Diacylglycerol Lipase in Mice Gastrointestinal Motility

Abstract

Recent studies have shown that the endocannabinoid system (CBS) plays a major in the control of certain physiological processes in some areas of the body such as the gastrointestinal tract, brain, and the adipose tissue. Several studies have shown that the CBS plays an important role in the control of gastrointestinal motility through endocannabinoids such as 2-arachidonoylglcerol (2-AG). The role of diacylglycerol lipase in the control of intestinal motility has not been well established. The enzyme is only thought to play a role in the conversion of diacylglycerol (DAG) to 2-AG. Thus the main objective of this study was to study the role of diacylglycerol lipase (DAGL) in the control of intestinal motility in mice. The study was carried out by measuring the effect of DAG, DAG blockers (orlistat, AM251, OMDM 188 and bethanechol) and other muscarinic antagonists (loperamide and Scopolamine) on the electrical contraction of the colonic and ileum slices from male C57BL/6 mice. The study was carried out using equipment by grass technology, West Warwick, RI, USA.

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The data obtained from the study were presented as ± S.E.M. of the experiments which indicates the number of individual mice. Multiple measurements were conducted using one-way ANOVA and Bonrerroni post-hoc testing. The results of the study indicated that indeed DAGL plays a role in the gastrointestinal motility of the C57BL/6 mice, specifically in the ileum when it dimishes mascular contraction. The physiological importance of slowing down the ileum motility by DAGL mediated 2-AG synthesis may be to aid in the absorption of vitamin B12, bile salts and other products that were not absorbed in the jejunum (Di Marzo & Piscitelli, 2011).

Introduction and Background

The connection between the gastrointestinal and central nervous systems is an area of immense research significance in the recent period. For years, investigators have been exploring the underlying mechanisms associated with the functional outcomes of this vital relationship. To this end, much emphasis was given to 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 much interest in this area (Di Marzo & Piscitelli, 2011). For their proper maintenance, the endocannabinoid system (ECS) involves enzymes 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).

There is a need for a thorough literature review to gain better insights about 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 the 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 localized 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). The agonists of CB1 receptors were also employed for the control of asthma and inhibiting the muscular pain related to spinal cord pain and (Pertwee,1999).

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Cannabinoid (CB1) receptor stimulation lessens the motility of GI neuronally, transient lower oesophageal sphincter relaxations (TLESRs) diarrhea, 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 in various animals, like mice, rats, guinea pigs and humans (Pertwee, 2001). Cannabinoid CB1 receptors lessen gastrointestinal motility through the inhibition of live contractile transmitter production (Pertwee, 2001).

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 is produced by the cannabinoid receptor agonist. 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). 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 lethanolamine (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 the 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.

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 degradation of the endocannabinoid (Spoto et al., 2006).

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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 the 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 glucose absorption and lipid formation (Vettor & Pagano, 2009). But the stimulation by CB1 results in lessened cellular synthesis of mitochondria (Vettor & Pagano, 2009).

The underlying mechanisms involved in gastrointestinal motility have led the scientists to develop certain protocols in order 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 a 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 gastrointestinal motility under various experimental conditions(Pozzoli & Poli, 2010).

Materials and Methods

The mice were killed by cervical dislocation. Segments of distal colon were removed, submerged in ice-cold oxygenated Kreb’s solution and then gently flushed to remove luminal contents. Four 1 cm segments from each mouse were then ligated at each end with 5-0 silk and suspended longitudinally in an organ bath (Kreb’s solution, 37°C, aerated with 95% O2, 5% CO2) connected to a FT03 force-displacement transducer (Grass Technologies, West Warwick, RI, USA) and positioned between a pair of parallel electrodes. This was followed by the application of 0.5 g tension and equilibration allowed to take place for 15 minutes. Amplification of the changes in tension was done using the P11T amplifier from Grass Technologies, West Warwick, RI, USA and the results were recorded on a computer using the PolyView software (Pozzoli & Poli, 2010). To examine muscle contractility the preparations were subjected to (EFS; 4 Hz; 24 V; stimulus duration 0.5 or 5 ms; train duration 10 s) provided by an S88X stimulator (Grass Technologies, West Warwick, RI, USA). “EFS of isolated smooth muscle strips caused twitch contractions, which were virtually abolished by TTX (10−6 mol L−1)” (Pozzoli & Poli, 2010). Cumulative concentrations of DAG or DAGL blockers were added to the organ bath at 15 min intervals. In some experiments the effects of DAG or DAGL blockers on bethanechol (10µM) induced contractility were examined. In a separate series of experiments, the effect of DAGL blockers on either scopolamine or loperamide model of decreased EFS contractility was investigated. The amplitude of EFS contractilities in the presence of different compounds was presented as a percentage of the initial contractions

Statistics

The results for the muscle contractility were presented as mean ± S.E.M. of experiments which indicates the number of individual mice. One-way or two-way analyses of variance (ANOVA) followed by Bonferroni posthoc testing were used for the analysis of multiple measurements. A value of p<0.05 was considered statistically significant. The findings were then tabulated and then represented graphically as shown in the results section.

