Evidence that ATP or a related purine is an excitatory neurotransmitter in the longitudinal muscle of mouse distal colon

Abstract

Background and purpose: This study analysed the contribution of the purinergic system to enteric neurotransmission in the longitudinal muscle of mouse distal colon.

Experimental approach: Motor responses to exogenous ATP and to nerve stimulation in vitro were assessed as changes in isometric tension.

Key results: ATP induced a concentration-dependent contraction, reduced by 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzene disulphonic acid (PPADS), suramin, P2Y purinoreceptor desensitisation with adenosine 5'-O-2-thiodiphosphate (ADPbetaS), and atropine, but unaffected by P2X purinoceptor desensitisation with alpha,beta-methylene ATP (alpha,beta-meATP) and by 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl ester (MRS 2395), a P2Y(12) selective antagonist. The response to ATP was increased by 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate (MRS 2179), a P2Y(1) selective antagonist, tetrodotoxin (TTX) or N(omega)-nitro-L-arginine methyl ester (L-NAME). ADPbetaS, a P2Y-purinergic agonist, induced muscular contraction, with the same pharmacological profile as the ATP-induced contraction. ADP, a natural ligand for P2Y(1) receptors, induced muscular relaxation, antagonized by MRS 2179 and by TTX or L-NAME. Nerve stimulation elicited a transient nitrergic relaxation, followed by contraction. Contractile responses was reduced by atropine, PPADS, suramin, P2Y purinoceptor desensitisation, but not by P2X purinoceptor desensitisation, MRS 2179 or MRS 2395. None of the purinergic antagonists modified the nerve-evoked relaxation.

Conclusions and implications: In the longitudinal muscle of mouse distal colon, ATP, through ADPbetaS-sensitive P2Y purinoceptors, contributed to the excitatory neurotransmission acting directly on smooth muscle and indirectly via activation of cholinergic neurons. Moreover, P2Y1 purinoceptors appear to be located on nitrergic inhibitory neurons. This study provides new insights into the role of purines in the mechanism inducing intestinal transit in mouse colon.

Figure 1

Left: original tracing showing the effects of ATP (1 mM), ADPβS (100 μM) and α,β-meATP (100 μM) on spontaneous mechanical activity of longitudinal muscle of mouse distal colon. Right: concentration–response curves to ATP (n=20 except for 3 mM ATP that is from four animals) and ADPβS (n=20 except for 300 μM ADPβS that is from four animals) in longitudinal muscle of mouse distal colon. Data are means±s.e.m. and are expressed in absolute values. ^*P<0.05 when the concentration–response curves were compared.

Figure 2

Concentration–response curves to ATP before and after PPADS (500 μM; n=4), suramin (100 μM; n=6), α,β-meATP (1 μM for 30 min, n=4), ADPβS (10 μM for 30 min; n=5), MRS 2179 (1 μM; n=4), MRS 2395 (1 μM; n=4) in longitudinal muscle of mouse distal colon. Data are means±s.e.m. and are expressed in absolute values. The values for the control curves are the means of the control data obtained before each treatment. ^*P<0.05 when the concentration–response curves were compared with those obtained in the respective control condition.

Figure 3

Concentration–response curves to ATP before and after atropine (1 μM; n=6) or indomethacin (10 μM; n=6), TTX (1 μM, n=5) or L-NAME (100 μM; n=4) in longitudinal muscle of mouse distal colon. Data are means±s.e.m. and are expressed in absolute values. The values for the control curves are the means of the control data obtained before each treatment. ^*P<0.05 when the concentration–response curves were compared with those obtained in the respective control condition.

Figure 4

Concentration–response curves to ADPβS before and after PPADS (500 μM; n=5), suramin (100 μM; n=6), α,β-meATP (1 μM for 30 min, n=4), ADPβS (10 μM for 30 min; n=5), MRS 2179 (1 μM; n=5), MRS 2395 (1 μM; n=4) in longitudinal muscle of mouse distal colon. Data are means±s.e.m. and are expressed in absolute values. The values for the control curves are the means of the control data obtained before each treatment. ^*P<0.05 when the concentration–-response curves were compared with those obtained in the respective control condition.

Figure 5

Concentration–response curves to ADPβS before and after atropine (1 μM; n=6) or indomethacin (10 μM; n=6), TTX (1 μM, n=5) or L-NAME (100 μM; n=6) in longitudinal muscle of mouse distal colon. Data are means±s.e.m. and are expressed in absolute values. The values for the control curves are the means of the control data obtained before each treatment. ^*P<0.05 when the concentration–response curves were compared with those obtained in the respective control condition.

Figure 6

Left: original tracing showing the effects of ADP (3 mM) on spontaneous mechanical activity of longitudinal muscle of mouse distal colon. Right: concentration–response curves to ADP before and after MRS 2179 (1 μM; n=4), TTX (1 μM, n=5) or L-NAME (100 μM; n=5) in longitudinal muscle of mouse distal colon. Data are means±s.e.m. and are expressed as a percentage reversal of the initial level of active force, 100% inhibition corresponding to total suppression of spontaneous contractions. The values for the control curves are the means of the control data obtained before each treatment. ^*P<0.05 when the concentration–response curves were compared with those obtained in the respective control condition.

Figure 7

Non-adrenergic, non-cholinergic (NANC) responses evoked by electrical field stimulation (EFS) in longitudinal colonic muscle preparations. Left: typical tracings showing the NANC-evoked responses induced by EFS in mouse colonic segments (0.5 ms, 4 Hz, supramaximal voltage for 10 s). Right: histograms showing the effects of the different drugs on the amplitude of the relaxation and of the contraction evoked by EFS (0.5-ms 4 Hz, supramaximal voltage for 10 s) in the longitudinal muscle of distal mouse colon. Data are means±s.e.m. (n=4–6 for each antagonist) and are expressed as a percentage of the respective control taken as 100%. The values for the control are the means of the control data obtained before each treatment. ^*P<0.05 when compared with the respective control. ^§P< 0.05 when compared with atropine.