NITROXYL ANION IN V ASORELAXATION: A POSSIBLE ENDOTHELIUM­ DERIVED HYPERPOLARIZING FACTOR AND V ASOPROTECTIVE MOLECULE. By Brandi Michele Wynne Submitted to the Faculty of the School of Graduate Studies of the Georgia Health Sciences University in partial fulfillment of the Requirements of the Degree of Doctor of Philosophy (Physiology) 2011 Nitroxyl anion in vasorelaxation: A possible endothelium-derived hyperpolarizing factor and vasoprotective molecule. This dissertation is submitted by Brandi Michele Wynne and has been examined and approved by an appointed committee of the faculty of the School of Graduate Studies of the Georgia Health Sciences University. The signatures which appear below verify the fact that all required changes have been incorporated and that the dissertation has received final approval with reference to content, form and accuracy of presentation. This dissertation is therefore in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Af>A \ 29 \2cJ 1\ Date Major Advisor It was observed that aorta from Angll hypertensive mice exhibit a preserved relaxation response to the HNO donor, AS, while mesenteric arteries exhibit a decreased AS­ mediated relaxation response. The role of voltage-gated potassium (Kv +) channels was explored as a possible mechanism for the decreased HNO vasorelaxation in the mesenteric arteries. And finally, using a model of ET-1 induced vascular dysfunction, a vasoprotective action of HNO was revealed. These data demonstrate that NO- is a mediator of vasodilation, which is lost in the mesenteric arteries during Angll hypertension possibly through Kv + channel dysfunction. This process may contribute to systemic hypertension. These data also reveal a therapeutic role for NO- donors, such as AS, which may lead to alternative treatment options during conditions where the use of organic nitrates are not effective. ACKNOWLEDGEMENTS I would like to thank my dad, G. Rush Wynne, for dealing with all the questions from 'the others' relating to when I will finish school and for making me memorize the alphabet. It has come in handy over the years. I am truly appreciative of all the help and support of my committee members; Drs. Adviye Ergul, Jessica Filosa, David Fulton, Mong Wang and my three readers, Drs. Alexis Stranahan, Nancy Kanagy and Richard White. Huge amounts of gratitude are owed Dr. Rita Tostes, a previous committee member who morphed into a 'big science sister' combined with 'personal cheerleader'. I think that meeting you during my graduate career was divine providence. I could add an entire laundry list of friends whose help I have received ( and involuntarily ~nlisted) over the years; however, thesis paper is rather expensive and I would need many days and many pages to describe in detail. You know who you are, thank you. And finally, copious quantities of gratitude are due to my mentor, Dr. R. Clinton Webb. Although I am positive that he has read these exact same statements in the acknowledgements sections of his many students, I must say it again. Thank you for being an inspiration to all of us. Thank you for being so generous. Thank you for allowing me to pursue and develop my own interests. And thank you, ~or being such a wonderful mentor. V TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ V LIST OF FIGURES ........................................... ~ ............................................................. viii LIST OF TABLES .............................................................................................................. X I. INTRODUCTION ........................................................................................................ 11 A. Statement of the problem and specific aims of the overall project. ....................... 11 B. Brief literature review and discussion of the rationale of the project. ..................... 3 Hypertension and Vascular Disease ................................................................................ 3 Vascular Smooth Muscle and the Endothelium .......................................................... 4 Molecular mechanisms ofsmooth muscle relaxation and contraction . ...................... 4 Regulation ofrelaxation responses in the vasculature: role ofthe endothelium ....... 6 Nitroxyl Anion ............................................................................................................. 9 Production ................................................................................................................... 9 Physiology and pharmacology ofHNO ..................................................................... l 0 HNO as a vasoprotective molecule ........................................................................... 12 II. MANUSCRIPTS ......................................................................................................... 14 A. Nitroxyl anion mediates relaxation in mesenteric arteries from angiotensin II hypertensive mice .......................................................................................................... 14 B. Aorta from angiotensin II hypertensive mice exhibit preserved nitroxyl anion mediated relaxation responses ....................................................................................... 3 8 VI C: Angeli's Salt, a nitroxyl anion donor, reverses endothelin-1 mediated vascular dysfunction in aorta....................................................................................................... 60 III. UNPUBLISHED DATA ........................................................... .-................................. 82 IV. DISCUSSION ............................................................................................................ 89 V. SUMMARY ................................................................................................................ 97 VI. REFERENCES .......................................................................................................... 99 APPENDIX ........... ~ ......................................................................................................... 107 Vascular smooth muscle cell signaling mechanisms for contraction to angiotensin II and endothelin-1. Wynne, B. M., Chiao, Chin-Wei, Webb, R. C. Journal ofAmerican Society ofHypertension. 2008 .................................................................................... 107 Mesenteric arteries from spontaneously hypertensive stroke prone rats exhibit an increase in NO-dependent vasorelaxation via iNOS. Wynne, B. M., Giachini, F. R., Labazi, H., Lima, V. V., Carneiro, F., Dorrance, A. M., Webb, R. 'C., Tastes, R. C .. 107 LIST OF FIGURES Figure 1. Chronic collection of MAP in conscience mice; sham and AngII-treated mice . ........................................................................................................................................... 31 Figure 2. Mesenteric arteries from AngII-hypertensive mice exhibit decreased ACh- and AS-mediated relaxation responses .................................................................................... 32 Figure 3. Nitroxyl anion is a mediator in endothelium-dependent relaxation in mesenteric arteries from both sham and AngII-treated mice .............................................................. 33 Figure 4. An inhibitor of voltage-gated potassium channels decreases relaxation in both sham and AngII-treated mice ............................................................................................ 35 Figure 5. Relaxation responses to the nitroxyl anion donor, Angeli's Salt, are mediated through soluble guanylate cyclase .................................................................................... 36 Figure 6. Protein expression of calcitonin-gene related peptide is decreased and soluble guanylate cyclase ~1 increased in mesenteric arteries from AngII-treated mice ............... 37 Figure 7. Aorta from AngII-hypertensive mice exhibit endothelial dysfunction ............. 53 Figure 8. Angeli's Salt-mediated relaxation responses were preserved in intact and denuded aorta from AngII hypertensive mice .................................................................. 54 Figure 9. Scavenging nitric oxide decreases ACh-mediated relaxation responses in aorta from AngII-treated and sham mice ................................................................................... 55 Figure 10. Nitroxyl anion does not primarily mediate endothelium dependent vasorelaxation in aorta from both sham and AngII-treated mice ..................................... 56 Figure 11. Voltage-gated potassium channel blockade decreases relaxation in aorta ...... 57 viii Figure 12. Relaxation responses to the nitroxyl anion donor, Angeli's Salt, are mediated through soluble guanylate cyclase .................................................................................... 58 Figure 13. Relaxation responses to the nitroxyl anion donor, Angeli's Salt, are reduced with nitric oxide and nitroxyl anion scavenging ............................................................... 59 Figure 14·. Aorta incubated with endothelin-1 exhibit vascular dysfunction .................... 76 Figure 15. Incubation with AS reverses ET-1 induced vascular dysfunction in mouse aorta ................................................................................................................................... 77 Figure 16. Incubation with the HNO donor, Angeli's