Laura Oliveira

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.

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.

Protein Kinase A and Cav1 (L-Type) Channels Are Common Targets to Facilitatory Adenosine A2A and Muscarinic M1 Receptors on Rat Motoneurons

At the rat motor endplate, pre-synaptic facilitatory adenosine A_2A and muscarinic M₁ receptors are mutually exclusive. We investigated whether these receptors share a common intracellular signalling pathway. Suppression of McN-A-343-induced M₁ facilitation of [³H]ACh release was partially recovered when CGS21680C (an A_2A agonist) was combined with the cyclic AMP antagonist Rp-cAMPS. Forskolin, rolipram and 8-bromo-cyclic AMP mimicked CGS21680C blockade of M₁ facilitation. Both Rp-cAMPs and nifedipine reduced augmentation of [³H]ACh release by McN-A-343 and CGS21680C. Activation of M₁ and A_2A receptors enhanced Ca²⁺ recruitment through nifedipine-sensitive channels. Nifedipine inhibition revealed by McN-A-343 was prevented by chelerythrine (a PKC inhibitor) and Rp-cAMPS, suggesting that Ca_v1 (L-type) channels phosphorylation by PKA and PKC is required. Rp-cAMPS inhibited [³H]ACh release in the presence of phorbol 12-myristate 13-acetate, but PKC inhibition by chelerythrine had no effect on release in the presence of 8-bromo-cyclic AMP. This suggests that the involvement of PKA may be secondary to M₁-induced PKC activation. In conclusion, competition of M₁ and A_2A receptors to facilitate ACh release from motoneurons may occur by signal convergence to a common pathway involving PKA activation and Ca²⁺ influx through Ca_v1 (L-type) channels.

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.

Tetanic failure due to decreased endogenous adenosine A2A tonus operating neuronal Cav1 (L-type) influx in Myasthenia gravis

J. Neurochem. (2011) 117, 797–811.

Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate

At the rat motor nerve terminals, activation of muscarinic M(1) receptors negatively modulates the activity of inhibitory muscarinic M(2) receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involved endogenous adenosine tonically activating A(1) receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [(3)H]-choline (2.5 microCi/ml). Selective activation of muscarinic M(1) and adenosine A(1) receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 microM) and R-N(6)-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [(3)H]-ACh release induced by muscarinic M(2) receptor activation with oxotremorine (10 microM). Attenuation of the inhibitory effect of oxotremorine (10 microM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M(1) autoreceptors, suggesting that suppression of M(2)-inhibiton by A(1) receptor activation is independent on muscarinic M(1) receptor activity. Conversely, the negative crosstalk between M(1) and M(2) autoreceptors seems to involve endogenous adenosine tonically activating A(1) receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 microM) by McN-A-343 (3 microM) was suppressed by the A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 microM). The results suggest that the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A(1) receptors at the rat motor endplate.

Deficits in Endogenous Adenosine Formation by Ecto-5′-Nucleotidase/CD73 Impair Neuromuscular Transmission and Immune Competence in Experimental Autoimmune Myasthenia Gravis

AMP dephosphorylation via ecto-5′-nucleotidase/CD73 is the rate limiting step to generate extracellular adenosine (ADO) from released adenine nucleotides. ADO, via A2A receptors (A2ARs), is a potent modulator of neuromuscular and immunological responses. The pivotal role of ecto-5′-nucleotidase/CD73, in controlling extracellular ADO formation, prompted us to investigate its role in a rat model of experimental autoimmune myasthenia gravis (EAMG). Results show that CD4+CD25+FoxP3+ regulatory T cells express lower amounts of ecto-5′-nucleotidase/CD73 as compared to controls. Reduction of endogenous ADO formation might explain why proliferation of CD4+ T cells failed upon blocking A2A receptors activation with ZM241385 or adenosine deaminase in EAMG animals. Deficits in ADO also contribute to neuromuscular transmission failure in EAMG rats. Rehabilitation of A2AR-mediated immune suppression and facilitation of transmitter release were observed by incubating the cells with the nucleoside precursor, AMP. These findings, together with the characteristic increase in serum adenosine deaminase activity of MG patients, strengthen our hypothesis that the adenosinergic pathway may be dysfunctional in EAMG. Given that endogenous ADO formation is balanced by ecto-5′-nucleotidase/CD73 activity and that A2ARs exert a dual role to restore use-dependent neurocompetence and immune suppression in myasthenics, we hypothesize that stimulation of the two mechanisms may have therapeutic potential in MG.

