Saoirse E O'Sullivan

Cannabinoids go nuclear: evidence for activation of peroxisome proliferator-activated receptors

Cannabinoids act at two classical cannabinoid receptors (CB1 and CB2), a 7TM orphan receptor and the transmitter‐gated channel transient receptor potential vanilloid type‐1 receptor. Recent evidence also points to cannabinoids acting at members of the nuclear receptor family, peroxisome proliferator‐activated receptors (PPARs, with three subtypes α, β (δ) and γ), which regulate cell differentiation and lipid metabolism. Much evidence now suggests that endocannabinoids are natural activators of PPARα. Oleoylethanolamide regulates feeding and body weight, stimulates fat utilization and has neuroprotective effects mediated through activation of PPARα. Similarly, palmitoylethanolamide regulates feeding and lipid metabolism and has anti‐inflammatory properties mediated by PPARα. Other endocannabinoids that activate PPARα include anandamide, virodhamine and noladin. Some (but not all) endocannabinoids also activate PPARγ; anandamide and 2‐arachidonoylglycerol have anti‐inflammatory properties mediated by PPARγ. Similarly, ajulemic acid, a structural analogue of a metabolite of Δ9‐tetrahydrocannabinol (THC), causes anti‐inflammatory effects in vivo through PPARγ. THC also activates PPARγ, leading to a time‐dependent vasorelaxation in isolated arteries. Other cannabinoids which activate PPARγ include N‐arachidonoyl‐dopamine, HU210, WIN55212‐2 and CP55940. In contrast, little research has been carried out on the effects of cannabinoids at PPARδ. In this newly emerging area, a number of research questions remain unanswered; for example, why do cannabinoids activate some isoforms and not others? How much of the chronic effects of cannabinoids are through activation of nuclear receptors? And importantly, do cannabinoids confer the same neuro‐ and cardioprotective benefits as other PPARα and PPARγ agonists? This review will summarize the published literature implicating cannabinoid‐mediated PPAR effects and discuss the implications thereof.

Cannabinoid activation of peroxisome proliferator-activated receptors: Potential for modulation of inflammatory disease

Cannabinoids act via cell surface G protein-coupled receptors (CB(1) and CB(2)) and the ion channel receptor TRPV1. Evidence has now emerged suggesting that an additional target is the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors. There are three PPAR subtypes alpha, delta (also known as beta) and gamma, which regulate cell differentiation, metabolism and immune function. The major endocannabinoids, anandamide and 2-arachidonoylglycerol, and ajulemic acid, a structural analogue of the phytocannabinoid Delta(9)-tetrahydrocannabinol (THC), have anti-inflammatory properties mediated by PPARgamma. Other cannabinoids which activate PPARgamma include N-arachidonoyl-dopamine, THC, cannabidiol, HU210, WIN55212-2 and CP55940. The endogenous acylethanolamines, oleoylethanolamide and palmitoylethanolamide regulate feeding and body weight, stimulate fat utilization and have neuroprotective effects mediated through PPARalpha. Other endocannabinoids that activate PPARalpha include anandamide, virodhamine and noladin ether. There is, as yet, little direct evidence for interactions of cannabinoids with PPARdelta. There is a convergence of effects of cannabinoids, acting via cell surface and nuclear receptors, on immune cell function which provides promise for the targeted therapy of a variety of immune, particularly neuroinflammatory, diseases.

The complexities of the cardiovascular actions of cannabinoids

The cardiovascular actions of cannbinoids are complex. In general they cause vasorelaxation in isolated blood vessels, while in anaesthetised animals they cause multiphasic responses which involve an early bradycardia and long‐lasting hypotension. However, in conscious animals, the picture is one of bradycardia followed by pressor responses. Clearly, the responses to cannabinoids are dependent on the experimental conditions and synthetic cannabinoids and endocannabinoids exhibit different pharmacologies. In terms of mechanisms involved in the vascular responses to cannabinoids, the following have been implicated: the involvement of ‘classical’ cannabinoid receptors, the involvement of a novel endothelial cannabinoid receptor, the release of nitric oxide, the release of endothelium‐derived hyperpolarising factor (EDHF), the activation of vanilloid receptors, metabolism of endocannabinoids to vasoactive molecules, and both peripheral inhibition and central excitation of the sympathetic nervous system.

Heterogeneity in the mechanisms of vasorelaxation to anandamide in resistance and conduit rat mesenteric arteries.

