Introduction Cannabis-based medicines have a number of therapeutic indications, including anti-inflammatory and analgesic effects. The endocannabinoid receptor system, including the cannabinoid receptor 1 (CB 1 ) and receptor 2 (CB 2 ) and the endocannabinoids, are implicated in a wide range of physiological and pathophysiological processes. Pre-clinical and clinical studies have demonstrated that cannabis-based drugs have therapeutic potential in inflammatory diseases, including rheumatoid arthritis (RA) and multiple sclerosis. The aim of this study was to determine whether the key elements of the endocannabinoid signalling system, which produces immunosuppression and analgesia, are expressed in the synovia of patients with osteoarthritis (OA) or RA.

Aims To investigate the regulation of cannabinoid receptors CB1 and CB2 on immune cells by proinflammatory cytokines and its potential relevance to the inflammatory neurological disease, multiple sclerosis (MS). CB1 and CB2 signalling may be anti-inflammatory and neuroprotective in neuroinflammatory diseases. Cannabinoids can suppress inflammatory cytokines but the effects of these cytokines on CB1 and CB2 expression and function are unknown. Methods Immune cells from peripheral blood were obtained from healthy volunteers and patients with MS. Expression of CB1 and CB2 mRNA in whole blood cells, peripheral blood mononuclear cells (PBMC) and T cells was determined by quantitative real time-polymerase chain reaction (qRT-PCR). Expression of CB1 and CB2 protein was determined by flow cytometry. CB1 and CB2 signaling in PBMC was determined by Western blotting for Erk1/2. Results Proinflammatory cytokines IL-1β, IL-6 and TNF-α (the latter likely NFκB-dependently) can up-regulate CB1 and CB2 on human whole blood and peripheral blood mononuclear cells (PBMC). We also demonstrate up-regulation of CB1 and CB2 and increased IL-1β, IL-6 and TNF-α mRNA in blood of MS patients compared with controls. Conclusion The levels of CB1 and CB2 can be up-regulated by inflammatory cytokines, which can explain their increase in inflammatory conditions including MS.

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1 The b 2 -agonist salmeterol is a potent relaxant of airway smooth muscle with a long duration of action. Previous studies of cyclic AMP accumulation, however, have indicated that salmeterol is a low ecacy b 2 -agonist when compared to isoprenaline. Here we have compared the properties of salmeterol and isoprenaline as stimulants of gene transcription in CHO-K1 cells transfected with the human b 2 -adrenoceptor to dierent levels (50 and 310 fmol mg protein 71 ). 2 Gene transcription was monitored using a secreted placental alkaline phosphate (SPAP) reporter gene under the transcriptional control of six cyclic AMP response element (CRE) sequences. 3 In the lower expressing cells (CHO-b 2 /6), salmeterol produced a maximal cyclic AMP response that was only 22% that of that obtained with isoprenaline. In contrast in the higher expressing cells (CHO-b 2 / 4), the two maxima were of similar magnitude. 4 Salmeterol was a more potent stimulant of gene transcription, producing the same maximal response as isoprenaline in both cell lines. Furthermore, in the CHO-b 2 /4 cells, Salmeterol was 50 fold more potent as a stimulant of SPAP secretion than of cyclic AMP accumulation. In contrast, isoprenaline was 24 fold less sensitive as a stimulant of SPAP secretion than of cyclic AMP accumulation. In the presence of serum (10%), the eects of both salmeterol and isoprenaline on gene transcription were augmented. 5 These data suggest that the low ecacy and/or long duration of action of salmeterol, favours a potent stimulation of gene transcription when compared to more ecacious but shorter-lived agonists such as isoprenaline.

