M. Alexandrina Timóteo

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.

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.

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.

Tetanic depression is overcome by tonic adenosine A2A receptor facilitation of L‐type Ca2+ influx into rat motor nerve terminals

Motor nerve terminals possess multiple voltage‐sensitive calcium channels operating acetylcholine (ACh) release. In this study, we investigated whether facilitation of neuromuscular transmission by adenosine generated during neuronal firing was operated by Ca2+ influx via ‘prevalent’ P‐type or via the recruitment of ‘silent’ L‐type channels. The release of [3H]ACh from rat phrenic nerve endings decreased upon increasing the stimulation frequency of the trains (750 pulses) from 5 Hz (83 ± 4 × 103 disintegrations per minute per gram (d.p.m. g−1); n= 11) to 50 Hz (30 ± 3 × 103 d.p.m. g−1; n= 5). The P‐type Ca2+ channel blocker, ω‐agatoxin IVA (100 nm) reduced (by 40 ± 10%; n= 6) the release of [3H]ACh evoked by 50‐Hz trains, while nifedipine (1 μm, an L‐type blocker) was inactive. Tetanic depression was overcome (88 ± 6 × 103 d.p.m. g−1; n= 12) by stimulating the phrenic nerve with 50‐Hz bursts (five bursts of 150 pulses, 20 s interburst interval). In these conditions, ω‐agatoxin IVA (100 nm) failed to affect transmitter release, but nifedipine (1 μm) decreased [3H]ACh release by 21 ± 7% (n= 4). Inactivation of endogenous adenosine with adenosine deaminase (ADA, 0.5 U ml−1) reduced (by 54 ± 8%, n= 5) the release of [3H]ACh evoked with 50‐Hz bursts. This effect was opposite to the excitatory actions of adenosine (0.5 mm), S‐(p‐nitrobenzyl)‐6‐thioinosine (5 μm, an adenosine uptake blocker) and CGS 21680C (3 nm, a selective A2A receptor agonist); as the A1 receptor agonist R‐N6‐phenylisopropyl adenosine (R‐PIA, 300 nm) failed to affect the release of [3H]ACh, the results indicate that adenosine generated during 50‐Hz bursts exerts an A2A‐receptor‐mediated tonus. The effects of ADA (0.5 U ml−1) and CGS 21680C (3 nm) were prevented by nifedipine (1 μm). Blocking tonic A2A receptor activation, with ADA (0.5 U ml−1) or 3,7‐dimethyl‐1‐propargyl xanthine (10 μm, an A2A antagonist), recovered ω‐agatoxin IVA (100 nm) inhibition and caused the loss of function of nifedipine (1 μm). Data indicate that, in addition to the predominant P‐type Ca2+ current triggering ACh release during brief tetanic trains, motoneurones possess L‐type channels that may be recruited to facilitate transmitter release during high‐frequency bursts. The fine‐tuning control of Ca2+ influx through P‐ or L‐type channels is likely to be mediated by endogenous adenosine. Therefore, tonic activation of presynaptic A2A receptors operating Ca2+ influx via L‐type channels may contribute to overcome tetanic depression during neuronal firing.

Influence of stimulation on Ca2+ recruitment triggering [3H]acetylcholine release from the rat motor-nerve endings

The influence of rat phrenic nerve stimulation frequency (5-50 Hz) and of pulse duration (0.04-1 ms) on Ca(2+) mobilization triggering [3H]acetylcholine release was investigated. The P-type voltage-dependent Ca(2+) channel (VDCC) blocker, omega-agatoxin IVA (100 nM), decreased [3H]acetylcholine release evoked by pulses of 0. 04-ms duration delivered at 5 Hz frequency. When the stimulus pulse duration was increased to 1 ms (5 Hz frequency) or the stimulation frequency to 50 Hz (0.04-ms duration), inhibition of [3H]acetylcholine release became evident after blockade of L-type VDCC, with nifedipine (1 microM), and/or depletion of thapsigargin-sensitive internal stores. The inhibitory effect of thapsigargin (2 microM) was still observed in Ca(2+)-free medium. Neither omega-conotoxin GVIA (1 microM) nor omega-conotoxin MVIIC (150 nM) modified neurotransmitter release. The results suggest that, depending on the stimulus paradigm, both internal (thapsigargin-sensitive) and external (either P- or L-type channels) Ca(2+) pools can be mobilized to promote acetylcholine release from motor nerve terminals.

