Adenosinergic System and BDNF Signaling Changes as a Cross-Sectional Feature of RTT: Characterization of Mecp2 Heterozygous Mouse Females.

Rett Syndrome is an X-linked neurodevelopmental disorder (RTT; OMIM#312750) associated to MECP2 mutations. MeCP2 dysfunction is seen as one cause for the deficiencies found in brain-derived neurotrophic factor (BDNF) signaling, since BDNF is one of the genes under MeCP2 jurisdiction. BDNF signaling is also dependent on the proper function of the adenosinergic system. Indeed, both BDNF signaling and the adenosinergic system are altered in Mecp2-null mice (Mecp2-/y), a representative model of severe manifestation of RTT. Considering that symptoms severity largely differs among RTT patients, we set out to investigate the BDNF and ADO signaling modifications in Mecp2 heterozygous female mice (Mecp2+/-) presenting a less severe phenotype. Symptomatic Mecp2+/- mice have lower BDNF levels in the cortex and hippocampus. This is accompanied by a loss of BDNF-induced facilitation of hippocampal long-term potentiation (LTP), which could be restored upon selective activation of adenosine A2A receptors (A2AR). While no differences were observed in the amount of adenosine in the cortex and hippocampus of Mecp2+/- mice compared with healthy littermates, the density of the A1R and A2AR subtype receptors was, respectively, upregulated and downregulated in the hippocampus. Data suggest that significant changes in BDNF and adenosine signaling pathways are present in an RTT model with a milder disease phenotype: Mecp2+/- female animals. These features strengthen the theory that boosting adenosinergic activity may be a valid therapeutic strategy for RTT patients, regardless of their genetic penetrance.

Sulfonamide Porphyrins as Potent Photosensitizers against Multidrug-Resistant Staphylococcus aureus (MRSA): The Role of Co-Adjuvants.

Sulfonamides are a conventional class of antibiotics that are well-suited to combat infections. However, their overuse leads to antimicrobial resistance. Porphyrins and analogs have demonstrated excellent photosensitizing properties and have been used as antimicrobial agents to photoinactivate microorganisms, including multiresistant Staphylococcus aureus (MRSA) strains. It is well recognized that the combination of different therapeutic agents might improve the biological outcome. In this present work, a novel meso-arylporphyrin and its Zn(II) complex functionalized with sulfonamide groups were synthesized and characterized and the antibacterial activity towards MRSA with and without the presence of the adjuvant KI was evaluated. For comparison, the studies were also extended to the corresponding sulfonated porphyrin TPP(SO3H)4. Photodynamic studies revealed that all porphyrin derivatives were effective in photoinactivating MRSA (>99.9% of reduction) at a concentration of 5.0 µM upon white light radiation with an irradiance of 25 mW cm-2 and a total light dose of 15 J cm-2. The combination of the porphyrin photosensitizers with the co-adjuvant KI during the photodynamic treatment proved to be very promising allowing a significant reduction in the treatment time and photosensitizer concentration by six times and at least five times, respectively. The combined effect observed for TPP(SO2NHEt)4 and ZnTPP(SO2NHEt)4 with KI seems to be due to the formation of reactive iodine radicals. In the photodynamic studies with TPP(SO3H)4 plus KI, the cooperative action was mainly due to the formation of free iodine (I2).

Bioprospecting antibiotic properties in photodynamic therapy of lipids from Codium tomemtosum and Chlorella vulgaris.

The growing resistance from pathogens against antibiotics has increased the research for new compounds and strategies with antibacterial potential. Lipids from algae are emerging as natural and potent bioactive molecules with interesting results regarding the inactivation of bacteria, viruses, and fungi. The combination of algae lipids with innovative strategies, such as antibacterial photodynamic therapy (aPDT) can enhance their antimicrobial potential. In this work, we aimed to evaluate the antibacterial potential in aPDT of total lipid extracts and polar lipid fractions from the green macroalga, Codium tomentosum, and the green microalga, Chlorella vulgaris on a Gram-positive bacteria Staphylococcus aureus. Total lipid extracts and polar lipid fractions were characterized by LC-MS. The results revealed that the total extracts of algae promote S. aureus inhibition after light irradiation, with a decrease of ca. 6 log10 (CFU/mL) after 15 min of treatment with both extracts of algae. The polar lipid fractions, composed by phospholipids, glycolipids and betaine lipids, from C. tomentosum and C. vulgaris also revealed antibacterial potential in combination with aPDT, but a decrease of ca. 6 log10 (CFU/mL) was reached at 60 min of treatment, later than with the total extracts. These results unveil algae lipids as antibacterial compounds in combination with aPDT displaying an alternative from natural origin to tackle pathogen resistance.

