The endocannabinoids anandamide and 2-arachidonoylglycerol, and the anandamide-congener, palmitoylethanolamide, are all substrates for the enzyme fatty acid amide hydrolase, and are endowed with anti-inflammatory actions exerted via cannabinoid receptors or, in the case of palmitoylethanolamide, also via other targets. We investigated the role of the endocannabinoid system during granuloma formation, a model of chronic inflammation sustained by neoangiogenesis, in rats. Granuloma was induced by subcutaneous lambda-carrageenin-soaked sponge implants on the back of male Wistar rats. After 96h, granulomas were detached and tissue formation was evaluated as wet weight; the endocannabinoid system was evaluated by the measurement of endocannabinoid levels, by LC-MS, and of cannabinoid receptor expression, by western blot analysis. Moreover, angiogenesis was evaluated by the measurement of both hemoglobin content and CD31 protein expression. Arachidonoylserotonin (AA-5-HT, 12.5-50mug/ml), an inhibitor of FAAH, and palmitoylethanolamide (PEA, 200-800mug/ml) were given locally only once at the time of implantation. Granuloma formation was accompanied by a significant decrease in endocannabinoid and palmitoylethanolamide levels paralleled by increased levels of the fatty acid amide hydrolase, responsible for their breakdown. Moreover, an increase of cannabinoid receptor expression was also observed. Pharmacological elevation of endocannabinoids and palmitoylethanolamide, obtained separately by arachidonoylserotonin and exogenous palmitoylethanolamide treatment, dose-dependently reduced inflammatory hallmarks including tumor necrosis factor-alpha as well as granuloma-dependent angiogenesis. The effect of arachidonoylserotonin was accompanied by near-normalization of 2-arachidonoylglycerol and palmitoylethanolamide levels in the tissue. These findings suggest that chronic inflammation might develop also because of endocannabinoid and palmitoylethanolamide tissue concentration impairment, the correction of which might be exploited to develop new anti-inflammatory drugs.

Source: pubmed.gov
Via Levels Of Endocannabinoids And Palmitoylethanolamide And Their Pharmacological

Background

The phytocannabinoid cannabidiol (CBD) exhibits antioxidant and antiinflammatory properties. The present study was designed to explore its effects in a mouse model of sepsis-related encephalitis by intravenous administration of lipopolysaccharide (LPS).

Methods

Vascular responses of pial vessels were analyzed by intravital microscopy and inflammatory parameters measured by qRT-PCR.

Results

CBD prevented LPS-induced arteriolar and venular vasodilation as well as leukocyte margination. In addition, CBD abolished LPS-induced increases in tumor necrosis factor-alpha and cyclooxygenase-2 expression as measured by quantitative real time PCR. The expression of the inducible-nitric oxide synthase was also reduced by CBD. Finally, preservation of Blood Brain Barrier integrity was also associated to the treatment with CBD.

Conclusions

These data highlight the antiinflammatory and vascular-stabilizing effects of CBD in endotoxic shock and suggest a possible beneficial effect of this natural cannabinoid.

Go to:
Background
Endotoxic shock (ES) is a life-threatening condition with mortality rates of 40-70% that usually takes place in seriously ill, immunologically compromised patients [1]. In ES, usually secondary to Gram-negative bacterial infection, there is a severe impairment of vascular, coagulant, immune and inflammatory responses of the host [2]. The lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, mediates many of the alterations leading to ES. LPS profoundly impairs endothelial functions, promoting intravascular coagulation, disruption of the endothelial wall and intense vasodilation and hypotension. The therapeutic usefulness of potent antiinflammatory agents as steroids remains controversial [2]. Thus, the search for effective treatments in ES is still demanding.

Encephalopathy is a common complication in ES patients, usually appearing very early in the pathologic process and determining the prognosis [3]. LPS injection, by inducing both endothelial and astrocytic cell dysfunction [4,5], is particularly harmful for brain circulation, impairing cerebrovascular autoregulation [3,6]. Autoregulatory responses of brain arteries and arterioles guarantee a constant cerebral perfusion during systemic blood pressure changes, being dependent on a normal endothelial function, in particular during hypotension [7]. LPS also disrupts the coupling of local cerebral blood flow (CBF) with the activity of underlying neurons [3,4].

Cannabidiol (CBD] is a phytocannabinoid with well-known antiinflammatory and antioxidant properties [8,9]. El-Remessy et al [10] recently reported that CBD prevented inflammatory and oxidative damage and preserved endothelial integrity in an experimental model of diabetic retinopathy. Furthermore, CBD preserves cerebral circulation in pathological conditions such as brain ischemia [11]. Recent data support the clinical use of CBD for the treatment of a variety of damaging conditions, including nephropathy and diabetic cardiomyopathy. In particular, the antioxidant properties of CBD seem to play a major role in the protective effects of this phytocannabinoid against the oxidative and nitrosative stress induced by chemoterapy agents and by high glucose conditions [12,13].

In the present work, we aimed to test the beneficial effects of CBD in brain circulation and inflammation in an in vivo model of sepsis after parenteral injection of LPS. To that end, we opened a cranial window in adult mice to study vascular responses by intravital microscopy. Former studies using this method have demonstrated that topic application of LPS altered arteriolar responses [14]; however, few studies have so far reported on the effect of i.v. injection of LPS. Such a difference is relevant as LPS cannot readily cross the blood brain barrier (BBB) [15] and because, during the actual septic condition, both endotoxin release and leukocyte activation take place inside the intravascular space [16].

Methods

Mice and preparation for intravital microscopy

Adult C57BL/6J mice were maintained in a temperature-controlled specific pathogen-free facility with a strict 12-hour light/dark cycle and with free access to food and water. All experiments were performed in accordance to international and local guidelines as approved by an internal committee (86/609/EEC). Mice were anesthetized with ketamine plus medetomidine (50 mg/kg and 1 mg/kg, respectively). After removing the skin and muscle, a custom-made device was attached to the cranial surface and then fitted to the microscope. Body temperature was continuously monitored by a rectal probe and maintained constant with a thermal blanket. Blood pressure (BP) was monitored by a cuff tail device with a photoelectric sensor (NIPREM 645, Cibertec, Madrid, Spain). A cranial window (2 mm of diameter) was then opened with a high-speed drill, to gain direct access to the brain parenchyma. The tissue was kept humid constantly by subsequent additions of 200 μl-drops of 0.9% saline. Staining of superficial endothelium and microglia was performed by topical administration of Griffonia simplicifolia conjugated with fluorescein (Vector Laboratories, Burlingame, CA, USA), in a 0.9% NaCl solution, for 30 min. At the beginning of the experiment, at 90 and at 180 min, a 50 μL blood sample was obtained by tail puncture to determine blood gases (i-STAT, Abbot Laboratories, NJ, USA). At the end of the experiment mice were killed by decapitation and brains harvested, frozen and conserved at -80C until use.

Drug administration

To clearly observe the cerebrovascular tree throughout the entire experiment, 100 μl of a 70000 MW Texas red-conjugated dextrane solution (Invitrogen, Carlsbad, CA, USA) was administered through the tail vein. This approach stains blood plasma while leaving nucleated cells unstained [17]. Afterwards, vehicle (Tween/saline, N = 7), LPS (Sigma, St Louis, MO, USA; 1 mg/kg, N = 8), LPS+CBD (1 mg/kg + 3 mg/kg respectively; Tocris Bioscience, Bristol, UK, N = 7) or CBD alone (3 mg/kg, n = 5) were administered i.v. through the tail vein in a total volume of 100 μl. Doses of LPS and CBD were chosen based on previous data [5,18]. A single dose of CBD was chosen because its long half-life time [18] makes it appropriate for experiments lasting for 3 h as ours.

Image acquisition and analysis

Observations were made using a Nikon 90i upright microscope coupled to a C1 scanhead confocal system with two laser sources (Arg 488 nm and He/Ne 543 nm). Once the area of interest was defined, 60 μm-thick stacks in the Z-axis (3 μm steps) were obtained with the Nikon EZ-C1 software, every 15 min for a total time of 180 min post drug administration. Three-dimensional constructs were analyzed and changes in the diameter of venules (internal diameter 39-112 μm) and third-order arterioles (internal diameter 14-50 μm) (at least 4 of each per animal) measured. Pial vessels of those diameters are considered as optimal for intravital studies on microvessel reactivity [19]. In addition, the total number of marginated cells (revealed as immobilized black dots inside the vessels) at each time point was counted and the accumulated amount was expressed per area unit (μm2). To that end, the total area corresponding to vessels was estimated in each field of observation by means of ImageJ (NIH) software.

BBB integrity

70000 MW dextrane is unable to leave the blood vessels under normal conditions, but diffuses into the brain parenchyma when BBB integrity is compromised. In order to measure this phenomenon, laser-scanning micrographs were analyzed and fluorescence intensity across a cross-section of edematous vessels was measured. This allowed the analysis of fluorochrome distribution inside and outside the affected vessels as an index of BBB damage [20].

