Diacylglycerol Lipase in Mice Gastrointestinal Motility Research Paper

Background

The endocannabinoid system (ECS) involves two types of cannabinoid receptors, CB(1) and CB(2). These are coupled to G proteins. The central and peripheral nervous system express CB(1) receptors whereas cells in involved in immunological machinery express CB(2) receptors. The ECS controls key physiological processes in the regions like gastrointestinal tract, brain, and adipose tissue. The ECS is more specific in its functional ability with regard to the control of motility in the gut through endocannabinoids such as anandamide and 2-Arachidonoylglycerol (2-AG). There is limited information on the role of Diacylglycerol lipase, a key enzyme involved in the synthesis of endocannabinoid, 2-arachidonoylglycerol on the role of gastrointestinal motility in mice.

• Objective: The main objective of the study is to perform a literature review to strengthen the description on the role of Diacylglycerol lipase in the control of the gastrointestinal motility in mice.
• Method: A thorough literature search was performed to collect the pertinent articles from the Medline databases. The key words used are Endocannabinoids, Endocannabinoid receptors and gastrointestinal motility, Endocannabinoid System, Enteric nervous system etc. Literatures other than English were excluded.
• Results: The search yielded appropriate articles giving significant information on the endocannabinoid system.
• Conclusion: Endocannabinoid system is a potential platform for executing various physiological processes important in the gastrointestinal tract, and central nervous system. The enteric nervous system is essential for inducing propulsive movements in the small intestine. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are t considered as the main endogenous agonists of cannabinoid receptors, which can regulate pharmacological effects of (-)-Delta9-tetrahydrocannabinol (THC). However, studies on the role of Diacylglycerol lipase are still limited and thorough investigations are needed for concrete information.

The connection between the gastrointestinal and central nervous system is in the area of immense research significance in the recent period. Since years, investigators have been exploring the underlying mechanisms associated with the functional outcomes of this vital relationship. To this end, much emphasis was given in exploring the effects of Cannabis on intestinal motility and its secreted products to determine the role of the endocannabinoid system in gut function and its disorders. The finding of the cannabinoid receptors and their endogenous ligands, endocannabinoids have stimulated the much interest in this area (Di Marzo & Piscitelli, 2011).

For their proper maintenance, the endocannabinoid system (ECS) involves enzymes and ligands and receptors (Storr & Sharkey, 2007). ECS participates in the regulation of satiety, inflammation motility, secretion, sensation and emesis (Storr & Sharkey, 2007). In the gastrointestinal tract and brain, the endocannabinoid levels differ depending on the situation with regard to certain conditions like inflammation, diarrhea etc(Izzo and Sharkey,2010).

The actual role of endocannabinoid system in maintaining the important physiologic conditions required by the body is still under investigation with regard to the inter association between the receptors and ligands or vice versa and its impact on the gastrointestinal system.

There is a need of a thorough literature review to gain better insights with regard to the deep understanding of this endocannabinoid system and the likely intervention strategies or benefit expected from the exploitation of this research area.

In detail Cannabis cures gastrointestinal (GI) aberrations like disorders of abdominal pain that involve inflammatory conditions and infections(Izzo and Sharkey,2010). The rationale of the therapy is due to the finding that Delta (9)-tetrahydrocannabinol (THC) is an important major component of Cannabis (Izzo and Sharkey, 2010).

Later Delta(9)-tetrahydrocannabinol receptors were discovered which are essential for endocannabinoid system. It has particular endogenous ligands cannabinoid receptors, and their biosynthetic and degradative enzymes (Izzo and Sharkey,2010). Various studies have described that the distribution of endocannabinoid system is rampant throughout the gut, with few regional differences and organ-specific functions. It mostly controls key functions like secretions of GI tract and its motility, nausea inflammation, transfer of ions and cell growth (Izzo and Sharkey, 2010).

Here certain targets at the cellular level were recognized which include immune and epithelial cells, and the enteric nervous system (Izzo and Sharkey, 2010). At the molecular level, the targets were endocannabinoid system and the cannabinoid receptors, peroxisome proliferator-activated receptor, GPR 55 and GPR119, and alpha receptors (Izzo and Sharkey,2010). It was described that Delta9-tetrahydrocannabinol, chemically prepared cannabinoids and endogenous cannabinoids, show their effects on the gastrointestinal tract by stimulating CB1 and CB2 receptors (Massa & Monory, 2006).

