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

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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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.

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.

Fine-tuning modulation of myenteric motoneurons by endogenous adenosine: on the role of secreted adenosine deaminase.

Besides the well-characterized inhibitory effect of adenosine in the gastrointestinal tract mediated by A1 receptors, we recently demonstrated that endogenously generated adenosine facilitates [3H]acetylcholine release from myenteric neurons through preferential activation of prejunctional A2A receptors. The co-existence of both receptor subtypes on cholinergic neurons prompted the question of how does adenosine discriminate between these receptors to regulate synaptic transmission in the longitudinal muscle-myenteric plexus (LM-MP) of the rat ileum. Electrical stimulation of the LM-MP increased the outflow of adenosine, inosine and hypoxanthine. Myenteric neurons seem to be the main source of endogenous adenosine, since blockade of action potentials with tetrodotoxin (1 microM) or omission of Ca2+ (plus EGTA, 1 mM) in the buffer essentially abolished nucleosides release, while adenosine outflow remained unchanged when smooth muscle contractions were prevented by nifedipine (1 microM). Inhibition of ecto-5'-nucleotidase by concanavalin A (0.1 mg ml-1) produced only a moderate decrease (approximately 25%) on adenosine accumulation in the LM-MP, indicating that the extracellular catabolism of released ATP might not be a major source of the nucleoside. Data using the acetylcholinesterase inhibitor, physiostigmine (10 microM), and several subtype-specific muscarinic receptor antagonists, 4-DAMP (100 nM), AF-DX 116 (10 microM) and muscarinic toxin-7 (1 nM), suggest that cholinergic motoneurons are endowed with muscarinic M3 autoreceptors facilitating the outflow of adenosine. Surprisingly, bath samples collected after stimulating the LM-MP exhibited a relatively high adenosine deaminase (ADA) activity (0.60+/-0.07 U ml-1), which increased in parallel with the accumulation of adenosine and its deamination products. Our findings are in keeping with the hypothesis that ADA secretion, along with a less-efficient dipyridamole-sensitive nucleoside transport system, may restrict endogenous adenosine actions to the synaptic region channelling to facilitatory A2A receptors activation. Such a local environment may also limit diffusion of exogenously added adenosine towards the active zones, as we showed that this constrain may be overcome by inhibiting ADA activity with erythro-9(2-hydroxy-3-nonyl) adenine (50 microM).

Tetanic depression is overcome by tonic adenosine A(2A) receptor facilitation of L-type Ca(2+) 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 Ca(2+) influx via 'prevalent' P-type or via the recruitment of 'silent' L-type channels. The release of [(3)H]ACh from rat phrenic nerve endings decreased upon increasing the stimulation frequency of the trains (750 pulses) from 5 Hz (83 +/- 4 x 10(3) disintegrations per minute per gram (d.p.m. g(-1)); n = 11) to 50 Hz (30 +/- 3 x 10(3) d.p.m. g(-1); n = 5). The P-type Ca(2+) channel blocker, omega-agatoxin IVA (100 nm) reduced (by 40 +/- 10%; n = 6) the release of [(3)H]ACh evoked by 50-Hz trains, while nifedipine (1 microM, an L-type blocker) was inactive. Tetanic depression was overcome (88 +/- 6 x 10(3) 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, omega-agatoxin IVA (100 nM) failed to affect transmitter release, but nifedipine (1 microM) decreased [(3)H]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 [(3)H]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 microM, an adenosine uptake blocker) and CGS 21680C (3 nM, a selective A(2A) receptor agonist); as the A(1) receptor agonist R-N(6)-phenylisopropyl adenosine (R-PIA, 300 nM) failed to affect the release of [(3)H]ACh, the results indicate that adenosine generated during 50-Hz bursts exerts an A(2A)-receptor-mediated tonus. The effects of ADA (0.5 U ml(-1)) and CGS 21680C (3 nm) were prevented by nifedipine (1 microM). Blocking tonic A(2A) receptor activation, with ADA (0.5 U ml(-1)) or 3,7-dimethyl-1-propargyl xanthine (10 microM, an A(2A) antagonist), recovered omega-agatoxin IVA (100 nM) inhibition and caused the loss of function of nifedipine (1 microM). Data indicate that, in addition to the predominant P-type Ca(2+) 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 Ca(2+) influx through P- or L-type channels is likely to be mediated by endogenous adenosine. Therefore, tonic activation of presynaptic A(2A) receptors operating Ca(2+) influx via L-type channels may contribute to overcome tetanic depression during neuronal firing.