Photodynamic inactivation of pathogenic Gram-negative and Gram-positive bacteria mediated by Si(IV) phthalocyanines bearing axial ammonium units.

The photodynamic inactivation (PDI) of microorganisms has gained interest as an efficient option for conventional antibiotic treatments. Recently, Si(IV) phthalocyanines (SiPcs) have been highlighted as promising photosensitizers (PSs) to the PDI of microorganisms due to their remarkable absorption and emission features. To increase the potential of cationic SiPcs as PS drugs, one novel (1a) and two previously described (2a and 3a) axially substituted PSs with di-, tetra-, and hexa-ammonium units, respectively, were synthesized and characterized. Their PDI effect was evaluated for the first time against Escherichia coli and Staphylococcus aureus, a Gram-negative and a Gram-positive bacterium, respectively. The photodynamic treatments were conducted with PS concentrations of 3.0 and 6.0 µM under 60 min of white light irradiation (150 mW.cm-2). The biological results show high photodynamic efficiency for di- and tetra-cationic PSs 1a and 2a (6.0 µM), reducing the E. coli viability in 5.2 and 3.9 log, respectively (after 15 min of dark incubation before irradiation). For PS 3a, a similar bacterial reduction (3.6 log) was achieved but only with an extended dark incubation period (30 min). Under the same experimental conditions, the photodynamic effect of cationic PSs 1a-3a on S. aureus was even more promising, with abundance reductions of ca. 8.0 log after 45-60 min of PDI treatment. These results reveal the high PDI efficiency of PSs bearing ammonium groups and suggest their promising application as a broad-spectrum antimicrobial to control infections caused by Gram-negative and Gram-positive bacteria.

Photoinactivation of Phage Phi6 as a SARS-CoV-2 Model in Wastewater: Evidence of Efficacy and Safety.

The last two years have been marked by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. This virus is found in the intestinal tract; it reaches wastewater systems and, consequently, the natural receiving water bodies. As such, inefficiently treated wastewater (WW) can be a means of contamination. The currently used methods for the disinfection of WW can lead to the formation of toxic compounds and can be expensive or inefficient. As such, new and alternative approaches must be considered, namely, photodynamic inactivation (PDI). In this work, the bacteriophage φ6 (or, simply, phage φ6), which has been used as a suitable model for enveloped RNA viruses, such as coronaviruses (CoVs), was used as a model of SARS-CoV-2. Firstly, to understand the virus's survival in the environment, phage φ6 was subjected to different laboratory-controlled environmental conditions (temperature, pH, salinity, and solar and UV-B irradiation), and its persistence over time was assessed. Second, to assess the efficiency of PDI towards the virus, assays were performed in both phosphate-buffered saline (PBS), a commonly used aqueous matrix, and a secondarily treated WW (a real WW matrix). Third, as WW is generally discharged into the marine environment after treatment, the safety of PDI-treated WW was assessed through the determination of the viability of native marine water microorganisms after their contact with the PDI-treated effluent. Overall, the results showed that, when used as a surrogate for SARS-CoV-2, phage φ6 remains viable in different environmental conditions for a considerable period. Moreover, PDI proved to be an efficient approach in the inactivation of the viruses, and the PDI-treated effluent showed no toxicity to native aquatic microorganisms under realistic dilution conditions, thus endorsing PDI as an efficient and safe tertiary WW disinfection method. Although all studies were performed with phage φ6, which is considered a suitable model of SARS-CoV-2, further studies using SARS-CoV-2 are necessary; nevertheless, the findings show the potential of PDI for controlling SARS-CoV-2 in WW.

ß3 Adrenoceptor-induced cholinergic inhibition in human and rat urinary bladders involves the exchange protein directly activated by cyclic AMP 1 favoring adenosine release.

