Lab 8: Gastrointestinal Motility: Measuring the effects of various conditions on smooth muscle contraction

1 Lab 8: Gastrointestinal Motility: Measuring the effects of various conditions on smooth muscle contraction Jana Wong Group Members: Bryan Lairmore, Jessica Chang, Amanda Perez-Stires TA: Nicholas Klug; Section 9 December 5, 2013

2 INTRODUCTION Every system in the human body contributes to maintaining homeostasis. The digestive system helps to maintain homeostasis through the strategic transfer of nutrients, waters, and electrolytes from food to the body. Within the digestive system, four processes are noted: motility, secretion, digestion, and absorption. This experiment focused solely on the motility of the digestive system, specifically intestinal motility. Intestinal motility is known as the muscular contractions that mix and move luminal contents forward in the digestive tract via peristalsis and segmentation. Peristalsis acts to propagate contents through the tract and segmentation acts to mix and break up contents thoroughly. Motility in the gastrointestinal system is regulated by the extrinsic autonomic nervous system, the intrinsic enteric nervous system, as well as hormones in the system (Sherwood, 2010, p.593). In this lab, students observed the effects of the autonomic nervous system on the enteric nervous system and how these effects played a role in intestinal motility. The sympathetic and parasympathetic systems act on smooth muscle cells and exert changes in intestinal contraction. This lab observes how motility changes in response to various drugs and environments. Pacemaker cells of the smooth muscle cause an electrical slow wave rhythm that has the potential to reach threshold. When this happens, contraction can occur due to action potential propagation in the muscle cells forming cross-bridges. This experiment observed changes in motility of an intestinal segment under the presence of adrenergic, cholinergic, and purinergic agents as well as in a calcium free environment. Epinephrine, an adrenergic agent that comes from the sympathetic nervous system, is expected to cause a decrease in intestinal motility. Methacholine, a cholinergic agent from the parasympathetic nervous system, is expected to cause an increase in intestinal motility.

3 Adenosine 5 -Diphosphate (ADP), a purinergic agent, is expected to cause a decrease in motility. To observe the effects of calcium on intestinal activity, observations were made in normal Ringer-Tyrode s solution versus in a Ca 2+ -free Ringer-Tyrode s solution. The calcium free solution was expected to cause significant decrease in intestinal motility. METHODS AND MATERIALS Detailed procedures for each part of the experiment can be found in the second edition NPB 101L Physiology Lab Manual (Bautista & Korder, 2009, p.75-81). In this lab, students used a small section of rabbit intestine to observe intestinal motility reactions in various mediums. The piece of intestine was put into normal Ringer-Tyrode s solution, normal Ringer-Tyrode s solution with added epinephrine, normal Ringer-Tyrode s solution with added methacholine as a cholinergic agent, normal Ringer-Tyrode s solution with added ADP as a purinergic agent, and a Ca 2+ -free Ringer-Tyrode s solution to be observed and analyzed in different conditions for intestinal motility properties. Variations in procedures from the lab manual included setting up the section of intestine differently, with one piece of string on each end of the section instead of having three pieces total. Another variation from the lab manual was recording gut activity for five to six minutes instead of ten minutes after the different agents were added to the normal Ringer s solution. Due to time constraints, these times may have been cut down even further as instructed by the TA. Data Analysis In analyzing data gathered from the experiment, BioPac software was used. Three main components were analyzed from the experiment frequency, amplitude, and baseline tension. In order to calculate frequency, the frequencies from ten consecutive wave cycles (??) were taken using the frequency tool in the BioPac software and then averaged for one mean frequency value

Frequency (Hz) / Amplitude (g) / Tension (g) 4 at that point in time. To calculate amplitude, the p-p tool in the Biopac program was used to calculate the value from the trough to the peak of one wave cycle and the average amplitude of five consecutive waves were taken to represent the amplitude at that point in time. Tension was calculated using the minimum tool on five consecutive waves in the BioPac program and the average of five measurements were taken to represent the tension at that point in time. RESULTS Normal Gut Activity To observe the normal intestinal motility patterns, the section of intestine was analyzed in normal Ringer-Tyrode s solution. This showed a frequency, amplitude, and baseline tension that remained around the same values throughout the whole observation period, as seen in Figure 1. It is seen in Figure 1a that the frequency of the trial stayed within 0.3-0.4Hz throughout 10 minutes. It is seen in Figure 1b that the amplitude stayed within 0.34-0.52g throughout the 10 minutes. Tension stayed within 0.29-0.37g throughout the trial, as seen in Figure 1c. 0.55 0.5 0.45 0.4 0.35 0.3 0.25 0 100 200 300 400 500 600 700 Times (seconds) Figure 1: Changes in normal gut activity plotted against time. Figure 1a: Changes in contraction frequency are plotted against time. Figure 1b: Changes in contraction amplitude are plotted against time. Figure 1c: Changes in baseline tension are plotted against time.