Quantification of markers of oxidative stress

Concentrations of 4-hydroxynonenal (HNE) and of malondialdehyde (MDA) as markers of oxidative stress, were measured in frozen brain tissue by ELISA (OxiSelect HNE-His Adduct and OxiSelect MDA Adduct, Cell Biolabs, San Diego, CA, USA).

COX-2, TNF-α and iNOS mRNA levels

mRNA levels of cyclooxygenase (COX)-2, tumor necrosis factor-alpha (TNF-α) and inducible nitric oxide synthase (iNOS) were quantified by qRT-PCR from frozen midbrains. Total RNA was extracted using the Tripure Isolation Reagent (Roche Diagnostics, Mannheim, Germany). Single-stranded complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany). PCR primers and TaqMan probes were designed by Tib Molbiol (Berlin, Germany) as shown in table ​table1.1. For normalization, 18S primers and probe number 55 from Universal ProbeLibrary probes 1-165 (Roche) were utilized. Gene expression was quantified using the Quantimix Easy Probes kit (Biotools, Madrid, Spain) in concert with a LightCycler thermocycler (Roche). Standard curves were calculated for quantification purposes using ten-fold serial dilutions of cDNA from brain mouse. The transcript amounts were calculated using the second derivate maximum mode of the LC-sotfware version 4.0. The specific transcript quantities were normalized to the transcript amounts of the reference gene 18S. All further calculations and statistical analyses were carried out with these values referred to as relative expression ratios.

Statistical analysis

Results are expressed as mean SEM of the indicated number of experiments. Changes in vessel diameter with time were compared between groups by 2-way ANOVA. Differences between groups in enzyme or protein expression were studied by 1-way ANOVA. Newman-Keuls post-hoc test was used for multiple comparisons. A p value of less than 0.05 was considered as statistically significant. Statistical analysis was performed using the 11.0.0 version of SPSS software (SPSS Inc.).

Results

Physiological data

Mice rectal temperature (37.5 0.6, 37.7 0.2 and 38.1 0.4C for VEH, LPS, and LPS+CBD, respectively, NS) measured in the first 30 min of the experiment indicated a mean decrease of 2C, remaining stable then. There were no differences between groups throughout the experiment.

BP remained stable throughout the entire experiment and showed no differences between groups (mean BP [range]: 137 14 [128-150], 141 12 [123-154] and 149 5 [130-155] mmHg for VEH, LPS and LPS+CBD, respectively, NS).

Blood gas values remained in the normal range until the end of the experiment, with no differences between groups (pH: 7.38 0.01, 7.35 0.02 and 7.39 0.04; pO2: 47.7 4.8, 45.6 5.1 and 45.6 2.7 mmHg; and pCO2: 66.3 9.3, 71.4 8.8 and 61.6 6.3 mmHg, for VEH, LPS and LPS+CBD, respectively, NS).

CBD counteracts LPS-induced vasodilation

LPS-induced sepsis-associated changes in cerebral blood flow are due to, among other factors, an excessive vasodilation [21]. As expected, LPS induced a sustained arteriolar vasodilation of up to 30 3%, starting at t15 min and peaking at t75 min (Figure ​(Figure1);1); LPS also induced a venular vasodilation of up to 15 2% starting at t75. CBD blunted the vasodilator effect of LPS, so that in LPS+CBD arteriolar and venular dilation accounted only for 10 2% and 5 2%, respectively (2-way ANOVA p < 0.05 vs. LPS, F = 3.48 and F = 4.22 for venules and arterioles, respectively).

CBD decreases LPS-induced cell margination

To study leukocyte margination and diapedesis, of paramount importance in LPS-induced sepsis, the intravascular space was stained with 70000 MW Texas red-conjugated dextrane. Blood cells thus appear as "ghosts" inside the vessels [17]. With this approach, only cells that are stationary or dramatically slowed by adhesive interactions with the vessel wall can be detected [17,22]. The great majority of these unstained cells are leukocytes, as they exceed in high number other nucleated cell types [17]. Leukocyte margination was not observed in VEH at any time studied, whereas in LPS-treated animals the density of marginated leukocytes was significantly elevated. LPS+CBD blunted this effect (Figure ​(Figure2)2) (2-way ANOVA p < 0.05, F = 1.87).

LPS treatment compromised BBB integrity, and this effect was prevented by CBD

Mice receiving LPS showed a clear disruption of the BBB, as revealed by extravasation of the fluorescently-labelled dextrane starting 45 min after administration (Figure ​(Figure3).3). This phenomenon has been previously employed for the quantification of BBB integrity [20]. In animals showing BBB alterations, CBD treatment dramatically reduced the extent of dextrane extravasation (p < 0.05 by 2-way ANOVA, F = 2.23) (Figure ​(Figure3).3). Interestingly, no vehicle-treated animals showed significant changes in BBB integrity.

CBD does not modify HNE or MDA concentrations

No differences were observed between groups in the concentration of HNE (1.37 0.4, 1.15 0.3 and 1.39 0.5 μg/ml, for VEH, LPS and LPS+CBD, respectively, NS) or MDA (3.2 0.3, 2.9 0.4 and 3.3 0.5 μg/ml, for VEH, LPS and LPS+CBD, respectively, NS) in brain tissue.

CBD reduces LPS-induced expression of COX-2, TNF-α, and iNOS

LPS triggers a massive inflammatory response involving cellular mediators such as cytokines and prostaglandins [21]. Thus, we aimed to quantify the expression of one crucial cytokine (TNF-α) and of some key enzymes (COX-2 and iNOS). Taken together, our results confirm the development of a proinflammatory environment in LPS-treated mice brain, with increases in mRNA levels for TNF-α and COX-2 (Figure ​(Figure4).4). iNOS expression was not modified in LPS vs vehicles although CBD-treated mice exhibited significantly lower expression of this enzyme. LPS-induced increases of TNF-α and COX-2 were dramatically reduced by CBD (Figure ​(Figure44).

No differences between VEH and animals treated with CBD alone were observed in any of the aforementioned determinations.

Discussion

CBD is a natural cannabinoid lacking psychotropic effects. This fact, together with its well-known antiinflammatory, antioxidant and neuroprotective effects, has focused research on its possible therapeutic relevance [8,23]. We here report that CBD counteracts some of the inflammatory responses associated to LPS in the mouse brain.

In the present work we observed that parenterally-introduced LPS induced dramatic arteriolar dilation, starting as early as 30 min after i.v. injection. Previous in vivo experiments on the effect of parenteral or intracerebral LPS administration (1 mg/kg or more) on brain circulation in rodents measured local or global CBF [3,6,15]. In those studies, brain hyperemia took place 1 h after LPS administration. We did not directly measure CBF, but the sequential and parallel increase in venular diameter after LPS administration likely reflected an increase in CBF as, due to the poor reactivity of the cerebral venous myocytes, brain venule diameter is mostly dependent on CBF [19]. Brain hyperemia after abnormal brain vasodilation represents a lack of autoregulation, due to endothelial and glial dysfunction [3]. Administration of CBD blunted the arteriolar dilation, so arteriolar diameter remained similar to control throughout the experiment. Besides, venular diameter after CBD administration remained similar to control. Altogether, these results suggest that in the presence of CBD, CBF remained similar to control.

On the other hand, the compromise of BBB integrity is a common feature of LPS-associated encephalitis and may be caused by the disruption of endothelial tight junctions mainly by the action of several cytokines. Increased BBB permeability leads to secondary lesions that worsen with increased duration of septic shock and correlate with poor outcome [16]. CBD reduced BBB alteration, in agreement with a recent report showing that CBD prevents endothelial cell inflammatory responses and preserves barrier functions in a murine model of experimental diabetes [24]. Furthermore, our data showing that CBD was able to prevent cellular margination match with recent observations in vivo in which CBD decreased the expression of adhesion molecules as well as chemotaxis in experimental models of inflammation and tissue injury [12,13].

LPS is known to induce COX-2 in neurons and glial cells, subsequently increasing COX metabolites of arachidonic acid, which contributes to LPS-induced cerebral hyperemia [15]. At high concentrations, CBD inhibits COX-2 activity [18], an effect dependent on the cell type, as it is not observed in tumoral cells [25]. Our results confirm previous observations by Costa et al [26] showing that oral administration of CBD diminishes carrageenan-induced paw inflammation in rat by decreasing COX activity and edema formation.

Since hemodynamic changes in ES are triggered by the massive inflammatory reaction induced by LPS as well as by the increased oxidative stress, CBD beneficial effects could also derive both from its antiinflammatory and antioxidant properties [27]. Binding of LPS to specific receptors in brain endothelial cells triggers a series of signaling events leading to the increase of cytokine production [28]. These cytokines participate in the disruption of BBB integrity and induce brain vessel dilation [21,29]. TNF-α is a major mediator in septic encephalopathy, as mice deficient in TNF-receptor 1 are more resistant to LPS-induced changes [30]. CBD exerts a potent immunosuppressive effect in vivo, reducing production of TNF-α and other cytokines from immune cells [8,12,13,18].

