Receptors and Ligands: Examining the effects of pharmaceutical compounds on Daphnia magna physiology Abstract The following study was conducted in order to determine the effects of four different pharmaceutical compounds on the heart rate of Daphnia specimens. After basal heart rate was observed and computed, four different solutions each containing either an adrenergic, muscarinic, or nicotinic agonist or antagonist was tested against individual Daphnia. Observations were made through a compound light microscope through the 10x objective.
It was found that nicotine decreased, caffeine increased, lidocaine decreased, epi decreased basal heart rate. This can be explained secondary to their effects as either receptor antagonists or agonists. It can be concluded, thus that chemical agents that exhibit these properties will have similar physiological results in organisms with homological pathways. Introduction Cell signaling and regulation is essential to how organisms’ physiology responds to both systemic and environmental changes.
Ligands may travel many distances to their target cell (such as with neurons) or may otherwise travel relatively short distances such as those ligands that bind to cell surfaces. Cell communication occurs at through three basic steps: reception, transduction, and response. Reception occurs when a ligand binds to a receptor and a conformational change takes place opening the binding site for other molecules such as enzymes. Transduction occurs as the signal follows a certain pathway to elicit a cascade of events.
For instance, both organisms have the onduction system located in the right atrium which ensures that cardiac events take place in the ordered sequence from sinoatrial node, atrioventricular node, bundle of His, and finally to the Purkinje fibers. Finally, the response is the physiological result of first two events. This may be the opening of a K+ or Na + ion channel or the cleaving of a tryptophan from a neurotransmitter via monoamine oxidase. This lab will be performed in order to determine the effects of four different rugs on the resting (basal) heart rate of Daphnia magna.
Though much smaller in overall size (approximately 1mm in length) and less complex than a typical chordate, many tissues are similar in composition and organ systems arranged in similar fashion, particularly the cardiac system. In this way, they make an ideal model organism for physiological comparisons to vertebrate cardiovascular systems. Though the heart of Daphnia are located dorsally as opposed to ventrally on most chordates, the composition and function is homologous. Because of these homologies, the physiological effects of the tested drugs on Daphnia specimens will be analogous to their effects on vertebrate physiologies.
The drugs being tested today will either agonistic or antagonistic effects on nicotinic and muscarinic acetylcholine receptors and adrenergic receptors. “Agonistic” drugs fully activate a receptor that it binds to. “Antagonistic” drugs will bind to the receptor, but will do so in a way that it interferes or completely blocks agonists. Nicotinic receptors are found in the central nervous system and transmit incoming and outgoing signals in the sympathetic and parasympathetic nervous system. They are also found on skeletal cells that receive acetylcholine necessary for muscle contraction.
Similar to nicotinic receptors, muscarinic receptors also play a vital role in regulating an organism’s “flight or fight” response with an increased sensitivity to muscarine over nicotine. Adrenergic receptors are also G-Coupled Protein Receptors, but are specific to the neurotransmitter catecholamine, most notably adrenaline and nor-adrenaline. The drugs that were tested were 0. 004% Epinephrine, 0. 01% Nicotine, 0. 5% Lidocaine, and 0. 4% Caffeine. The hypothesis was as follows: epinephrine solution, an adrenergic agonist, will increase daphina’s heart rate.
Nicotine, a nicotinic agonist, will increase Daphnia’s heartrate. Lidocaine’s target receptor is voltage gated ion channels to which it acts as an antagonist. It is unknown as to whether it also affects g-coupled protein receptors. Daphnia treated with lidocaine will exhibit depressed heart rate. Lastly, it was predicted that caffeine, an adrenergic antagonist will increase heart rate. Resources used include journal articles published by the National Institutes of Health, the American Journal of Obstetrics and Gynecology, and web-sources by text book author and physiologist Dr. Richard Klabunde.
Materials and Methods A Daphnia specimen was obtained, placed into a well plate, and treated with detain to restrict movement allowing for careful examination under a light microscope using the 10x objective. Heart was located and basal heart rate was measured in beats per minute. This count was repeated three times and the basal heart rate then averaged. Next, this Daphnia was removed from well and allowed to “rest” in a second well of deionized water until it once again became active. It was subsequently removed from the “wash” well, placed in a third well and then was treated with 1 drop of the chosen drug.
Once the drug had taken effect over approximately one minute, the Daphnia specimen was treated with detain and observed under the compound light microscope and a new drug induced heartrate was measured over a period of one minute. Once calculations were made, the Daphnia specimen was removed from the “drug well” and placed into a rehab chamber. This process was repeated three times for three different Daphnia specimen. Treatments were made with 0. 004% Epinephrine, 0. 01% Nicotine, 0. 5% Lidocaine, and 0. 4% Caffeine. Basal and drug induced heart rates were averaged to obtain a single experimental value.
Group BHR Nicotine Lidocaine Caffeine Epinephrine Standard Deviation Series 1: n=3 147. 04 66. 67 95. 34 169. 34 205. 34 48 Series 2: n=12 239. 24 92. 85 180. 1 145. 2 285. 6 67 Results Fig 1. Average basal heart rate and drug induced heart rate for two different sample sizes along with standard deviation of appropriate samples. O Chart 1. Sample size n=3 of drug treated samples compared to basal heart rate (BHR). Chart 2. Sample size n=3 comparing basal heart rate (BHR) to drug treated samples. O Chart 3. Comparison of averaged experimental data between groups 3 and , drug induced heart rate with standard error bars.
Group BHR Nicotine Lidocaine Caffeine Epinephrine Standard Deviation Series 1: n=3 147. 04 66. 67 95. 34 169. 34 205. 34 48 Series 2: n=12 239. 24 92. 85 180. 1 145. 2 285. 6 67 Results Fig 1. Average basal heart rate and drug induced heart rate for two different sample sizes along with standard deviation of appropriate samples. O Chart 1. Sample size n=3 of drug treated samples compared to basal heart rate (BHR). Chart 2. Sample size n=3 comparing basal heart rate (BHR) to drug treated samples. O Chart 3. Comparison of averaged experimental data between groups 3 and , drug induced heart rate with standard error bars.