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The Effects of Linoleic Acid on Rubidium Uptake by Sodium Potassium Pumps in Rat Myocardial Cells

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The Effects of Linoleic Acid on Rubidium Uptake by Sodium Potassium Pumps in Rat Myocardial Cells Minnesota State University, Mankato Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato All Graduate Theses, Dissertations, and Other Graduate Theses, Dissertations, and Other Capstone Projects Capstone Projects 2017 The Effects of Linoleic Acid on Rubidium Uptake by Sodium Potassium Pumps in Rat Myocardial Cells Mohammed Ahmed Gaafarelkhalifa Minnesota State University, Mankato Follow this and additional works at: https://cornerstone.lib.mnsu.edu/etds Part of the Biology Commons Recommended Citation Gaafarelkhalifa, M. A. (2017). The Effects of Linoleic Acid on Rubidium Uptake by Sodium Potassium Pumps in Rat Myocardial Cells [Master’s thesis, Minnesota State University, Mankato]. Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato. https://cornerstone.lib.mnsu.edu/etds/744/ This Thesis is brought to you for free and open access by the Graduate Theses, Dissertations, and Other Capstone Projects at Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato. It has been accepted for inclusion in All Graduate Theses, Dissertations, and Other Capstone Projects by an authorized administrator of Cornerstone: A Collection of Scholarly and Creative Works for Minnesota State University, Mankato. 1 The Effects of Linoleic Acid on Rubidium Uptake by Sodium Potassium Pumps in Rat Myocardial Cells By Mohammed A. Gaafarelkhalifa Mentored by Dr. Michael Bentley A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biology Minnesota State University, Mankato Mankato, Minnesota November 9th, 2017 2 November 7th, 2017 The Effects of Linoleic Acid on Rubidium Uptake by Sodium Potassium Pumps in Rat Myocardial Cells Mohammed Gaafarelkhalifa This thesis has been examined and approved by the following members of the student’s committee. ________________________________ Dr. Michael Bentley Advisor ________________________________ Dr. Penny Knoblich Committee Member ________________________________ Dr. David Sharlin Committee Member 3 Abstract The Effects of Linoleic Acid on Rubidium Uptake by Sodium Potassium Pumps in Rat Myocardial Cells Name: Mohammed Gaafarelkhalifa Degree: Master of Biology Institution: Minnesota State University, Mankato Date of Submission: November 9th, 2017 Congestive Heart failure is amongst the leading causes of death in the United States. It is treated through a variety of different therapeutic targets including the inhibition of sodium potassium pumps. Drugs currently used in this pathway of treatment such as Digitalis have adverse toxic effects. This study examines the inhibitory effects of linoleic acid (L.A) on sodium potassium pumps in isolated rat cardiomyocytes using the incorporation of rubidium detected by SEM/EDS technology as the index for sodium- potassium pump activity. Isolated heart cells were incubated in the following treatments: Rb-Krebs buffer without treatment (negative control), Ouabain+ Rb-Krebs buffer (positive control), 10-3 M L.A+ Rb-Krebs buffer, and 10-6 M L.A+ Rb-Krebs buffer. The results established Rb-Krebs as the negative control with a mean of 1.279 +/- 0.075 for Rb % average of 10 replicates with 10 cells analyzed in each replicate. Ouabain + Rb- Krebs was established as the positive control for inhibition of rubidium uptake with a mean value of 0.701 +/- 0.073. It was found that the 10-6 M, and not 10-3 M, concentration of linoleic acid exhibited inhibitory effects of rubidium uptake with a mean value of 0.805 +/- 0.121 and 0.903 +/- 0.095, respectively. 4 Acknowledgements I am pleased, honored, and forever-thankful to have had Dr. Michael Bentley as my thesis advisor, and I am sincerely grateful for all of his mentoring, support and teachings throughout my research experience in his lab. I would like to also genuinely thank my committee members Dr. David Sharlin for teaching me “How to research” and providing me with endless support and extremely valuable techniques, and Dr. Penny Knoblich for teaching me all that I know about cardiovascular physiology and for generously providing me with laboratory equipment throughout my research. Lastly, I would like to thank the Department of Biological Sciences for granting me this wonderful opportunity, and for providing me with financial and facility support and resources. 5 Table of Contents Introduction …………………………………………………………………………p. 6 Literature Review …………………………………………………………………..p. 8 Congestive Heart Failure …………………………………………………….p. 9 Sodium Potassium Pump ……………………………………………………p. 10 Calcium and Muscle Cell Contraction ……………………………………....p. 12 Glycosides and Enzymatic Inhibition …………………………………….