In the enteric nervous system, CB1 receptors were identified in sensory endings of spinal and vagal neurons, and CB2 receptors are localised in the immune system; and their exact function is still unclear (Massa & Monory, 2006). Whenever the normal hygienic GI system gets altered, the endocannabinoid system conveys protection to the GI tract, like production of increased enteric and gastric secretion and inflammation (Massa & Monory, 2006). Hence, to ensure protection the endocannabinoid system serves as a promising therapeutic tool against various motility and secretion related disorders, inflammatory bowel and functional bowel diseases and other GI tract disorders (Massa & Monory, 2006). Thus, ECS has been greatly considered for pharmacological studies with possible implications for the design of novel drug targets.

Next, to date there were two cannabinoid CB1 receptor agonists described to be clinically relevant (Pertwee,1999). These areas already described D 9 – tetrahydrocannabinol (THC) and nabilone and they were being used as appetite stimulants or as antiemetics(Pertwee,1999).CB1 receptor agonists were also employed for inhibiting spinal cord injury, muscular spasm associated with bronchial asthma, spinal cord injury or multiple sclerosis and the relief of chronic pain. (Pertwee,1999).

Cannabinoid (CB1) receptor stimulation lessens the motility of GI neuronally, transient lower oesophageal sphincter relaxations (TLESRs) diarrhoea, pain, and emesis, and favors eating (Sanger, 2007). Whereas CB2 receptor stimulation undergoes through immune cells to lessen inflammation (Sanger, 2007). The presence of cannabinoid CB1 receptors was well reported in the enteric nervous system of not only humans but also various animals, like mouse, rat, and guinea pig and humans (Pertwee, 2001). Cannabinoid CB1 receptors lessen gastrointestinal motility through the inhibition of a live contractile transmitter production (Pertwee, 2001).

Signs of this depressant effect are, in the whole organism, delayed gastric emptying and inhibition of the transit of non-absorbable markers through the small intestine and, in isolated strips of ileal tissue, inhibition of evoked acetylcholine release, peristalsis, and cholinergic and non-adrenergic non-cholinergic (NANC) contractions of longitudinal or circular smooth muscle. The identifying features of this action are muscular contraction of cholinergic and non-adrenergic non-cholinergic (NANC) origin.

The muscles may be either circular smooth muscle or longitudinal. The other recognizing characteristics are peristaltic movements, suppression of activated acetylcholine release, slow rate of gastric emptying (Pertwee, 2001). As a result, contractile movements will rapidly occur that are obtained through electrical stimulation and which enable the release of contractile transmitters in experimental animals (Pertwee, 2001).

The CB1 receptors present in the enteric and brain regions contribute to the intestinal transit and GI tract emptying through their depressing action on the agonists of cannabinoid receptors (Pertwee, 2001). This also leads to a reduction in the production of gastric acid in response to CB1 receptor activation. Further, the effects of cannabinoid receptor agonists on gastric emptying are also found in rodents (Pertwee, 2001). Therefore, gastrointestinal motility produced by the cannabinoid receptor agonists may be resisted by strategies involving pretreatment of Cannabinoid (Pertwee, 2001).

The identifying characteristics produced by the action of agonist SR141716A and CB1 selective antagonist have led to small intestine enhanced motility in rodents. This may indicate CB1receptor activity that is previously influenced by effector mechanisms through their prelinking in the enteric nervous system (Pertwee, 2001). Animal studies have tested the rat gastric fundus for its neuromuscular function induced by the cannabinoids (Storr et al., 2002). Here, the rat gastric fundus was tested for the impact of relaxant and contractile nerves (Storr et al., 2002). It was found that the cannabinoid receptor agonists such as anandamide and methanandamide were not successful in affecting the smooth muscle activity (Storr et al., 2002).

This made it clear that the rat gastric fundus is easily influenced by the potential effects of cannabinoid inhibitory and excitatory neurotransmission (Storr et al., 2002). The localization of cannabinoid receptors on neuronal structures and involvement of more than one receptor type was shown in this study(Storr et al., 2002). The muscular system present in the gut provides a good platform for the action of cannabinoids (Bitar, 2003). Smooth muscles in the intestinal wall generate tonic contractions that enable the proper dimension of organ to sustain loads generated by food bolus and powerful contractions that generate shortening of muscle to force the bolus along the gastrointestinal tract (Bitar, 2003). Such activities are controlled by smooth muscle mechanical and intrinsic electrical properties (Bitar, 2003).

