitter release from phrenic nerve endings of the rat

1 The effects of the adenosine analogues, 5′‐N‐ethyl‐carboxamide adenosine (NECA), R‐N6‐phenylisopropyladenosine (R‐PIA), 2‐chloroadenosine (CADO), and CGS 21680C on electrically evoked tritium outflow from preparations loaded with [3H]‐choline and on evoked endplate potentials (e.p.ps), as well as the ability of the xanthines, 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX) and PD 115,199 to antagonize the effects of the adenosine analogues, were investigated in phrenic nerve‐diaphragm preparations. 2 NECA, R‐PIA and CADO decreased, in a concentration‐dependent manner, the evoked tritium outflow from preparations loaded with [3H]‐choline. NECA and R‐PIA were about equipotent and more potent than CADO. 3 DPCPX shifted to the right in a near parallel fashion the concentration‐response curve for the inhibitory effect of R‐PIA on evoked tritium outflow. 4 In the presence of DPCPX, NECA increased, rather than decreased, evoked tritium outflow. PD 115,199 antagonized, in a concentration‐dependent manner, this excitatory effect of NECA. 5 CGS 21680C, in low nanomolar concentrations, increased evoked tritium outflow, an effect also antagonized by PD 115,199. 6 CGS 21680C increased, and R‐PIA decreased, the amplitude of e.p.ps recorded from preparations paralysed with tubocurarine. Both effects could be observed in the same endplate. 7 It is concluded that both inhibitory (probably A1) and excitatory (probably A2) adenosine receptors coexist at the rat neuromuscular junction, modulating the evoked release of acetylcholine.

Functional Transient Receptor Potential Vanilloid 1 is Expressed in Human Urothelial Cells

PURPOSE We investigated the expression and functional status of TRPV1 receptor in human urothelial cells. MATERIAL AND METHODS Human urothelium was cultured and TRPV1 receptor expression was studied by immunocytochemistry and reverse transcriptase-polymerase chain reaction. The influence of inflammatory mediators on TRPV1 mRNA levels was also studied. Functional assays (cobalt uptake measurements and whole cell voltage clamp records) were used to study the response of urothelial cells to capsaicin, temperature, low pH and inflammatory mediators. Capsaicin induced adenosine triphosphate release from urothelial cells was assessed by bioluminescence. RESULTS TRPV1 protein and mRNA were detected in human urothelial cells and mRNA more than tripled in the presence of inflammatory mediators. Nerve growth factor treatment alone did not affect TRPV1 mRNA expression. Capsaicin (100 nM and 1 microM) and heat (41C and 45C) evoked cobalt uptake and inflammatory mediators lowered the temperature threshold for TRPV1 activation to 37C. Capsaicin (1 microM) induced TRPV1 desensitization to further applications of the agonist. In whole cell patch clamp experiments 1 microM capsaicin and a heat ramp from 37C to 50C caused inward currents. The same concentration of capsaicin induced the release of about 7 fmol adenosine triphosphate per mg. CONCLUSIONS TRPV1 receptors expressed by human urothelial cells respond to capsaicin and thermal stimuli. Capsaicin evoked release of adenosine triphosphate suggests that human urothelial TRPV1 is involved in the afferent branch of the micturition reflex. Inflammatory mediators decrease the TRPV1 thermal threshold of activation to body temperature and increase its expression. This finding may be relevant for symptoms associated with cystitis.

Modulation by adenosine of both muscarinic M1-facilitation and M2-inhibition of [^3H]-acetylcholine release from the rat motor nerve terminals

The crosstalk between adenosine and muscarinic autoreceptors regulating evoked [3H]‐acetylcholine ([3H]‐ACh) release was investigated on rat phrenic nerve‐hemidiaphragm preparations. Motor nerve terminals possess facilitatory M1 and inhibitory M2 autoreceptors that can be activated by McN‐A‐343 (1–30 µm) and oxotremorine (0.3–100 µm), respectively. The muscarinic receptor antagonist, dicyclomine (3 nm−10 µm), caused a biphasic (inhibitory/facilitatory) effect, indicating that M1‐facilitation prevails during 5 Hz stimulation trains. Concomitant activation of AF–DX 116‐sensitive M2 receptors was partially attenuated, as pretreatment with M1 antagonists, muscarinic toxin 7 (MT‐7, 0.1 nm) and pirenzepine (1 nm), significantly enhanced inhibition by oxotremorine. Activation of A2A‐adenosine receptors with CGS 21680C (2 nm) (i) potentiated oxotremorine inhibition, and (ii) shifted McN‐A‐343‐induced facilitation into a small inhibitory effect. Conversely, the A1‐receptor agonist, R‐N6‐phenylisopropyl adenosine (R‐PIA, 100 nm), attenuated the inhibitory effect of oxotremorine, without changing facilitation by McN‐A‐343. Synergism between A2A and M2 receptors is regulated by a reciprocal interaction with facilitatory M1 receptors, which may be prevented by pirenzepine (1 nm). During 50 Hz‐bursts, facilitation (M1) of [3H]‐ACh release by McN‐A‐343 disappeared, while the inhibitory (M2) effect of oxotremorine became predominant. This muscarinic shift results from the interplay with A2A receptors, as it was precluded by the selective A2A receptor antagonist, ZM 241385 (10 nm). In conclusion, when the muscarinic M1 positive feedback loop is fully operative, negative regulation of ACh release is mediated by adenosine A1 receptors. During high frequency bursts, tonic activation of A2A receptors promotes M2 autoinhibition by braking the M1 receptor operated counteraction.