In order to address mechanistic differences between arterial vessel types, we have compared the vasorelaxant actions of anandamide in resistance (G3) and conduit (G0) mesenteric arteries. Anandamide produced concentration‐dependent relaxations of pre‐constricted G3 arteries with a maximal response that was significantly greater than seen in G0. The CB1 receptor selective antagonists SR141716A (100 nM) and AM251 (100 nM) caused reductions in the vasorelaxant responses to anandamide in both arteries. Maximal vasorelaxant responses to anandamide were reduced in both arteries after treatment with capsaicin to deplete sensory neurotransmitters (10 μM for 1 h). Vasorelaxation to anandamide was not affected by the nitric oxide synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME, 300 μM) in either artery. Only responses in G3 arteries were sensitive to removal of the endothelium. In G3 vessels only, vasorelaxation to anandamide was reduced by inhibition of EDHF activity with a combination of charybdotoxin (100 nM) and apamin (500 nM) in the presence of L‐NAME (300 μM) and indomethacin (10 μM). Antagonism of the novel endothelial cannabinoid receptor (O‐1918, 1 μM) caused a reduction in the sensitivity to anandamide in G3 but not G0. G3, but not G0, vessels showed a small reduction in vasorelaxant responses to anandamide after inhibition of gap junctional communication with 18α‐GA (100 μM). These results demonstrate that there are differences in the mechanisms of vasorelaxation to anandamide between conduit and resistance mesenteric arteries. In small resistance vessels, vasorelaxation occurs through stimulation of vanilloid receptors, CB1 receptors, and an endothelial receptor coupled to EDHF release. By contrast, in the larger mesenteric artery, vasorelaxation is almost entirely due to stimulation of vanilloid receptors and CB1 receptors, and is endothelium‐independent.

Characterisation of the vasorelaxant properties of the novel endocannabinoid N-arachidonoyl-dopamine (NADA).

We have investigated the vascular effects of N‐arachidonoyl‐dopamine (NADA), a novel endocannabinoid/vanilloid. NADA caused vasorelaxant effects comparable to those of anandamide in small mesenteric vessels (G3), the superior mesenteric artery (G0) and in the aorta. In G3, addition of NG‐nitro‐L‐arginine methyl ester (300 μM) or the dopamine (D1) receptor antagonist (SCH23390, 1 μM) did not affect responses to NADA. In the presence of 60 mM KCl, after de‐endothelialisation, or after K+ channel inhibition with charybdotoxin (100 nM) and apamin (500 nM), relaxant responses to NADA were inhibited. In G3, pretreatment with the vanilloid receptor (VR) agonist capsaicin (10 μM) or the VR antagonist capsazepine (10 μM) reduced vasorelaxation to NADA. In G3, application of the CB1 antagonist SR141716A at 1 μM but not 100 nM reduced the potency of NADA. Another CB1 antagonist, AM251 (100 nM and 1 μM), did not affect vasorelaxation to NADA. After endothelial denudation, SR141716A (1 μM) did not reduce the responses further. A combination of capsaicin and SR141716A (1 μM) reduced vasorelaxation to NADA further than with capsaicin pretreatment alone. The novel endothelial cannabinoid (CB) receptor antagonist O‐1918 opposed vasorelaxation to NADA in G3. In the superior mesenteric artery (G0), vasorelaxation to NADA was not dependent on an intact endothelium and was not sensitive to O‐1918, but was sensitive to capsaicin and SR141716A or AM251 (both 100 nM). The results of the present study demonstrate for the first time that NADA is a potent vasorelaxant. In G3, the effects of NADA are mediated by stimulation of the VR and the novel endothelial CB receptor, while in G0, vasorelaxation is mediated through VR1 and CB1 receptors.

Cannabinoids mediate opposing effects on inflammation‐induced intestinal permeability

BACKGROUND AND PURPOSE Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion and intestinal motility. The ability to modulate intestinal permeability in inflammation may be important in therapy aimed at maintaining epithelial barrier integrity. The aim of the present study was to determine whether cannabinoids modulate the increased permeability associated with inflammation in vitro.

Pharmacological Effects of Cannabinoids on the Caco-2 Cell Culture Model of Intestinal Permeability

Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion, and intestinal motility. However, the effects of cannabinoids on intestinal permeability have not yet been established. The aim of the present study is to examine the effects of cannabinoids on intestinal permeability in an in vitro model. Caco-2 cells were grown until fully confluent on inserts in 12-well plates. Transepithelial electrical resistance (TEER) measurements were made as a measure of permeability. EDTA (50 μM) was applied to reversibly increase permeability (reduce TEER). The effects of cannabinoids on permeability in combination with EDTA, or alone, were assessed. Potential target sites of action were investigated using antagonists of the cannabinoid (CB)1 receptor, CB2 receptor, transient receptor potential vanilloid subtype 1 (TRPV1), peroxisome proliferator-activated receptor (PPAR)γ, PPARα, and a proposed cannabinoid receptor. When applied to the apical or basolateral membrane of Caco-2 cells, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) enhanced the speed of recovery of EDTA-induced increased permeability. This effect was sensitive to cannabinoid CB1 receptor antagonism only. Apical application of endocannabinoids caused increased permeability, sensitive to cannabinoid CB1 receptor antagonism. By contrast, when endocannabinoids were applied basolaterally, they enhanced the recovery of EDTA-induced increased permeability, and this involved additional activation of TRPV1. All cannabinoids tested increased the mRNA of the tight junction protein zona occludens-1, but only endocannabinoids also decreased the mRNA of claudin-1. These findings suggest that endocannabinoids may play a role in modulating intestinal permeability and that plant-derived cannabinoids, such as THC and CBD, may have therapeutic potential in conditions associated with abnormally permeable intestinal epithelium.