Animal studies involving porcine enteric nervous system have also shed light on the medical relevance of cannabinoid receptors. Researchers have extracted 2-arachidonylglycerol from the gut samples and detected sites in small intestine which facilitate binding of CB1-cannabinoids (Kulkarni-Narla &Brown, 2000). It is widely believed that the digestive tract of Humans and Pig is similar. Hence, using immunohistochemical approaches, CB1-cannabinoid receptors (CB1-R) were confined to the pig enteric nervous system (Kulkarni-Narla &Brown, 2000). Here anti-CB1-R antisera, was experimentally developed to enable them to react with CB1-R near the region of N-terminal epitopes (Kulkarni-Narla &Brown, 2000).

By the approach of secondary immunofluorescence antisera were later utilized to localize receptor immunoreactivity (Kulkarni-Narla &Brown, 2000). Colon and ileum of animal porcine were observed for immunological response towards CB1-R especially in the region of submucosal and myenteric plexuses enriched with ganglions (Kulkarni-Narla &Brown, 2000). There was significant immunological affinity to choline acetyltransferase (ChAT) by all the ileal CB1-R neurons (Kulkarni-Narla &Brown, 2000).

In addition, these neurons were found in the close vicinity to various ileal segments (Kulkarni-Narla &Brown, 2000). The immunoreactivity was shown by neurons to ChAT in the distal colon. In contrast, there was no immunological reaction in the neurons of colon and ileum towards nitric oxide synthase or peptides that are vasoactive in the intestine region (Kulkarni-Narla &Brown, 2000). This made it clear that porcine enteric nervous system has cholinergic neurons that are of CB1-R (Kulkarni-Narla &Brown, 2000).

The gastrointestinal functions are well regulated by Cannabinoids. The effect of endocannabinoid, 2-arachidonoylglycerol (2-AG) has been proven in this regard (Darmani, 2002). By employing an ashrew (Cryptotis parva) emetic model, researchers have administered 2-AG in a dose dependant manner which enhanced vomiting frequency and subsequently the vomiting animals were recorded (Darmani, 2002).

Other cannabinoids, methanandamide and anandamide have led to a emetogenic response also in a dose-dependent (Darmani, 2002). However, they were able to withhold the induced emetic effects temporarily. Vomiting was also prevented by nonemetic doses of SR 141716A (Darmani, 2002). But again vomiting was induced by 2-AG metabolite arachidonic acid. Thus, the effects of cannabinoids on vomiting have been shown to vary (Darmani, 2002). It was described that a cyclooxygenase inhibitor known as Indomethacin, was shown to impede the emetogenic effects of both 2-AG (Darmani, 2002). CP 55,940 and arachidonic acid and 2-AG. It was concluded that 2-AG will induce the emetic effects lower doses with regard to its locomotor suppressant actions(Darmani, 2002). CB(1) receptors facilitate the antiemetic effects of tested cannabinoids and the emetic potential of 2-AG (Darmani, 2002).

Thus 2-AG is considered as an potential endogenous emetogenic cannabinoid important for vomiting (Darmani, 2002). There is need to emphasize the enteric nervous system as it serves as an intestinal driving force for movements and their regulation(Kunze and Furness, 1999). During the process of digestion stimulation of intrinsic primary afferent neurons (IPANs) occurs due to the impact of intestinal components (Kunze and Furness, 1999).

These IPANS thus become physiologically enriched and get localized in the intrinsic myenteric ganglia (Kunze and Furness, 1999). It was described that the IPANS are randomly distributed in the small intestine with a length of 650mm in animals like guinea pig (Kunze and Furness, 1999). They possess communication among themselves via decreased excitatory transmission and finally result in the development of self-reinforcing assemblies (Kunze and Furness, 1999). The neurons in increased numbers communicate when a stimulus is given with chemicals in the lumen or to a muscular tension(Kunze and Furness, 1999).

Numerous assemblies of IPANs in thousands get activated by physiological responses (Kunze and Furness, 1999). Hence, associations exist in the form of connections between interneurons, muscle motor neurons, and IPANS (Kunze and Furness, 1999). It was further reported in the small intestine of guinea pig that contraction of circular muscle occurs eventually oral-to-anal extent manner (Kunze and Furness, 1999). An electrical syncytium results due to effect of the motor neuron innervations on smooth muscle cells (Kunze and Furness, 1999). The connection between endocannabinoid system and immunity is also matter of much clinical relevance. The rationale for this study is investigation on the health impacts of smoking marijuana and of endogenous cannabimimetic ligands in the immune system (Klein, Lane, Newton & Friedman, 2000).