LPS is one the most important stimuli for the induction of iNOS in brain cells [15]. iNOS induction leads to massive NO production, inducing endothelial cytotoxicity by direct damage and by increasing oxidative stress and inflammation [31], and impairing cerebrovascular autoregulation [3]. However, due to the limited ability of LPS to cross the BBB, the effect of parenteral LPS on brain iNOS mRNA levels could not be observed before 6 h after injection [5]. In agreement, we did not find an increase of iNOS expression in brain during our 3 h period of study after LPS injection. Thus, the CBD-induced decrease of iNOS expression likely corresponded with the prevention by CBD of iNOS induction in brain due to the experimental procedure. A similar effect on iNOS induction has been described for CBD newborn mice brains after manipulation to perform oxygen-glucose deprivation of forebrain slices [32]. CBD is known to prevent iNOS expression through inhibition of MAPK and NF-κB signaling [12,13,33,34], an observation that may be especially relevant in advanced stages of circulatory shock, when iNOS contribution to NO production seems to be maximal [35]. Remarkably, peroxynitrite formation (known to participate in pathophysiological alterations of shock) has been found to follow a similar time course to iNOS expression after challenge with LPS in rats (reviewed in 35).

We did not observe any difference between groups in brain concentration of oxidative stress markers as HNE or MDA. LPS administration leads to a brief transient increase of oxidative stress markers in brain, observed shortly after injection [36]. Nevertheless, the sustained and significant increase of these markers is observed 6 h after LPS administration, thus beyond our experimental period, and lasts for at least 24 h, being mainly due to cytokine-induced activation of microglial cells [36].

Finally, the complex pharmacological profile of CBD may explain some of our data [reviewed in [37]]. Thus, and although the possible mediation of cannabinoid receptors has not been analyzed in the present experiments, it is important to note that recent reports suggest that CBD effects on LPS-induced inflammation are receptor-independent [34]. However, CBD antagonizing properties on CB1 receptors might underlie some of the observed effects, as CB1 receptor blockade prevents the primary hypotensive response to LPS [27]. Furthermore, CB1 receptor blockade has been proposed to improve survival in ES [38]. In addition, CBD might also partially activate CB2 receptors, which play a crucial role in the regulation of the immune response against sepsis in an animal model of cecal ligation and puncture [39]. Furthermore, its activity as a CB2 receptor inverse agonist could partially account for these actions since CB2 receptor inverse agonism reduces clinical signs of inflammation and cell migration [40]. Finally, CBD may also alter inflammatory processes by targeting the abnormal CBD receptor [41], as this receptor partially mediates the hypotensive effects of anandamide and other cannabinoids [42,43].

Conclusions

In conclusion, CBD blunted LPS-induced changes in vessel diameter and permeability as well as leukocyte margination, effects that were associated with modulation of cytokine and NO production. However, more studies on the optimal dosage regime, timing of effectiveness and response in other models of sepsis are warranted before considering CBD as a candidate for treatment in humans.

Source, Graphs and Figures: pubmed.gov
Via Cannabidiol Reduces Lipopolysaccharide-induced Vascular Changes And Inflammation

Abstract
Stemming from the centuries-old and well known effects of Cannabis on intestinal motility and secretion, research on the role of the endocannabinoid system in gut function and dysfunction has received ever increasing attention since the discovery of the cannabinoid receptors and their endogenous ligands, the endocannabinoids. In this article, some of the most recent developments in this field are discussed, with particular emphasis on new data, most of which are published in Neurogastroenterology & Motility, on the potential tonic endocannabinoid control of intestinal motility, the function of cannabinoid type-1 (CB1) receptors in gastric function, visceral pain, inflammation and sepsis, the emerging role of cannabinoid type-2 (CB2) receptors in the gut, and the pharmacology of endocannabinoid-related molecules and plant cannabinoids not necessarily acting via cannabinoid CB1 and CB2 receptors. These novel data highlight the multi-faceted aspects of endocannabinoid function in the GI tract, support the feasibility of the future therapeutic exploitation of this signaling system for the treatment of GI disorders, and leave space for some intriguing new hypotheses on the role of endocannabinoids in the gut.

Introduction
Apart from being the most widely used recreational drug in the Western world since the 1960s, Cannabis has been very popular in Chinese and ayurvedic traditional medicines for centuries.1Cannabis preparations were applied as palliatives to treat a wide array of health problems, including gastrointestinal (GI) disorders, and extracts from this plant were still indicated for diarrhea a century ago, whereas anecdotal reports exist for their use during dysentery and cholera.2,3 Although the medicinal as well as psychoactive properties of Cannabis were both ascribed, until a few years ago, to the same major component of this plant, i.e. (−)-Δ9-tetrahydrocannabinol (THC), we now know that several other cannabinoids with fewer psychotropic actions, such as, for example, cannabidiol, may contribute to its pharmacology (reviewed in4) (Fig. 1). Nevertheless, studies on the molecular mechanism of action of THC were instrumental in identifying in vertebrates an endogenous signaling system, known as the endocannabinoid system (ECS). This system is active in several tissues, including the GI tract, and comprises at least two G-protein-coupled receptors, the cannabinoid CB1 and CB2 receptors, their endogenous ligands, the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) (Fig. 1), and proteins for the metabolic regulation of endocannabinoid levels (reviewed in5). It has also become increasingly clear that endocannabinoids, and anandamide in particular, can activate non-CB1, non-CB2 receptors, the most studied of which is the Transient Receptor Potential Vanilloid type-1 (TRPV1) channel, and that several other endocannabinoid-like molecules, often exhibiting low affinity for CB1 and CB2 receptors, also occur in mammals (reviewed in6). Furthermore, we now know that cannabinoids can interact with proteins of the ECS and other targets, in particular TRPV1 and other TRP channels, to the point that many researchers now consider these channels as part of the ECS.6 However, the physiological and pathological significance of these latter discoveries, particularly in the gut, has not yet been investigated. In the present article, we review some of the most recent developments in the research of the function of the ECS in the GI tract, with particular emphasis on data published in the Journal.

CB1 RECEPTORS: FROM MOTILITY TO PAIN AND INFLAMMATION
A CB1 ?tone? controlling intestinal motility: to be or not to be?
Anatomical and functional evidence suggests the presence of CB1 receptors in neurons of the myenteric plexus in a variety of species, including humans. Activation of prejunctional CB1 receptors reduces excitatory enteric transmission (mainly cholinergic transmission) in different regions of the GI tract, thereby leading to inhibition of motility (reviewed in7). There has been a debate as to whether, under physiological conditions, endocannabinoids tonically activate CB1 receptors to control small intestine and colon motility. Initial studies had suggested this possibility based on the observation that: (i) endocannabinoid levels in several districts of the GI tract are sufficient to constitutively activate CB1 receptors; (ii) CB1 antagonists increase motility, which parallels both their stimulation of electrically induced contractions of the guinea pig ileum in vitro and the finding of increased motility in CB1 receptor knockout mice; and (iii) blockade of endocannabinoid catabolism with selective inhibitors reduces intestinal and colonic motility.7 Importantly, as shown in a paper published in the Journal, CB1 antagonists as well as ?knockout? of CB1 also modulate other neurophysiological correlates of small intestine propulsion, such as the ascending neuronal contraction following electrical field stimulation of the rat ileum.8 The use of this set-up allowed the measurement of peristaltic activity and to separate the aboral stimulation site from the oral one, and led the authors to confirm that endocannabinoids and CB1 receptors are physiologically involved in the control of small intestine motility by inhibiting activity at the neuromuscular junction.8,9 Finally, an elegant in vitro study, published again in the Journal,10 showed how, in primary cultures of guinea pig myenteric neurons, CB1 receptor antagonists increase, and agonists decrease, spontaneous network activity as well as the number of: (i) synaptic vesicles being recycled during electrical stimulation; (ii) synaptophysin-immunopositive release sites; and (iii) mitochondria transported towards enteric fiber terminals, which are all specific indicators of prejunctional synaptic activity of myenteric neurons. The effects of the agonists could also be reproduced with two inhibitors of anandamide inactivation, thus again pointing to a constitutive control of myenteric neuron activity by the ECS.10

The conclusion from studies using cannabinoid antagonists that endocannabinoids exert tonic modulation of CB1 to inhibit motility was recently questioned on the basis that these compounds are not ?neutral? antagonists, but behave as inverse agonists in vitro at concentrations not too far from those corresponding to their Ki?s for CB1 receptors. This would suggest that the observed stimulation of motility by these compounds is not the result of their antagonizing the effects of endocannabinoid levels, but instead is due to their stabilization of a receptor conformation that has stronger affinity for the inactive form of the G-protein. It was argued that such possibility could be investigated using a new generation of ?neutral? CB1 antagonists now available,11,12 as these compounds would produce stimulation of GI transit only in the presence of endocannabinoid levels sufficiently high to activate CB1 receptors. Indeed, Storr et al., reported that one such compound, AM4113, unlike the widely used inverse agonist AM251, was devoid of any stimulatory activity on electrically induced contractions of the mouse ileum in vitro, although, somehow paradoxically, it did enhance upper intestinal transit, whereas it produced no stimulation of whole gut transit.11 The issue of ?neutral?vs?inverse agonism? is tricky and very difficult to investigate in vivo, and a mathematical model has been proposed recently according to which all supposedly ?neutral? antagonists would exert inverse agonism in vitro provided that a sufficiently high, but still specific, concentration is used.13 Nevertheless, a somehow conservative interpretation of these results is that, while there might still be a tonic ECS controlling motility in the upper GI tract, further investigations are needed for the large intestine, although a study showed the depressant effect of an inhibitor of endocannabinoid inactivation on colonic propulsion.14

