Relative contribution of ecto-ATPase and ecto-ATPDase pathways to the biphasic effect of ATP on acetylcholine release from myenteric motoneurons

Abstract

Background and purpose: The relative contribution of distinct ecto-nucleotidases to the modulation of purinergic signalling may depend on differential tissue distribution and substrate preference.

Experimental approach: Extracellular ATP catabolism (assessed by high-performance liquid chromatography) and its influence on [(3)H]acetylcholine ([(3)H]ACh) release were investigated in the myenteric plexus of rat ileum in vitro.

Key results: ATP was primarily metabolized via ecto-ATPDase (adenosine 5'-triphosphate diphosphohydrolase) into AMP, which was then dephosphorylated into adenosine by ecto-5'-nucleotidase. Alternative conversion of ATP into ADP by ecto-ATPase (adenosine 5'-triphosphatase) was more relevant at high ATP concentrations. ATP transiently increased basal [(3)H]ACh outflow in a 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP)-dependent, tetrodotoxin-independent manner. ATP and ATPgammaS (adenosine 5'-[gamma-thio]triphosphate), but not alpha,beta-methyleneATP, decreased [(3)H]ACh release induced by electrical stimulation. ADP and ADPbetaS (adenosine 5'[beta-thio]diphosphate) only decreased evoked [(3)H]ACh release. Inhibition by ADPbetaS was prevented by MRS 2179 (2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate diammonium salt, a selective P2Y(1) antagonist); blockade of ADP inhibition required co-application of MRS 2179 plus adenosine deaminase (which inactivates endogenous adenosine). Blockade of adenosine A(1) receptors with 1,3-dipropyl-8-cyclopentyl xanthine enhanced ADPbetaS inhibition, indicating that P2Y(1) stimulation is cut short by tonic adenosine A(1) receptor activation. MRS 2179 facilitated evoked [(3)H]ACh release, an effect reversed by the ecto-ATPase inhibitor, ARL67156, which delayed ATP conversion into ADP without affecting adenosine levels.

Conclusions and implications: ATP transiently facilitated [(3)H]ACh release from non-stimulated nerve terminals via prejunctional P2X (probably P2X(2)) receptors. Hydrolysis of ATP directly into AMP by ecto-ATPDase and subsequent formation of adenosine by ecto-5'-nucleotidase reduced [(3)H]ACh release via inhibitory adenosine A(1) receptors. Stimulation of inhibitory P2Y(1) receptors by ADP generated alternatively via ecto-ATPase might be relevant in restraining ACh exocytosis when ATP saturates ecto-ATPDase activity.

Figure 1

Effect of ATP (30 µmol·L⁻¹) on tritium outflow from the longitudinal muscle-myenteric plexus of the rat ileum. The time course of tritium outflow taken from a typical experiment is shown. Tritium outflow (ordinate) is expressed as a percentage of the total radioactivity present in the tissue at the beginning of the collection period (Fractional Release, %). The release of [³H]acetylcholine ([³H]ACh) in response to electrical field stimulation (EFS; 200 pulses of 1 ms duration delivered at a 5 Hz frequency) was elicited twice during the periods indicated (S₁ and S₂). ATP (30 µmol·L⁻¹) was applied 15 min before S₂ (as represented by the horizontal bar). The time course of tritium outflow in a control situation, that is, in the absence of ATP, is also shown for comparison. Note that ATP (30 µmol·L⁻¹) transiently increased the resting tritium outflow, but decreased the release of [³H]ACh from the stimulated myenteric plexus.

Figure 2

Time course of extracellular (A) ATP, (B) ADP and (C) AMP metabolism in the myenteric plexus of the rat ileum. Adenine nucleotides (30 µmol·L⁻¹) were added at time zero to the preparations, and samples (75 µL) were collected from the bath at indicated times on the abscissa. Each collected sample was analysed by high-performance liquid chromatography to separate and quantify ATP, ADP, AMP, adenosine (ADO), inosine (INO) and hypoxanthine (HXP). Averaged results obtained in (A) six, (B) four and (C) four experiments; the vertical bars represent SEM and are shown when they exceed the symbols in size. In control conditions, without the addition of ATP, ADP or AMP, only inosine monophosphate could be detected in the bath, reaching a maximum concentration (1.65 µmol·L⁻¹) at the end of 45 min. In (D), are represented the semi-logarithmic progress curves obtained by polynomial fitting of the catabolism of ATP (30 µmol·L⁻¹, filled circles), ADP (30 µmol·L⁻¹) and AMP (30 µmol·L⁻¹). Note that ATP, ADP and AMP linearly disappeared from the bath; the calculated half-degradation times appear in the text.