It has been already described about the existence of two major cannabinoid receptors subtypes. Brain is the primary site for CB1expression and peripheral region is the site for CB2. Here several ligands exist for these receptors depending on cannabinoid structure which were later manufactured chemically (Klein et al., 2000). Similarly, various compounds dependant on noncannabinoid structure and those obtained from eicosanoids have been manufactured and investigated (Klein et al., 2000). On the other hand, highly selective receptor antagonists were also given priority for investigations (Klein et al., 2000).

Ligands with small affinity lead to anti-inflammatory properties via suppression of acute phase cytokines and TNF-alpha(Klein et al., 2000). Ligands like THC and marijuana have facilitated the observation of the diminishing action of cytokines like, interleukins 6, 12, interferon and granulocyte macrophage colony stimulating factor when subjected to their action (Klein et al., 2000). This made the cannabinoid inhibiting action pharmacologically interesting and various experiment have gradually scaled up.

In the recent period, studies were reported on the characterization of the diacylglycerol lipase (DAGL), which produces 2-AG from diacylglycerol (DAG) substrates and N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), which manufactures AEA from N arachidonoylphosphatidy ethanolamine (NArPE) (Bari et al., 2006). Hence, the function of these chemically processed routes in controlling the endocannabinoid tone in vivo may play role in the treatment models (Bari et al., 2006). Cannabinoids belong to a distinct structural category of lipophilic molecules that unite with cannabinoid receptors.

Endogenous cannabinoids are better considered as signaling lipids and they possess esters of long-chain polyunsaturated fatty acids and amides as their main constituents (Oz, 2006). They were reported to be manufactured from cell membrane lipid precursors through G-protein-dependent or Ca (2+) processes and possess cannabinoid-like function by binding to cannabinoid receptors (Oz, 2006). But, these receptors are not utilized by the endocannabinoids in yielding their outcome (Oz, 2006). Pharmacologically characterizing the endocannabinoids, their levels modify the useful features of various types of K(+) channels, Na(+) channels and voltage-gated ion channels including Ca(2+) channels (Oz, 2006). Also glycine receptors, ligand-gated ion channels like serotonin type 3, and nicotinic acetylcholine (Oz, 2006).

Further, endocannabinoids were also described to modify the proteins involved in neurotransmissions and transfer of ions (Oz, 2006). The useful characteristic features of G-protein-coupled receptors for various kinds of neuropeptides and neurotransmitters are changed by effects of endocannabinoids (Oz, 2006). However, the pathways responsible for the effects of endocannabinoids on receptors still need clarification (Oz, 2006). This could give insightful information on the efficacy of molecular targets for endocannabinoids and reflects new points for cannabinoids to change either the neuronal system response or neurons excitability (Oz, 2006).

This has strengthened the earlier reports on the action of Endocannabinoids on inhibition and excitation. They serve as messengers to prevent the neurotransmitter release at the stage of depolarization mediated inhibition or excitation. This process is also known as depolarization-induced suppression of inhibition (DSI) or excitation (DSE).To check this effect, researchers have determined the inhibiting action of endocannabinoids on N-type voltage-dependent Ca2+ channels by stimulating G(i/o)-protein-coupled CB1 cannabinoid receptors (CB1R) (Guo & Ikeda,2004). This is based on a pathway connected to DSI/DSE(Guo & Ikeda,2004).

Here, endocannabinoids such as cannabimimetic aminoalkylindole WIN 55,212-2 (WIN), anandamide (AEA)] and the2-arachidonyl glycerol ether (2-AGE),[2-arachidonylglycerol (2-AG) were used (Guo & Ikeda,2004).Rats were prior injected with cDNA encoding a human CB1R and then the mentioned endocannabinoids prevented whole-cell Ca2+ currents in rat sympathetic neurons (Guo & Ikeda,2004). It was described that agonist-mediated Ca2+ current inhibition was prevented by pertussis toxin and (PTX) a selective CB1R antagonist [SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride] (Guo & Ikeda,2004).. The potential of WIN was greater followed by 2-AG and then AEA. CB1R expression in high proportion increased the AEA efficiency (Guo & Ikeda,2004).

Thus the three endocannabinoids have been demonstrated to stimulate the heterologous expression of G-protein-coupled inwardly rectifying potassium (GIRK) channels and GIRK1/4, in sympathetic neurons (Guo & Ikeda,2004).

The association between endocannabinoids and presynaptic function is important and influential. Hence, cannabinoids were described to exhibit the dual role with regard to the neuromodulatory action in the central nervous system linked with the prevention of G-protein coupled cannabinoid receptor (CB1R) facilitated prevention of voltage-gated calcium channels (VGCCs) (Lozovaya, Min, Tsintsadze &Burnashey, 2009). In contrast, cannabinoids could also alter the properties of irrespective of CB1R activation and the action of cannabinoid-VGCC interaction is under investigation (Lozovaya et al., 2009).