Results

The C57BL/6 mice were killed and the segments of the distal colon removed and placed in the ice-cold oxygenated Krebs solution to maintain the physiological condition of the muscle tissues. 5-0 silk ligation was carried out at each end of the four 1 cm segments from each mouse. The muscle contractility results from a series of cumulative concentrations of DAG, Scopolamine, and loperamide or DAGL blockers were obtained, tabulated and used to produce the graphs shown. The graphs indicate the percentage contractility versus the levels of DAG, loperamide or scopolamine and the DAG-blockers. The first three graphs were used as control for the experiments.

In the first experiment, it’s shown that the Inhibitory effects of DAG are blocked by Orlistat and AM251 (Fig 4). In the second series, the effects of DAG (from 1uM to 10uM) in the ileum are completely reversed by OMDM188 at 1uM (Fig 5). In the third series, the results indicate how increasing levels of scopolamine (from 1nM to 100nM) suppresses the ileum contractility in the presence of Orlistat at 5uM, OMDM at 188 uM and AM251 at 100nM (Fig 6). In the fourth series, the results indicate how scopolamine (from 1nM to 100nM) minimally inhibits colon contractility enhanced by vehicle, orlistat at 5uM and OMDM188 at 1uM (Fig 7). In the last series, increase in the level of loperamide (from 10 nM to 1uM) causes an increase in the reversal of the effects of orlistat at 5 uM (Fig 8).

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effect of orlistat and bethanechol on the contractility of the ileum and the colon
Figure 1: effect of orlistat and bethanechol on the contractility of the ileum and the colon

The figure above shows that orlistat and bethanechol do not change contractility in the ileum and colon.

Effect of OMDM 188 on colon and ileum contractility
Figure 2: Effect of OMDM 188 on colon and ileum contractility

The figure above shows that OMDM does not change contractility in the ileum and colon in the absence of DAG

The effect of DAG and bethanechol on ileum and colon contractility
Figure 3: The effect of DAG and bethanechol on ileum and colon contractility

The figure above shows that DAG (from 5uM to 10 uM) results in a diminished contractility in the ileum and the colon to a lesser extent. The introduction of bethanechol blocks the effect of DAG in the ileum to return the electrical contractility nearly to 100%.

Ileum contractility under DAG, Orlistat + DAG and AM251 and DAG; colon DAG contractility
Figure 4: Ileum contractility under DAG, Orlistat + DAG and AM251 and DAG; colon DAG contractility

1) The results above (figure 4) indicate that electrical contractility in the ileum of the C57BL/6 mice is further reduced by simultaneous increases in the levels of Stearoyl-2-Arachidonoyl-sn-Glycerol (DAG). However, the effects of DAG are reduced by Orlistat and AM 251 which shows consistently higher levels of electrical contractility even with increasing levels of DAG. Stearoyl-2-Arachidonoyl-sn-Glycerol (DAG) does not affect the electrical contractility of the colon in the C57BL/6 mice.

 Ileum contractility under DAG and OMDM 188+DAG
Figure 5: Ileum contractility under DAG and OMDM 188+DAG

The results in figure 5 show that the effect of 2-Stearoyl-2-Arachidonoyl-sn-Glycerol (DAG) in inhibiting the contraction of the ileum in the C57BL/6 mice is completely blocked by OMDM 188. Increase in the level of DAG from 1uM to 10uM does not change the effect of OMDM 188 which is kept constantly at 1uM.

Effect of vehicle, Orlistat, OMDM 188 and AM251 on scopolamine inhibited ileum contractility
Figure 6: Effect of vehicle, Orlistat, OMDM 188 and AM251 on scopolamine inhibited ileum contractility

In the figure above the electrical muscle contractility in the ileum of the C57BL/6 mice is inhibited by scopolamine. The inhibitory effect is increased by the increasing level of scopolamine (from 1nM to up to 100nM). Orlistat (5uM) completely reverses the effect of scopolamine at 1uM. Orlistat (5uM) and OMDM 188(1uM) have little effect on the inhibitory effect of scopolamine at higher levels. AM251 (100nM) reverses the effect of scopolamine (at 10nM) by a higher margin as compared to Orlistat and OMDM 188.

