Cannabinoid signalling

• Main Author: Demuth, Dirk Geoffrey
• Published: University of Hertfordshire 2004
• Subjects: 615.7827
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415855

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.