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.

Dual effects of adenosine on acetylcholine release from myenteric motoneurons are mediated by junctional facilitatory A2A and extrajunctional inhibitory A1 receptors

The coexistence of both inhibitory A1 and facilitatory A2 adenosine receptors in the rat myenteric plexus prompted the question of how adenosine activates each receptor subtype to regulate cholinergic neurotransmission. Exogenously applied adenosine (0.3–300 μM) decreased electrically evoked [3H]acetylcholine ([3H]ACh) release. Blocking A1 receptors with 1,3‐dipropyl‐8‐cyclopentylxanthine (10 nM) transformed the inhibitory action of adenosine into a facilitatory effect. Adenosine‐induced inhibition was mimicked by the A1 receptor agonist R‐N6‐phenylisopropyladenosine (0.3 μM), but the A2A agonist CGS 21680C (0.003 μM) produced a contrasting facilitatory effect. Increasing endogenous adenosine levels, by the addition of (1) the adenosine precursor AMP (30–100 μM), (2) the adenosine kinase inhibitor 5′‐iodotubercidin (10 μM) or (3) inhibitors of adenosine uptake (dipyridamole, 0.5 μM) and of deamination (erythro‐9(2‐hydroxy‐3‐nonyl)adenine, 50 μM), enhanced electrically evoked [3H]ACh release (5 Hz for 40 s). Release facilitation was prevented by adenosine deaminase (ADA, 0.5 U ml−1) and by the A2A receptor antagonist ZM 241385 (50 nM); these compounds decreased [3H]ACh release by 31±6% (n=7) and 37±10% (n=6), respectively. Although inhibition of ecto‐5′‐nucleotidase by α,β‐methylene ADP (200 μM) or by concanavalin A (0.1 mg ml−1) attenuated endogenous adenosine formation from AMP, analysed by HPLC, the corresponding reduction in [3H]ACh release only became evident when stimulation of the myenteric plexus was prolonged to over 250 s. In summary, we found that endogenously generated adenosine plays a predominantly tonic facilitatory effect mediated by prejunctional A2A receptors. Extracellular deamination and cellular uptake may restrict endogenous adenosine actions to the neuro‐effector region near the release/production sites.

Evidence that the presynaptic A2a-adenosine receptor of the rat motor nerve endings is positively coupled to adenylate cyclase

The action of the A2a-adenosine analogue, CGS 21680C, on electrically evoked [3H]acetylcholine ([3H]-ACh) release, and its interaction with forskolin (an activator of adenylate cyclase), MDL 12,330A (an irreversible inhibitor of adenylate cyclase), rolipram (an inhibitor of cyclic AMP specific phosphodiesterase), dibutyryl- (db-cAMP) and 8-bromo- (8-Br-cAMP) cyclic AMP analogues (substances that mimic intracellular actions of cyclic AMP), were investigated using rat phrenic nerve-hemidiaphragm preparations.CGS 21680C facilitated [3H]ACh release. Forskolin (but not 1,9-dideoxy forskolin), rolipram, db-cAMP and 8-Br-cAMP also increased evoked neurotransmitter release in a concentration-dependent manner. When the evoked [3H]-ACh release that is dependent on stimulation of the adenylate cyclase/cyclic AMP transduction system was supramaximally stimulated by these compounds, CGS 21680 C (3 μmol/l) could not further increase [3H]-ACh release. Phosphodiesterase inhibition with low concentrations (⩽ 30 μmol/l) of rolipram significantly potentiated the augmenting effect of CGS 21680C (1 μmol/l) on evoked [3H]ACh release. MDL 12,330A (an irreversible inhibitor of adenylate cyclase) decreased evoked [3H]-ACh release. The irreversible blocking action of MDL 12,330A on [3H]-ACh release was overcome by by-passing cyclase activation with db-cAMP and 8-Br-cAMP, but could not be overcome with FSK or CGS 21680 C. The inhibitory effect of MDL 12,330A on evoked [3H]-ACh release was not mimicked by nifedipine.It is concluded that the increase in [3H]-ACh release caused by CGS 21680C results from activation of an A2a-adenosine receptor positively linked to the adenylate cyclase/cyclic AMP system.

