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Intestinal Motility with Adrenergic, Cholinergic, Purinergic, and Calcium Free Environments Luis Salgado-Salazar Matthew Stein, Marc Hyman NPB101L 006, Cheryl Dykstra-Aiello December 4, 2013

Introduction

The gastrointestinal (GI) tract is the means by which people digest, absorb, direct through motility, and secrete all nutrients eaten. Motility in the GI tract is the response involving highly regulated muscular contraction influenced by the different endocrine and/or neuronal sensations. The motor activity executed by the GI tract performs segmentation contractions, peristaltic contractions, and allows space for a digestive material reservoir. Different types of substances are introduced to the smooth muscle in the GI tract that elicit a basic response processed by either the autonomic nervous system or the enteric nervous system. For this experiment, an adrenergic, cholinergic, and purinergic agent were introduced in the environment of a piece of intestinal segment in order to evaluate the differing rate of muscle contraction and how they affect smooth muscle contraction strength. The nervous system has essential control over the responses to the stimuli affecting the GI tract. Intrinsic neuronal processing happens through the activity of the enteric nervous system (ENS) and its various types of neurons that work in synergy to traverse the input information to efferent neurons at the smooth muscle locations. Efferent neurons release neurotransmitters that may ether increase contractility or relax the smooth muscle. Extrinsic neuronal processing is influenced entirely by the autonomic nervous system (ANS) and this affects the local neuronal processing of the ENS. The ANS may induce parasympathetic activity to increase contractility or sympathetic activity to inhibit or slow the contractility. Nevertheless, the interstitial cells of Cajal (ICC) are the pacemaker cells of the GI tract initiating cyclic slow-waves and its basal electrical

rhythm (BER). The ICC relay the electrical activity to the smooth muscle cells through gap junctions. A rabbits isolated intestinal segment was observed in vitro under four different controlled environments and data was gathered from the intestine segments electrical signals. To observe smooth muscle contractions electrical rhythm frequency/rate and strength/tension, the intestinal segment was immersed in solutions with normal calcium-containing Ringer-Tyrodes (Ca2+ R-T) solution, epinephrine Ca2+ R-T solution (EPI Ca2+ R-T solution) , methacholine Ca2+ R-T solution (Met Ca2+ R-T solution), adenosine diphosphate (ADP) Ca2+ R-T solution, and a calcium clear Ringer-Tyrode (Ca2+C R-T). The conclusive evidence from the experiment should have pointed towards how calcium influx in smooth muscle cells in the GI tract elicit stronger contractions. Any chemical pathway in the bodys physiology preventing calcium release would thereby slow or prevent contraction.

Materials and Methods

Specific details and procedures may be found in NPB 101L Systemic Physiology Lab Manual, 2nd Edition, Bautista & Korber, Exercise 8, Intestinal Motility, pages 75-82. The smooth muscles strength and rate of contraction was measured using the electrical signal input transmitted to the transducer. The rabbit intestine segment was separately immersed in chambers while an air bubble valve delivered 1-3 bubbles per second. An experimental change that differs from the lab manual includes a different reaction chamber preparation. Instead of

following lab manual instructions to fill all reaction cuvettes with Ca2+ R-T solution, three cuvettes were filled with Ca2+ R-T solution where as one was filled with Ca2+C R-T.

Results

Data collection began when normal gut activity was recorded using the Ca2+ R-T solution with the intestinal segment. The baseline and amplitude results were measured in grams but are equivalent to millivolts (mV) when calibrated using the transducer equipment. Thirteen cycles were considered when determining the results for all parameters. Using the Bio Pac Software, the average of 5 P-P from the 13 cycles were obtained to get the amplitude, the Delta T option was used to get the time frame, frequency was calculated by dividing 13 by the difference in time, and baseline tension was calculated using the average of 5 Max units from those same 13 waves (Table 1).

Table 1: The time, frequency, amplitude, and baseline tension results from the intestinal response to Ca2+ R-T solution.The time was measured in seconds, frequency was measured in hertz, and both amplitude and baseline tension were measured in mV.
Timeframe (sec) Frequency (Hz) Amplitude (mV) Baseline (mV) 43.6 0.298 15.6 17.4

Gut activity was observed and measured in EPI Ca2+ R-T solution, Met Ca2+ R-T solution, ADP Ca2+ R-T solution, and in Ca2+C R-T solution. Each solution had its own baseline tension recorded prior to the EPI, Met, or ADP modification. Figure 1 illustrates a slight decrease in frequencies for the EPI and Met Ca2+ R-T solutions. A 7.5% fall in EPI and a 2.5% fall in the Met Ca2+ R-T solution. Also, there was a 9.6% decline in frequency for the ADP solution and a 42.4% decline in frequency for the Ca2+C R-T solution (Figure 1).