Effect of vehicle, Orlistat, OMDM 188 and AM251 on scopolamine inhibited colon contractility
Figure 7: Effect of vehicle, Orlistat, OMDM 188 and AM251 on scopolamine inhibited colon contractility

These results above indicate that scopolamine minimally inhibits contractility in the colon under both orlistat and OMDM 188. Scopolamine (up to 100nM) only inhibits the contractility effects of orlistat (at 5uM) and OMDM (at 1uM) by about 30%.

Effect of Orlistat on Loperamide inhibited contractility
Figure 8: Effect of Orlistat on Loperamide inhibited contractility

The results above show that Orlistat (5 uM) causes the highest percentage of contraction which is about 100% when the level of loperamide is 10 nM. The control shows low muscle activity at 10nM loperamide. However, as the loperamide increases the effect of orlistat is reduced as shown. At 1 uM loperamide, the muscle contraction is diminished to a point below 25% which is much lower than what is seen when a similar level of loperamide is used with the control. Thus the increase in the level of loperamide increases the inhibition of Orlistat.

Discussion

The endocannabinoid system (ECS) has been cited to play a role in intestinal motility through endocannabinoids such as the anandamide and 2-Arachdonolglycerol (2-AG). The role of Diacylglycerol lipase, which can be described as a key enzyme that catalyzes the synthesis of endocannabinoid, 2-arachidonoylglycerol is not clearly known. Previous data and the results of this study suggest that DAGL plays an important role in intestinal motility. The data above shows that DAG causes a deactivation of the muscarinic receptors in the gastrointestinal wall. The role of DAG as a muscarinic antagonist is facilitated by the enzyme Diacylglycerol lipase. To test this assertion, the effects of DAG in the colon and ileum slices of the C57BL/6 mice were studied along with known muscarinic agonists. The study also included other muscarinic antagonists including loperamide and scopolamine.

The results of the present study have shown that endocannabinoid, 2-arachidonoylglycerol (DAG) results in reduced muscle contractility in the ileum and not the colon.

1-Stearoyl-2-arachidonoyl-sn-glycerol (DAG)
Figure 9: 1-Stearoyl-2-arachidonoyl-sn-glycerol (DAG)

Figure 3 shows that DAG exerts its inhibitory effects on the C57BL/6 mice ileum from 1uM to 5uM where the amplitude electrical contraction of the ileum wall is reduced to 60% at 1uM and to 40% at 5uM. However, the same figure shows that DAG does affect the electrical contractility of slices from the colon of the same mouse strain. Increase in the levels of DAG from 1uM to 10uM results in a slight fall in the contractility of the colon by about 5%. This indicates that 2-arachidonoylglycerol plays a role in the regulation of the contractility of the ileum muscles.

To further understand the effect of DAG in gastrointestinal motility, other inhibiting drugs (muscarinic receptor antagonists) such as loperamide and scopolamine were studied along with other known DAGL blockers such as orlistat, OMDM 188, AM 251 and bethanechol (muscarinic receptor agonists). The first control carried out (Figure 1) shows that orlistat does not interfere with the electrical contractility of the ileum and the colon. Studies carried out previously show that orlistat blocks gastric, pancreatic and carboxyl ester lipases thereby preventing the hydrolysis of triglycerides to free fatty acids and monoglycerides. Unconfirmed sources also indicate that orlistat may be playing a role in increasing gastric emptying. Indeed this study has shown that orlistat increases muscle contractility in both the ileum and colon by blocking the effect of DAGL, a key enzyme in the endocannabinoid system. This is clearly shown in figure 4 whereby the comparative analysis of the effects of DAG and DAG + Orlistat in the ileum shows that orlistat blocks the effect of DAG by inhibiting the DAGL.