Pancreatitis, irritable bowel syndrome and septic ileus: is CB1 the ?bad guy??
Seminal studies carried out in the mid-2000s (reviewed in15) showed for the first time that the ECS, both in terms of endocannabinoid levels and CB1 receptor expression, is up-regulated with pro-homeostatic and protective function during several different types of experimental small intestine and colon inflammation, and that such up-regulation occurs also in human inflammatory bowel diseases (IBD). Recent reports (see below) have highlighted the role that CB2 receptors may also play in the taming of colonic inflammation and its consequences on motility, a possibility that, given the potential central side effects of CB1 receptor agonists, opens the way to the possible use of non-psychotropic CB2 agonists for the treatment of IBD, along with compounds that inhibit endocannabinoid inactivation. The anti-inflammatory effects of CB1/CB2 agonists have been recently studied also in the pancreas, as described in an article published in the Journal, through experiments carried out both in vitro, in isolated pancreatic acini, and in vivo, in experimental pancreatitis in rats.16 The authors showed that the cannabinoid receptor agonist, WIN55,212-2, inhibits the release of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) from acinar cells obtained from untreated rats, and reduced serum amylase, pancreatic edema and IL-6 and MCP-1 acinar content in rats with caerulein-induced pancreatitis, whilst also improving pancreatic damage in these animals. Interestingly, however, these protective effects were observed in vivo only when the CB1/CB2 agonist was given before the inflammatory stimulus, whereas when WIN55,212-2 was administered afterwards, the pancreatitis was worsened. While the protective effect observed with pretreatment was antagonized by a selective CB2 receptor blocker, the worsening effect was instead antagonized by a CB1-selective blocker.16 The authors suggested that, in the context of pancreatitis in vivo, CB1 activation might concur to oxidative stress or exert chemoattractant activity on macrophages, thus contributing to inflammation. An alternative explanation, however, might lie in the previous observation that, in the same experimental model of pancreatitis, TRPV1 channels participate in inflammation via a sensory mechanism leading to the production of pro-inflammatory peptides,17 and the same has also been reported for TRP channels of ankirin-1 type (TRPA1) in mice.18 The possibility exists that WIN55,212-2 might worsen pancreatitis through the sensitization of TRPV1 either via a direct interaction with this channel in a complex with TRPA1,19 or, indirectly, by activating CB1 receptors.20,21

In view of the very efficacious effects of endocannabinoid-based drugs in animal models of visceral pain, the role of the ECS in the control of irritable bowel syndrome (IBS) has also been proposed (reviewed in22). This hypothesis is supported by the recent finding of an association between a polymorphism in the Cnr1 gene encoding for CB1 receptors and the occurrence of IBS in the Korean population.23 Two studies published in the Journal have now addressed this possibility, using completely different approaches. Yuece et al., investigated the effect of CB1 agonists and antagonists/inverse agonists on afferent nerve discharges from rat myenteric neurons stimulated with either serotonin or bradykinin, two mediators known to activate sensory GI afferents and participate in visceral sensitivity.24 The results were intriguing and perhaps surprising in as much as the authors reported different effects of the agonist WIN55,212-2 (the activity of which on peristaltic activity was shown by the same group to be mostly mediated by CB1 receptors8) depending on the type of the stimulus, and possibly in a direction opposite to what expected. While WIN55,212-2 enhanced the effect of serotonin and did not influence that of bradykinin, the CB1 inverse agonist SR141716A (rimonabant) reduced the effect of bradykinin without affecting that of serotonin. Although counterintuitive, the findings with rimonabant might help to explain some anti-inflammatory effects observed in vivo with this compound in mice treated with lipopolysaccharide (LPS).25 The lack of effect of WIN55,212-2 on bradykinin, and its stimulation of the serotonin effect, instead, might argue against the possible therapeutic use of CB1 agonists in IBS, although of course studies in more specific animal models of this disorder should be carried out before reaching this conclusion. Interestingly, however, in the other study on this issue published very recently in the Journal, THC failed to produce any relief of visceral sensitivity after rectal distension in both healthy volunteers and IBS patients.26

Another GI disorder that might be ameliorated by antagonizing, rather than enhancing, the activity of CB1 is ileus, a pathological state consisting of decreased intestinal motility following peritonitis, surgery, or other noxious situations. Mascolo et al., showed that, in acetic acid-induced ileus in mice, reduced intestinal motility was accompanied by increased levels of anandamide compared with control mice, and by overexpression of CB1 receptors in myenteric nerves.27 Importantly, reduced transit was alleviated by rimonabant, but not by a CB2-selective antagonist, and was worsened by VDM11, a selective inhibitor of anandamide cellular uptake.27 In an article published in the Journal, Li et al., show that not only CB1, but also CB2 receptors might participate in LPS-induced ileus in rats, a model of septic ileus.28 In this case, the authors monitored not only upper intestinal motility but also spontaneous jejunal myoelectrical activity and IL-6 and tumor necrosis factor (TNF)-α release, and found that antagonism not only of CB1, but also of CB2 receptors, prevented LPS-induced reduction of myoelectrical activity and of upper GI transit. CB1 and CB2 antagonists also tended to reduce the elevation of IL-6 induced by a low dose of LPS.28 These data indicate that, contrary to ileus induced by a chemical irritant, also CB2 receptors participate in the etiopathology of septic ileus, possibly because of their role in inflammation. Furthermore, they also confirm the role of CB2 receptors in regulating intestinal motility under inflammatory conditions (or perhaps not just [see below]?).

Gastric motility
Although initially neglected, the study of the role of the ECS in the control of gastric motility has been recently investigated in several studies, two of which published in the Journal. The existence of a CB1 tone controlling gastric emptying was first suggested by data indicating that: (i) anandamide inhibits this function in a way counteracted by the CB1 receptor antagonist rimonabant, but not by the CB2 receptor antagonist SR144528 or by TRPV1 antagonist 5′-iodoresiniferatoxin; (ii) inhibition of anandamide degradation by fatty acid amide hydrolase (FAAH) also reduces gastric emptying in a way partly reduced by rimonabant; and (iii) rimonabant per se increases gastric motility.29 Interestingly, the inhibitory effect on gastric transit by CB1 activation, as recently investigated by the use of WIN55,212-2 and the CB1 antagonst AM251, does not undergo tolerance following chronic stimulation, unlike the inhibition of upper intestinal or colorectal transit, or the psychotropic effects of chronic CB1 agonism.30 This finding should open the way to future mechanistic studies investigating the molecular bases of this lack of tolerance, which might be due, for example, to impaired CB1 receptor internalization following repeated stimulation in the stomach. Furthermore, since delayed gastric transit may contribute to satiety and emesis, the authors suggested that the lack of tolerance to inhibition of gastric motility following chronic administration with CB1 agonists might reduce the efficacy of these compounds as anti-anorexiant and anti-emetic therapies.31 Nevertheless, WIN55,212-2 was recently shown to inhibit gastric myoelectric function, in terms of reduction of the frequency of antral pacemaker activity, both in vehicle- and apomorphine-treated ferrets.31 Although no CB1 antagonist was used in this study to ascertain the involvement of CB1 receptors in the effects of the compound, these data provided further substantiation to the well-known anti-emetic actions of CB1 receptor activation (reviewed in32), and in fact WIN55,212-2 was found by the authors to inhibit also the apomorphine-induced emetic response.31 On the other hand, contrary to previous findings obtained in the ferret using a different pro-emetic stimulus,33 the authors found that the FAAH inhibitor URB597 did not reduce retches and vomits induced by the non-selective dopamine receptor agonist.31

CB2 RECEPTORS: INFLAMMATION AND BEYOND
The role of the CB2 receptor in the GI tract has been investigated more recently than that of its cognate cannabinoid receptor (reviewed in34). It is now clear that CB2 receptors can become activated by elevated endocannabinoid levels in several types of experimental colitis, and that mutated mice lacking this receptor are more sensitive to the inflammatory effects of trinitrobenzene sulfonic acid.35,36 More recent data, published in the Journal, suggest a wider role of this receptor than just the control of gut inflammation. Hillsley et al., reported that the CB2-selective agonist, AM1241, is capable of blocking bradykinin-induced elevation of mesenteric afferent nerve activity, a neurophysiological correlate of small intestine sensitivity, monitored in vivo in the mouse jejunum.37 This inhibitory effect was fully antagonized by a CB2 antagonist and was absent in CB2−/− mice. Given the role of bradykinin in pain and inflammation, this finding was interpreted by the authors as further confirmation of the analgesic and anti-inflammatory effects of CB2 agonists during IBD. However, whilst the observed effect was clearly of peripheral nature, no experiment was performed in order to assess whether AM1241 was acting at the level of sensory neurons or immune cells.37 The former possibility should not be excluded since there is evidence, albeit still controversial, that some sensory fibers involved in pain perception do express CB2 receptors.38

