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THE PRESYNAPTIC REGULATION OF ISOLATED NEONATAL RAT CAROTID BODY TYPE I CELLS BY HISTAMINE. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By DREW C. BURLON B.S., University of Dayton, 2006 2009 Wright State University WRIGHT STATE UNIVERSITY SCHOOL OF GRADUATE STUDIES August 11th, 2009 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Drew Burlon ENTITLED The Presynaptic Regulation of Neonatal Rat Carotid Body Type I Cells by Histamine BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science. ___________________________ Christopher N. Wyatt, Ph.D. Thesis Director ___________________________ Timothy C. Cope, Ph.D. Department Chair Signatures of Committee on Final Examination ______________________________ Christopher N. Wyatt, Ph.D. ______________________________ Robert W. Putnam, Ph.D. ______________________________ Kathrin L. Engisch , Ph.D. ______________________________ Joseph F. Thomas, Ph.D. Dean, School of Graduate Studies ABSTRACT Burlon, Drew C. M.S., Department of Neuroscience, Cell Biology and Physiology, Wright State University, 2009. The Presynaptic Regulation of Isolated Neonatal Rat Carotid Body Type I Cells by Histamine. It has been previously shown that Carotid Body Type I cells have the ability to synthesize, package and release histamine in response to hypoxia, thereby contributing to the modulation of respiration within the rat. Here, isolated neonatal rat carotid body type I cells were used to identify the presynaptic effects of histamine and the specific receptor subtypes that modulate them. Although all four histamine receptor subtypes are expressed on the type I cells, and preliminary data showed promising results, further data proved that the activation of these receptors with histamine or selective agonists caused 2+ no rise in intracellular calcium ([Ca ]i) and histamine did not augment calcium entry. Thus activation of histamine receptors on type I cells is unlikely to provide a presynaptic positive feedback mechanism during chemotransduction and any excitatory role attributed to the actions of histamine is likely to come from a postsynaptic effect on the carotid sinus nerve (CSN). Some of the data from this thesis have been accepted for publication in the journal Respiratory Physiology and Neurobiology: Burlon, D. C., Jordan, H. L. & Wyatt, C. N. (2009) Presynaptic regulation of isolated neonatal rat carotid body type I cells by histamine. Resp. Physiol and Neurobiol. 168, 218-223 iii TABLE OF CONTENTS Page I. INTRODUCTION ……………………………………………………………… 1 II. LITERATURE REVIEW ………………………………………………………. 4 General Anatomy………………………………………………………………... 5 History …………………………………………………………………………... 7 Chemotransduction by the Carotid Body (CB)…………………………………. 11 Role of the Type I cell as a Chemoreceptor…………………………………….. 18 a. The Mitochondrial Hypothesis……………………………………….. 13 b. The Membrane Hypothesis…………………………………………... 17 Neurotransmission within the Carotid Body……………………………………. 19 a. Excitatory Neurotransmitters: Post-Synaptic……………………….... 22 b. Excitatory Neurotransmitters: Pre-Synaptic………………………….. 25 c. Inhibitory Neurotransmitters: Pre-Synaptic………………………….. 26 Evidence for Histamine as an Excitatory Neurotransmitter within CB………… 27 H1 Receptor…………………………………………………………………….. 28 H2, H3 and H4 Receptors………………………………………………………. 29 Summary……………………………………………………………………....... 30 iv TABLE OF CONTENTS (CONT’D) III. MATERIALS AND METHODS……………………………………………….. 33 Dissection and Dissociation of CB Type I cells………………………………… 34 Immunocytochemistry…………………………………………………………... 36 Deconvolution Microscopy……………………………………………………... 37 FURA-2-AM……………………………………………………………………. 40 Ca2+ Imaging……………………………………………………………………. 44 Perfusion of Isolated Type I cells………………………………………………. 47 Statistical Analysis……………………………………………………………… 47 IV. RESULTS………………………………………………………………………. 48 Immunocytochemistry………………………………………………………….. 49 2+ Effect of Histamine on [Ca ]i………………………………………………….. 55 2+ 2+ Dependence of Histamine evoked rise in [Ca ]i on extracellular Ca ………... 58 2+ Effect of Histamine agonists on [Ca ]i………………………………………… 61 Concentration Dependence of Histamine Response……………………………. 70 2+ Effects of Histamine and Agonists on [Ca ]i with the Addition of O.7 ND filter. 75 V. DISCUSSION………………………………………………………………….. 8 Results Summary……………………………………………………………….. 88 Location of Histamine Receptors………………………...…………………….. 88 Cells Damaged By Enzyme Dissociation Process……………………………… 90 Artifact…………………………………………………………………………. 95 v TABLE OF CONTENTS (CONT’D) VI. FUTURE DIRECTIONS………………………………………………………. 96 VII. BIBLIOGRAPHY……………………………………………………………… 98 vi LIST OF FIGURES Page FIGURE 1. Structure and ultrastructure of the carotid body……………………..... 