The effects of Δ9-tetrahydrocannabinol in rat mesenteric vasculature, and its interactions with the endocannabinoid anandamide

1 Δ9‐tetrahydrocannabinol (THC) produces varying effects in mesenteric arteries: vasorelaxation (third‐order branches, G3), modest vasorelaxation (G2), no effect (G1) and vasoconstriction (the superior mesenteric artery, G0). 2 In G3, vasorelaxation to THC was inhibited by pertussis toxin, but was unaffected by the CB1 receptor antagonist, AM251 (1 μM), incubation with the TRPV1 receptor agonist capsaicin (10 μM, 1 h), the TRPV1 receptor antagonist capsazepine (10 μM) or de‐endothelialisation. 3 In G3, vasorelaxation to THC was inhibited by high K+ buffer, and by the following K+ channel inhibitors: charybdotoxin (100 nM), apamin (500 nM) and barium chloride (30 μM), but not by 4‐aminopyridine, glibenclamide or tertiapin. 4 In G3, THC (10 and 100 μM) inhibited the contractile response to Ca2+ in a Ca2+‐free, high potassium buffer, indicating that THC blocks Ca2+ influx. 5 In G0, the vasoconstrictor responses to THC were inhibited by de‐endothelialisation and SR141716A (100 nM), but not by the endothelin (ETA) receptor antagonist FR139317 (1 μM). 6 THC (1 and 10 μM) antagonised vasorelaxation to anandamide in G3 but not G0. THC did not antagonise the noncannabinoid verapamil, capsaicin or the CB1 receptor agonist CP55,940. THC (10 and 100 μM) inhibited endothelium‐derived relaxing factor (EDHF)‐mediated responses to carbachol in a manner similar to the gap junction inhibitor 18α‐glycyrrhetinic acid. 7 These data show that THC causes vasorelaxation through activation of K+ channels and inhibition of Ca2+ channels, and this involves non‐CB1, non‐TRPV1 but G‐protein‐coupled receptors. In G0, THC does not cause relaxation and at high concentrations causes contractions. Importantly, THC antagonises the effects of anandamide, possibly through inhibition of EDHF activity.

Cannabinoids in experimental stroke: a systematic review and meta-analysis.

Cannabinoids (CBs) show promise as neuroprotectants with some agents already licensed in humans for other conditions. We systematically reviewed CBs in preclinical stroke to guide further experimental protocols. We selected controlled studies assessing acute administration of CBs for experimental stroke, identified through systematic searches. Data were extracted on lesion volume, outcome and quality, and analyzed using random effect models. Results are expressed as standardized mean difference (SMD) with 95% confidence intervals (CIs). In all, 144 experiments (34 publications) assessed CBs on infarct volume in 1,473 animals. Cannabinoids reduced infarct volume in transient (SMD −1.41 (95% CI −1.71), −1.11) P<0.00001) and permanent (−1.67 (−2.08, −1.27), P<0.00001) ischemia and in all subclasses: endocannabinoids (−1.72 (−2.62, −0.82), P=0.0002), CB1/CB2 ligands (−1.75 (−2.19, −1.31), P<0.00001), CB2 ligands (−1.65 (−2.09, −1.22), P<0.00001), cannabidiol (−1.20 (−1.63, −0.77), P<0.00001), Δ9-tetrahydrocannabinol (−1.43 (−2.01, −0.86), P<0.00001), and HU-211 (−2.90 (−4.24, −1.56), P<0.0001). Early and late neuroscores significantly improved with CB use (−1.27 (−1.58, −0.95), P<0.00001; −1.63 (−2.64, −0.62), P<0.002 respectively) and there was no effect on survival. Statistical heterogeneity and publication bias was present, median study quality was 4 (range 1 to 6/8). Overall, CBs significantly reduced infarct volume and improve functional outcome in experimental stroke. Further studies in aged, female and larger animals, with other co-morbidities are required.

The sites of action of bumetanide in man

Abstract Some pharmacological effects of bumetanide were investigated in 8 healthy volunteers. It was a very potent diuretic, as judged by the fact that, at peak effects during water diuresis, 20% of filtered sodium was excreted in the urine. During water diuresis it reduced free water clearance by approximately 25% and during hydropoenia it reduced the reabsorption of solute free water to a similar extent, indicating a major site of action on the ascending limb of the loop of Henle. A significant phosphaturia was induced during the period of maximum diuresis suggesting an additional action on the proximal tubule. The excretion of urinary titratable acid and ammonium as well as potassium were increased, indicating that the drug did not have a significant effect on sodium transport in the distal tubule. Glomerular filtration rate was not affected but the drug caused a decrease in the renal clearance of uric acid.