Although it efficaciously counteracts alterations of intestinal motility during inflammatory conditions,39 activation of CB2 is known not to affect this function in healthy animals. This is true also for gastric emptying, although a recent study, published in the Journal, seems to cast some doubts over this last concept. Indeed, whilst most reports investigating the selective CB2 inverse agonist, SR144528, in the context of gastric emptying found no effect of this compound per se, and no antagonism of the inhibitory effects of CB1/CB2 agonists, Abalo et al.40 showed that this compound, at a rather selective dose (1 mg kg−1, i.p.), significantly potentiates the inhibitory effect of the CB1/CB2 agonist, WIN55,212-2, while exerting a little, and not-statistically significant, inhibitory effect per se. SR144528 enhancement of WIN55,212-2-induced inhibition of gastric emptying was so strong to result also in delayed emptying of the small intestine, cecum and colon, and it is certainly surprising that such a phenomenon had never been reported before. However, the authors reported that another CB2 inverse agonist, AM630, was not endowed with the same property, thus leaving open the possibility that SR144528 acts via a non-CB2-mediated mechanism.40 Alternatively, it is possible that the use by Abalo and colleagues of radiographic methods to study GI transit, and of longer observation periods, unmasked a previously undetected and intriguing tonic stimulatory function of CB2 receptors on gastric motility, which could be exploited for the development of new satiety- and weight loss-inducing drugs from CB2 antagonists. Indeed, CB2−/− mice are resistant to weight gain following a high fat diet,41 which in mice also leads to higher levels of the endocannabinoid 2-AG and lower levels of CB1 receptor expression in the stomach.29

ENDOCANNABINOID-RELATED MOLECULES AND PHYTOCANNABINOIDS: NEW MECHANISMS AWAITING TO BE DISCOVERED
The identification of anandamide opened the way to the finding of several anandamide-like molecules that are metabolically related to this endocannabinoid but act mostly via non-CB1 and non-CB2-mediated mechanisms.6 One of the most studied of these compounds is oleoylethanolamide (OEA) (Fig. 1), an anorexigen mediator acting mostly at peroxisome proliferator-activated receptor-α (PPAR-α) nuclear receptors and, to some extent, TRPV1 channels (reviewed in42). Oleoylethanolamide was originally reported to inhibit small intestine motility43 in a manner insensitive to a TRPV1 antagonist and only partly attenuated by a CB1 antagonist. This effect was shared with other fatty acid amides with little affinity for cannabinoid, PPAR-α and TRPV1 antagonists, and suggested to be mediated in part by inhibition of FAAH through substrate competition, thus potentially leading to elevated levels of endocannabinoids in the small intestine.43 A study recently appeared in the Journal, using different types of mutated mice in which CB1, CB2, or PPAR-α receptors are absent, showed the lack of involvement of these proteins in the effect of OEA, despite the finding of PPAR-α immunoreactivity in the myenteric plexus of the stomach, duodenum, jejunum, ileum and distal colon of the mouse.44 Moreover, a glucagon-like peptide-1 receptor antagonist did not reverse the inhibitory effect of OEA, which, however, was statistically significant in this study only at i.p. doses fourfold higher than those used in the previous study.43,44 Interestingly, OEA also inhibits gastric transit, again in a manner not antagonized by cannabinoid, PPAR-α or TRPV1 receptor antagonists, and since the levels of this compound are increased in the stomach of mice subjected to a chronic high fat diet, this effect was suggested to be responsible for the decreased gastric transit observed in these mice, and perhaps to contribute also to a part of the satiety-inducing effects of OEA.45

The realization that another cannabinoid constituent of Cannabis, namely cannabidiol, possess potential therapeutic properties,4 suggested the thorough pharmacological exploration of several non-THC cannabinoids also in the GI tract. Cannabidiol has very low affinity for CB1 and CB2 receptors, but was reported to exert either functional enhancement or counteraction of CB1-mediated effects, and to inhibit some of the processes through which endocannabinoids are inactivated, and FAAH in particular.46 This compound was recently investigated in models of upper intestinal motility disturbances induced by inflammatory stimuli. Thus, cannabidiol reversed croton oil-induced small intestine hypermotility,47 and worsened LPS-induced hypomotility.48 While the former effect, based on experiments with CB1 and FAAH inhibitors, was suggested to be due to indirect activation of CB1 receptors subsequent to inhibition of FAAH activity,47 the effect on LPS-induced hypomotility was accompanied by reduction of FAAH expression in the small intestine (which is up-regulated by LPS), and again antagonized by CB1 receptor blockade.48 Thus, by acting as an indirect agonist at CB1 receptors, cannabidiol reproduces some of the effects of selective inhibitors of anandamide hydrolysis or reuptake and ameliorates small intestine motility whilst worsening LPS-induced ileus. Interestingly, cannabidiol also produces anti-inflammatory effects in experimental models of colitis.49,50 These effects are particularly strong in the mouse and, however, at least in this species, do not seem to involve FAAH inhibition.49

Phytocannabinoids have been recently defined as ?any plant-derived natural product capable of either directly interacting with cannabinoid receptors or sharing chemical similarity with cannabinoids, or both?.51 With this definition in mind, salvinorin A (SA) (Fig. 1), the major active ingredient of Salvia divinorum and potent κ-opioid receptor (KOR) agonist, which produces central effects in vivo that are partly antagonized by CB1 blockers,52 but does not bind with appreciable affinity to either CB1 or CB2 receptors,53 should not be considered a ?phytocannabinoid?. Nevertheless, the GI effects produced by this hallucinogenic compound are antagonized by CB1 inverse agonists. Capasso et al., showed that SA counteracts croton oil-induced hypermotility in a manner attenuated by both KOR and CB1 antagonists,53 whereas Fichna et al. published the results of a thorough investigation of the effects of this compound on GI transit in vivo and in vitro, and on neurogenic ion transport in vitro, in healthy mice.54 These authors observed that SA inhibits contractions of the mouse stomach, ileum, and colon in vitro, and prolongs colonic propulsion and slows upper GI transit in vivo, without affecting gastric emptying. It also reduces veratridine-, but not forskolin-, induced epithelial ion transport. The effects of SA on colonic motility in vitro were mediated by both KOR and CB1 receptors, as they were inhibited by the antagonists nor-binaltorphimine and AM251, respectively. Perhaps even more intriguing was the finding that AM630, a CB2-selective inverse agonist, also inhibited these effects. However, in the colon in vivo, SA actions were almost uniquely mediated by KOR. Finally, the effects of SA on veratridine-mediated epithelial ion transport were inhibited by both nor-binaltorphimine and AM630.54 These data, bearing in mind the lack of affinity of SA for CB1 and CB2 receptors,53 point to the existence of a functional cross-talk between KOR and cannabinoid receptors. This possibility is also suggested by the recent finding that CB1 antagonism attenuates the activation of KOR by a selective agonist in a GTPγS binding assay, although SA does not substitute for THC in mice trained to discriminate this compound.55 It is possible that KOR and CB1 or CB2 receptors form heterodimers with pharmacology different from that of the homodimers, and this could be also a unique way through which CB2 receptors may participate in upper GI motility and epithelial ion transport. Alternatively, KOR and CB1 or CB2 receptors might cross-talk at the level of their signal transduction cascades, as was recently suggested for CB1 and δ-opioid receptors,56 and previously reported for CB1 and μ-opioid receptors (reviewed in57).

Conclusions
The reports published in Neurogastroenterology & Motility and included in this special collection, together with related studies published in other journals over the last 2 years, confirm that the ECS and related emerging signaling systems may play a fundamental role in the control of all aspects of GI physiology and pathology. As with pathological states affecting other vital functions,5 the available data allow us to predict that strategies that either enhance or curb the activity of the ECS might be both employed for future therapies targeting various GI disorders. Furthermore, the new data discussed in this article allow for speculations on what could be novel physiological and pathological functions in the GI tract of the ECS, particularly at the level of CB2 receptors and TRP channels, and of endocannabinoid-related molecules, while opening the way also to future investigations on non-THC cannabinoids and plant natural products that do not necessarily directly modify the activity of CB1 and CB2 receptors. Future research will tell us if these ?gut feelings? about the ECS will eventually translate into new knowledge of basic and clinical importance.

Source: Wiley.com
Via Gut Feelings About The Endocannabinoid System

Background

Cannabinoid 2 receptor (CB2R) agonists attenuate inflammatory pain but the precise mechanism implicated in these effects is not completely elucidated. We investigated if the peripheral nitric oxide-cGMP-protein kinase G (PKG)-ATP-sensitive K+ (KATP) channels signaling pathway triggered by the neuronal nitric oxide synthase (NOS1) and modulated by opioids, participates in the local antinociceptive effects produced by a CB2R agonist (JWH-015) during chronic inflammatory pain.

