Cell Communication: Receptors and Ligands

Abstract

The connection between ligand binding to eliciting physiological responses is a fundamental mechanism that is a major characteristic of organisms. In this lab, we observed Daphnia Magna’s heart rateunder the influence of various type of drugs, and were able to measure the effects of ligand binding through evaluating their basal and treated heart rates of each Daphnia Magna. The purpose of this study was to observe the changes between the Daphnia’s basal and treated heart rates in order to determine the ligand’s activated effects on the circulatory system of the Daphnia. Our results showed a significant difference between the basal and treated heart rates of the Daphnia and we were able to conclude that ligand binding was responsible for this change. We also determined atropine, nicotine, lidocaine are all significant antagonists, whereas caffeine is not as statistically significant of an antagonist.

Introduction

Cell communication is a process, where cell detects and responds to signals in its environment. Cell communication allows millions of cells to communicate and work together to perform bodily processes that are essential for the cells to survive. Both multicellular and unicellular organisms are relied on cell-cell communication. The principle of cell signaling is based on the fact that cellular communication involves converting signals that carry information from one form to another. During cell communication, the signaling cell releases a particular type of signal that is then detected by the target cell. For example, in animal, cells can communicate through direct contact or by secreting local regulators such as growth factors or neurotransmitters. Also, in animal cells send and receive signals and travels to their target cells in varies of ways, such as, cell releases their signals, and then binds to the targeted cells, and while other cells signal and travels for longer amount of time.  There are three stages of cell communication; reception, transduction, and response. In reception, a signal molecule known as the ligand binds to a receptor protein, which causes the shape of the protein to change. In transduction, cascades of intracellular molecular interactions occurs, and relay signals from receptors to target molecules in the cell. In response, the specific change in cellular behavior occurs by cytoplasmic machinery, that then targets effector proteins. 

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Furthermore, ligand binding is a significant biological mechanism that acts throughout the various organisms; for example, single celled to multivariate ligand binding acts through enzymatic processes to transmit signals eliciting different responses depending on the stimulus involved (1). The system that we used to observe were Daphnia Magna, these small protists are usually found in ponds and contain the same types of tissues as any vertebrate of epithelial, connective, nervous tissues (1).  In addition, the circulatory system is also similar to vertebrae and can be observe under a light microscope easily [1]. Therefore, each of the Daphnia’s basal and heart rates before and after treatment of the drugs were easily measured. The four drugs that we tested were; lidocaine, epinephrine, nicotine, and caffeine. These four drugs were determined to be either agonists or antagonists through our experimental methods. Nicotine, and Epinephrine are known to be antagonistic drugs however, caffeine is an antagonist for adenosine, which is a neural repressor, and may have agonistic effect [3]. Although, if the effects from the drugs change the rate between the basal and treated heart rates of the Daphnia then the ligand pathways would be responsible for the physiological change because of the chemical stimulant.  

 The main purpose of this lab was to be able to identify and measure the effects of the stimulants or depressants on the ligand pathways, and determine their overall effects on the Daphnia. The drugs are classified as being either agonists or antagonists, also the diffidence between the drugs and their target receptors were determined, by quantifying the ligand binding of the organisms to respond and stimuli and as a result it may increase or decrease their chances of survive depending the effect of the drug.

Methods and Materials

In the laboratory experience, at least twelve unique Daphnia specimens were obtained, and kept in water that has no effect on the Daphnia. The Daphnia’s were observed under a compound microscope to evaluate their initial condition. Also we had to make sure that the microscope was not kept at too bright a light as it could kill the Daphnia, if there are kept for a long time. All of the Daphnia were set in a well plate, one Daphnia per well so that any misperception of the identity of the Daphnia could be avoided.

Using a transfer pipette, a single Daphnia specimen from the culture was obtained. Then a small drop of detain solution was place in the well of a depression slide and the Daphnia was positioned within the drop, so that the Daphnia can be immobilized. Then the slide was place onto the compound microscope, using the 4X or 10X objective. The slide was properly adjusted so that the Daphnia can be easily located. After the Daphnia was found in the compound microscope, then identified the dorsal heart of the Daphnia.

Measuring basal heart rate

After the heart was located of the Daphnia, a resting heart rate or basal heart rate of the Daphnia was obtained. The heart rate was counted for about 10-15 seconds each time. After counting, the slide that contained Daphnia was removed from the microscope, and the slide was place in the table to cool. The steps were repeated for each Daphnia two times, but then the roles were switched the tapper became the timekeeper and vice versa. Then the average for the two basal heart rate measurement for each Daphnia was calculated.

Measuring drug-induced heart rate

After obtaining the basal heart rate, the Daphnia was gently lift using a reagent spatula from the Detain resin, and was place into a depression well, which contained fresh water. Using a pipette, one drop of the chosen drug solution was placed into the depression well. The Daphnia was recapture with the spatula, and was place into the drug solution, allowing the drug to exert for one minute. Then the Daphnia was captured and small drop of Detain was placed into the Daphnia. The average of two heart rate were calculated for each of the Daphnia. These steps were repeated for each of the Daphnia.