BACKGROUND AND PURPOSE: The mechanism by which ß3 receptor agonists (e.g. mirabegron) control bladder overactivity may involve adenosine release from human and rat detrusor smooth muscle. Retrograde activation of adenosine A1 receptors reduces ACh release from cholinergic bladder nerves. ß3 -Adrenoceptors usually couple to adenylyl cyclase. Here we investigated, which of the cAMP targets, protein kinase A or the exchange protein directly activated by cAMP (EPAC) could be involved in this cholinergic inhibition of the bladder. EXPERIMENTAL APPROACH: [3 H]ACh and adenosine release from urothelium-denuded detrusor strips of cadaveric human organ donors and rats were measured by liquid scintillation spectrometry and HPLC, respectively. In vivo cystometry was also performed in urethane-anaesthetized rats. KEY RESULTS: The exchange protein directly activated by cAMP (EPAC) inhibitor, ESI-09, prevented mirabegron- and isoprenaline-induced adenosine release from human and rat detrusor strips respectively. ESI-09, but not the PKA inhibitor, H-89, attenuated inhibition of [3 H]ACh release from stimulated (10 Hz) detrusor strips caused by activating ß3 -adrenoceptors, AC (forskolin) and EPAC1 (8-CTP-2Me-cAMP). Isoprenaline-induced inhibition of [3 H]ACh release was also prevented by inhibitors of PKC (chelerythrine and Go6976) and of the equilibrative nucleoside transporter 1 (ENT1; dipyridamole and NBTI), but not by PLC inhibition with U73122. Pretreatment with ESI-09, but not with H-89, prevented the reduction of the voiding frequency caused by isoprenaline and forskolin in vivo. CONCLUSION AND IMPLICATIONS: Data suggest that ß3 -adrenoceptor-induced inhibition of cholinergic neurotransmission in human and rat urinary bladders involves activation of an EPAC1/PKC pathway downstream cAMP production resulting in adenosine outflow via ENT1.

An Insight Into the Potentiation Effect of Potassium Iodide on aPDT Efficacy.

Antimicrobial photodynamic therapy (aPDT) is gaining a special importance as an effective approach against multidrug-resistant strains responsible of fatal infections. The addition of potassium iodide (KI), a non-toxic salt, is recognized to increase the aPDT efficiency of some photosensitizers (PSs) on a broad-spectrum of microorganisms. As the reported cases only refer positive aPDT potentiation results, in this work we selected a broad range of porphyrinic and non-porphyrinic PSs in order to gain a more comprehensive knowledge about this aPDT potentiation by KI. For this evaluation were selected a series of meso-tetraarylporphyrins positively charged at meso positions or at ß-pyrrolic positions and the non-porphyrinic dyes Methylene blue, Rose Bengal, Toluidine Blue O, Malachite Green and Crystal Violet; the assays were performed using a bioluminescent E. coli strain as a model. The results indicate that KI has also the ability to potentiate the aPDT process mediated by some of the cationic PSs [Tri-Py(+)-Me, Tetra-Py(+)-Me, Form, RB, MB, Mono-Py(+)-Me, ß-ImiPhTPP, ß-ImiPyTPP, and ß-BrImiPyTPP] allowing a drastic reduction of the treatment time as well as of the PS concentration. However, the efficacy of some porphyrinic and non-porphyrinic PSs [Di-Py(+)-Me opp , Di-Py(+)-Me adj , Tetra-Py, TBO, CV, and MG] was not improved by the presence of the coadjuvant. For the PSs tested in this study, the ones capable to decompose the peroxyiodide into iodine (easily detectable by spectroscopy or by the visual appearance of a blue color in the presence of amylose) were the most promising ones to be used in combination with KI. Although these studies confirmed that the generation of 1O2 is an important fact in this process, the PS structure (charge number and charge position), aggregation behavior and affinity for the cell membrane are also important features to be taken in account.

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 S100ß-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.

The Effect of Ischemia and Reperfusion on Enteric Glial Cells and Contractile Activity in the Ileum.