Methodology/Principal Findings

In wild type (WT) and NOS1 knockout (NOS1-KO) mice, at 10 days after the subplantar administration of complete Freund’s adjuvant (CFA), we evaluated the antiallodynic (von Frey filaments) and antihyperalgesic (plantar test) effects produced by the subplantar administration of JWH-015 and the reversion of their effects by the local co-administration with CB2R (AM630), peripheral opioid receptor (naloxone methiodide, NX-ME) or CB1R (AM251) antagonists. Expression of CB2R and NOS1 as well as the antinociceptive effects produced by a high dose of JWH-015 combined with different doses of selective L-guanylate cyclase (ODQ) or PKG (Rp-8-pCPT-cGMPs) inhibitors or a KATP channel blocker (glibenclamide), were also assessed. Results show that the local administration of JWH-015 dose-dependently inhibited the mechanical and thermal hypersensitivity induced by CFA which effects were completely reversed by the local co-administration of AM630 or NX-ME, but not AM251. Inflammatory pain increased the paw expression of CB2R and the dorsal root ganglia transcription of NOS1. Moreover, the antinociceptive effects of JWH-015 were absent in NOS1-KO mice and diminished by their co-administration with ODQ, Rp-8-pCPT-cGMPs or glibenclamide.

Conclusions/Significance

These data indicate that the peripheral antinociceptive effects of JWH-015 during chronic inflammatory pain are mainly produced by the local activation of the nitric oxide-cGMP-PKG-KATP signaling pathway, triggered by NOS1 and mediated by endogenous opioids. These findings suggest that the activation of this pathway might be an interesting therapeutic target for the treatment of chronic inflammatory pain with cannabinoids.

Introduction

The activation of both cannabinoid receptors 1 (CB1R) and 2 (CB2R) reduce nociception in numerous animal pain models [1]?[3]. However, while the analgesic potential derived from the stimulation of CB1R is accompanied with several central site-effects, the administration of selective CB2R agonists reduces nociception without causing those effects [4]. As a consequence, the peripheral antinociceptive effects produced by selective CB2R agonists after local inflammation have been demonstrated in several works [2], [5]?[7]. It is well known that CB2R are mainly located in the peripheral nervous system, but although an increased expression of these receptors has been recently demonstrated in the dorsal root ganglia and paw of animals with acute (2 hours) peripheral inflammation [8], the probable changes in their peripheral expression after chronic inflammatory pain remains to be fully elucidated. Moreover, the possible mechanisms implicated in the peripheral actions of CB2R agonists during chronic inflammatory pain have not been evaluated.

Several studies have shown that nitric oxide, synthesized by neuronal nitric oxide synthase (NOS1), mediates numerous inflammatory pain symptoms [9]?[10] and the local antinociceptive effects of opioids during inflammation is mainly produced by the activation of the peripheral nitric oxide-cGMP-protein kinase G (PKG)-ATP-sensitive K+ (KATP) channels signaling pathway [11]?[13]. Recent studies also demonstrated that the activation of CB1R stimulates the cGMP production in neuronal cells [14], that the antinociceptive effects produced by a CB1 endocannabinoid are mainly mediated by the nitric oxide-cGMP pathway activation [15] and that the inactivation of the nitric oxide-cGMP-PKG peripheral pathway enhanced the peripheral antinociceptive effects of CB2R agonists during neuropathic pain [16]. Some works have been also shown that the antinociceptive effects produced by AM1241 (a specific CB2R agonists) were mediated through the release of endogenous opioid peptides from CB2R-expressing cells [17]?[18]. Even so, the participation of the local endogenous opioid peptides as well as the nitric oxide-cGMP-PKG-KATP signaling pathway in the peripheral antinociceptive effects produced by JWH-015 during chronic inflammatory pain is not known.

Thus, in order to study if the nitric oxide synthesized by NOS1 could modulate the local effects of CB2R agonists during chronic peripheral inflammation we evaluated the mechanical antiallodynic and thermal antihyperalgesic effects of the subplantar administration of 2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone (JWH-015), in wild type (WT) and NOS1-KO mice, at 10 days after the complete Freund’s adjuvant (CFA)-injection. The receptor specificity of these effects and the possible participation of the peripheral endogenous opioids in the effects produced by JWH-015 after inflammatory pain were assessed by evaluation their reversion with specific a CB2R (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone; AM630), a CB1R (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM251) or a peripherally acting opioid receptor (naloxone methioide; NX-ME) antagonist. The mRNA and protein levels of CB2R in the dorsal root ganglia and paw as well as of NOS1 in the dorsal root ganglia of WT mice, with and without peripheral inflammation, were also assessed. Finally, to evaluate if the cGMP-PKG-KATP peripheral pathway activation could modulate the local effects produced by JWH-015, the antiallodynic and antihyperalgesic effects produced by this agonist co-administered with different doses of a selective soluble guanylate cyclase (1H-[1], [2], [4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ) or a PKG ((Rp)-8-(para-chlorophenylthio)guanosine-3′,5′-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs) inhibitor as well as by a selective KATP channel blocker (glibenclamide), were also determined.

Results

Effects of the subplantar administration of JWH-015 in the mechanical allodynia and thermal hyperalgesia induced by CFA

In a mouse model of CFA-induced inflammatory pain [19], our results show that the subplantar administration of JWH-015 into the ipsilateral paw dose-dependently inhibited the mechanical allodynia (Fig. 1A) and thermal hyperalgesia (Fig. 1B) induced by the inflammatory agent. Thus, the mechanical antiallodynic and thermal antihyperalgesic effects produced by different doses of JWH-015 (15-300 g) in the ipsilateral paws of CFA-injected WT mice were significantly higher than those obtained in their corresponding vehicle treated groups (p<0.01; Student’s t test). The subplantar administration of JWH-015 or vehicle did not produce any significant effect on the contralateral paw of these animals (data not shown).

Reversion of the antinociceptive effects of JWH-015 by AM630, NX-ME or AM251 after chronic inflammatory pain

The administration of CFA induced a significant mechanical allodynia (Fig. 2A) and thermal hyperalgesia (Fig. 2B) in the ipsilateral paw as compared to their corresponding contralateral paw (p<0.001; paired Student’s t test). The antiallodynic (Fig. 2A) and antihyperalgesic (Fig. 2B) effects produced by a high dose of JWH015 in the ipsilateral paw of CFA-injected WT mice were completely reversed by their subplantar co-administration with a selective CB2R (AM630) or a peripheral opioid receptor (NX-ME) antagonist (p<0.001; paired Student’s t test compared to their corresponding contralateral paw). The subplantar administration of AM251 (a selective CB1R antagonist) was unable to revert the local antiallodynic and antihyperalgesic effects produced by JWH-015 (p<0.05; one way ANOVA, followed by Student Newman Keuls, compared with the ipsilateral paw of the vehicle treated group). The subplantar administration of the agonist alone or combined with the different tested antagonists did not produce any significant effect in the contralateral paw as compared to vehicle. In addition, the subplantar administration of AM630, NX-ME, AM251 or vehicle alone in CFA-injected mice did not show any significant effect on the two different nociceptive responses evaluated in this study (data not shown).

The mRNA and protein levels of CB2R in the dorsal root ganglia and paw of WT mice with and without chronic inflammatory pain

The mRNA and protein levels of CB2R in the ipsilateral side of the dorsal root ganglia (A and B) and paw (D and E) from WT mice, with (CFA) and without (naive) inflammatory pain, are shown in Figure 3. While peripheral inflammation did not alter the dorsal root ganglia mRNA and protein expression of CB2R, it significantly increased their expression in the paw (p<0.030 Student’s t test as compared to naive animals).

The mRNA levels and protein levels of NOS1 in the dorsal root ganglia of WT mice with and without chronic inflammatory pain

The mRNA and protein levels of NOS1 in the ipsilateral side of the dorsal root ganglia from WT mice with (CFA) and without (naive) inflammatory pain are shown in Figure 4A and 4B, respectively. Our results showed that inflammatory pain significantly enhanced the mRNA expression of NOS1, but not their protein levels, in the ipsilateral side of CFA injected mice as compared to naive (p<0.032, Student’s t test).

The role of nitric oxide synthesized by NOS1 in the local antinociceptive effects produced by JWH-015 during chronic inflammatory pain

The role played by nitric oxide synthesized by NOS1 in the local antinociceptive effects produced by JWH-015 during peripheral inflammation was evaluated by comparing the antiallodynic (Fig. 5A) and the antihyperalgesic (Fig. 5B) effects produced by a high dose of this agonist (150 g) in WT and NOS1-KO mice at 10 days after CFA injection. Our results show that the subplantar injection of CFA induces a reduced mechanical allodynia (p<0.05; one way ANOVA, followed by Student Newman Keuls test) and a similar thermal hyperalgesia in the ipsilateral paw of NOS1-KO mice as compared to WT. The subplantar administration of JWH-015 only reversed these effects in WT mice, but not in NOS1-KO (p<0.001, paired Student’s t test, comparing ipsilateral vs. contralateral paw). Moreover, the effects produced by JWH-015 in the ipsilateral paw of NOS1-KO mice are significantly lower to that those produced by this drug in the ipsilateral paw of WT mice (p<0.05; one way ANOVA, followed by Student Newman Keuls test). In both genotypes the subplantar administration of JWH-015 or vehicle did not have any significant effect on the contralateral paw of these animals.