Frequency (Hz) / Amplitude (g) / Tension (g) 5 Gut Activity in the Presence of Epinephrine In the next part, intestinal motility in the presence of an adrenergic agent was observed by adding epinephrine. It is seen in Figure 2 that baseline activity in frequency, amplitude, and tension dropped significantly when epinephrine was added. Epinephrine was added to the normal Ringer-Tyrode s solution at 480 seconds. At this point, the contraction frequency dropped from 0.31Hz to 0.18 Hz, amplitude dropped from 0.33g to 0.05g, and baseline tension dropped from 0.16g to 0.05g, all seen in Figure 2. 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 Epinephrine added 0 100 200 300 400 500 600 700 800 Time (seconds) Figure 2: Changes in gut activity plotted against time before, during, and after the addition of epinephrine. Figure 2a: Changes in contraction frequency are plotted against time. Figure 2b: Changes in contraction amplitude are plotted against time. Figure 2c: Changes in baseline tension are plotted against time. Gut Activity in the Presence of a Cholinergic Agent Next, intestinal motility in the presence of methacholine was observed. In the presence of a cholinergic agent, baseline activity of tension made a significant increase as seen in Figure 3. One drop of methacholine was added at 280 seconds and another three drops were added at 360 seconds. It can be seen in Figure 3a that contraction frequency did not change very much with

Frequency (Hz) / Amplitude (g) / Tension (g) 6 the additions, but in figure 3b, it can be seen that contraction amplitude dropped 0.1g at the first addition, and then increased significantly from 0.13g to 0.29g after the second addition. In Figure 3c, it can be seen that baseline tension made a significant increase from 0.18g to 0.81g after the first addition and continued to stay high after the second addition. 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1 drop MCh 3 drops MCh 0 100 200 300 400 500 600 Time (seconds) Figure 3: Changes in gut activity plotted against time before, during, and after the additions of methacholine. Figure 3a: Changes in contraction frequency are plotted against time. Figure 3b: Changes in contraction amplitude are plotted against time. Figure 3c: Changes in baseline tension are plotted against time. Gut Activity in the Presence of a Purinergic Agent In order to observe the effects of a purinergic agent, ADP was added to observe changes in gut activity. In Figure 4, it can be seen baseline activity had a decreasing trend with the addition of ADP. The purinergic agent was added to normal Ringer-Tyrode s solution at 580 seconds. At this point, contraction frequency stayed in a similar range, as seen in Figure 4a, but contraction amplitude saw a significant decrease as seen in Figure 4b. Baseline tension saw a general decreasing trend throughout the trial, but there was no significant change after ADP was added, shown in Figure 4c.

Frequency (Hz) / Amplitude (g) / Tension (g) 7 0.3 0.25 0.2 0.15 0.1 0.05 0 ADP added 0 200 400 600 800 1000 Time (seconds) Figure 4: Changes in gut activity plotted against time before, during, and after the addition of ADP. Figure 4a: Changes in contraction frequency are plotted against time. Figure 4b: Changes in contraction amplitude are plotted against time. Figure 4c: Changes in baseline tension are plotted against time. Gut Activity in Ca 2+ -free Ringer-Tyrode s Solution In order to observe the effects on intestinal motility in a calcium-free environment, the section of intestine was placed in a Ca 2+ -free Ringer-Tyrode s solution to be compared to activity in normal Ringer-Tyrode s solution. At 300 seconds, the intestine was taken from the normal Ringer-Tyrode s solution and put into the Ca 2+ -free Ringer-Tyrode s solution, and at 540 seconds, the intestine was taken out of the Ca 2+ -free Ringer-Tyrode s solution and put back into the normal Ringer-Tyrode s solution. It is seen in Figure 5 that baseline activity decreased while in the Ca 2+ -free Ringer-Tyrode s solution compared to the normal Ringer-Tyrode s solution. In Figure 5a, frequency is seen to decrease from 0.23Hz to 0.17Hz, while in Figure 5b, contraction amplitude decreases from 0.22g to 0.10g as it goes from normal environment to a calcium-free environment. Baseline tension did not seem to change much as it decreased from 0.03g to 0.02g, as seen in figure 5c. Once the intestine was moved back into the normal Ringer-Tyrode s