7 FIGURE 2. Isolation of a canines head connected with anastamoses to the isolated trunk of another animal………………………………………………... 10 FIGURE 3. Schematic showing the AMPK theory in detail……………………….. 15 FIGURE 4. Schematic showing neurotransmission within a glomoid in the carotid body…………………………………………………………………… 20 FIGURE 5. Representative image of deconvolution microscopy…………………. 37 FIGURE 6. Fluorescence emission of Fura-2 at differing wavelengths…………… 41 FIGURE 7. Screenshot from the Metafluor acquisition program………………….. 44 FIGURE 8. Histamine receptor subtype immunofluorescence in isolated carotid body type I cells……………………………………………………………... 50 FIGURE 9. Type I cells stained for Tyrosine Hydroxylase………………………... 52 FIGURE 10. Various effects of 100μM histamine on type I cell Fura-2 fluorescence ratio……………………………………………………………………. 55 FIGURE 11. Effects of 100µM histamine on type I cell Fura-2 Fluorescence ratios... 58 FIGURE 12. Pharmacological stimulation of the H1 histamine receptor subtype…... 61 FIGURE 13. Pharmacological stimulation of the H2 histamine receptor subtype…... 63 FIGURE 14. Pharmacological stimulation of the H3 histamine receptor subtype…... 65 FIGURE 15. Pharmacological stimulation of the H4 histamine receptor subtype…... 67 vii LIST OF FIGURES (CONT’D) FIGURE 16. Dose response experiments performed on individual type I glomus cells with no neutral density filter (300μM and 100μM Histamine)...……… 70 FIGURE 17. Dose response experiments performed on individual type I glomus cells with no neutral density filter (10μM and 0μM Histamine)……………. 72 FIGURE 18. Neutral density filter selection………………………………………… 76 FIGURE 19. Effects of 300μM histamine on type I cells with the incorporation of the 0.7 ND filter……………………………………………………………. 78 FIGURE 20. Pharmacological stimulation of the H1 histamine receptor subtype, with the incorporation of 0.7 ND filter……………………………………… 80 FIGURE 21. Pharmacological stimulation of the H2 histamine receptor subtype, with the incorporation of a 0.7 ND filter……………………………………. 82 FIGURE 22. Pharmacological stimulation of the H3 histamine receptor subtype, with the incorporation of a 0.7 ND filter……………………………………. 84 FIGURE 23. Graph representing intact G-protein receptor complexes in the plasma membrane of Type I cells……………………………………………… 92 FIGURE 24. Graph determining whether or not histamine amplifies Ca2+ influx by regulating voltage gated calcium channels…………………………….. 94 viii ACKNOWLEDGEMENTS I would really like to thank Dr. Christopher Wyatt for his relentless support of me during the arduous process of completing this thesis project. He was incredibly handsome and brilliant, which I think are the two best qualities to have in a primary investigator. Also, a special thanks to Robert Putnam, Ph.D and Kathrin Engisch, Ph.D for listening to my preliminary talks and providing great feedback in areas that needed improvement; it helped my thesis proceed much more smoothly. I would also really like to thank Barbara Barr for her lab awareness and protocol expertise as well as Andrew Jenkins and Heidi Jordan. And last but not least, I need to thank my family: Dan, Konnie, Brett, Jessica, Dave, Aidan, Evan, Julia and Megan. If they weren’t in my life it just wouldn’t be as cool. ix CHAPTER I INTRODUCTION 1 Why do we need to breathe? Is it an important function? What controls our breathing? Ever since oxygen became a freely accessible molecule on planet earth, organisms have been using it to fuel cellular respiration. Cellular respiration, or the process of using oxygen to produce ATP in mitochondria, is what allowed life to flourish and diversify on earth. Because of oxygen’s role as the final electron acceptor in the electron transport chain, single as well as multi cellular organisms have the ability to increase their energy production. In animals, respiration is the process through which oxygen is exchanged in the lungs for carbon dioxide, a byproduct of cellular respiration. By inhaling air into our lungs, oxygen is delivered to the blood stream. The blood then transports oxygen to all the cells of the body, and allows for cellular respiration to take place. This fundamental process is pivotal for human life to exist as we know it. For millennia man has known breathing, or respiration, was integral for survival. What was not known however, were the structures and processes involved with the control of our breathing. Toward the beginning of the 20th century breathing was discovered to be influenced by specialized organs that sensed changes in the composition + of the body fluids (PO2, PCO2, or [H ]), which reflect O2 demands and CO2 production (Heymans, 1930; Gonzalez et al., 1994). These organs were designated the peripheral arterial chemoreceptors. Later, during the middle of the century chemoreceptor sites within the brain