Involvement of the peripheral nitric oxide?cGMP?PKG-KATP signaling pathway in the local antiallodynic and antihyperalgesic effects produced by JWH-015 during chronic inflammatory pain

The role of the peripheral nitric oxide-cGMP-PKG-KATP signaling pathway in the local mechanical antiallodynic and thermal antihyperalgesic effects produced by JWH-015 in CFA-injected WT mice was assessed by evaluating the effects produced by 150 g of this agonist co-administered with different dose of ODQ, Rp-8-pCPT-cGMPs or glibenclamide.

Our results showed that the local antiallodynic and antihyperalgesic effects produced by JWH-015 in the ipsilateral paw of CFA-injected WT mice were significantly inhibited by their peripheral co-administration with different doses of ODQ (Fig. 6, A-B), Rp-8-pCPT-cGMPs (Fig. 6, C-D) or glibenclamide (Fig. 6, E-F) in a dose-dependent manner (p<0.001, one way ANOVA followed by Student Newman Keuls test). While the local co-administration of JWH-015 plus ODQ, Rp-8-pCPT-cGMPs or glibenclamide did not have any significant effect on the contralateral paw of CFA-injected mice (data not shown). Our results also indicated that the subplantar administration of the highest tested doses of ODQ (3 g), Rp-8-pCPT-cGMP (5 g), glibenclamide (10 g) did not produce any significant antiallodynic or antihyperalgesic effect in the ipsilateral or contralateral paw of CFA-injected WT mice.

Discussion

In this study, we showed for first time that the local administration of JWH-015 dose-dependently inhibited the mechanical allodynia and thermal hyperalgesia induced by CFA through the activation of the peripheral nitric oxide-cGMP-PKG-KATP channel signaling pathway, triggered by NOS1 and mediated by opioids. Indeed, the local mechanical antiallodynic and thermal antihyperalgesic effects produced by a high dose of JWH-015 were blocked by NX-ME, annulled in NOS1-KO mice and dose-dependently diminished by their co-administration with different doses of ODQ, Rp-8-pCPT-cGMPs and glibenclamide. Our results also show that chronic inflammatory pain increases the paw expression of CB2R as well as to the dorsal root ganglia transcription of NOS1.

Several works demonstrated that the local administration of CB2R selective agonists attenuates the thermal and mechanical hypersensitivity induced by carrageenan or CFA in different models of acute (hours to two days) inflammatory pain [2], [8], [20]. Our results support and expand this hypothesis in a chronic model of inflammatory pain at 10 days after CFA injection. The CB2R specificity of the inhibitory effects induced by JWH-015 was demonstrated by the complete reversion of their effects with the local co-administration with a selective CB2R, but not a CB1R, antagonist. In addition, the fact that the highest dose of JWH-015 did not produce any significant effect in the contralateral paw of CFA-injected mice denotes the peripheral site of action of this drug.

Our data also show that although chronic inflammatory pain did not alter the peripheral mRNA or protein levels of CB2R in the dorsal root ganglia, it increases their expression in the paw. This is in accordance with the unchanged expression of these receptors observed in the dorsal root ganglia of animals with bone-cancer induced chronic pain [3] as well as to the increased expression of those observed in the paw of animals with acute inflammatory pain [8]. Thus, our results support these data and expand theme to chronic inflammatory pain conditions.

It is known that the antinociceptive effects produced by a specific CB2R agonist (AM1241) are mediated through the release of β-endorphins which appear to act at -opioid receptors located on the terminals of primary afferent neurons to produce peripheral antinociception during acute inflammation and bone cancer pain [3], [17]?[18]. Our results demonstrated that the antiallodynic and antihyperalgesic effects produced by JWH-015 were completely reversed by their local co-administration with a peripherally acting opioid receptor antagonist. These findings revealed that during chronic inflammatory pain the opioid-mediated antinociception derived from the activation of peripheral CB2R by JWH-015 is also functional.

In accordance with the literature [21], our results also demonstrated that chronic inflammatory pain induced a modest increase in the dorsal root ganglia transcription of NOS1, which did not correlate with an increased protein expression probably related to the much higher sensitivity of the real-time PCR assay compared to the western blot. Several works have been demonstrated that the local antinociceptive effects produced by -opioid receptor agonists during inflammation are mainly mediated by the release of nitric oxide synthesized by NOS1 [19], [22]. Thus, and taking account that JWH-015 produces their antinociceptive effects by the activation of peripheral opioid receptors, we have evaluated if this opioid-mediated antinociception induced by CB2R activation is also produced via NOS1 by using knockout mice. The fact that the local administration of JWH-015 did not block the mechanical and thermal hypersensitivity induced by CFA in NOS1-KO animals suggests that nitric oxide synthesized by NOS1 also participates in the local antinociceptive effects produced by this agonist during chronic inflammatory pain.

The possible involvement of the peripheral cGMP-PKG-KATP channel signaling pathway in the local effects of a CB2R agonist after chronic inflammatory pain was also evaluated. Interestingly, and in contrast to neuropathic pain [16], the local pharmacological blockage of the nitric oxide-cGMP-PKG-ATP signaling pathway diminished the peripheral antiallodynic and antihyperalgesic effects of a CB2R agonist after CFA injection. That is, the inhibitory effects induced by JWH-015 were dose-dependently diminished by their peripheral co-administration with ODQ, Rp-8-pCPT-cGMPs or glibenclamide. Therefore, and similarly to what occurs with a CB1 endocannabinoid [15], the peripheral analgesia induced by JWH-015 under chronic inflammatory conditions depends on the activation of the local nitric oxide-cGMP-PKG-KATP channel signaling pathway.

In summary, our data demonstrate that the peripheral nitric oxide-cGMP-PKG-KATP signaling pathway, triggered by NOS1 and mediated by local endogenous opioids, participates in the antinociceptive effects produced by JWH-015 and suggest that the activation of this pathway might be an interesting therapeutic target for the treatment of chronic inflammatory pain with cannabinoids.

Materials and Methods

Ethics statement

Animal procedures were conducted in accordance with the guidelines of the European Communities, Directive 86/609/EEC regulating animal research and approved by the local ethical committee of our Institution (Comissi d’Etica en l’Experimentaci Animal i Humana de la Universitat Autnoma de Barcelona, #00801).

Animals

Male NOS1-KO mice (C57BL/6J background) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA), while WT mice with the same genetic background (C57BL/6J) were acquired from Harlan Laboratories (Barcelona, Spain). All mice weighing 21 to 25 g were housed under 12-h/12-h light/ dark conditions in a room with controlled temperature (22C) and humidity (66 %). Animals had free access to food and water and were used after a minimum of 6 days acclimatization to the housing conditions. All experiments were conducted between 9:00 AM and 5:00 PM.

Induction of chronic inflammation

Chronic inflammatory pain was induced in WT and NOS1-KO mice by the subplantar injection of 30 l of complete Freund’s adjuvant (CFA; Sigma) into the right hind paw under brief anesthetic conditions with isoflurane according to the method described by Larson et al. [23]. All experiments were performed at 10 days after CFA injection. At this time point, all of these animals developed a local inflammatory reaction, allodynia to mechanical stimuli and hyperalgesia to noxious thermal stimuli as previously reported by our group [19].

Nociceptive behavioral tests

Mechanical allodynia
The mechanical allodynia was quantified by measuring the hind paw withdrawal response to von Frey filament stimulation. In brief, animals were placed in a Plexiglas box (20 cm high, 9 cm diameter) with a wire grid bottom through which the von Frey filaments (North Coast Medical, Inc., San Jose, CA, USA) bending force range from 0.008 to 3.5 g, were applied by using a modified version of the up?down paradigm, as previously reported by Chaplan et al. [24]. The filament of 0.4 g was used first and the 3.5 g filament was used as a cut-off. Then, the strength of the next filament was decreased or increased according to the response. The threshold of response was calculated from the sequence of filament strength used during the up?down procedure by using an Excel program (Microsoft Iberia SRL, Barcelona, Spain) that includes curve fitting of the data. Clear paw withdrawal, shaking or licking of the paw were considered nociceptive-like responses. Both ipsilateral and contralateral hind paws were tested. Animals were allowed to habituate for 1 h before testing in order to allow an appropriate behavioral immobility.

Thermal hyperalgesia
The thermal hyperalgesia was assessed as previously reported by Hargreaves et al. [25]. Paw withdrawal latency in response to radiant heat was measured using the plantar test apparatus (Ugo Basile, Italy). Briefly, mice were placed in Plexiglas boxes (20 cm high 9 cm diameter) positioned on a glass surface. The heat source was positioned under the plantar surface of the hind paw and activated with a light beam intensity, chosen in preliminary studies to give baseline latencies from 8 to 10 s in control mice. A cut-off time of 12 s was used to prevent tissue damage in absence of response. The mean paw withdrawal latencies from the ipsilateral and contralateral hind paws were determined from the average of 3 separate trials, taken at 5 min intervals to prevent thermal sensitization and behavioral disturbances. Animals were habituated to the environment for 1 h before the experiment to become quiet and to allow testing.

