Sn v9 . 1:: . g . a: . . 4.3 figufimf .fiwd PF 1 .:. . 531.. in .11: L . an... mmyy u.. . in; . . . En: . ‘3.- 5...... 1:» Lzluag. L n. .5 .t‘ .13. I“: \‘1 . t I In! In? .1. t: .l. . 1|. ‘3! 81...!!! .31.... ~ thy '- V 1? 2 :3l .31..)an igiuv‘ . 4...: isthYJQuut thug I I. ta.- 4 aflmrfl‘dmmw .- I II? .4 Ermwm I. #35 . Sum ‘37:. a. t|ul.\x \l. 3 4K. 2.. .. :5 . A... xii!!!“ 1. ‘ ‘5'. V . 1.... Jain? . m. , ~Wm? ... , “.ng .. i: . ; anyway . ...r....T ... . y ., 5.9.; . A 2.. . Hr mam "03 LIBRARY Michigan State University This is to certify that the dissertation entitled ROLE OF NADPH OXIDASE IN PERIPHERAL SYMPATHETIC AND SENSORY NEURONS IN HYPERTENSION presented by XIAN CAO has been accepted towards fulfillment of the requirements for the degree In Neuroscience Mfl/fl/flflfi/ Maj (Prbfpé pésor’s Signature 77/515775 fi/5Q (7 V Date/ MSU is an affirmative-action, equal—opportunity employer PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:[Prol/Acc8Pres/CIRC/Daleoue indd ROLE OF NADPH OXIDASE IN PERIPHERAL SYMPATHETIC AND SENSORY NEURONS IN HYPERTENSION By Xian Cao A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Neuroscience 2008 ABSTRACT ROLE OF NADPH OXIDASE IN PERIPHERAL SYMPATHETIC AND SENSORY NEURONS IN HYPERTENSION By Xian Cao The peripheral sympathetic division of the autonomic nervous system and the primary sensory nervous system modulate the splanchnic circulation. Abnormalities of these neurons, such as neurotransmitter mishandling, may contribute to neurogenic hypertension. NADPH oxidase is a superoxide anion (02")—producing enzyme that contributes to elevated vascular reactive oxygen species (ROS) production in hypertension. lts enzymatic activity has also been detected in prevertebral sympathetic ganglia. However, the role of NADPH oxidase in peripheral neurons in hypertension is not clear. This thesis is aimed to 1) localize NADPH oxidase in peripheral sympathetic and sensory ganglion neurons; 2) investigate the regulation of NADPH oxidase in sympathetic and sensory neurons in deoxycorticosterone acetate (DOCA)—salt hypertension; 3) examine the functional effects of ROS in sympathetic neurons by measuring norepinephrine transporter (NET) expression in response to NADPH oxidase- derived 02" induced by endothelin-1 (ET-1). Localization of NADPH oxidase. NADPH oxidase subunits are present in prevertebral sympathetic ganglia and sensory dorsal root ganglia (DRG). The NADPH oxidase protein expression was localized to not only the neuronal cell bodies in the ganglia but also the perivascular nerve fibers originating from these neurons. The localization of NADPH oxidase to the two distinct functional compartments of peripheral neurons indicates that ROS may modulate neuronal properties via multiple discrete mechanims in the same neurons. Regulation of NADPH oxidase. In DOCA-salt hypertension, the NADPH oxidase in sympathetic ganglia was found to be regulated differently compared to sensory ganglia. NADPH oxidase activity and expression were increased in sympathetic ganglia in hypertension but decreased in hypertensive DRG. The opposing regulation of NADPH oxidase in sympathetic and sensory neurons may have an impact on their innervation of the vasculature in hypertension. Function of NADPH oxidase in sympathetic neurons. ET-1 increased 02“ production in PC12 cells, while also inducing a transient decrease in NET mRNA expression. Neither an induction of 02“ generation nor a downregulation of NET by ET—1 was observed in p22"“°" knockdown PC12 cells. These results indicate that NADPH oxidase derived ROS production in sympathetic neurons may modulate gene expression of the proteins that are critically involved in catecholamine handling, and thus contribute to hypertension. In conclusion, NADPH oxidase is present in peripheral sympathetic and sensory ganglion neurons and nerve fibers. The enzyme is differently regulated in these two ganglia in DOCA—salt hypertension. NADPH oxidase derived-ROS decrease NET mRNA expression, which may contribute to perturbed sympathetic vascular innervation in hypertension. These findings may shed light on novel roles of NADPH oxidase and ROS in the peripheral neurons relating to the pathogenesis