Next, certain essential proteins ensure smooth function of muscular contraction which may be helpful for gastric motility. Calmodulin is a vital protein necessary for smooth muscle contraction (Bitar, 2003; Walsh,1994). It binds to four Ca²⁺ ions whenever their is an increased smooth muscular [Ca²⁺] leading the formation of Ca²⁺-CaM complex(Bitar, 2003; Walsh,1994). This complex stimulates an enzyme myosin light chain kinase (MLCK). It is a transferase kind of enzyme that ensures phosphorylation of the regulatory light chains that are associated with myosin II molecule (Bitar, 2003; Walsh, 1994). This function results in the conformational variation on the myosin molecule that leads to increase of its ATPase activity (Bitar, 2003; Walsh, 1994).

Myosin while in the phosphorylated state caps the myosin filaments and could bind or lead to ‘form cross-bridges’ with the opposing actin filaments(Bitar, 2003; Walsh,1994). This interaction between myosin and actin indicates a ‘power-stroke’ motion that enables the two filaments to slide in opposite directions (Bitar, 2003; Walsh, 1994). During the stage of ATP hydrolysis occurring from the myosin head, the cross bridge gets broken and the filaments get back to their native locations(Bitar, 2003; Walsh,1994). Thus a series of successive cross bridges constitute the underlying mechanism of smooth muscle contractions (Bitar, 2003; Walsh, 1994).

Further, intestinal smooth muscles exist in 7 layers of which two are found the gastrointestinal system (Kunze & Furness,1999). These are named as the serosa, longitudinal muscle, myenteric plexus, circular muscle, submucosal plexus, and the mucosa beginning from the outermost layer (Kunze & Furness,1999). Small intestine is responsible for vital functions of digestion and absorption of nutrients. To accomplish this task small intestine possesses two classes of motor activity such as churning and propulsion of luminal content (Kunze & Furness,1999).

Segmental-nonpropulsive contractions occur in the small intestine to enable the process of churning, a motor activity by which the bolus is mixed mechanically. Circular muscles perform these contractions and these muscles contract in segments that are over hanged by relaxed segments of muscle that receive the bolus (Kunze & Furness, 1999). However, both longitudinal and circular muscles are required for peristaltic propulsion. In an upstream segment, the propulsive process of bolus gets initiated (Kunze & Furness,1999). This is the site for relaxation of the longitudinal muscle and contraction of the circular muscle (Kunze & Furness, 1999).

Followed by this downstream segment site favors contraction of the longitudinal muscle and relaxation of the circular muscle (Kunze & Furness,1999). This process occurs frequently in a cyclic manner and the bolus is finally propelled in an aboral path(Kunze & Furness,1999). It can be assumed that the interaction of endocannabinoids with the receptors and channels may occur through some check points. It is unknown whether these checkpoints are regulating the action of endocannabinoid in a dose dependant manner as observed in some experiments. Several receptors may respond simultaneously to give rise to an effect that could lead to some physiological change.

Experimental studies have been in progress to exploit the potential of cannabinoids to modify the intestinal movements by lessening the cholinergic neurotransmission facilitated by CB(1) receptors (Baldassano, Zizzo, Serio, & Mulè, 2009).

In a study, a novel interactive role of endocannabinoids in more complicated circuits and systems is highlighted (Baldassano et al., 2009). Here emphasis was given in checking the mouse ileum longitudinal muscle spontaneous contractility with regard to the interactions between purines and cannabinoid CB(1) receptors(Baldassano et al., 2009). Isomeric contractions were noted while studying the longitudinally oriented mice ileal mechanical activity (Baldassano et al., 2009). It was found that mouse ileum spontaneous contractions were lessened by the selective CB(1) receptor agonist, N-(2-chloroethyl)5,8,11,14-eicosaetraenamide (ACEA).

But, atropine or tetrodotoxin (TTX) did not exhibit this effect (Baldassano et al., 2009). Compounds like Theophylline or adenosine 5′[beta-thio] diphosphate trilithium salt induced desensitization of P2Y have not prevented the inhibition.