Results

Epinephrine Avg

Nicotine Avg

Lidocaine Avg

Caffeine

 Avg

Basal

202

94

138

126

Treated

116

72

66

180

Figure 1. This table shows the raw data points for the average basal (resting heart rate) and treated (drug induced heart rate) Daphnia heart rates in 15 second intervals. The averages were tabulated by taking a standard mean of the respective Resting and Treated heart rates per 15 second intervals of each Daphnia.

In this laboratory experience, we were able to retrieve data from all the Daphnia that was test, and found a significant increase of heart rate in the Daphnia treated with epinephrine and caffeine. However, the nicotine and lidocaine had the most significant differences from rested to treated heart rate, and the heart rate decreased.  This can be shown in Figure 1. The most significant decreases occurred in lidocaine; drop of 72 beats/15 second, and the nicotine also showed an average drop of 22 beats/15 second. The Figures 2-7 represents the data graphically per each Daphnia, illustrating the greater magnitude in change in the heart rate after being treated with the four type of drugs epinephrine, nicotine, lidocaine, and caffeine.

Figure 2. This graph shows the effects of epinephrine on heart rate by using the average heart rate of 4 trails. Graph illustrates that the heart rate after epinephrine treatment had increased.

Figure 3. This graph shows the effects of nicotine on heart rate by using the average heart rate of 4 trails. Graph illustrates that the heart rate after nicotine treatment had slightly decreased.

Figure 4. This graph shows the effects of lidocaine heart rate by using the average heart rate of 4 trails. Graph illustrates that the heart rate after lidocaine treatment had decreased.

Figure 5. This graph shows the effects of caffeine on heart rate by using the average heart rate of 3 trails. Graph illustrates that the heart rate after caffeine treatment had increased.

Figure 6. This graph shows the average of basal (resting heart rate) and the treated heart rate of all four drugs

epinephrine, nicotine, lidocaine, and caffeine that our group found during the experiment. 

Figure 7. This graph shows the average of basal (resting heart rate) and the treated heart rate of all four drugs

epinephrine, nicotine, lidocaine, and caffeine without the group data.

As a result, the graphs above represent the data for each of the Daphnia, illustrating significant decrease or increase in heart rate of the Daphnia after being treated with the four drugs. This data was consistent with previous studies showing that epinephrine, lidocaine, and caffeine had antagonistic effects. However, caffeine had an agonistic effect since it slightly decreased from basal to treated heart rate.

Discussion

The data presented in the result section, demonstrates that the drugs cause the Daphnia’s heart rate to decreased after the drugs were induced in the solution. Since, the ligand binding with the drugs cause the heart rate to decrease. From our group’s data, we can conclude that the

that epinephrine and lidocaine are antagonistic drugs; which cause a depressant effect on the heart rate of the Daphnia, whereas we cannot conclude that caffeine and nicotine are antagonistic drugs since nicotine slightly change between their basal and treated heart rates, and the heart rate was Daphnia significantly increased after being treated with caffeine. Also, during our experiment we went through some difficulties when trying to measure the heart rate for the third and fourth time because most of the Daphnia were inactive or died after being induced with nicotine and caffeine, so we didn’t have proper data. However, many researchers suggest that caffeine is certainly an agonistic drug, since it has stimulating properties (4). Since, Daphnia do not possess the same amount of caffeine ligand receptors as does an average human cell, therefore we can predict that caffeine is an agonistic drugs. Although nicotine is accepted as an antagonistic drug it may not have had such distinguishing effects on the Daphniafor similar reasons as the caffeine. 

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 Overall, the observance of effects of the drugs on ligand binding pathways resulted in the change of a physiological state of an organism, Daphnia, in this experience most of the drugs that was test classified as antagonistic except caffeine. Since, the data clearly supports that the ligand pathways are significant to all organisms, because it regulates systems that are fundamental to survive such as in circulation. In the experiment, we were able to induce results that supported our hypothesis provided conclusive evidence that the ligand mechanisms are responsible for these physiological changes in organisms. Although, further research should be done to evaluate how the stimulants may play in the overall physiological response of organisms, many of Daphnis were inactive or died after the drugs induced.

References:

Woodham, Hadiya (2017). Cell Biology and Physiology Laboratory Manual, (pg. 85-94)

University of Kent. Study guide: Cell Communication. Retrieved from   https://moodle.kent.ac.uk/external/mod/book/tool/print/index.php?id=2396

Masaru. 2011. Muscarinic Acetylcholine Receptors. [Abstract]. Bentham Science; Current Pharmaceutical Design. 3573-3581

G. Fisone. 2003. Caffeine as a psychomotor stimulant: mechanism of action. [Abstract]. Cellular and Molecular Life Sciences. 61(7-8): 857-872.