of hypertension. DEDICATION To my parents Lijiang Cao, Chunmei Yang and my husband Xuerui Yang iv ACKNOWLEDGEMENTS The year of 2003 turns out to be the most important year in my career life. After taking an overnight greyhound trip, with the passion of becoming a research scientist, I arrived at Michigan State University on a snowy day and knocked on Dr. David L. Kreulen’s office door. It was then in August of 2003, I came back to Michigan State University as an incoming graduate student in Neuroscience Program and kicked off my science journey in Dr. Kreulen’s lab. I have always been grateful to myself for what I chose to do in 2003. I feel more grateful to Dr. Kreulen, who became my research mentor and accompanied me with all the patience and kindness in this wonderful and painful world. Five years later now, I am sitting here finishing my doctoral disstertation. Sometimes I still cannot believe that I am truly becoming a PhD scientist. I cannot help thinking back of that girl in the greyhound bus. Thanks to Dr. Kreulen, the dream does come true. Thank you for being such a great advisor not only in science but also in life! Thanks for feeling my growing pain when I was frustrated! Thanks for sharing my happiness when I succeeded! I may not be your greatest student, but you will always be my greatest advisor! In addition to Dr. Kreulen, I would also like to thank Dr. James Galligan, Dr. Greg Fink, Dr. Hongbing Wang and Dr. Lorraine Sordillo who served on my dissertation committee. Thank you for your valuable time and effort in helping with my dissertation project! Special thanks to Dr. Sordillo who kindly taught me the technique of RNA interference and patiently guided me throughout the project optimization and troubleshooting. Many thanks to Neuroscience program director Dr. Cheryl Sisk. Thanks for including me in the program and being so supportive for my graduate study in the last five years. This is such a wonderful family. I am very proud of being a Neuroscience Alumni. Friendship has always been one most important part in my life. I feel extremely lucky to be able to work with the crew of Dr. Kreulen’s lab. Not only we work together, but also we make the best friends with each other. Erica Wehnlvein, who recently became Dr. Wehrwein, was my deskmate in the lab for five years. Thanks for being such a great friend! I will always remember the days we spent together in the lab, in the dancing class, in the football game, in the coffee shop and in the Thai restaurant. I would also like to thank Xiaohong Wang, who came to the lab at the same time with me. It has truly been a great pleasure to work with you. There is much camaraderie among other lab members as well: Lindsay Parker, Anna Wright, Mohammad Esfahanian, Carmen Affonso, Ian Behr, Arun Nagaraju, Josh Mastenbrook, and Amy Albin. Michigan State University and the last five years mean a lot to me not only because this is the place I get my PhD, this is also the place that I met my husband Xuerui Yang. You are the greatest thing that happened to me. You made my life colorful ever since we met. Thank you for being there for me! Last but not the least, I would like to thank my parents Lijiang Cao and Chunmei Yang. Thanks for giving me endless unconditional love throughout the years! Countless times when l was crying over the phone, you told me to be strong; times when I was cheering for my success, you told me to be humble; times when I felt lonely and hopeless, you assured me that I am never alone in vi the world because you are always there with me. You didnot teach me science, but you taught me the meaning and the beauty of life! Thank you! vii TABLE OF CONTENTS LIST OF TABLES .................................................................................... ix LIST OF FIGURES .................................................................................. x KEY TO SYMBOLS AND ABBREVIATIONS ............................................... xii CHAPTER 1 INTRODUCTION .................................................................................... 1 References ................................................................................... 