The general aim of the study was to investigate the signalling pathways utilised by cannabinoids. Cannabinoid CB1 receptor stimulation in DDT, MF-2 smooth muscle cells induces a rise in [Ca2], which is dependent on extracellular Cat' and modulated by thapsigargin-sensitive stores and MAP kinase suggesting capacitative Ca2+ entry (CCE). Non-capacitative calcium entry (NCCE) stimulated by arachidonic acid (AA) partly mediates histamine Hl receptor-evoked increases in [Ca2+]; in DDTI MF-2 cells. In the current study both Ca 2+ entry mechanisms and a possible link between MAP kinase activation and increasing [Ca2+];, were investigated. In the whole-cell patch clamp configuration, the cannabinoid receptor agonist CP 55,940 evoked a transient Cat+-dependent K+ current, which was not blocked by inhibitors of CCE, 2-APB and SKF 96365, although SKF 96365 did inhibit the outward current evoked by the refilling component of the response to histamine. AA but not its metabolites evoked a transient outward current and inhibited the response to CP 55,940 in a concentration-dependent manner. CP 55,940 induced a concentration-dependent release of AA, which was inhibited by the CB1 receptor antagonist SR 141716A. The non-specific Ca2+ channel blockers, La3+ and Gd3+, inhibited the CP 55,940-induced current at concentrations that had no effect on thapsigargin-evoked CCE. La3+ also inhibited AA-mediated currents. The effect of CP 55,940 on AA release was abolished by phospholipase A2 inhibition with quinacrine. This compound also inhibited outward currents mediated by CP 55,940. The data supports the possibility that in DDT, MF-2 cells AA is an integral component of the CBI receptor signalling pathway, upstream of NCCE and, via PLA2, downstream of MAP kinase. In a parallel line of work the present study aimed to identify the signalling events that might mediate a cannabinoid-induced inhibition of neurotransmission in the myenteric plexus, leading to a reduction in intestinal motility. Myenteric neurons were grown in primary culture enabling electrophysiological recordings to be made from individual cells to study the effects of cannabinoids on ion conductance. Immunohistochemistry validated these neurons as a model for those in situ, demonstrating that all CB1 receptor-positive cells express the cholinergic marker choline acetyltransferase. CP 55,940 was not shown to activate G-protein inwardly rectifying K+ channels but did inhibit evoked Ca2+ currents in myenteric cultures, a signalling mechanism that may underlie the CB1 receptor-mediated inhibition of neurotransmitter release from presynaptic sites. Nicotinic ACh (nACh) receptors are also expressed on cultured myenteric neurons. Stimulation of these receptors by nicotine evoked a transient inward current, which was inhibited by CP 55,940 and the endogenous cannabinoid anandamide, in an SR 14716A-insensitive manner. In fact, SR 141716A alone inhibited currents mediated by nACh receptors. PEA, a cannabinoid ligand whose effects are thought to occur independently of CB1/ CB2 receptor activation, also inhibited nicotine-induced currents. Pertussis toxin, a Gil,, inhibitor, did not reverse the cannabinoid-induced inhibition of nicotinic currents. In addition, CP 55,940 inhibited the sustained inward current evoked by 5-11T application in cultured myenteric neurons. The results suggest that cannabinoids inhibit nACh channels through a CB1 receptor-independent pathway in myenteric neurons, which would lead to a reduction in excitatory neurotransmission in the intact myenteric plexus. The inhibitory effect on the 5-HTinduced sustained inward current also suggests a cannabinoid-evoked inhibition of currents possibly mediated by the 5-HT1p receptor.

No description available.

Type 1 cannabinoid receptors (CB1R) have a well established role in modulating GABAergic signalling with the central nervous system, and are thought to be the only type present at GABAergic presynaptic terminals. In the medial entorhinal cortex (mEC), some cortical layers show high levels of ongoing GABAergic signalling (namely layer II) while others show relatively low levels (layer V). Using whole-cell patch clamp techniques, I have, for the first time, demonstrated the presence of functional CB1R in both deep and superficial layers of the mEC. Furthermore, using a range of highly specific ligands for both CB1R and CB2R, I present strong pharmacological evidence for CB2Rs being present in both deep and superficial layers of the mEC in the adult rat brain. In brain slices taken at earlier points in CNS development (P8-12), I have shown that while both CB1R and CB2R specific ligands do modulate GABAergic signalling at early developmental stages, antagonists/ inverse agonists and full agonists have similar effects, and serve only to reduce GABAergic signalling. These data suggest that the full cannabinoid signalling mechanisms at this early stage in synaptogenesis are not yet in place. During these whole-cell studies, I have developed and refined a novel recording technique, using an amantidine derivative (IEM1460) which allows inhibitory postsynaptic currents to be recorded under conditions in which glutamate receptors are not blocked and network activity remains high. Finally I have shown that bath applied CB1 and CB2 receptor antagonists/ inverse agonists are capable of modulating kainic acid induced persistent oscillatory activity in mEC. Inverse agonists suppressed oscillatory activity in the superficial layers of the mEC while it was enhanced in the deeper layers. It seems likely that cannabinoid receptors modulate the inhibitory neuronal activity that underlies network oscillations.

An increased understanding of the peripheral cannabinoid receptor (CB2) is required due to the CB2 receptor's emerging involvement in a number of disease states. New fluorescent technologies are capable of generating information about the CB2 receptor systems that has been unachievable using existing pharmacological methods i.e. radioisotope techniques. Our work, to develop fluorescently labelled CB2 receptor ligands, will provide cannabis researchers a unique pharmacological tool to use in conjunction with these emerging fluorescent technologies. This will aid the understanding of cellular actions of cannabinoids and accelerate the discovery of novel CB2 selective drugs. We report on the design, synthesis, and biological evaluation of novel fluorescent ligands targeted towards the CB2 receptor. The fluorescent ligands were designed and synthesised by conjugating recognized high affinity selective CB2 ligands (JTE2-3, JTE2-6 & JWH015) to appropriate fluorescent dyes via a chemical linker. Positioning of the fluorescent dye upon the pharmacophore was guided using established SAR data and supplemented by in-house molecular modelling experiments. Our results showed that modification of JTE2-3 and JTE2-6 with dansyl and BODIPY fluorophores resulted in fluorescent ligands which displayed poor affinity to the CB2 receptor and consequently were unsuccessful when used in fluorescent confocal microscopy experiments. Furthermore, our studies revealed important species selectivity, associated with JTE2-3, which was previously unrecognised. Using de novo drug design on JWH015, we synthesised and tested a 3-naphthyl modified fluorescent conjugate, 3-Gly-NBD-JWH015. The compound retained limited affinity to the CB2 receptor, but fluorescent confocal microscopy did not reveal specific receptor membrane binding. Further experiments using 3-naphthyl precursor compounds, displaying less bulky 3-substituents, demonstrated that limited modification to the 3-naphthyl position of JWH015 was tolerated and provided a first insight to the SAR at this position.