Teresa Magalhães-Cardoso

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.

Corpus Cavernosum from Men with Vasculogenic Impotence Is Partially Resistant to Adenosine Relaxation due to Endothelial A 2B Receptor Dysfunction

Although adenosine has been implicated in penile erection in human males, the receptor subtype responsible for adenosine regulation of human corpus cavernosum (HCC) smooth muscle tone is still a matter of debate. Using selective adenosine agonists and antagonists, we aimed at characterizing the adenosine receptors mediating relaxation of precontracted (with 1 μM phenylephrine) HCC strips. HCC specimens were collected from control subjects (organ donors) and from patients with severe vasculogenic erectile dysfunction (ED). In control subjects, adenosine and 5′-N-ethyl-carboxamide adenosine (NECA) fully relaxed HCC. The selective A2A receptor agonist 2-[4-(2-p-carboxy ethyl)phenylamino]-5′-N-ethylcarboxamido adenosine (CGS21680C) produced only a partial relaxation (30-50%) of HCC, which could be further enhanced by simultaneous application of 100 μM NECA. The selective A2B receptor antagonist N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-il)phenoxy] acetamida (MRS1706) (10 nM) attenuated NECA-induced relaxation without affecting CGS21680C action. The A2A receptor antagonist 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol (ZM241385) (50 nM) consistently reduced the actions of both agonists. In contrast to CGS21680C, NECA-induced relaxation was attenuated when endothelial production of NO and prostanoids was reduced by 100 μM NG-nitro-l-arginine and 10 μM indomethacin, respectively. HCC strips from patients with vasculogenic ED were partially resistant to NECA but kept relaxation to CGS21680C; the remaining effect was sensitive to blockade of A2A receptors with 50 nM ZM241385. Data suggest that adenosine regulates HCC smooth muscle tone through the activation of two receptor populations, CGS21680C-sensitive (A2A) and -insensitive (A2B) receptors, located on smooth muscle fibers and on endothelial cells, respectively. Endothelial dysfunction may be correlated with a loss of adenosine A2B receptor activity in penile vessels from men with vasculogenic ED.

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.