Structure of orlistat
Figure 10: Structure of orlistat

Available literature on orlistat shows that orlistat is used therapeutically to treat or prevent obesity by inhibiting the absorption of dietary fat. The fat absorption is inhibited by blocking the pancreatic lipases. The current study was interested in studying the role of diacylycerol lipase in the control of gastrointestinal motility in mice (Izzo and Sharkey,2010). From the orlistat study results it can be said that diacylycerol lipase inhibits or reduces intestinal motility to facilitate the absorption of dietary fat. Figure 4 shows that when DAG is administered alone, the electrical contractility of the ileum is diminished progressively from a DAG level of 1uM to 10uM. Note that the contractility is not completely diminished but it only slows down. When DAG is administered with orlistat (at 5uM), the inhibitory effect of DAG is abolished. Thus if diacylycerol lipase (DAGL) catalyzes the synthesis of DAG as seen in the literature review then it actually plays a role in the reduction of gastrointestinal motility to facilitate the absorption of fat from the diet (Di Marzo & Piscitelli, 2011). Through previous research findings, it has been noted that lipolytic products that are contained in the dietary fat and the 2-monoacylglycerols that are formed by the action of luminal lipases on triglycerides form a very important part of gastric emptying regulation(Storr & Sharkey, 2007). The free fatty acids formed are re-synthesized to form cylomicrons that enter the lymph to trigger paracrine processes that involve the vagal afferent signals. These cause a reduction in the food intake, release of gastrointestinal hormone and suppression of gastric emptying (Izzo and Sharkey,2010). Orlistat and some other blockers inhibit the production of free fatty acids (FFA) and 2-monoacylglycerol by covalently bonding on the gastric and pancreatic lipases (Massa & Monory, 2006). The results established by this study confirm the findings of previous studies which have led to the utilization of orlistat in the acceleration of gastric emptying. In this study however, the effects of orlistat are studied in one lipid, DAG, which is acted upon by DAGL in order to begin the process that will result in the formation of free fatty acids and thus cause the slowing down of gastric emptying. Evidence from previous research indicates that the CB1 simulation results in the slowing down of motility throughout the gut including the colon (Pertwee,1999). However, in the current experiment, CB1 stimulation by DAG administration has only resulted in reduced ileum contractility.

The function of DAG is also very often found in the central nervous system where 2-arachdonoyglycerol (2-AG), an endogeneous agonist of the CB1 system is usually created from arachidonic acid that contains dicylgycerol (DAG) (Sanger, 2007). The formation of 2-AG is said to be mediated by phospholipase C (PLC) and DAGL(Pertwee, 2001)..

Other DAGL blockers have also been investigated in this study to confirm the effects of DAG on gastrointestinal motility and by extension provide an explanation for the role of DAGL on the same. AM251 had the same effect on DAG as seen with orlistat. Figure 4 shows that AM251 exhibits the antagonistic properties that are shown by orlistat. Studies carried out on the ileum of the C57BL/6 mice show that AM251 antagonizes DAG activity, probably by blocking DAGL. Indeed the available literature shows that AM251 is an inverse agonist that exerts its action at the CB1 cannabinoid receptor (Storr et al., 2002)..

AM251
Figure 11: AM251

OMDM 188 is the third DAGL blocker that was investigated in the study. Figure 2 shows that electrical contractility of the ileum and colon is not changed by administration of OMDM 188. However, in figure 5 it’s seen that OMDM 188 (at 1uM) reverses the effects of DAG in the ileum. Whereas DAG causes diminished electrical contractility when administered alone, the effect is completely when administered with OMDM 188. Thus OMDM 188 is antagonist that exerts its effect on gastrointestinal motility by blocking DAGL. Available literature describes OMDM as an endocannabinoid analog that has been designed to selectively inhibit the uptake of arachidonoyl ethanolamide (AEA) (Storr et al., 2002).

Structure of OMDM 188
Figure 12: Structure of OMDM 188

The same agonist effect was seen on administration bethanechol, Bethanechol is a conventional drug that often exerts its action by helping gastric muscles to contract faster and thus enable a faster food movement between esophagus and the stomach. Bethanechol is described as a parasympathomimetic choline ester that acts by selectively stimulating the muscarinic receptors(Klein, Lane, Newton & Friedman, 2000). The presence of muscarinic receptors in the bladder and abdomen has facilitated the therapeutic application of bethanechol to expel urine and increase gastrointestinal motility. In the present study, the effects of DAG on the ileum wall were reversed by bethanechol. This was achieved through competitive binding of bethanechol to the muscarinic receptors that are bound by DAG to cause reduced muscle contractility in the ileum wall.

