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Title: Studies on cannabinoid effects in intestinal tissue and in neuroblastoma cells
Author: Sones, William Rupert
ISNI:       0000 0001 3470 7029
Awarding Body: University of Hertfordshire
Current Institution: University of Hertfordshire
Date of Award: 2007
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The growing numbers of putative receptors and allosteric sites in which cannabinoids have been observed to act upon has called in to question the established action of cannabinoids through solely the CB1 receptor in the guinea-pig ileum myenteric plexus. Previous work within our laboratory demonstrated cannabinoid inhibition of nicotine induced currents in cultured myenteric neurones through a mechanism expressing different pharmacological properties to those acknowledged as being ('. possessed by the CBI· receptor. To examine-this the effects of cannabinoid receptor ligands were examined on nicotine evoked contractions of the guinea-pig ileum myenteric plexus-longitudinal muscle preparation and on nicotine evoked currents in primary cultures ofmyenteric neurones. Contraction evoked by 100 JlM nicotine was inhibited by the cannabinoid agonist CP 55,940 (ICso 215 nM) but inhibition was not blocked by the CB1 receptor selective antagonist SR141716. Nicotinic log concentration-response curves showed a combination of some rightward displacement and decrease in maximal contraction ,. upon exposure to CP 55,940 suggesting non-competitive inhibition. In the presence of the voltage-gated sodium channel antagonist tetrodotoxin (TTX), both CP 55,940 and SR141716 inhibited contraction and acted additively when applied in conjunction to produce greater inhibition of nicotine evoked contraction. Some degree of stereoselectivity was observed for the cannabinoid agonist WIN 55,212-2 over the stereoisomer WIN 55,212-3, but in the presence ofTTX both stereoisomers displayed equal potency in inhibiting nicotine evoked contraction. Contractions evoked in the presence of the nicotinic receptor antagonist tubocurarine and those evoked by 10 JlM nicotine showed greater CP 55,940 inhibition (ICso 0.74 and 0.55 nM respectively) than that observed with 100 JlM nicotine. CP 55,940 inhibition of contraction evokedby 10 IlM nicotine displayed SR141716 sensitivity with log concentration-response. curves showing a parallel rightward shift (lCso 19.5 nM). Whole cell patch clamp of cultured myenteric neurones confirmed cannabinoid inhibition of nicotine evoked currents and displayed similar levels of inhibition by both active and inactive WIN 55,212 stereoisomers. Blockade of the adenylate cyclase/cAMP intracellular messaging system by 8-Br-cAMP did not affect CP 55,940 inhibition. Results suggest that cannabinoids may act through both CBI receptor dependent and independent mechanisms to inhibit nicotine evoked contraction in the myenteric (' plexus. The mechanisms through which cannabinoids act demonstrated some degree of selectivity dependent upon the level of stimulus evoked by nicotine, with contraction evoked by 100 IlM nicotine demonstrating CBI receptor independence whilst contraction evoked by 10 IlM nicotine showed SR141716 sensitivity. Blockade of action potential propagation by TTX showed a CB1 receptor independent inhibition of contraction occurring within the nerve endings of motor neurones. Whole cell recording confirmed previous work with the lack of WIN 55.212 stereoselectivity corroborating CBI receptor independent inhibition. The inhibition by cannabinoids of electrically induced contraction of the guinea-pig is well accepted. However, the intracellular mechanisms through which cannabinoid receptors act have yet to be established. The effects of cannabinoid receptor ligands on Ca2+ currents were examined in primary cultures of guinea-pig ileum myenteric AH neurones using the whole-cell patch clamp technique. Membrane depolarisation evoked a high-voltage activated calcium current (lca) that was reduced by 65.6% by c.o-conotoxin GVIA (100 nM). Exposure to the cannabinoid agonists CP 55,940 (10 JlM) and WIN 55,212-2 (10 JlM) reduced peak 1ca by 79.7% and 74.7% respectively. The inhibitory effects of CP 55,940 were concentration . dependent over the range 0.3 - 10 JlM with an ICso of 0.54 JlM, and blocked by pertussis toxin (lOO ng.mrl , 18 hr equilibration). WIN 55,212-3 (lO JlM), the (-)- enantiomer of WIN 55,212-2, evoked significantly less inhibition tha~ that produced by WIN 55,212-2, reducing peak lea by 43.1 %. The CBl receptor selective antagonist, SR141716 (l and 10 JlM), had no effect on CP 55,940 (l and 10 JlM) inhibition, whilst another CBl receptor selective antagonist, AM251 (lO JlM), and the CB2 receptor selective antagonist SR144528 (1 Jl~) produced only slight blockade of CP 55,940 (1 JlM) inhibition. In combination, SR141716 (1 JlM) and SR144528 (0.1 JlM) produced complete blockade of CP 55,940 (1 JlM) inhibition. When tested on their own, SR141716 (lO JlM) and the putative 'silent antagonist' 0-2050 (lO JlM) reduced lea by 53.0% and 82.3% respectively. Results suggest that in primary cultures of guinea-pig ileum myenteric AH neurones, cannabinoids inhibit N-type calcium channels through Gilo coupled receptors, comprising of either a combination of CBt and CB2 receptors, or involve a novel ,. cannabinoid receptor. Cannabinoids and opioids have been shown to possess similar pharmacological properties, including analgesia, addiction and euphoria. The powerful analgesic effects of opioids make them vital in the treatment of patients, but their undesirable effects limit their use. The interaction of cannabinoids and opioids opens the possibility of achieving similar or greater beneficial effects whilst reducing undesirable effects through the use oflower amounts ofdrug. The interaction between cannabinoid and opioid receptor ligands.was examined on electrically evoked contractions of the guinea-pig ileum myenteric plexus-longitudinal muscle preparation, and their action in both primary myenteric and NG108-15 neuronal cell cultures investigated. Further examination of the action of cannabinoids on opioidwithdrawal evoked contractions ofthe whole ileum was performed. Both morphine and CP 55,940, when applied independently, produced a concentration-dependent inhibition of electrically evoked contraction of the MPLM preparation. With prior application of CP 55,940, at concentrations of 1 and 10 nM which produced inhibition of 12.0 ± 2.9% (n = 7) and 26.8 ± 5.6% (n = 11) respectively, morphine displayed similar co?~entration-dependent characteristics as displayed in the absence of CP 55,940, suggesting a purely additive interaction. 100 nM CP 55,940 inhibited all morphine evoked inhibition. An inhibition of 40.8± 7.5% (n = 9) was produced by 100 nM CP 55,940 in the absence of morphine, whilst in combination with 1 JlM morphine, which produced maximum inhibition, an inhibition of 46.8 ± 6.8% (n = 9) was observed. Maximal inhibition produced by morphine in the presence of 100 nM CP 55,940 (46.8 ± 6.8%, n = 9) was smaller that that produced in the presence of 10 nM CP 55,940 (62.7 ± 3.4%, n = 11). Prior application of 100 nM and 1 JlM morphine prevented 1 JlM WIN 55,212-2 inducing inhibition of contraction. SR141716 (100 nM) significantly increased the size of electrically evoked contraction and increased inhibition produced by morphine. Opioidwithdrawal contractions of whole ileum were not inhibited by CP 55,940. Both potassium and calcium currents were not affected by morphine or DADLE. In NG108-15 neurones calcium currents were inhibited by DADLE and WIN 55,212-2 in a concentration-dependent manner. In the presence of a concentration of WIN 55,212-2 that produced maximal inhibition, DADLE, also at a concentration producing maximal inhibition, produced a significantly reduced inhibition.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available