Frequency (Hz) / Amplitude (g) / Tension (g) 8 solution, there was a significant increasing trend in frequency, amplitude, and tension between 540 seconds and 880 seconds. 0.6 0.5 0.4 0.3 0.2 0.1 moved into Ca-free solution moved back to normal Ringer's 0 0 200 400 600 800 1000 Time (seconds) Figure 5: Changes in gut activity plotted against time before, during, and after placement in Ca 2+ -free Ringer's solution. Figure 5a: Changes in contraction frequency are plotted against time. Figure 5b: Changes in contraction amplitude are plotted against time. Figure 5c: Changes in baseline tenstion are ploted against time. DISCUSSION The gastrointestinal (GI) tract functions for digestion, absorption, secretion, and motility. In doing this, it digests and assimilates nutrients and removes wastes from the body. The GI tract is comprised of various specialized layers that all act uniquely for different functions but as one whole unit. The outermost layer of the GI tract is a connective tissue called the serosa. It connects to the abdominal cavity and secretes a lubricating fluid that helps prevent abrasion among the digestive organs (Sherwood, 2010, p.593). The muscularis externa, the next layer of the digestive tract, is the main layer of smooth muscle that is composed of an outer longitudinal smooth muscle layer and an inner circular smooth muscle layer. Longitudinal muscles contract to cause a shortening of the digestive tract while circular muscles contract to cause a decrease in

9 diameter of the tract, working together to create segmentation and peristalsis. Next, the submucosa provides distensibility and elasticity to the tract as a layer connective tissue. In this layer also lie large blood vessels and lymph vessels that branch out into inner and outer layers of the digestive tract. The next, most inner, layer of the digestive tract is the mucosa, composed of the mucous membrane, the lamina propria, and the muscularis mucosa. This layer that lines the lumen is composed of many folds that help increase the available surface area for absorption (Sherwood, 2010, p.591). The digestive tract is controlled by both the autonomic nervous system (ANS) as well as the enteric nervous system (ENS). The ENS contains two main nerve networks, the submucosal plexus and the myenteric plexus, located in the submucosa and between the longitudinal and circular muscles in the muscularis externa respectively (Sherwood, 2010, p.593). These two nerve networks play a role in GI activity regulation. Intestinal motility generally refers to segmentation and peristalsis of the digestive tract. The pacemaker cells of the system, interstitial cells of Cajal (ICC), generate slow wave potentials, creating the basic electrical rhythm (BER), that allow for these actions to occur (Sherwood, 2010, p.621).this slow wave movement brings the membrane toward and away the threshold potential controlled by the pacemaker cells, which in turn control the rate of intestinal motility (Sherwood, 2010, p.594). Segmentation occurs when muscles contract and relax in a rhythmic manner, which helps to break down and thoroughly mix the contents in the lumen. Peristalsis occurs when muscles contract and relax strategically to move luminal contents down the digestive tract. The BER travels down adjacent smooth muscle cells. Once threshold is reached, action potentials are generated, which can cause a series of contractions.

10 In this study, gut activity was measured while exposed to normal Ringer-Tyrode s solution, epinephrine (adrenergic agent), methacholine (cholinergic agent), adenosine-5 - diphosphate (purinergic agent), and Ca 2+ -free Ringer-Tyrode s solution. In normal GI activity, action potentials propagate down, resulting in contractions along the digestive tract and causing luminal contents to travel down the tract in a similar manner. Segmentation helps in breaking down and mixing the contents. In order to help further break down and propagate luminal contents down the digestive tract, slow waves of BER need to propagate down the GI tract and reach threshold in order to signal a series of contractions. When threshold is reached, depolarization occurs as cell receives an influx of calcium via voltage gated calcium channels. Hyperpolarization occurs with an efflux of potassium and the closing of calcium channels (Sherwood, 2010, p.594). Once the calcium-dependent potassium channels close, one slow wave cycle is complete. In this part of the experiment normal Ringer-Tyrode s solution provided the necessary calcium concentration for the intestine to show contractile responses. As seen in Figure 1, the section of intestine showed a relatively constant contraction frequency, amplitude, and baseline tension throughout the trial in normal Ringer-Tyrode s solution. Sympathetic stimulation to the GI system causes a release of epinephrine. After epinephrine administration, a dramatic decrease in BER was observed. Epinephrine, an adrenergic agent, caused an inhibitory effect because of its stimulation of β-adrenoreceptors (Jun, Choi, Yeum, et al., 2004, p.671). Epinephrine binds to β2-receptors on the membrane of smooth muscle cells, which in turn activates the G-protein cascade that eventually activates adenylyl cyclase, increasing cyclic AMP in the cell (Guan, Amend, & Strader, 1995, p.492). This increase of cyclic AMP activates protein kinase A, which inactivates myosin light chain kinase and