However significantly reversal produced by compounds like 8,8′-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)bis(1,3,5-naphthalenetrisulphonic acid)] (P2X receptor antagonist), alpha,beta-methyleneadenosine5′-triphosphate lithium salt (alpha,beta-MeATP) desensitization of P2X receptor and pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (P2 receptor antagonist) (Baldassano et al., 2009).Thus, CB(1) receptors when stimulated negatively alter endogenous purinergic effects, mediated by P2X receptors, on cholinergic neurons(Baldassano et al., 2009).

This strategy serves as an additional feature after CB(1) receptors exert inhibiting effect on the release of acetylcholine release from cholinergic nerve endings(Baldassano et al., 2009). Hence, cannabinoids offer great advantage in altering the purinergic transmission interneuronally. Berthoud, Carlson and Powley (1991) have described that in rats gastric, celiac, and hepatic abdominal vagal nerves collectively innervate gastrointestinal territories.

This was done by exploring particular vagotomic pathways like determination of cervical vagal stimulated motility responses overall the region of gut axis and examining dorsal motor nucleus when carbocyanine dye DiI injected was injected for tracing anterograde (Berthoud et al., 1991). The gastrointestinal (GI) regions have responded well to the motility due to the involvement of vagal terminals that were Dil labeled (Berthoud et al., 1991). Thus, the gastric branches play role in evoking response to motility especially in the proximal duodenum and stomach (Berthoud et al., 1991). Here, duodenum gets mainly innervated by the hepatic root with some endings reaching the intestines and the distal stomach (Berthoud et al., 1991). This strengthens the association between the vagal nerve innervations and GI tract. In addition, sites from colon to cecum were innervated by celiac

branches which in turn combines with vagal branches to influence the duodenal region innovation (Berthoud et al., 1991). It is important to conceive that the innervations of vagal branches of the abdominal region could not only advance the knowledge on the rat gastrointestinal tract but also would draw the practical investigations into the main track with the help of several biochemical, immunohistochemical or molecular markers.

Energy maintenance is a key feature of the endocannabinoid system (Matias & Di Marzo, 2006). This is well accomplished by CB1 and CB2 receptors through the directions of the anabolic and catabolic pathways set for the, N-arachidonoylethanolamine (anandamide) and 2-arachidonoyl glycerol (2- AG) (Matias & Di Marzo, 2006). This discovered certain catabolic and metabolic enzymes which are nothing but the hydrolytic enzymes that identify endocannabinoids as substrates (Matias & Di Marzo,2006).

The enzymes get activated by adequately available CB1 receptor and tissue concentrations present in huge proportions in peripheral organs, brain gastrointestinal tract which are important for maintaining energy homeostasis (Matias & Di Marzo,2006). Endocannabinoids could thus ensure the proper control of gastrointestinal functional biochemistry. Briefly, peripheral and hypothalamic neuropeptides and endocrine secretions participating in the balance of energy and diet control endocannabinoid inactivating and biosynthetic pathways (Matias & Di Marzo, 2006). In contrast endocannabinoids, facilitate the ingestion of nutrients efficiently by controlling the specific mediators (Matias & Di Marzo,2006).

Deviations arising from this particular interconnection could lead to the formation of eating disorders (Matias & Di Marzo, 2006). Thus, endocannabinoids were specially recognized as endogenous lipids meant for regulating the energy balance where they serve as active mediators. It is well known that the transient receptor potential vanilloid type-1 (TRPV1) which is a non-selective cation, capsaicin-sensitive channel is influenced by binding with the AEA (Spoto et al., 2006).

Enzymes namely 1-selective diacylglycerol lipase (DAGL) and N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD) act on endocannabinoids and contribute to their manufacture (Spoto et al., 2006).

Similarly, enzymes such as monoglyceride lipase (MGL), and the fatty acid amide hydrolase (FAAH) contribute to the endocannabinoids degradation (Spoto et al., 2006).

This offered significant experiments in human adipose tissue, endocannabinoid system by employing healthy subjects (Spoto et al., 2006). Here, the process involved biochemical and molecular biology analyses on the subcutaneous abdominal adipose tissues to determine the expression of the enzymes and binding efficiency of adipose tissue (Spoto et al., 2006). HPLC analyses revealed and quantified the identification of AEA and 2-AG levels (Spoto et al., 2006).

On the other hand the presence of endocannabinoids and the related compounds was revealed by the real time PCR and immunofluorescence assays, respectively (Spoto et al., 2006). In addition, other binding experiments and activity assays performed for vanilloid and cannabinoid receptors have shed significant insights on the functional activities (Spoto et al., 2006). This made it clear that human adipose tissue could possess potential to bind AEA and 2-AG and also could facilitate the operation of biochemical armory to act on endocannabinoids (Spoto et al., 2006). Since endocannabinoids play important role in maintaining the energy balance through functional alterations of hypothalamic circuits, they are considered vital for lipogenesis and fatty acid metabolism (Vettor & Pagano, 2009).