4 CHAPTER 2 LITERATURE REVIEW AND RATIONALE .................................................. 6 References ................................................................................... 16 CHAPTER 3 LOCALIZATION OF NADPH OXIDASE IN SYMPATHETIC AND SENSORY GANGLION NEURONS AND PERIVASCULAR NERVES .................................. 23 References ................................................................................... 64 CHAPTER 4 DIFFERENTIAL REGULATION OF NADPH OXIDASE IN SYMPATHETIC AND SENSORY GANGLIA IN DOCA-SALT HYPERTENSION ............................ 71 References ................................................................................. 108 CHAPTER 5 ENDOTHELIN-1 TRANSIENTLY DOWNREGULATES NOREPINEPHRINE TRANSPORTER VIA THE ACTIVATION OF P22PHOX-CONTAINING NADPH OXIDASE IN PC12 CELLS .................................................................. 114 References ................................................................................. 147 CHAPTER 6 CONCLUSION AND DISCUSSION ........................................................ 153 References ................................................................................. 182 viii LIST OF TABLES CHAPTER 3 Table 3.1: Primer sequences for NADPH oxidase subunits Nox, Nox2, Nox4, p47°h°" and p229” and B-actin ................................................................ 30 Table 3.2: Antibodies for immunohistochemical staining ................................ 31 CHAPTER 4 Table 4.1: Primer sequences for NADPH oxidase subunits Nox1,Nox2, Nox4, p47""°x and p22”“°", B-actin and GAPDH ..................................................... 79 CHAPTER 5 Table 5.1: Target sequences for NADPH oxidase subunit p22°“°" for shRNA constructs .......................................................................................... 121 ix LIST OF FIGURES CHAPTER 2 Figure 2.1: Schematic diagram of NADPH oxidase structure .............................. 12 CHAPTER 3 Figure 3.1: NADPH oxidase subunits in celiac ganglia (CG), dorsal root ganglia (DRG) and PC12 cells .......................................................................... 35 Figure 3.2: Immunolocalization of p22"“°" in rat inferior mesenteric ganglion (IMG) ............................................................................................................. 38 Figure 3.3: Colocalization of p22""°x and NeuN in rat inferior mesentetic ganglia (IMG) ................................................................................................. 39 Figure 3.4: lmmunostaining of NeuN in cultured celiac ganglia (CG) neurons...40 Figure 3.5: Immunolocalization of p47°"°" and p22"“°" in celiac ganglia (CG) neurons ............................................................................................. 42 Figure 3.6: Immunolocalization of p22phox in dorsal root ganglia (DRG) ............ 45 Figure 3.7: Immunoreactivity of p47'°"°x and p22"“°" in NGF-differentiated PC12 cells and cultured celiac ganglia (CG) neurons ........................................... 46 Figure 3.8: P47""°x and p22”"°" colocalize with neuropeptide Y in nerve fibers in cultured celiac ganglia (CG) neurons ........................................................ 50 Figure 3.9: P47""°x and p22phox colocalize with tyrosine hydroxylase (TH) in nerve fibers in cultured celiac ganglia (CG) neurons ............................................. 51 Figure 3.10: P47°"°" and p22!“x colocalize with neuropeptide Y (NPY) in periarterial nerve fibers in mesenteric arteries ............................................. 52 Figure 3.11: P47°"°" and p22phox colocalize with CGRP in periarterlal nerve fibers in mesenteric arteries ............................................................................ 55 Figure 3.12: Schematic diagram of proposed mechanisms for the function of NADPH oxidase at the perivascular nerve terminal ...................................... 61 CHAPTER 4 Figure 4.1: NADPH oxidase subunits are expressed in dorsal root ganglia (DRG) and celiac ganglia (CG) ............................................................................ 84 Figure 4.2: NADPH oxidase activity in DRG and CG .................................. 