Endogenous adenosine prevents post-tetanic release facilitation mediated by α3β2 nicotinic autoreceptors

We investigated the modulatory role of endogenous adenosine on tetanic-induced (50 Hz for 5 s) nicotinic facilitation of [ 3 H]acetylcholine release (5 Hz for 50 s) from rat motoneurons. Adenosine deaminase (0.5 U/ml) and the adenosine A2A receptor antagonist, 3,7-dimethyl-1propargyl xanthine (DMPX, 30 AM), facilitated post-tetanic [ 3 H]acetylcholine release. Release inhibition caused by tubocurarine (1 AM), dihydro-h-erythroidine (1 AM) and a-conotoxin MII (0.1 AM) was attenuated after tetanic preconditioning. Nicotinic inhibitory action was fully restored after adenosine A2A receptor block by DMPX or adenosine deaminase. DMPX (10 AM) caused a leftward shift of the inhibitory dose–response curves for d-tubocurarine (0.1–1 AM), dihydro-h-erythroidine (0.03–10 AM) and a-conotoxin MII (1–300 nM) on posttetanic twitch amplitude. In contrast, the post-tetanic twitch depression caused by a-bungarotoxin (3–100 nM, which had no effect on transmitter release) was attenuated by DMPX (10 AM). It is concluded that activation of adenosine A2A receptors by endogenously generated adenosine prevents the post-tetanic release facilitation mediated by nicotinic a3h2 autoreceptors. D 2003 Elsevier Science B.V. All rights reserved.

Adenosine activating A2A-receptors coupled to adenylate cyclase/cyclic AMP pathway downregulates nicotinic autoreceptor function at the rat myenteric nerve terminals

In addition to the somatodendritic region, myenteric motoneuron terminals are endowed with nicotinic autoreceptors. We aimed at investigating the effect of nicotinic receptor (nAChR) activation on [3H]-acetylcholine ([3H]-ACh) release from longitudinal muscle-myenteric plexus of the rat ileum and to evaluate whether this could be modulated by adenosine, an endogenous neuromodulator typically operating changes in intracellular cyclic AMP. The nAChR agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 1-30 microM, 3 min) increased [3H]-ACh release in a concentration-dependent manner. DMPP (30 microM)-induced [3H]-ACh outflow was attenuated by hexamethonium (0.1-1 mM), tubocurarine (1-5 microM), or by removing external Ca2+ (plus EGTA, 1 mM). In contrast to veratridine (0.2-10 microM)-induced [3H]-ACh release, the DMPP (30 microM)-induced outflow was resistant to tetrodotoxin (1 microM) and cadmium (0.5 mM). Pretreatment with adenosine deaminase (0.5 U/mL) or with the adenosine A(2A)-receptor antagonist, ZM 241385 (50 nM), enhanced nAChR-induced transmitter release. Activation of A(2A) receptors with CGS 21680C (3 nM) reduced the DMPP-induced release of [3H]-ACh. CGS 21680C (3 nM) inhibition was prevented by MDL 12,330A (10 microM, an adenylate cyclase inhibitor) and by H-89 (10 microM, an inhibitor of protein kinase A), but was potentiated by rolipram (300 microM, a phosphodiesterase inhibitor). DMPP-induced transmitter release was decreased by 8-bromo-cyclic AMP (1 mM, a protein kinase A activator), rolipram (300 microM), and forskolin (3 microM, an activator of adenylate cyclase). Both MDL 12,330A (10 microM) and H-89 (10 microM) facilitated DMPP-induced release of [3H]-ACh. The results indicate that nAChR-induced [3H]-ACh release is triggered by the influx of Ca2+, independent of voltage-sensitive calcium channels, presumably directly through nAChRs located on myenteric axon terminals. It was also shown that endogenous adenosine, activating A(2A) receptors coupled to the adenylate cyclase/cyclic AMP transducing system, is tonically downregulating this nAChR-mediated control of [3H]-ACh release.

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.