…p. 13 Linoleic Acid as an Inhibitor ………………………………………………..p. 14 Rubidium as a Potassium Congener ………………………………………...p. 17 Langendorff Apparatus ……………………………………………………...p. 17 Principles of Scanning Electron Microscopy ……………………………….p. 18 Principles of Energy Dispersive Spectroscopy ……………………………..p. 19 Materials and Methods ……………………………………………………………p. 23 Results ………………………………………………………………………………p. 34 Viability of Cells …………………………………………………………….p. 34 The Effects of Linoleic Acid on Cardiomyocyte Rubidium………………....p. 35 Discussion ………………………………………………………………………….p. 38 Work Cited …………………………………………………………………….......p. 47 Appendices …………………………………………………………………………p. 50 Appendix A: Cardiomyocyte Rubidium over time…………………..…........p. 50 Appendix B: Optimum Electron Beam Spot Size ………………………...…p. 53 Appendix C: Optimum Voltage of Electron Beam …...…………………..…p. 55 Appendix D: Ouabain effect on Cardiomyocyte Rubidium ………………...p. 57 Appendix E: Carbon Stub Trial……………………………………………...p. 59 Appendix F: Linoleic Acid Trials Raw Data……………………………...…p. 61 Appendix G: Standard Krebs Buffer Recipe……………………………...…p. 71 6 Introduction The sodium potassium ATPase, also known as the sodium potassium pump, is a cellular membrane protein located in all animal cells (2). Its main function is to transport potassium ions into the cell and to transport sodium ions out against their respective concentration gradients. This action serves the cell directly in several ways such as maintenance of cell volume, allowing the cells to create a cellular membrane resting potential, and serving as a transducer for cellular signals (2). Indirectly, functions of the sodium potassium pump include interactions which couple its activity with specialized cellular processes. One such interaction couples the pump’s activity with the sodium- calcium exchange (NCX) channel (2). Calcium ions are necessary for cardiomyocytes (heart muscle cells) to generate heart muscle contractions (10). Inhibition of sodium potassium pump activity has been a treatment route for congestive heart failure (CHF) (10). This inhibition requires specific molecules such as glycosides (23), for example digoxins and Ouabain, which have been used as prescription medicine and home remedies, respectively, to treat heart failure and other heart problems for over 200 years (10). The therapeutic dose range for digoxins, pharmaceutically recognized as digitalis, is narrow, falling between 0.8 -1.8 ng/ml of blood (19). CHF patients under digoxin treatment face issues of drug toxicity. Moreover, digoxins may exhibit drug-to-drug interactions with drugs that relieve symptoms and complications of CHF (8). Such drugs include diuretics used to relieve fluid retention and may exacerbate digoxin toxicity. Symptoms of digoxin toxicity include gastrointestinal complications, arrhythmias, and breathing difficulty amongst many other severe symptoms (23). Linoleic acid is a naturally occurring fatty acid derived from several plants including Nigella sativa. Nigella sativa has been a staple of traditional medicine for centuries (1). Often ingested orally, the seeds of Nigella sativa are used to alleviate a 7 variety of different aliments including respiratory infections and hypertension (1). Early studies suggest that linoleic acid inhibits sodium potassium pumps (12, 25). Toxicologically, linoleic acid appears to be safe for human consumption, and is included in a variety of dietary supplements (27). It exhibits no apparent toxic effects in doses as high as 3.2 grams of linoleic acid per kilogram of body mass. However, it is important to note that linoleic acid has a variety of chemically produced isomers and metabolic derivatives, some of which have toxic effects (27). The most biologically-active linoleic acid isomers, cis-9,trans-11 and trans-10,cis-12 isomers, are considered safer for human consumption (30) and are used in this study. The hypothesis of this thesis is that linoleic acid decreases sodium potassium pump activity. The pump activity was determined by cardiomyocyte rubidium weight percentages. Rubidium is a congener for potassium and is actively transported into heart muscle cells by sodium potassium ATPase (18, 21). Furthermore, cardiomyocyte rubidium weight percent may be measured by Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) (4, 18, 21). There were four main experimental objectives in this thesis. First, isolation of viable adult rat cardiomyocytes by the digestive enzyme collagenase was optimized through preliminary trials. Second, cardiomyocyte rubidium weight percent over time was determined by using Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS). Third, inhibitory effect of Ouabain on rubidium uptake was determined by a decrease in cardiomyocyte rubidium weight percentages through SEM and EDS analysis. Finally,
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