Interaction between cannabinoid CB1 receptors and endogenous ATP in the control of spontaneous mechanical activity in mouse ileum

Abstract

Background and purpose: Although it is well accepted that cannabinoids modulate intestinal motility by reducing cholinergic neurotransmission mediated by CB(1) receptors, it is not known whether the endocannabinoids are involved in more complex circuits and if they interact with other systems. The aim of the present study was to examine possible interactions between cannabinoid CB(1) receptors and purines in the control of spontaneous contractility of longitudinal muscle in mouse ileum.

Experimental approach: The mechanical activity of longitudinally oriented ileal segments from mice was recorded as isometric contractions.

Key results: The selective CB(1) receptor agonist, N-(2-chloroethyl)5,8,11,14-eicosaetraenamide (ACEA) reduced, concentration dependently, spontaneous contractions in mouse ileum. This effect was almost abolished by tetrodotoxin (TTX) or atropine. Inhibition by ACEA was not affected by theophylline (P1 receptor antagonist) or by P2Y receptor desensitization with adenosine 5'[beta-thio]diphosphate trilithium salt, but was significantly reversed by pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (P2 receptor antagonist), by P2X receptor desensitization with alpha,beta-methyleneadenosine 5'-triphosphate lithium salt (alpha,beta-MeATP) or by 8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino) bis(1,3,5-naphthalenetrisulphonic acid)] (P2X receptor antagonist). Contractile responses to alpha,beta-MeATP (P2X receptor agonist) were virtually abolished by TTX or atropine, suggesting that they were mediated by acetylcholine released from neurones, and significantly reduced by ACEA.

Conclusion and implications: In mouse ileum, activation of CB(1) receptors, apart from reducing acetylcholine release from cholinergic nerves, was able to modulate negatively, endogenous purinergic effects, mediated by P2X receptors, on cholinergic neurons. Our study provides evidence for a role of cannabinoids in the modulation of interneuronal purinergic transmission.

Figure 1

Typical tracings showing the effects of atropine (1 µmol·L⁻¹) or neostigmine (10 µmol·L⁻¹) on the spontaneous contractions of mouse ileum longitudinal muscle.

Figure 2

Typical tracings showing the inhibitory effects of the selective CB₁ receptor agonist N-(2-chloroethyl)5,8,11,14-eicosaetraenamide (ACEA; 10 µmol·L⁻¹) on the spontaneous mechanical activity of mouse ileal longitudinal muscle in control conditions or after SR141716A (100 nmol·L⁻¹), a CB₁ receptor antagonist, tetrodotoxin (TTX; 1 µmol·L⁻¹) or atropine (1 µmol·L⁻¹).

Figure 3

Concentration-response curves for the inhibitory effects induced by N-(2-chloroethyl)5,8,11,14-eicosaetraenamide (ACEA) in the control, in the presence of theophylline (10 µmol·L⁻¹), a P1 receptor antagonist or pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (PPADS; 50 µmol·L⁻¹), a P2 receptor antagonist. The inhibitory response is expressed as percent change of the resting activity (−100% corresponds to the abolition of spontaneous activity). All values are means ± standard error of the mean (n = 5 for each treatment). *P < 0.05 compared with control value.

Figure 5

Inhibitory effects induced by NF279, on the amplitude of the spontaneous contractions in the control (n = 8), in the presence of tetrodotoxin (TTX; 1 µmol·L⁻¹) (n = 4) or in the presence of atropine (1 µmol·L⁻¹) (n = 4) and on the increase of the spontaneous contractions induced by neostigmine (10 µmol·L⁻¹) (n = 4). The ordinate shows the amplitude of the contractions expressed as percent change of the resting activity. All values are means ± standard error of the mean. *P < 0.05 compared with control value. #P < 0.05 compared with neostigmine.

Figure 4

Concentration-response curves for the inhibitory effects induced by N-(2-chloroethyl)5,8,11,14-eicosaetraenamide (ACEA) in the control, after P2X receptor desensitization with α,β-methyleneadenosine 5′-triphosphate lithium salt (α,β-MeATP; 50 µmol·L⁻¹ for 30 min), after P2Y receptor desensitization with adenosine 5′[β-thio]diphosphate trilithium salt (ADPβS; 10 µmol·L⁻¹ for 30 min) or in the presence of NF279 (1 µmol·L⁻¹), a P2X receptor antagonist. The inhibitory response is expressed as percent change of the resting activity (−100% corresponds to the abolition of spontaneous activity). All values are means ± standard error of the mean (n = 4 for each treatment). *P < 0.05 compared with control value.

Figure 6

Concentration-response curves for the inhibitory effects induced by adenosine, a P1 receptor agonist, in the control or in the presence of theophylline (10 µmol·L⁻¹), a P1 receptor antagonist. The inhibitory response is expressed as percent change of the resting activity (−100% corresponds to the abolition of spontaneous activity). All values are means ± standard error of the mean (n = 4). *P < 0.05 compared with control value.

Figure 9

Concentration-response curves for the contractile effects induced by α,β-methyleneadenosine 5′-triphosphate lithium salt (α,β-MeATP), P2X receptor agonist in the control or after N-(2-chloroethyl)5,8,11,14-eicosaetraenamide (ACEA; 10 µmol·L⁻¹). All values are means ± standard error of the mean (n = 4) and are reported as a percentage of the maximal effect. *P < 0.05 compared with control value.

Figure 8

Concentration-response curves for the contractile effects induced by α,β-methyleneadenosine 5′-triphosphate lithium salt (α,β-MeATP), a P2X receptor agonist in the control, after P2X receptor desensitization with α,β-MeATP (50 µmol·L⁻¹ for 30 min), in the presence of NF279 (1 µmol·L⁻¹), tetrodotoxin (TTX; 1 µmol·L⁻¹) or atropine (1 µmol·L⁻¹). All values are means ± standard error of the mean (n = 5 for each treatment) and are reported as a percentage of the maximal effect. *P < 0.05 compared with control value.

Figure 7

Relaxant or contractile effects evoked by ATP (10 µmol·L⁻¹–1 mmol·L⁻¹), a P2 receptor agonist, in mouse ileal longitudinal muscle in control and after pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (PPADS; 50 µmol·L⁻¹), a P2 receptor antagonist, desensitization of P2Y receptors with adenosine 5′[β-thio]diphosphate trilithium salt (ADPβS; 10 µmol·L⁻¹ for 30 min) or desensitization of P2X receptors with α,β-methyleneadenosine 5′-triphosphate lithium salt (α,β-MeATP; 50 µmol·L⁻¹ for 30 min). All values are means ± standard error of the mean. (n = 5 for each treatment) and are reported as a percentage of the maximal effect. *P < 0.05 compared with control value.

Figure 10

Inhibitory effects induced by N-(2-chloroethyl)5,8,11,14-eicosaetraenamide (ACEA; 10 µmol·L⁻¹) on the contractions evoked by EFS (supramaximal voltage, 0.5 ms pulse duration, in trains of 5 s, 8 Hz) in the absence or in the presence of 8,8′-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino) bis(1,3,5-naphthalenetrisulphonic acid)] (NF279; 1 µmol·L⁻¹). All values are means ± standard error of the mean (n = 5) and are expressed as a percentage of the respective control taken as 100%. *P < 0.05 compared with control value. #P < 0.05 compared with ACEA or NF279.