This could be because of the evidence indicating the presence of EC system in adipose tissues (Vettor & Pagano, 2009). The potential targets for EC’s are fat producing cells (Vettor & Pagano, 2009). Here much important CB1 receptors activate the EC mediated system that favors the glucose absorption and lipid formation (Vettor & Pagano, 2009). But the stimulation by CB1 results in lessened cellular synthesis of mitochondria (Vettor & Pagano, 2009).

This could be due to endothelial nitric oxide synthase (eNOS) suppressed activity by CB1 (Vettor & Pagano, 2009). The total of the above mentioned effects is prevented by a compound known as rimonabant, a CB1 antagonist(Vettor & Pagano, 2009). This indicated that decrease in lipid production and the transient suppression of food intake contribute to the weight-lessening effect of CB1 and also high oxidative metabolism and mitochondrial biogenesis that compensates or overcomes the inhibitory effects of ECs whose concentrations are in high proportions in fat tissues of obese humans and also rodents (Vettor & Pagano, 2009).

Hence, endocannabinoids contribute to the lipid metabolism through the concerted action of all its receptors at various pathways (Vettor & Pagano, 2009). There is a need to know about the characterization of Diacylglycerol lipase as this enzyme may hold vital clues for lipid metabolism (Giudicellia, Combes-Pastréa & Boyera,1974). Earlier, with the aid of emulsified glycerol di[9,10-3H] oleate as substrate, studies have been focused on Diacylglycerol lipase activity of human adipose tissue (Giudicellia et al.,1974). Here, glycerol-water enabled chemical stabilization of the Diacylglycerol lipase has led to its pure form (Giudicellia et al., 1974).

When enzymatic hydrolysis was checked it was revealed that glycerol lipases were able to possess similar proportion of intrinsic characteristics (Giudicellia et al., 1974). As a result, it has indicated that glycerol lipases in di and tri forms, of human adipose tissue are confined to an enzymatic protein reflecting the efficiency of several crude methodologies (Giudicellia et al., 1974). It was described that gastrointestinal tract possesses a well furnished enteric nervous system (ENS) which is a system of ganglia that functions devoid of brain and spinal cord. Hence ENS is better known as “little brain” or the “second brain” (Powley,2000).

This could be because of the association between ENS and the central nervous system (CNS) where ENS could assist motor activity and a plethora of local reflexes when the viscera were isolated (Powley,2000).

Vast network of vagal connections spread from the brain and nodose ganglia and then to interpenetrate the enteric plexuses (Powley,2000). These connections indicate the ENS and CNS are tied perfectly in the neural network in which CNS outward paths may have pervasive effects on ENS functions which may have extensive inputs to the CNS through vagal afferent nerves (Powley, 2000). Since ENS is connected to the CNS through vagus and spinal nerves, it is considered to strongly innervate the gastrointestinal system and regulates most of its functions in an autonomous manner (Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000).

Brain and spinal cord receive the gut signals from the primary afferent nerves (Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000). These nerves have their cell bodies found in the nodose and dorsal root ganglia. The submucosal and myenteric plexus is formed of a plethora of neurons that could be assigned to one of three groups namely motor, sensory, and inter-neurons (Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000).

Further these groups are dived into various subdivisions. It is alraduy known that motor-neurons could be either excitatory or inhibitory (Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000). Their activity can result in the modulation of vaso/secretomotor-neurons and circular/longitudinal muscle layers(Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000).

Motor-neurons occur mostly in the myenteric plexus and they are categorized as uniaxonal or Dogiel type I neurons based on their morphological features (Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000). Inter-neurons and motor-neurons are identical and are only found in the myenteric plexus and also deviate into two classes such as ascending and descending(Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000). The ascending-neurons are cholinergic and are related to the propulsive reflex (Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000).

Whereas descending inter-neurons are complex and are divided into 3 divisions, one of which is involved in the migrating myoelectric complexes (MMCs) (Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000). Primary afferent neurons or Sensory-neurons are divided into mucosal chemoreceptors, stretch response neurons, and mucosal mechanoreceptors(Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000). The first two divisions are identified in the myenteric plexus, and the other receptors are found in the submucosal plexus. Here, sensory neurons are named as Dogiel type II in regards with their characteristic multi-axonal morphology (Duncun, Davison & Sharkey, 2005; Kunze & Furness, 1999; Powley,2000).