1 Constitutive activity of the b 2 -adrenoceptor, which is sensitive to inhibition by an inverse agonist such as ICI 118551, has been readily demonstrated in recombinant systems expressing constitutivelyactive mutant receptors or over-expressing the wild-type b 2 -adrenoceptor. Here we demonstrate the presence of constitutive b 2 -adrenoceptor activity in BC3H1 cells which endogenously express this receptor. . 4 This non-competitive eect of ICI 118551 in BC3H1 cells was also observed when either salbutamol was used as agonist, or the incubation period with isoprenaline was extended to 30 min. 5 The possibility that these eects of ICI 118551 are due to an interaction with dierent anity states (R, R* and R') of the receptor is discussed.

Background and purpose: The uracil nucleotides UDP and UTP have been reported to activate P2Y 2 , P2Y 4 and P2Y 6 receptors to cause vasoconstriction. We have performed a comparative analysis of these receptors in endothelium-denuded smooth muscle from porcine isolated coronary and ear arteries, using pharmacological and molecular tools. Experimental approach: Tissue segments were used to construct non-cumulative concentration response curves for UTP and UDP, in the absence and presence of the P2 receptor antagonists PPADS or suramin. RT-PCR and immunoblot analyses were employed to define gene expression and immunoreactivity for P2Y 2 , P2Y 4 and P2Y 6 receptors. Key results: In the coronary artery, UTP-evoked contractile responses were reduced in the presence of suramin, but not PPADS, while the smaller responses to UDP were unaffected by either antagonist. In the ear artery, contractile responses to UDP were much smaller than those to UTP; responses to UTP were inhibited by both PPADS and suramin. RT-PCR suggested predominant expression of P2Y 2 receptors in the coronary artery, while P2Y 4 and P2Y 6 receptor gene expression appeared equivalent in both tissues. Immunoblot analyses provided evidence for P2Y 6 receptors in both tissues, with equivocal evidence of P2Y 2 and P2Y 4 receptor immunoreactivities. Conclusions and implications:We conclude that UTP-evoked contraction of porcine coronary artery smooth muscle appears to be predominantly P2Y 2 -mediated, while the ear artery appears to express a uracil nucleotide-sensitive P2 receptor(s) which fails to fit readily into the current classification.

BACKGROUND AND PURPOSEThe P2Y14 receptor is the newest member of the P2Y receptor family; it is Gi/o protein-coupled and is activated by UDP and selectively by UDP-glucose and MRS2690 (2-thiouridine-5′-diphosphoglucose) (7-10-fold more potent than UDP-glucose). This study investigated whether P2Y14 receptors were functionally expressed in porcine isolated pancreatic arteries. EXPERIMENTAL APPROACHPancreatic arteries were prepared for isometric tension recording and UDP-glucose, UDP and MRS2690 were applied cumulatively after preconstriction with U46619, a TxA2 mimetic. Levels of phosphorylated myosin light chain 2 (MLC2) were assessed with Western blotting. cAMP concentrations were assessed using a competitive enzyme immunoassay kit. KEY RESULTSConcentration-dependent contractions with a rank order of potency of MRS2690 (10-fold) > UDP-glucose ≥ UDP were recorded. These contractions were reduced by PPTN {4-[4-(piperidin-4-yl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthoic acid}, a selective antagonist of P2Y14 receptors, which did not affect responses to UTP. Contraction to UDP-glucose was not affected by MRS2578, a P2Y6 receptor selective antagonist. Raising cAMP levels and forskolin, in the presence of U46619, enhanced contractions to UDP-glucose. In addition, UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP levels. Removal of the endothelium and inhibition of endothelium-derived contractile agents (TxA2, PGF2α and endothelin-1) inhibited contractions to UDP glucose. Y-27632, nifedipine and thapsigargin also reduced contractions to the agonists. UDP-glucose and MRS2690 increased MLC2 phosphorylation, which was blocked by PPTN. CONCLUSIONS AND IMPLICATIONSP2Y14 receptors play a novel vasocontractile role in porcine pancreatic arteries, mediating contraction via cAMP-dependent mechanisms, elevation of intracellular Ca 2+ levels, activation of RhoA/ROCK signalling and MLC2, along with release of TxA2, PGF2α and endothelin-1.

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