Cannabinoid receptor 1 signalling dampens activity and mitochondrial transport in networks of enteric neurones

Abstract

Cannabinoid (CB) receptors are expressed in the enteric nervous system (ENS) and CB(1) receptor activity slows down motility and delays gastric emptying. This receptor system has become an important target for GI-related drug development such as in obesity treatment. The aim of the study was to investigate how CB(1) ligands and antagonists affect ongoing activity in enteric neurone networks, modulate synaptic vesicle cycling and influence mitochondrial transport in nerve processes. Primary cultures of guinea-pig myenteric neurones were loaded with different fluorescent markers: Fluo-4 to measure network activity, FM1-43 to image synaptic vesicles and Mitotracker green to label mitochondria. Synaptic vesicle cluster density was assessed by immunohistochemistry and expression of CB(1) receptors was confirmed by RT-PCR. Spontaneous network activity, displayed by both excitatory and inhibitory neurones, was significantly increased by CB(1) receptor antagonists (AM-251 and SR141716), abolished by CB(1) activation (methanandamide, mAEA) and reduced by two different inhibitors (arachidonylamide serotonin, AA-5HT and URB597) of fatty acid amide hydrolase. Antagonists reduced the number of synaptic vesicles that were recycled during an electrical stimulus. CB(1) agonists (mAEA and WIN55,212) reduced and antagonists enhanced the fraction of transported mitochondria in enteric nerve fibres. We found immunohistochemical evidence for an enhancement of synaptophysin-positive release sites with SR141716, while WIN55,212 caused a reduction. The opposite effects of agonists and antagonists suggest that enteric nerve signalling is under the permanent control of CB(1) receptor activity. Using inhibitors of the endocannabinoid degrading enzyme, we were able to show there is endogenous production of a CB ligand in the ENS.

Early Emergence of Neural Activity in the Developing Mouse Enteric Nervous System

“…Calcium imaging. Changes in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) were assayed by Fluo-4 imaging as described previously Boesmans et al, 2009;Gomes et al, 2009). Cells were loaded with Fluo-4AM (5 M; Invitrogen) in HEPES solution (in mM: 148 NaCl, 5 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 glucose, 10 HEPES, with pH adjusted to 7.4 using NaOH) for 20 min, and then rinsed for 20 min with fresh solution before recording.…”

TRP channels in neurogastroenterology: opportunities for therapeutic intervention

“…Also the interaction of TRPV1 with other signalling systems, especially the protease-activated receptor (PAR) has to be addressed in more detail (Amadesi et al, 2004;. Also the relationship between TRPV1 and the cannabinoid system in the GI tract (Sanger, 2007) is of increasing interest as it has been shown that endocannabinoids are produced within the ENS (Boesmans et al, 2009;Hong et al, 2009). Interference with TRPV1 function for pure analgesic purposes furthermore warrants a better understanding of the physiological role of TRPV1 in other than neuronal tissues (Wong and Gavva, 2009).…”

Effect of rimonabant on oesophageal motor function in man

“…[36][37][38] Endocannabinoids can potentially control this vago-vagal reflex pathway at many points as CB1 receptors are expressed on vagal afferents, in the brain stem, on interneurons in the wall of the gastrointestinal tract and on postganglionic fibres innervating smooth muscle in the gastrointestinal tract. [39][40][41][42] In the dog, rimonabant only increased the number of TLESRs, whereas their characteristics and oesophageal motility were unchanged, and this was considered an argument in favour of a central site of action. 30 The observation that fasting LES pressure was not affected by rimonabant in the present study, whereas the postprandial drop in LES pressure and the occurrence of TLESRs were inhibited could also argue in favour for a The pre-treatment with rimonabant was not associated with a postprandial increase of acid reflux episodes as after placebo.…”

“…Indeed, we and others previously provided evidence for a continuous endocannabinoid tone acting on CB1 to submit neurotransmitter release in the enteric nervous system. [39][40][41][42] Rimonabant may thus enhance the release of, for instance, acetylcholine from intrinsic motor neurons, which could contribute to the observed longer duration of peristaltic contractions and higher postprandial LES pressure. The absence of any change in symptoms from the gastrointestinal tract could also be viewed as an argument against a primarily central site of action.…”