The underlying mechanisms involved in the gastrointestinal motility have led the scientists to develop certain protocols to better evaluate the motor activity of different gastric and intestinal muscle preparations and the effects of drugs that modify such activity (Pozzoli & Poli, 2010). The protocols developed influence several characteristics that reflect motility in the GI tract contractile behaviors especially in regions like duodenum and ileum (Pozzoli & Poli, 2010).

These preparations mimic the motility variations of the gut wall found naturally in animals (Pozzoli & Poli, 2010). The protocols help in better assessing the efficiency of the tissue response with regard to the determination of gut motility affecting compounds(Pozzoli & Poli, 2010). They also facilitate network among the contractile pathways and the output gained indirectly through the secretion changes of myenteric neurotransmitters (Pozzoli & Poli, 2010). Therefore, the protocols are made worth fitting for evaluating new components that become preclinically available and to assess the functional toxicity rendered by environmental or alimentary pollutants, and the relevant mechanisms (Pozzoli & Poli, 2010). This study could better help in devising the appropriate methodology for assessing the gastrointestinal motility under various experimental conditions(Pozzoli & Poli, 2010).

More probably, the receptors and enzymes involved in strengthening the interconnection between the GI tract and the enteric nervous system could be better explored by the protocols (Pozzoli & Poli, 2010). For example, drug-stimulated motility might enable to study the efficacy of regional nerve response. Large studies are needed to better explore the motility machinery of the GI tract Endocannabinoid system offers a great platform for studying such effects as it is intertwined with various important branching connections involving gastric and central nervous system.

Elphick and Eqertova (2001) have described a model that furnishes insights on cannabinoid signalling relevant to nervous system which is essential for smooth muscle contractility and movement. The present concern is that the role of ECS in the gastrointestinal system is innervated by the needs much understanding and investigations due to scarcity of the information. The location of CB1 receptors was assigned to the myenteric plexus of the enteric nervous system (ENS) which is localized in the wall of the gut. Myenteric neurons are large connections of neurons named as ENS which is a part of the autonomic nervous system and it innervates the gastrointestinal system.

It can be assumed that, since CB1 receptors are present in the gastrointestinal tract, certain endogenous ligands would be operating the process of stimulating receptors, precursor enzymes and the metabolic enzymes.

Proposal can be framed keeping in view that DAG lipase, crucial for the manufacture of CB1 ligand 2-AG is localized in the ENS and controls gut function. Finally, research question would arise on whether DAGL would play a role in control of gastrointestinal motility in vitro and in vivo or not. In view of the above information, it can be summarized that endocannabinoid system (ES) has emerged as a pioneer in the control of vital physiological processes and have implications for treating pain, obesity, multiple sclerosis, anxiety and psychiatric disorders(Rodríguez de Fonseca et al., 2005).

It accomplishes the key roles through specially designed receptors namely cannabinoid type 1 receptor (CB1 receptor) and cannabinoid type 2 receptor (CB2 receptor), endogenous ligands, and proteins involved in the synthesis and inactivation(Jean-Gilles, Gran & Constantinescu, 2010). ECS has gained momentum for energy homeostasis with its potential to alter caloric expenditure, transfer of nutrients and metabolism of cellular components (Silvestri, Ligresti & Di Marzo, 2011).

ECs could be considered as lipid mediators as they are also involved in lipid metabolism (Kim , Li & Watkins, 2011).ES takes part in the control of immune responses in the nervous system. Stimulation of cannabinoid system is related to therapeutic effects that may be mediated by the down-regulation of cytokine expression (Jean-Gilles, Gran & Constantinescu, 2010). Delta9-tetrahydrocannabinol (THC), chemically prepared cannabinoids and endogenous cannabinoids, influence the gastrointestinal function through a variety of receptor and signal transuding interaction.

Voltage gated calcium channels, ATP activity and cross bridge formation by myosin filaments contribute to underlying mechanism of smooth muscle contractions which influences the gastrointestinal tract function. The connection between ES and CNS is vital for excitatory and inhibitory functions of ES receptors.

Various dichotomies of nervous system innervate gastric regions at specific locations like colon, ileum and duodenum. This prompted the researchers to undertake to various experiments by employing model animals. Biochemical processes involving lipid metabolism is tightly interconnected with ES where the activity of key enzymes like Diacylglcerol lipase (DAG) needs much research attention. On the whole endocannabinoid system has potential implications for exploring the key machineries of a spectrum of physiological process related with gastrointestinal motility.

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