Frequency of Intestine Segment Contraction

0.4

Frequency (Hz)

0.3

0.2

0.1

EPI

Met

ADP

No Ca2+

Solution Type

Before

After

Figure 1: The frequencies for pre/post solution modification are shown for EPI, Met, ADP, and calcium free solutions. The before bars are to the left and the after bars are to the right of the figure, respective to each substance modification.

Figure 2 shows an amplitude decrease in all the experiments. The amplitude fell 74.7% in the EPI Ca2+ R-T solution, 38.8% in the Met Ca2+ R-T solution, 83.4% in the ADP Ca2+ R-T solution, and 64.2% in the Ca2+C R-T solution (Figure 2).

Amplitude for Intestine Segment Contraction

30.0

Amplitude (mV)

22.5

15.0

7.5

EPI

Met

ADP

No Ca2+

Solution Type

Before

After

Figure 2: Amplitude of the contractions for the before and after timeframe in the EPI, Met, ADP, and calcium free solutions. Amplitude was measured in mV with the careful calibrations of the transducer.

As seen in Figure 3, the baseline tension for all the experiment variables decreased except for the Met Ca2+ R-T solution. Baseline tension fell 84.4% for the EPI Ca2+ R-T solution, fell 83.1% for the ADP Ca2+ R-T solution, and fell 62.4% for the Ca2+C R-T solution. However, the Met Ca2+ R-T solution increased by 33.0% (Figure 3).

Baseline Tension for Intestine Segment Contraction

50.0 Baseline tension (mV)

37.5

25.0

12.5

EPI

Met

ADP

No Ca2+

Solution Type

Before

After

Figure 3: Baseline tension, measured in mV, is compared before and after the solution modification in EPI, Met, ADP, and Calcium free experiments.

Discussion The gastrointestinal smooth muscle response to epinephrine differed from the response to methacholine. Results demonstrated that when epinephrine was introduced to the intestinal segment, baseline tension decreased by 84.4% where as baseline tension for methacholine increased by 33%. Frequency decreased in both epinephrine and methacholine solutions by 7.5% and 2.5% respectively. The expected results from the experiment would agree with the results from EPI Ca2+ R-T solution but not entirely with the Met Ca2+ R-T solution because the methacholine solution frequency was expected to increase. The sympathetic nervous system (SNS) controls the release of epinephrine and the parasympathetic nervous system (PNS) controls the release of methacholine. Both EPI and methacholine are external influences and part

of the extrinsic ANS pathway to stimulate receptors on the effector cells of the smooth muscles (SM) of the GI tract (Sherwood, 2010, p. 595). These autonomic neuronal pathways function to inhibit or excite the SM contraction. In vitro, methacholine can activate all effector cells on the intestine segment where as in vivo, this cholinergic stimulus may not have such a high response (Bhetwal et al., 2013, p. 2981). This may explain why epinephrine and methacholine had such high percentage differences for baseline tension. The results from the ADP Ca2+ R-T solution demonstrated that purinergic regulation of GI tract motility functions to inhibit smooth muscle contraction. ADP mimics ATP and therefore stimulates the P2Y purinergic receptors on the cell membrane that send a signal to activate Gprotein complex and phospholipase A2 (PLA2). The G-protein complex activates phospholipase C that in turn initiates the reaction of phosphotidylinositol biphosphate (PIP2) into inositol triphosphate (IP3). IP3 causes an increased activity of phosphokinase C by increasing the sarcoplasmic calcium through elevated calcium permeability on the membrane. All this leads to a decreased phosphorylation of the myosin light chain (MLC) by its myosin light chain kinase (MLCK) and so cytosolic calcium decreases causing the muscle to relax. The experiment results show a drop in baseline tension, amplitude, and frequency. These results are what was expected from the ADP modified Ca2+ R-T solution. The idea that ADP is part of the SNS aligns with the effects that other SNS neurotransmitters, like EPI, have on the GI SM cells. ADP has an inhibitory effect on the SM due to the SNS/purinergic signaling (Burnstock, 2006, p. S175). The GI has smooth muscle cells instead of skeletal muscle cells. The structure of skeletal muscle differs from that of smooth muscle because skeletal muscle is striated and SM is unstriated/squamous. The organization in skeletal muscle is made up of thin and thick filaments