Adenosine activating A(2A)-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.

Blockade of neuronal facilitatory nicotinic receptors containing alpha 3 beta 2 subunits contribute to tetanic fade in the rat isolated diaphragm.

Nicotinic receptor (nAChR) subtypes involved in pre- and postjunctional actions underlying tetanic fade were studied in rat phrenic-nerve hemidiaphragms. We investigated the ability of subtype-specific nAChR antagonists to depress nerve-evoked contractions and [(3)H]-acetylcholine ([(3)H]-ACh) release. Muscle tension was transiently increased during brief high frequency trains (50 Hz for 5 sec). The rank potency order of nAChR antagonists to reduce tetanic peak tension was alpha-bungarotoxin > d-tubocurarine >> mecamylamine > hexamethonium. Reduction of maximal tetanic tension produced by dihydro-beta-erythroidine (0.03-10 microM), methyllycaconitine (0.003-3 microM), and alpha-conotoxin MII (0.001-0.3 microM) did not exceed 30%. Besides reduction of peak tension d-tubocurarine (0.1-0.7 microM), mecamylamine (0.1-300 microM), and hexamethonium (30-3,000 microM) also caused tetanic fading. With alpha-conotoxin MII (0.001-0.3 microM) and dihydro-beta-erythroidine (0.03-10 microM), tetanic fade was evident only after decreasing the safety factor of neuromuscular transmission (with high magnesium ions, 6-7 mM). The antagonist rank potency order to reduce evoked (50 Hz for 5 sec) [(3)H]-ACh release from motor nerve terminals was alpha-conotoxin MII (0.1 microM) > dihydro-beta-erythroidine (1 microM) approximately d-tubocurarine (1 microM) > mecamylamine (100 microM) > hexamethonium (1,000 microM). When applied in a concentration (0.3 microM) above that producing tetanic paralysis, alpha-bungarotoxin failed to affect [(3)H]-ACh release. Data obtained suggest that postjunctional neuromuscular relaxants interact with alpha-bungarotoxin-sensitive nicotinic receptors containing alpha1-subunits, whereas blockade of neuronal alpha3beta2-containing receptors produce tetanic fade by breaking nicotinic autofacilitation of acetylcholine release.

Endogenous adenosine prevents post-tetanic release facilitation mediated by alpha3beta2 nicotinic autoreceptors.

We investigated the modulatory role of endogenous adenosine on tetanic-induced (50 Hz for 5 s) nicotinic facilitation of [3H]acetylcholine release (5 Hz for 50 s) from rat motoneurons. Adenosine deaminase (0.5 U/ml) and the adenosine A(2A) receptor antagonist, 3,7-dimethyl-1-propargyl xanthine (DMPX, 30 microM), facilitated post-tetanic [3H]acetylcholine release. Release inhibition caused by tubocurarine (1 microM), dihydro-beta-erythroidine (1 microM) and alpha-conotoxin MII (0.1 microM) was attenuated after tetanic preconditioning. Nicotinic inhibitory action was fully restored after adenosine A(2A) receptor block by DMPX or adenosine deaminase. DMPX (10 microM) caused a leftward shift of the inhibitory dose-response curves for d-tubocurarine (0.1-1 microM), dihydro-beta-erythroidine (0.03-10 microM) and alpha-conotoxin MII (1-300 nM) on post-tetanic twitch amplitude. In contrast, the post-tetanic twitch depression caused by alpha-bungarotoxin (3-100 nM, which had no effect on transmitter release) was attenuated by DMPX (10 microM). It is concluded that activation of adenosine A(2A) receptors by endogenously generated adenosine prevents the post-tetanic release facilitation mediated by nicotinic alpha3beta2 autoreceptors.

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 microm) and oxotremorine (0.3-100 microm), respectively. The muscarinic receptor antagonist, dicyclomine (3 nm-10 microm), 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.