87 Figure 4.3: P22°“°" mRNA level is higher in CG in HT animals compare to NT controls, and p47p“°" mRNA is lower in HT DRG than in NT DRG .................. 89 Figure 4.4: Western blot data from ganglia homogenate reveals that p22”"°", p47p“°" and Rec-1 are present in DRG and CG, and are differentially regulated in HT and NT animals 94 Figure 4.5: P47°“°" protein is redistributed in the CG neurons in HT animals compared to NT controls .. .................................................................................. 97 Figure 4.6: lmmunohistochemistry reveals p47p“°" localization in DRG neurons ....................................................................................................... 102 CHAPTER 5 Figure 5.1: Plasma-based expression of shRNA-p22 ................................. 120 Figure 5.2: ET-1 induces a transient decrease of NET mRNA expression in PC12 cells ........................................................................................ 126 Figure 5.3: Apocynin abolishes the decrease of NET mRNA in response to ET-1 in PC12 cells ...................................................................................... 127 Figure 5.4: Actinomycin D abolished NET mRNA decrease in response to ET-1 in PC12 cells ........................................................................................ 128 Figure 5.5: P22”“°" mRNA expression level is suppressed by RNA interference in PC12 cells ........................................................................................ 130 Figure 5.6: P22phox immunoreactivity in shRNA-p22 PC12 cells ................... 132 Figure 5.7: ET-1-incduced 02" production in PC12 cells is attenuated in shRNA- p22 cells .......................................................................................... 135 Figure 5.8: NET mRNA is reduced by ET-1 in normal PC12 cells but not shRNA- p22 cells .......................................................................................... 140 CHAPTER 6 Figure 6.1: Topological model of rat NET protein ..................................... 174 Figure 6.2: Diagram of NADPH oxidase in the sympathetic and sensory innervation of mesenteric blood vessels in hypertension ............................. 181 xi Angfl ATCC BP BLAST CBB cDNA CG CGRP Cl Ct Cfl DEPC DHE DMSO DNA DOCA DRG EDTA ET-1 GAPDH HEPES HRP KEY TO SYMBOLS AND ABBREVIATIONS angiotensin II american type culture collection blood pressure basic local alignment and search tool Coomassie brilliant blue complementary DNA celiac ganglia calcitonin gene related peptide chloride cycle threshold armol diethylpyrocarbonate dihydroethidium dimethylsulfoxide deoxyribonucleic acid deoxycorticosterone acetate dorsal root ganglia ethylenediaminetetraacetic acid endothelin-1 glyceraldehyde phosphate dehydrogenase 4-(2-hydroxyethyl) piperazine-1 ethanesulfonic acid horseradish peroxidase xii Kd KO KRH MA mRNA MA MV Na NADPH NaCl NCBI NE NET NGF no RT NOX NPY NT hypertensive immunoglobin G inferior mesenteric ganglion immunoprecipitation immunoreactivity kilo Dalton knockout mouse Krebs-Ringer-Hepes mesenteric artery messenger ribonucleic acid Mesenteric artery mesenteric vein sodium reduced nicotinamide-adenine dinucleotide phosphate sodium chloride national center for biotechnology information norepinephrine norepinephrine transporter nerve growth factor no reverse transcriptase control non-phagocytic oxidase neuropeptide Y normotensive xiii NTC 02" PAGE PBS PC12 PCR PKC PMSF PVDF qPCR Rac REST RNA ROS RT-PCR S6c SDS SHR TAE buffer TEMED TH VMAT2 no template control superoxide anion polyacrylamide gel electrophoresis phosphate-buffered saline rat pheochromocytoma cell line polymerase chain reaction protein kinase C phenol methane sulfanyl fluoride polyvinylidene Fluoride quantitative real time polymerase chain reaction small ras related G protein relative expression software tool ribonucleic acid reactive oxygen species reverse transcription polymerase chain reaction sarafotoxin 6c sodium dodecyl sulfate spontaneously hypertensive rat tris-glacial acetic acid-EDTA buffer tetramethylethylenediamine tyrosine hydroxylase vesiscular monoamine transporter 2 wild type xiv CHAPTER 1: INTRODUCTION Approximately 65 million