11 inhibits calcium channels, ultimately decreasing and inhibiting contraction of smooth muscle. This was evident in the experiment as intestinal motility decreased with the addition of epinephrine, as seen in Figure 2. On the contrary, after methacholine administration, a significant increase in intestinal activity was observed. Methacholine has similar effects on smooth muscle activity as acetylcholine, another cholinergic agent. When a cholinergic agent is released due to parasympathetic stimulation, it binds to muscarinic receptors that activate the G-protein cascade, which in turn activates phospholipase C, ultimately hydrolyzing PIP2 into IP3 and DAG. IP3 then goes on to bind to IP3-receptors in the sarcoplasmic reticulum to open a channel to allow calcium ions to flow out of the sarcoplasmic reticulum and into the cytosol. This increase of cytosolic calcium allows more calcium-calmodulin complexes to be formed to activate myosin light chain kinase, ultimately increasing cross-bridge formation and muscle contraction. This increase in smooth muscle activity is evident in Figure 3, as baseline tension increased significantly after the addition of methacholine. A similar study was done on the effects of epinephrine and acetylcholine on various animal intestines. In this study, results were consistent with what was observed in this experiment. The overall effect of acetylcholine was continuous contraction whereas epinephrine showed relaxation of the intestine, thus decrease in contractility (Bernheim, 1934, p.59). The effects of epinephrine and methacholine encircle the effects that the autonomic nervous system has on intestinal motility. Increases in either the sympathetic system or parasympathetic system can cause a decrease or increase in gut activity due to activation or inhibition of calcium channels that ultimately affect MLCK activity for cross-bridge formation. This is seen through the variation of contractility, which is dependent on the concentration of Ca 2+ inside the cell

12 which helps determine the frequency of slow waves, thus controlling the rate of segmentation and peristalsis (Al-Shboul, 2013, p.4). ADP was used to mimic the effect of ATP on intestinal motility, which ultimately causes an inhibitory result. In doing so, neurotransmitters were released which activated the purinergic pathway, inhibiting gut motility and causing a relaxation in the gut segment (Westfall, Todorov, & Mihaylova-Todorova, 2002, p. 441). ADP induces transmission for excitatory as well as inhibitory reflexes, causing rapid hyperpolarization of the membrane to relax the muscle (Bornstein, 2008, p.202). ADP binds to the 2-γ purinergic receptor, which activates the G-protein cascade in order to activate PLC and PIP2. At this point, DAG and IP3 are both activated by PIP2 and unlike the cholinergic pathway, IP3 activates only local calcium channels open for an influx of calcium ions. While this occurs, a rapid efflux of potassium ions causes calcium channels to close and a rapid hyperpolarization of the cell. The calcium concentration and activated DAG work to increase phosphokinase-c (PKC) activity. PKC works to phosphorylate myosin light chains (MLC) where MLCK does not act, decreasing activity on the MLC and causing a decrease in contraction amplitude. This is consistent with data obtained, as seen in Figure 4, as contraction amplitude shows a significant decrease due to the inhibitory nature of ADP. A similar study was done with purinergic and nitrergic agents resulting in a similar inhibition pattern. In the study, agents were introduced to the gut and peristaltic contractions were decreased, but not completely gone (Hwang, Blair, Durnin, et al., 2012, p.1967). The effect of a calcium free environment on intestinal motility was observed by using Ca2+-free Ringer-Tyrode s solution in comparison to gut activity in normal Ringer-Tyrode s solution. It has already been established that calcium concentration plays a large role in smooth