and so is the case in SM with the exception that SM does not have troponin in its thin filament (Sherwood, 2010, p. 292). Since SM does not have troponin, contraction is reached when calcium binds with a troponin-like protein called calmodulin. Calcium-Calmodulin complex activate a myosin cross bridge with a phosphorylated myosin light chain (Sherwood, 2010, p. 293). Skeletal muscle contraction happens when actin and myosin are free to interact when calcium chemically alters the thick filament. (Sherwood, 2010, p. 292). The results from this experiment prove this, as seen by the decrease in strength of contraction in Ca2+C R-T solution. As described earlier, the autonomic system modifies the contraction of the smooth muscle cells through SNS and PNS stimulation. That is done by the extrinsic input of the sympathetic and parasympathetic fibers where afferent sensory nerves modify the activity of the ENS. (Prins, 2011, p. S8). The intrinsic input to the GI SM cells comes from the ENS, which encompasses the submucosal plexus and the myenteric plexus (Prins, 2011, p. S9). The intrinsic neuronal pathways relies on many different types of neurons that function as a chain reaction to excite or inhibit Sm contraction. Sensory neurons respond to specific stimuli in the gut and activate interneurons that send the information to efferent neurons (Sherwood, 2010, p. 595). The output neurons release neurotransmitters that can increase muscle contraction as seen in the increased strength of contraction in Met Ca2+ R-T solution experiment. They may also slow down or inhibit muscle contraction as seen by the EPI Ca2+ R-T solution experiment. Excitation-contraction coupling in GI SM occurs when there is depolarization due to the large increase of cytosolic calcium. The extracellular calcium comes in through voltage-gated Ca2+ channels and through intracellular stores from the sarcoplasmic reticulum (Hill-Eubanks et al., 2011, p. 9). Ca2+ binds with calmodulin to initiate the myosin induced cross bridge

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contraction. Therefore, the lack of Ca2+ as is the case in the Ca2+C R-T solution, prevents a strong contraction from the intestinal segment because there is no extracellular Ca2+ . The SM cells only have the intracellular storage of Ca2+ in the sarcoplasmic reticulum. So, Ca2+ induced Ca2+ release (CICR) is the only thing pushing Ca2+ into the cytosol for depolarization. Nevertheless, CICR would end because it requires the activation of IP3 or high influx of Ca2+ from the extracellular plasma (Bolton et al., 1999, p. 96). There was enough Ca2+ in the cytosol to initiate CICR.

Conclusion As seen by the results, the EPI Ca2+ R-T solution demonstrated that adrenergic substances reduced the SM contraction in the GI tract. The Met Ca2+ R-T solution experiment showed the opposite effect where SM contraction in the GI tract was strengthened by the stimulation of cholinergic substances. The ADP Ca2+ R-T solution showed that SM contraction was diminished due to the phosphorylation of MLCK. The Ca2+C R-T solution experiment proved that SM contraction in the GI tract is greatly reduced because the lack of Ca2+ in the extracellular environment cannot trigger the CICR and IP3 mechanisms. The rate at which contraction is induced was reduced in all experiments. This was expected from the purinergic, adrenergic, and Ca2+ clear substances. However, this was not expected from the cholinergic substance of methacholine. The slight decrease of 2.5% in the Met Ca2+ R-T solution may have been due to the reduced storage of Ca2+ in the sarcoplasmic reticulum since this was the second to last experiment done on the intestinal segment.

References

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1. Sherwood, Lauralee, and Robert Thomas Kell. "The Digestive System." Muscle Physiology. Human Physiology: From Cells to Systems. Toronto: Nelson Education, 2010. 292, 293, 595. Print. 2. Bhetwal, Bhupal P. "Ca2+ Sensitization Pathways Accessed by Cholinergic Neurotransmission in the Murine Gastric Fundus." The Journal of Physiology512.12 (2013): 2981. Print. 3. Burnstock, Geoffrey. "Purinergic Signalling." British Journal of Pharmacology 147 (2006): S175. Nature Publishing Group. Web. 1 Dec. 2013. 4. Prins, Arina. "The Brain-gut Interactions: The Conversation and Implications." S Afr J Clin Nutr (2011): S8-S9. Little Company of Mary Medical Centre. Web. 1 Dec. 2012. 5. Hill-Eubanks, D. C., M. E. Werner, T. J. Heppner, and M. T. Nelson. "Calcium Signaling in Smooth Muscle." Cold Spring Harbor Perspectives in Biology 3.9 (2011): A004549. Print. 6. Bolton, T. B., S. A. Prestwich, A. V. Zholos, and D. V. Gordienko. "Excitation-Contraction Coupling In Gastrointestinal And Other Smooth Muscles." Annual Review of Physiology 61.1 (1999): 85-115. Print. 7. Bautista, Erwin, Korber, Julia. "Exercise 8: Intestinal Motility." NPB 101L Systemic Physiology. Mason: Cenage Learning, 2009. 75-82. Print.