L‐Citrulline inhibits [3H]acetylcholine release from rat motor nerve terminals by increasing adenosine outflow and activation of A1 receptors

Nitric oxide (NO) production and depression of neuromuscular transmission are closely related, but little is known about the role of L‐citrulline, a co‐product of NO biosynthesis, on neurotransmitter release.

Tuning adenosine A1 and A2A receptors activation mediates l-citrulline-induced inhibition of [3H]-acetylcholine release depending on nerve stimulation pattern

The influence of nerve stimulation pattern on transmitter release inhibition by L-citrulline, the co-product of NO biosynthesis by nitric oxide synthase (NOS), was studied in the rat phrenic nerve-hemidiaphragm. We also investigated the putative interactions between NOS pathway and the adenosine system. L-citrulline (10-470 microM), the NOS substrate L-arginine (10-470 microM) and the NO donor 3-morpholinylsydnoneimine (SIN-1, 1-10 microM), concentration-dependently inhibited [(3)H]-acetylcholine ([(3)H]-ACh) release from rat motor nerve endings. Increasing stimulus frequency from 5 Hz-trains to 50 Hz-bursts enhanced [(3)H]-ACh release inhibition by l-arginine (47 microM) and L-citrulline (470 microM), whereas the effect of SIN-1 (10 microM) remained unchanged. NOS inhibition with N(omega)-nitro-L-arginine (100 microM) prevented the effect of L-arginine, but not that of L-citrulline. Adenosine deaminase (2.5 U/ml) and the adenosine transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine (10 microM), attenuated release inhibition by L-arginine and L-citrulline. With 5 Hz-trains, blockade of A(1) receptors with 1,3-dipropyl-8-cyclopentyl xanthine (2.5 nM), but not of A(2A) receptors with ZM241385 (10nM), reduced the inhibitory action of l-arginine and L-citrulline; the opposite was verified with 50 Hz-bursts. Blockade of muscarinic M(2) autoreceptors with AF-DX116 (10 nM) also attenuated the effects of L-arginine and L-citrulline with 50 Hz-bursts. L-citrulline (470 microM) increased basal adenosine outflow via the equilibrative nucleoside transport system sensitive to NBTI (10 microM), without significantly (P>0.05) changing the nucleoside release subsequent to nerve stimulation. Data indicate that NOS-derived L-citrulline negatively modulates [(3)H]-ACh release by increasing adenosine outflow channelling to A(1) and A(2A) receptors activation depending on the stimulus paradigm. While adenosine acts predominantly at inhibitory A(1) receptors during 5 Hz-trains, inhibition of ACh release by L-citrulline at 50 Hz-bursts depends on the interplay between adenosine A(2A) and muscarinic M(2) receptors.

Amplification of neuromuscular transmission by methylprednisolone involves activation of presynaptic facilitatory adenosine A2A receptors and redistribution of synaptic vesicles.

The mechanisms underlying improvement of neuromuscular transmission deficits by glucocorticoids are still a matter of debate despite these compounds have been used for decades in the treatment of autoimmune myasthenic syndromes. Besides their immunosuppressive action, corticosteroids may directly facilitate transmitter release during high-frequency motor nerve activity. This effect coincides with the predominant adenosine A2A receptor tonus, which coordinates the interplay with other receptors (e.g. muscarinic) on motor nerve endings to sustain acetylcholine (ACh) release that is required to overcome tetanic neuromuscular depression in myasthenics. Using myographic recordings, measurements of evoked [(3)H]ACh release and real-time video microscopy with the FM4-64 fluorescent dye, results show that tonic activation of facilitatory A2A receptors by endogenous adenosine accumulated during 50 Hz bursts delivered to the rat phrenic nerve is essential for methylprednisolone (0.3 mM)-induced transmitter release facilitation, because its effect was prevented by the A2A receptor antagonist, ZM 241385 (10 nM). Concurrent activation of the positive feedback loop operated by pirenzepine-sensitive muscarinic M1 autoreceptors may also play a role, whereas the corticosteroid action is restrained by the activation of co-expressed inhibitory M2 and A1 receptors blocked by methoctramine (0.1 μM) and DPCPX (2.5 nM), respectively. Inhibition of FM4-64 loading (endocytosis) by methylprednisolone following a brief tetanic stimulus (50 Hz for 5 s) suggests that it may negatively modulate synaptic vesicle turnover, thus increasing the release probability of newly recycled vesicles. Interestingly, bulk endocytosis was rehabilitated when methylprednisolone was co-applied with ZM241385. Data suggest that amplification of neuromuscular transmission by methylprednisolone may involve activation of presynaptic facilitatory adenosine A2A receptors by endogenous adenosine leading to synaptic vesicle redistribution.