BACKGROUND: We investigated the effects of ischemia followed by different periods of reperfusion (I/R) on immunoreactive S100ß-positive glial and Hu-immunoreactive neurons co-expressing the P2X2 receptor in the myenteric plexus of the rat ileum. METHODS: The ileal artery was occluded for 35 min with an atraumatic vascular clamp. The animals were killed 24 h, 72 h, and 1 week after ischemia. Sham animals were not submitted to ileal artery occlusion. The relative density, size, and co-localization of P2X2 receptor-expressing cells in relation to S100ß-immunoreactive glial and Hu-immunoreactive neuronal cells were evaluated. Additionally, we analyzed the effects of I/R on gastrointestinal transit and ileum contractile activity. RESULTS: The cellular density of P2X2 receptor and neuronal Hu immunoreactivity/cm(2) decreased after I/R, whereas glial S100ß immunoreactivity/cm(2) increased. No significant differences between sham and I/R groups were observed regarding the perikarya area of Hu-positive neurons. The area of S100ß-immunoreactive glial cells increased by 24.1 % 1 week after I/R compared with the 24 h group. Methylene blue progression along the small intestine decreased (P < 0.05) from 24.5 ± 2.3 % in the sham group to 17.2 ± 2.0 % 1 week post-ischemia. We noted a significant (P < 0.05) decrease in the maximal contraction amplitude triggered by electrical field stimulation in the presence of ATP in preparations submitted to 24 h of I/R. CONCLUSIONS: Changes in the P2X2 receptor density parallel myenteric neuronal loss following I/R of the rat ileum. This, together with the increase in the activated (oversized) glial cells, may contribute to decreased GI motility after I/R.

Improving the MDR reversal activity of 6,17-epoxylathyrane diterpenes.

Aiming to optimize macrocyclic lathyrane-type diterpenes as effective Pgp modulators, the phytochemical study of the methanolic extract of Euphorbia boetica aerial parts was carried out. Two new macrocyclic 6,17-epoxylathyrane-type diterpenes, named epoxyboetiranes A (1) and B (2), along with three known analogues (3-5) were isolated. Epoxyboetirane A (1), a triacetate isolated in large amounts, was hydrolyzed to give epoxylathyrol (6). In order to study the effect of the substitution pattern of the macrocyclic scaffold on MDR reversal, 6 was acylated with aroyl, phenylacetyl, cinnamoyl and alkanoyl chlorides/anhydrides, yielding eight new esters, epoxyboetiranes C-J (7-14). The ability of compounds 1-14 as P-glycoprotein (Pgp, ABCB1) modulators was evaluated through combination of transport and chemosensitivity assays, using L5178Y mouse T lymphoma cell line transfected with the human MDR1 gene. In the transport assay, excepting 1, 3 and 6, the compounds, at non-cytotoxic concentrations, displayed strong MDR reversing activity in a dose-dependent mode, exhibiting all the new acyl derivatives (7-14) a many fold increase in the activity when compared with 1. Apart from 11 and 12, all compounds exhibited remarkable synergistic effects in combination with doxorubicin. An ATPase assay, using membrane vesicles from mammalian cells overexpressing Pgp, was also performed with two representatives of the modulators (4 and 5). The results suggest that both compounds compete with substrates for the Pgp drug-binding sites.

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 A(2A) 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.

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.

Mechanisms of P-gp inhibition and effects on membrane fluidity of a new rifampicin derivative, 1,8-dibenzoyl-rifampicin.

PURPOSE: To assess P-glycoprotein (P-gp)-modulation ability and the mechanisms of P-gp inhibition mediated by a new synthetic rifampicin derivative, 1,8-dibenzoyl-rifampicin (DiBenzRif), in an in vitro model of the blood-brain barrier (BBB), RBE4 cells, and in membrane mimetic models (liposomes). METHODS: P-gp expression (western blot) and activity [rhodamine 123 accumulation studies] were assessed until 72h of exposure to DiBenzRif. The effects on intracellular ATP levels and on P-gp ATPase activity were studied using luciferin-luciferase bioluminescence assay. Membrane fluidity changes were tracked by steady-state anisotropy measurements. Non-P-gp-related rhodamine 123 accumulation was evaluated using liposomes prepared with the main lipids present in RBE4 cell membranes. RESULTS: A significant increase in intracellular rhodamine 123 content was observed in DiBenzRif-treated cells at all tested time-points. This effect was associated with a significant reduction in ATP intracellular levels, the inhibition of P-gp ATPase activity and a significant increase in membrane fluidity. DiBenzRif also favoured rhodamine 123 accumulation in a liposomal model of RBE4 cells, suggesting that it may be useful in increasing intracellular levels of substances that passively diffuse into the cells. CONCLUSION: DiBenzRif-induced inhibitory effect on P-gp increases xenobiotic accumulation in BBB cells, which may contribute to the development of therapeutic adjuvants to enhance brain penetration of drugs.

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.

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).