American adults are classified as hypertensive. Hypertension is known to be a risk factor for a variety of cardiovascular diseases, including atherosclerosis, heart failure and stroke, and accounts for 6% of adult deaths worldwide. The neuronal regulation of splanchnic circulation is crucial in determining systemic blood pressure and therefore is important in hypertension development. The splanchnic circulation is composed of gastric, small intestinal, colonic, pancreatic, hepatic and splenic circulation (11). It stores 38% of total blood, of which up to 64% can be mobilized by the direct stimulation of sympathetic nerves (7). The splanchnic circulation is innervated by both the sympathetic division of autonomic nervous system (prevertebral sympathetic ganglion neurons, including celiac ganglia (CG), superior and inferior mesenteric ganglia (IMG)) and by the spinal sensory nerves (dorsal root ganglia neurons (DRG)). Elevated sympathetic nervous system activation is one of the key pathophysiological changes observed in hypertension (5). It is characterized by diminished norepinephrine transporter (NET) reuptake in sympathetic neurons and elevation of plasma norepinephrine (NE) (6; 13). On the other hand, alterations in the neuronal properties of peripheral sensory neurons that innervate the splanchnic circulation were also found to be significant in some types of hypertension (8; 16; 17). Although the exact mechanisms of how these changes in sympathetic or sensory neurons Contribute to hypertension are not clear, a possible role of reactive oxygen species (ROS) was suggested in both scenarios (2) (15)- lncreased generation of ROS is associate with many forms of hypertension (14), including deoxycorticosterone acetate (DOCA)—sa|t hypertension (1), in which endothelin-1 (ET-1) contributes to the pathogenesis of hypertension secondary to a low-renin state (10; 12). Among several enzyme systems that catalyze ROS production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is believed to be the predominant source of superoxide (02"), the precursor of all other ROS, in hypertension (9). ROS play essential roles in central nervous system -mediated regulation of cardiovascular function (18). In the peripheral nervous system, 02" levels are elevated in CG and IMG of DOCA-salt hypertensive rats compared with normotensive rats and this 02" is produced by NADPH oxidase (3) (4). However, the mechanisms underlying the elevation of NADPH oxidase-derived 02'" and the physiological consequences of increased ROS in peripheral neurons are not fully understood. The goal of this study is to 1) examine the expression and the regulation of NADPH oxidase in peripheral sympathetic and sensory neurons in DOCA-salt 2 hypertension; 2) evaluate the functional effects of increased ROS in sympathetic neurons by measuring NET regulation induced by R08. Reference List . Beswick RA, Dorrance AM, Leite R and Webb RC. NADH/NADPH oxidase and enhanced superoxide production in the mineralocorticoid hypertensive rat. Hypertension 38: 1107-1111, 2001. . Campese VM, Ye S, Zhong H, Yanamadala V, Ye Z and Chiu J. Reactive oxygen species stimulate central and peripheral sympathetic nervous system activity. Am J Physiol Heart Circ Physiol 287: H695-H703, 2004. . Dai X, Cao X and Kreulen DL. Superoxide anion is elevated in sympathetic neurons in DOCA-salt hypertension via activation of NADPH oxidase. Am J Physiol Heart Circ Physiol 290: H1019-H1026, 2006. . Dai X, Galligan JJ, Watts SW, Fink GD and Kreulen DL. Increased 02*- production and upregulation of ETB receptors by sympathetic neurons in DOCA-salt hypertensive rats. Hypertension 43: 1048-1054, 2004. . Esler M, Lambert G and Jennings G. Increased regional sympathetic nervous activity in human hypertension: causes and consequences. J Hypertens Suppl 8: 853-857, 1990. . Goldstein 08. Plasma catecholamines and essential hypertension. An analytical review. Hypertension 5: 86-99, 1983. . Greenway CV. Role of splanchnic venous system in overall cardiovascular homeostasis. FASEB J 42: 1678-1684, 1983. . Kawasaki H, Saito A and Takasaki K. Changes in calcitonin gene-related peptide (CGRP)—containing vasodilator nerve activity in hypertension. Brain Res 518: 303-307, 1990. . Lassegue B and Griendling KK. Reactive oxygen species in hypertension; An update. Am J Hypertens 17: 852-860, 2004. 