Structure of bethanechol
Figure 13: Structure of bethanechol

Comparative studies were carried out using loperamide and scopolamine which have muscarinic antagonist effects similar to DAG. Loperamide is a known drug that usually acts by slowing down the intestinal motility and by interfering with the movement of water and electrolytes through the bowel. The drug usually abolishes the peristaltic movement of the gut muscles. Loperamide is described as a non-selective calcium channel blocker that binds to opioid mu-receptors (Klein, Lane, Newton & Friedman, 2000). Recent studies have shown that when loperamide is administered in higher doses it can bind to calmodulin. In the present study, it was seen that loperamide exerts the same effect on the electrical contractility of the C57BL/6 mice ileum muscles as DAG. However, loperamide exerts a more strong effect that is not inhibited by similar levels of orlistat that abolished the effect of DAG. Tiny concentrations of loperamide (from 10nM to 1uM) are able to prevent the effects of orlistat at 5uM.

Structure of loperamide
Figure 14: Structure of loperamide

Scopolamine can describe as a tropane alkaloid drug that causes a number of effects including muscarinic antagonist effects (Oz, 2006). The drug is sourced from plants that belong to the family of solonaceae where it occurs as a secondary metabolite. Its mode of action is through competitive antagonism at the muscarinic acetylcholine receptors, particularly the M1 receptors (Kunze and Furness, 1999). Therefore, the drug is classified as both an anti-cholinergic and anti-muscarinic. In the case of gastrointestinal tract, the drug is often used to treat cramping (Oz, 2006). In the present study scopolamine was used in both colon and ileum slices from the male C57BL/6 mice. Scopolamine exerted the same effect as loperamide where it was able to prevent or diminish electrical contraction even when muscarinic agonists ( orlistat at 5uM, OMDM 188 at 1uM and AM 251 at 100nM) were administered along. Unlike DAG, scopolamine was seen to diminish electrical contractility to a more pronounced extent.

scopolamine
Figure 15: scopolamine

The results from this study have shown that DAG causes inhibition of muscular contraction in the ileum and not the colon. The information regarding the selective inhibition of DAG is not clearly understood as the CB1 receptors that form the binding sites for DAG products are present along the whole course of the gastrointestinal tract. Some studies have tried to reveal generate the understanding of the mechanism of pre and post-synaptic inhibition (Kunze and Furness, 1999). It has been shown that the inhibition of the excitatory neurotransmission that is elicited by the CB1 receptor both in the gastrointestinal tract and elsewhere could be mediated by the several subtypes of lipases. Indeed previous studies have shown that the endocannabinoids can be considered as a family of lipid-derived messengers that are usually involved in a variety of short-range signaling events in different parts of the body (Darmani, 2002). Past physiological experiments have suggested that the lipid mediators are often released from neurons in the postsynaptic region. The lipid mediators are thought to diffuse across the synaptic cleft, and bind to CB1 cannabinoid receptors that are located on the presynaptic terminals and thus cause the regulation of calcium and potassium. It has also been shown that the transient or persistent depression of the excitatory neurotransmission by the cholinergic muscarinic receptors is often through a CB1 dependent pathway. The retrograde signaling is most thought to result from the postsynaptic release of the 2-arachidonoylglycerol (2-AG). Many studies have shown that 2-AG is the most common form of endocannabinoid substance that is present in the mammalian CNS. DAG lipase plays a lead role in the production of the 2-AG, which is thought to take place in the following manner; an enzyme phospholipase C (PLC)-catalyzes the cleavage of the membrane phosphatidylinositol-4,5-bisphosphate (PIP2) to give rise to 1,2-diacylglycerol (Izzo and Sharkey, 2010). The DAG that results is catalyzed by diacylglycerol lipase (DAGL). The DAG is thus hydrolyzed to 2-AG by DAGL and the formed 2-AG is broken down via monoacylglycerol lipase located in the excitatory and inhibitory axon terminals (Izzo and Sharkey, 2010).

To understand the functions of 2-AG in the contractility of the gastrointestinal wall, specifically in the retrograde signaling is important if the molecular mechanism of the inhibition is to be understood and by extension, the functions of the different lipases that are involved in the inhibition process. The present study provides evidence of silencing the effect of DAG in the ileum of the C57BL/6 mice using cholinergic muscarinic agonists such as AM 251 and orlistat. Through these studies it is apparent that DAG plays an important role in the suppression of motility in the ileum wall as shown in the C57BL/6 mice studies. The physiological implication of this may not be to enable the absorption of food substances in the ileum. Products absorbed from the ileum include vitamin B12 and bile salts, together with other foods that escape absorption in the jejunum (Izzo and Sharkey, 2010). The finding specifically pertains to the C57BL/6 mice as the enzyme may play additional roles in the motility of other parts of the gut in other mammals.

Understanding the role of DAGL in gastrointestinal motility is of great medical importance as it may be altered in pathophysiological conditions (Izzo and Sharkey, 2010).

References

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