Figure 9

Schematic representation of the modulatory role of ATP and its metabolites on [³H]ACh release from myenteric neurons of the rat ileum. The myenteric plexus contains the enzymes responsible for the catabolism of ATP released from activated smooth muscle cells as well as from stimulated myenteric neurons. ATP is converted preferentially into AMP via ecto-ATPDase (EC 3.6.1.5), which is then sequentially dephosphorylated into ADO by ecto-5′-nucleotidase (5′-NTase, EC 3.1.3.5); the alternative pathway, conversion of ATP into ADP via ecto-ATPase (EC 3.6.1.3), only becomes relevant upon increasing the extracellular concentration of ATP. Understanding the effective contribution of the ATP breakdown via ecto-ATPase (generating ADP) and via ecto-ATPDase (bypassing ADP formation) is of central importance to predict the fine tuning of purinergic control of gut motility. Data suggest that ATP transiently activates facilitatory P2X receptors mediating spontaneous [³H]ACh release, while ATP metabolites, like ADP and ADO, interplay to control evoked transmitter release by activating inhibitory P2Y₁ and A₁ receptors respectively. [³H]ACh, [³H]acetylcholine; ADO, adenosine; ADPβS, adenosine 5′[β-thio]diphosphate; ATPase, adenosine 5′-triphosphatase; ATPDase, adenosine 5′-triphosphate diphosphohydrolase; R-PIA, R-N⁶-phenylisopropyl adenosine.

Figure 3

Semi-logarithmic progress curves obtained for the extracellular catabolism of ATP in the absence and in the presence of the ecto-ATPase (adenosine 5′-triphosphatase) inhibitor, ARL 67156 (6-N,N-diethyl-D-β,γ-dibromomethylene-D-adenosine-5-triphosphate) (100 µmol·L⁻¹). ATP (30 µmol·L⁻¹) was added at time zero to the preparations in the absence and in the presence of ARL 67156 (100 µmol·L⁻¹). Samples (75 µL) were collected at the times indicated on the abscissa and retained for high-performance liquid chromatography to separate and quantify (A) ATP, (B) ADP, (C) AMP and (D) adenosine (ADO). Both progress curves were obtained from the same preparations; in the absence of ARL 67156 (100 µmol·L⁻¹), time-matched results did not significantly (P > 0.05) differ from the control situation (Figure 2A). Semi-logarithmic curves were obtained by polynomial fitting from an average of six experiments; for the sake of clarity bars representing SEM are not shown. Note that ARL 67156 (100 µmol·L⁻¹) reduced (A) ATP catabolism and delayed (B) the formation of ADP, without affecting the profile of (C) AMP and (D) ADO generation. In control conditions, without the addition of ATP, only inosine monophosphate could be detected in the bath, reaching a maximum concentration (1.15 µmol·L⁻¹) at the end of 45 min.

Figure 4

Kinetics of extracellular ATP breakdown and metabolite formation upon increasing the initial concentration of the substrate. ATP (10–100 µmol·L⁻¹) was added at time zero to the preparations. Samples (75 µL) collected at the times indicated on the abscissa were retained for high-performance liquid chromatography to separate and quantify (A) ATP, and its metabolites (B) ADP or AMP and (C) adenosine (ADO) plus inosine (INO). In (A), semi-logarithmic progress curves were obtained by polynomial fitting of the catabolism of ATP (10–100 µmol·L⁻¹). Note that the kinetics of ATP breakdown was not affected by the concentration of the initial substrate. In (B), shown is the relative amount of ADP and AMP as compared with the total amount of nucleotides [(ATP + ADP + AMP)] in the bath following 15 min incubation with ATP (10–100 µmol·L⁻¹). In (C), the ratio [nucleosides][total nucleotides]⁻¹ following 15 min incubation with ATP (10–100 µmol·L⁻¹) as a direct measure of the activity of ecto-5′-nucleotidase is shown. Data shown are pooled from the number of experiments shown in parentheses. The vertical bars represent SEM and are shown when they exceed the symbols in size.

Figure 5

Extracellular ATP transiently activates P2X receptors mediating spontaneous [³H]ACh release from myenteric nerve terminals. In (A), the ordinates represent spontaneous tritium outflow (as Fractional Release %; see Methods) induced by increasing extracellular ATP concentrations (1–300 µmol·L⁻¹). ATP was applied as indicated in Figure 1. Each column represents pooled data from five to six experiments. The vertical bars represent SEM. In (B) and (C), shown is the time course of [³H]ACh release induced by ATP (100 µmol·L⁻¹, arrow) in the absence and in the presence of (B) two non-selective P2X antagonists, PPADS (10 µmol·L⁻¹) and TNP-ATP (10 µmol·L⁻¹), (C) TTX (1 µmol·L⁻¹, an action potential generation blocker) and Tyrode's solution without Ca²⁺ plus EGTA (1 mmol·L⁻¹). Tritium outflow (ordinates) is expressed as Fractional Release, %. The abscissa indicates the times at which the samples were collected. Each point is pooled data (±SEM) from five to six experiments. None of the drugs changed spontaneous tritium outflow. [³H]ACh, [³H]acetylcholine; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; PPADS, pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) tetrasodium salt; TNP-ATP, 2′,3′-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate; TTX, tetrodotoxin.