13 muscle contraction. Once the intestine segment was taken out of normal Ringer-Tyrode s solution, the only calcium available to the cell was the remaining intracellular calcium. This caused a significant decrease in motility of the muscle. Without the extracellular supply of calcium from normal Ringer-Tyrode s solution, there is no way that the muscle can obtain enough calcium to remain at a constant rate of contraction due to its inability to form crossbridges for contraction. Results from the experiment remained generally consistent with this phenomenon. There were some unexpected results such as baseline tension not decreasing significantly from the transfer from normal Ringer-Tyrode s solution to a calcium free environment, and tension increasing an enormous amount from the transfer from calcium free back to normal solution, seen in Figure 5c. This could have been due to error in transferring the gut segment as the knot slipped during the first transfer and needed to be retied during the second transfer. This error resulted in inconsistent baseline control activity. Aside from this error, Figure 5 shows that most of the results were consistent with a similar study done addressing the role of calcium influx in slow-wave potentials. This study concluded that gastrointestinal motility is caused by integrated calcium ion management instead of solely release from intracellular calcium stores (Ward, Ordog, Koh, Abu Baker, et al, 200, p.355). This all is consistent with the need for calcium ions for cross-bridge formation that lead to contraction of muscle cells. In conclusion, gastrointestinal motility is affected differently in various environments. When sympathetic activity is stimulated, GI motility shows a decrease in activity, but when parasympathetic activity is stimulated, GI motility increases. Epinephrine and ADP both showed inhibitory effects, decreasing contraction of the muscle. On the other hand, methacholine showed

14 an excitatory effect, increasing the contraction of the muscle. These effects help regulate homeostasis in the whole body, not just the digestive system through responses to environment, such as fight-or-flight or rest-and-digest.

15 REFERENCES: Al-Shboul, O.A. (2013). The Importance of Interstitial Cells of Cajal in the Gastrointestinal Tract. Saudi J Gastroeneterol, 19 (1), 3-15. Bautista, E., and Korber, J. 2009. NPB 101L Physiology Lab Manual (2 nd ed.). 75-82. Department of Neurobiology, Physiology and Behavior, University of California, Davis. Bernheim, F. (1934). Interaction of acetylcholine and epinephrine on the isolated small intestines of various animals. Journal of Pharmacology and Experimental Therapeutics, 51 (1), 59-67. Bournstein, J.C. (2008). Purinergic mechanisms in the control of gastrointestinal motility. Purinergic Signaling, 4, 197-212. Guam, X.M., Amend, A., & Strader, C.D. (1995). Determination of structural domains for G protein coupling and ligand binding in beta 3-adrenergic receptor. Molecular Pharmacology, 48 (3), 492-498. Hwang, S.J., Blair, P.J., Durnin, L., Mutafova-Yambolieva, V., Sanders, K.M., & Ward, S.M. (2012). P2Y1 purinoreceptors are fundamental to inhibitory motor control of murine colonic excitability and transit. Journal of Physiology, 590 (8), 1957-1972. Jun, J.Y., Choi, S., Yeum, C.H., Chang, I.Y., Park, C.K., Kim, M.Y., Kong, I.D., So, I., Kim, K.W., & You, H.J. (2004). Noradrenaline inhibits pacemaker currents through stimulation of β1-adrenoreceptors in cultured interstitial cells of Cajal from murine small intestine. British Journal of Pharmacology, 141, 670-677. Sherwood, L. Human Physiology: From Cells to Systems (7 th ed.). California: BrooksCole, Cengage Learning. Ward, S.M,, Ordog, T., Koh, S.D., Abu Baker, S., Jun, J.Y., Amerberg, G., Monaghan, K.,

16 Sanders, K.M. (2000). Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria. Journal of Physiology, 525 (2), 355-361. Westfall, D.P., Todorov, L.D., & Mihaylova-Todorova, S.T. (2002). ATP as a Cotransmitter in Sympathetic Nerves and its Inactivation by Releasable Enzymes. Journal of Pharmacology and Experimental Therapeutics, 303 (2), 439-444.

17 APPENDICES: Sample calculations: Freq = an average of 10 frequencies taken from Biopac: (0.414421+0.355669+0.372606+0.391236+0.363953+0.355694+0.372633 +0.363953+0.381708+0.340205)/10 = 0.371208 Amplitude (Tension) = an average of 5 amplitudes (or tensions) taking from Biopac: (0.331942+0.392295+0.331942+0.362119+0.311824)/5 = 0.364024. Raw Data: Normal gut activity Epinephrine added

18 1 drop Methacholine added 3 drops Methacholine added ADP added Normal Ringer-Tyrode s to Ca 2+ -free Ringer-Tyrode s