ATP released via pannexin-1 hemichannels mediates bladder overactivity triggered by urothelial P2Y6 receptors

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 video-microscopy 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.

Potentiation by tonic A2a-adenosine receptor activation of CGRP-facilitated [3H]-ACh release from rat motor nerve endings

1 The effect of calcitonin gene‐related peptide (CGRP) on [3H]‐acetylcholine ([3H]‐ACh) release from motor nerve endings and its interaction with presynaptic facilitatory A2a‐adenosine and nicotinic acetylcholine receptors was studied on rat phrenic nerve‐hemidiaphragm preparations loaded with [3H]‐choline. 2 CGRP (100–400 nm) increased electrically evoked [3H]‐ACh release from phrenic nerve endings in a concentration‐dependent manner. 3 The magnitude of CGRP excitation increased with the increase of the stimulation pulse duration from 40 μs to 1 ms, keeping the frequency, the amplitude and the train length constants. With 1 ms pulses, the evoked [3H]‐ACh release was more intense than with 40 μs pulse duration. 4 Both the nicotinic acetylcholine receptor agonist, 1,1‐dimethyl‐4‐phenylpiperazinium, and the A2a adenosine receptor agonist, CGS21680C, increased evoked [3H]‐ACh release, but only CGS21680C potentiated the facilitatory effect of CGRP. This potentiation was prevented by the A2a adenosine receptor antagonist, PD 115,199. 5 Adenosine deaminase prevented the excitatory effect of CGRP (400 nm) on [3H]‐ACh release. This effect was reversed by the non‐hydrolysable A2a‐adenosine receptor agonist, CGS 21680C. 6 The nicotinic antagonist, tubocurarine, did not significantly change, whereas the A2‐adenosine receptor antagonist, PD 115,199, blocked the CGRP facilitation. The A1‐adenosine receptor antagonist, 1,3‐dipropyl‐8‐cyclopentylxanthine, potentiated the CGRP excitatory effect. 7 The results suggest that the facilitatory effect of CGRP on evoked [3H]‐ACh release from rat phrenic motor nerve endings depends on the presence of endogenous adenosine which tonically activates A2a‐adenosine receptors. Since both CGRP and A2a‐adenosine receptors are positively coupled to the adenylate cyclase/cyclic AMP system, cooperation between these receptors might occur at the second messenger transduction system level.

Ecto‐AMP Deaminase Blunts the ATP‐Derived Adenosine A2A Receptor Facilitation of Acetylcholine Release at Rat Motor Nerve Endings

At synapses, ATP is released and metabolised through ecto‐nucleotidases forming adenosine, which modulates neurotransmitter release through inhibitory A1 or facilitatory A2A receptors, according to the amounts of extracellular adenosine. Neuromuscular junctions possess an ecto‐AMP deaminase that can dissociate extracellular ATP catabolism from adenosine formation. In this study we have investigated the pattern of ATP release and its conversion into adenosine, to probe the role of ecto‐AMP deaminase in controlling acetylcholine release from rat phrenic nerve terminals. Nerve‐evoked ATP release was 28 ± 12 pmol (mg tissue)−1 at 1 Hz, 54 ± 3 pmol (mg tissue)−1 at 5 Hz and disproportionally higher at 50 Hz (324 ± 23 pmol (mg tissue)−1). Extracellular ATP (30 μm) was metabolised with a half time of 8 ± 2 min, being converted into ADP then into AMP. AMP was either dephosphorylated into adenosine by ecto‐5′‐nucleotidase (inhibited by ATP and blocked by 200 μmα,β‐methylene ADP) or deaminated into IMP by ecto‐AMP deaminase (inhibited by 200 μm deoxycoformycin, which increased adenosine formation). Dephosphorylation and deamination pathways also catabolised endogenously released adenine nucleotides, since the nerve‐evoked extracellular AMP accumulation was increased by either α,β‐methylene ADP (200 μm) or deoxycoformycin (200 μm). In the presence of nitrobenzylthioinosine (30 μm) to inhibit adenosine transport, deoxycoformycin (200 μm) facilitated nerve‐evoked [3H]acetylcholine release by 77 ± 9 %, an effect prevented by the A2A receptor antagonist, ZM 241385 (10 nm). It is concluded that, while ecto‐5′‐nucleotidase is inhibited by released ATP, ecto‐AMP deaminase activity transiently blunts adenosine formation, which would otherwise reach levels high enough to activate facilitatory A2A receptors on motor nerve terminals.