Figure 6

Effects of exogenously added adenine nucleotides on [³H]ACh release from myenteric neurons stimulated with EFS. The release of [³H]ACh in response to EFS (200 pulses of 1 ms duration delivered at a 5 Hz frequency) was elicited twice (S₁ and S₂). (A) Nucleotide triphosphates (ATP, ATPγS and α,β-MeATP) and (B) diphosphates (ADP, ADPβS and 2-MeSADP) were added 15 min before S₂ in a 30 µmol·L⁻¹ concentration. The ordinates are percentage changes in S₂/S₁ ratios compared with controls. Each column represents pooled data (±SEM) from an n number of experiments shown in parentheses. *P < 0.05 (one-way anova followed by Dunnett's modified t-test) when compared with zero percentage of change. 2-MeSADP, 2-methylthio-adenosine diphosphate; [³H]ACh, [³H]acetylcholine; EFS, electrical field stimulation; α,β-MeATP, α,β-methylene adenosine 5′-triphosphate; ADPβS, adenosine 5′[β-thio]diphosphate; ATPγS, adenosine 5′-[γ-thio]triphosphate.

Figure 7

Effect of exogenously added ADP on [³H]ACh release from myenteric neurons stimulated by EFS in the absence and in the presence of the selective P2Y₁ receptor antagonist, MRS 2179 (0.3 µmol·L⁻¹) and of ADA (0.5 U·mL⁻¹). The release of [³H]ACh in response to EFS (200 pulses of 1 ms duration delivered at a 5 Hz frequency) was elicited twice (S₁ and S₂). ADP (100 µmol·L⁻¹) was added 15 min before S₂; MRS 2179 (0.3 µmol·L⁻¹) and/or ADA (0.5 U·mL⁻¹) were added to the incubation media at the beginning of the release period (time zero) and were present throughout the assay, including S₁ and S₂. The ordinates are percentage changes in S₂/S₁ ratios compared with controls. The average S₂/S₁ ratios in the presence of MRS 2179 (0.3 µmol·L⁻¹, 0.87 ± 0.04, n = 6) and of ADA (0.5 U·mL⁻¹, 0.85 ± 0.06, n = 4) (without ADP) were not significantly (P > 0.05) different from the control value (0.83 ± 0.11, n = 4; data not shown). Each column represents pooled data (±SEM) from an n number of experiments shown in parentheses. *P < 0.05 (one-way anova followed by Dunnett's modified t-test), significant differences compared with the effect of ADP in the absence of MRS 2179 and/or ADA. [³H]ACh, [³H]acetylcholine; ADA, adenosine deaminase; EFS, electrical field stimulation; MRS 2179, 2′-deoxy-N⁶-methyl adenosine 3′,5′-diphosphate diammonium salt.

Figure 8

Crosstalk between inhibitory P2Y₁ and A₁ receptors regulating [³H]ACh release from myenteric neurons stimulated by EFS. The release of [³H]ACh in response to EFS (200 pulses of 1 ms duration delivered at a 5 Hz frequency) was elicited twice (S₁ and S₂). The ordinates are percentage changes in S₂/S₁ ratios compared with controls. (A) ADPβS (0.3–30 µmol·L⁻¹) and (B) R-PIA (30–300 nmol·L⁻¹) were added 15 min before S₂; MRS 2179 (0.3 µmol·L⁻¹) and DPCPX (10 nmol·L⁻¹) were added to the incubation media at the beginning of the release period (time zero) and were present throughout the assay, including S₁ and S₂. The average S₂/S₁ ratios in the presence of MRS 2179 (0.3 µmol·L⁻¹, 0.87 ± 0.04, n = 6) and of DPCPX (10 nmol·L⁻¹, 0.89 ± 0.07, n = 6) alone were not significantly (P > 0.05) different from the control value (0.83 ± 0.11, n = 4; data not shown). Each column represents pooled data (±SEM) from an n number of experiments shown in parentheses. *P < 0.05 (one-way anova followed by Dunnett's modified t-test), significant differences compared with the inhibitory effects of (A) ADPβS (30 µmol·L⁻¹) and (B) R-PIA (300 nmol·L⁻¹) in control conditions respectively. In (C), ADPβS (30 µmol·L⁻¹) was applied 15 min before S₂ in the absence and in the presence of R-PIA (300 nmol·L⁻¹, applied in S₁ and S₂), and vice versa. Each column represents pooled data (±SEM) from the number of experiments shown in parentheses. *P < 0.05 (one-way anova followed by Dunnett's modified t-test), significant differences compared with the inhibitory effects of ADPβS (30 µmol·L⁻¹) or R-PIA (300 nmol·L⁻¹) in the absence of the other modulator respectively. [³H]ACh, [³H]acetylcholine; ADPβS, adenosine 5′[β-thio]diphosphate; DPCPX, 1,3-dipropyl-8-cyclopentyl xanthine; EFS, electrical field stimulation; MRS 2179, 2′-deoxy-N⁶-methyl adenosine 3′,5′-diphosphate diammonium salt; R-PIA, R-N⁶-phenylisopropyl adenosine.