Molecular experiments

Tissue isolation
Animals were sacrificed at 0 (nave) and 10 days after CFA-injection by cervical dislocation. Three ganglia from the lumbar section (L3 to L5) of the ipsilateral site from WT and NOS1-KO mice were removed immediately after sacrifice, frozen in liquid nitrogen and stored at −80C until assay. Samples from four to five animals were pooled together to obtain enough RNA or protein levels for performing the real time-PCR or Western blot analysis, respectively. In these experiments nave mice that did not receive any injection were used as controls.

Total RNA extraction and reverse transcription
Tissues were homogenized in ice-cold with a homogenizer (Ultra-Turf, T8; Ika Werke, Staufen, Germany) and the total RNA was extracted with TRIzol reagent (Invitrogen, Renfrewshire, England). The amount of the purified RNA (A260/A280 ratio was ≥1.9) was determined by spectrophotometry. In all experiments, 1 g of total RNA was reverse transcribed into cDNA using SuperScript II RNAse H- reverse transcriptase (Invitrogen, Renfrewshire, UK) in a final volume of 10 l. Negative controls were performed in which all of the components were included except reverse transcriptase.

TaqMan probe real-time polymerase chain reaction (PCR)
The expression of NOS1 and CB2R was determined by real-time PCR using the pre-developed mice TaqMan gene expression assay: Mm0435189_m1 for NOS1 and Mm00438286_m1 for CB2R (Applied Biosystems, CA, USA). A probe against GAPDH (Mm 99999915_g1) was used as endogenous control and reactions without RNA were included as negative controls to ensure the specificity. PCR reactions were set up in 96-well plates containing the corresponding cDNA, 0.9 mol/L of each forward and reverse primers, 0.25 mol/L of TaqMan MGB probe and a final concentration of 1x universal master mix (Applied Biosystems, CA, USA), which provides the PCR buffer, MgCl2, dNTPs, and the thermal stable AmpliTaq Gold DNA polymerase. The assay was conducted using the Applied Biosystems ABI PRISM 7000 Sequence Detection System. All samples were assayed in duplicate. Relative expression of the target gene was calculated by means of the comparative threshold cycle method [26].

Western blot analysis
The CB2R protein levels in the dorsal root ganglia and paw tissue and the NOS1 protein levels in the dorsal root ganglia were analyzed by Western blot. Tissues were homogenized in buffer (50 mM Tris-Base, 150 nM NaCl, 1% NP-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 Triton X-100, 0.1% SDS, 1 mM Na3VO4, 25 mM NaF, 0.5 % protease inhibitor cocktail, 1% phosphatise inhibitor cocktail). All reactive were purchased at Sigma (St. Louis, MO, USA) with the exception of NP-40 from Calbiochem (Biosciences, La Jolla, CA, USA). The crude homogenate was solubilized 1 hour at 4C, sonicated for 10 seconds and centrifugated at 4C for 15 min at 700 g. For the CB2R, 50 g of total protein, were mixed with 4 laemmli loading buffer and then loaded onto 4% stacking/10% separating SDS-polyacrylamide gels. The proteins were electrophoretically transferred onto PVDF membrane during 2 hours, blocked with PBS +10% BSA, and subsequently incubated overnight at 4C with a polyclonal rabbit anti-CB2R antibody (1500, Abcam, Cambridge, UK). For the NOS1, 100 g of total protein, were mixed with 4 laemmli loading buffer and then loaded onto 4% stacking/5% separating SDS-polyacrylamide gels. The proteins were electrophoretically transferred onto PVDF membrane overnight, blocked with PBS +10% BSA, and subsequently incubated overnight at 4C with a polyclonal rabbit anti-NOS1 antibody (1100, BD Transduction, BD Transduction Laboratories, San Diego, CA, USA).

The proteins were detected by an horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and visualized by chemiluminescence reagents provided with the ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and exposure onto hyperfilm (GE, Healthcare). Membranes were stripped and reproved with a monoclonal rabbit anti-β-actin antibody (110.000, Sigma, St. Louis, MO, USA). β-actin was used as a loading control. The intensity of blots was quantified by densitometry.

Experimental protocol

In a model of CFA-induced inflammatory pain in mice [19], we investigated the mechanical antiallodynic and thermal antihyperalgesic effects of the subplantar administration of various doses of a selective CB2R agonist, JWH-015 (15?300 g) or vehicle in the ipsilateral and contralateral paw of WT mice at 10 days after CFA injection. All animals were tested in each paradigm at pre and post drug administration. The specificity and the possible participation of the endogenous opioids in the local antinociception produced by a high dose (150 g) of JWH-015 was assessed by evaluating the reversibility of their effects with the peripheral co-administration of 60 g of AM630 (a selective CB2R antagonist), 150 g of AM251 (a selective CB1R antagonist) or 150 g of NX-ME (a peripheral opioid receptor antagonist). This dose of JWH-015 was selected based upon their high efficacy in inhibiting the mechanical allodynia and thermal hyperalgesia induced by peripheral inflammation and the doses of the antagonists according to previous studies in the literature [2]?[3], [27]. The mRNA and protein levels of CB2R in the dorsal root ganglia and paw as well as of NOS1 in the dorsal root ganglia from the ipsilateral site of nave and CFA-injected WT mice by using real time PCR and Western blot analysis, were also assessed. In another set of experiments, the involvement of nitric oxide synthesized by NOS1 in the local antinociceptive effects of JWH-015 during peripheral inflammatory pain was investigated by using knockout mice. Thus, the effects of the subplantar administration of 150 g of JWH-015 or vehicle on the mechanical allodynia (von Frey filaments) and thermal hyperalgesia (plantar test) induced by peripheral inflammation in NOS1-KO mice, were also evaluated. Finally, the possible involvement of the peripheral nitric oxide-cGMP-PKG-KATP signaling pathway in the local mechanical and thermal antinociceptive effects of JWH-015 was also evaluated. For this purpose in WT mice at 10 days after CFA injection, the local antinociceptive effects produced by a high dose of JWH-015 (150 g) combined with different doses of ODQ (0.3?3.0 g) a selective soluble guanylate cyclase inhibitor [28], Rp-8-pCPT-cGMPs (0.3?5.0 g) a PKG inhibitor [29] or glibenclamide (3.0?10.0 g) a KATP channel blocker [30], selected according to previous studies in the literature [31], were also determined.

Drugs

JWH-015, AM630 and AM251 were obtained from Tocris (Ellisville, MI). ODQ, Rp-8-pCPT-cGMPs, glibenclamide and NX-ME were purchase from Sigma-Aldrich (St. Louis, MO). JWH-015 and ODQ were dissolved in dimethyl sulfoxide (DMSO) at 10% solution in saline while AM630, AM251 and glibenclamide were dissolved in DMSO at 50% solution in saline. NX-ME and Rp-8-pCPT-cGMPs was dissolved in saline solution (0.9% NaCl). All drug combinations were diluted in the highest required concentration of DMSO. All drugs alone or combined were injected in a final volume of 30 l. In all experiments, drugs were administered into the plantar side of the right paw, 20 min before behavioral testing. For each group treated with a drug the respective control group received the same volume of vehicle.

Statistical analysis

Data are expressed as mean standard error of the mean (SEM). For each test, the comparison of the effects produced by different doses of JWH-015 in the contralateral and ipsilateral paws was compared to the effects produced by vehicle in the same paw by using a Student’s t test. For each test, the reversion of the antinociceptive effects produced by JWH-015 in the ipsilateral and contralateral paws of WT mice was analyzed by comparing the effects produced by the subplantar administration of vehicle, JWH-015 alone or co-administered with AM630, NX-ME or AM251 by using a one-way ANOVA followed by the Student-Newman-Keuls test. The comparison of the effects produced by the different treatments in the contralateral and ipsilateral paws of CFA-injected mice was evaluated by using a paired Student’s t test.

Changes in the expression of NOS1 and CB2R in the ipsilateral sites of the dorsal root ganglia and/or paw from nave and CFA-injected WT mice were analyzed by using a Student’s t test.

For each genotype, the analysis of the antinociceptive effects produced by a high dose of JWH-015 or vehicle in the ipsilateral paw as compared to their corresponding contralateral paw was performed by using a paired Student’s t test. For each paw, the comparison of the effects produced by JWH-015 or vehicle in WT and NOS1-KO mice was evaluated by using a one way ANOVA followed by Student Newman Keuls test. The comparison of the antiallodynic and antihyperalgesic effects produced by a high dose of JWH-015 subplantarly administered alone or combined with different doses of selective inhibitors (ODQ and Rp-8-pCPT-cGMPs) or a blocker (glibenclamide) in the contralateral and ipsilateral paws of CFA-injected WT mice was performed by using a one way ANOVA followed by the Student Newman Keuls test.

A value of p<0.05 was considered as a significant.

Source, Graphs and Figures: Pubmed.gov
Via The Antinociceptive Effects Of JWH-015 In Chronic Inflammatory Pain Are Produced