Date of Award

Summer 5-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology and Therapeutics

First Advisor

Dr. Murat Oz

Abstract

Endogenous cannabinoids (endocannabinoids) exert a wide range of biological effects. In addition to having their well-known neurobehavioral effects, a role for the major endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA), in the cardiovascular system in various pathological conditions has been reported. The aim of this thesis is to explore the effects of AEA on contractility, Ca2+ signaling, and action potential (AP) characteristics in rat ventricular myocytes. A video edge detection system was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1 μM) caused a significant decrease in the amplitude of electrically-evoked myocyte shortening. The effect of AEA was not altered in the presence of pertussis toxin (PTX), AM251 and SR141716 (CB1 antagonists) or AM630 and SR 144528 (CB2 antagonists). AEA also caused a significant decrease in the amplitudes of electrically-evoked Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically-evoked Ca2+ transients following caffeine application were not altered. In the whole-cell mode of patch-clamp technique, AEA (1 μM) significantly decreased the duration of APs. The inhibition was not altered in the presence of PTX, AM251 and AM630. Furthermore, AEA inhibited voltage-activated inward Na+ (INa) and Ca2+ (IL,Ca) currents; major ionic currents shaping the APs in ventricular myocytes, in a voltage and PTX-independent manner. Cardiac Na+/Ca2+ exchanger (NCX1)-mediated currents were also suppressed by AEA. The effect of AEA was not influenced by the inhibition of fatty acid amide hydrolase (FAAH) or in the presence of PTX, AM251 and AM630 or following the inclusion of GDP-β-S in pipette solution. The results of this study indicate for the first time that impaired Ca2+ signaling underlies the negative inotropic actions of AEA in rat ventricular myocytes, and that the direct interaction of AEA with ion channel(s) shaping APs, mediates, at least in part, the effects of AEA on myocyte contractility. In addition, the results indicate for the first time that, under normal conditions, AEA can directly inhibit the activity of NCX1 in ventricular myocytes. In view of the massive release of various N-acylethanolamines (NAEs), including AEA, during cardiac ischemia and hypoxic conditions, further understanding of their mechanism(s) of action and target proteins is essential in the development of better treatment modalities under pathological conditions.

Acknowledgments

First and foremost, I would like to express my special appreciation and gratitude to my supervisor Dr. Murat Oz. He has been a tremendous mentor for me. He taught and guided me with remarkable patience, enthusiasm and understanding. I would like to thank him for encouraging my research and for allowing me to grow as a research scientist. His guidance and advice on my research have been priceless. I sincerely thank the members of my thesis advisory committee, Prof. Chris Howarth, Prof. Sehamuddin Galadari and Dr. Rajesh Mohanraj for their brilliant comments, invaluable suggestions and encouragement throughout my graduate studies. I would also like to thank them for giving me the chance to work in their laboratories and for making me so welcome. In addition, I will always be thankful to Dr. Dymtro Isaev from Bogomoletz Institute of Physiology, Kiev, for his support and unfailing guidance. Everyone in the laboratory has been helpful and made the past four years highly enjoyable. Special thanks go to Mr. Anwar Qureshi for his generous help in cardiac cell isolation and contractility studies. I would like to thank Dr. Nurulain for always being there to give help and for his useful suggestions and for helping me with statistical analysis. I would also like to express my deep thanks to Dr. Faisal Thayyullathil for his generous guidance in conducting the biochemical studies and for always being such a helpful person. Many thanks go to my friends and colleagues who always supported me, specially, Abrar Ashoor, Khawla Salim, Kholoud Arafat, Elham Al Kubaisy, Mohammad Mahgoub and Ramez Ali.

Words cannot express how grateful I am to my mother and father for all of the sacrifices that they have made on my behalf. I would not have got to this point without you. Special thanks go to my brothers Ahmad, Amjad and Ameer for their continuous encouragement. My mother-in-law, your prayers for me were what has sustained me thus far. My deep appreciation goes to my husband Mohammad for his continuous help during the course of my study. He was always my support in the moments when there was no one to answer my queries.

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