10. Letizia C, Cerci S, De TG, D'Ambrosio C, De CA, Coassin S and Scavo D. 11. 12. 13. 14. 15. 16. 17. 18. High plasma endothelin-1 levels in hypertensive patients with low-renin essential hypertension. J Hum Hypertens 11: 447-451, 1997. Parks DA and Jacobson ED. Physiology of the splanchnic circulation. Arch Intern Med 145: 1278-1281, 1985. Schiffrin EL. Role of endothelin-1 in hypertension and vascular disease. Am J Hypertens 14: 838-898, 2001. Schlaich MP, Lambert E, Kaye DM, Krozowski Z, Campbell DJ, Lambert G, Hastings J, Aggarwal A and Esler MD. Sympathetic augmentation in hypertension: role of nerve firing, norepinephrine reuptake, and Angiotensin neuromodulation. Hypertension 43: 169-175, 2004. Sedeek MH, Llinas MT, Drummond H, Fortepiani L, Abram SR, Alexander BT, Reckelhoff JF and Granger JP. Role of reactive oxygen species in endothelin-induced hypertension. Hypertension 42: 806-810, 2003. Song WZ, Chen AF and Wang DH. Increased salt sensitivity induced by sensory denervation: role of superoxide. Acta Pharmacol Sin 25: 1626-1632, 2004. Wang DH, Li J and Qiu J. Salt-sensitive hypertension induced by sensory denervation: introduction of a new model. Hypertension 32: 649-653, 1998. Xu D, Wang XA, Wang JP, Yuan QX, Fiscus RR, Chang JK and Tang JA. Calcitonin gene-related peptide (CGRP) in normotensive and spontaneously hypertensive rats. Peptides 10: 309-312, 1989. Zimmerman MC and Davisson RL. Redox signaling in central neural regulation of cardiovascular function. Prog Biophys Mol Biol 84: 125-149, 2004. CHAPTER 2: LITERATURE REVIEW AND RATIONALE Essential Hypertension Hypertension, or high blood pressure (BP), generally means systolic BP consistently over 140 mmHg and/or diastolic BP over 90 mmHg. Essential, primary, or idiopathic hypertension is defined as high BP in which secondary causes such as renovascular disease, renal failure, pheochromocytoma, aldosteronism, or other causes of secondary hypertension or mendelian forms (monogenic) are not present. Essential hypertension accounts for 95% of all cases of hypertension (6). It is a common and powerful independent predisposing factor for development of coronary heart disease, stroke, peripheral arterial disease, and heart failure. Essential hypertension is heterogeneous and likely has many related contributing factors including, but not limited to, elevated sympathetic nervous system activity (49); altered properties of sensory innervation (20; 21); increased reactive oxygen species (ROS) production (31); and enhanced circulating endothelin-1 (ET-1) levels (48). Elevated Sympathetic Nervous System Activation in Hypertension Sympathetic nervous system activation has been implicated in both human hypertensive patients and hypertension animal models (40). The important role of peripheral sympathetic nervous system in hypertension is indicated by the fact that the arterial BP in DOCA-salt hypertensive animals can be decreased by ganglionic blockade with hexamethonium to a greater level than that in normotensive animals (18). Generally used as a measurement of sympathetic nerve activity, plasma norepinephrine (NE) levels were shown to be elevated in hypertensive individuals (17; 22). This indicates that altered NE handling in the peripheral sympathetic neurons may be attributed to faulty BP regulation. Mechanisms for the increased spillover of NE include sympathetic hyperinnervation, epinephrine cotransmission, increased nerve firing rates, and dysfunction of NE reuptake via neuronal NE transporter (NET) (16). NET is a plasma membrane protein that belongs to the large gene family of Na+ICI- dependent neurotransmitter transporters. Transporters in this family are responsible for clearance of NE, dopamine, and serotonin from the neuroeffector junction (57). NET is present in prevertebral sympathetic ganglion neurons and nerve terminals (35). NET is also localized to sensory neurons in dorsal root ganglia (DRG) (30) although its function in sensory neurons is unclear. NET-mediated reuptake of NE increases when elevated nerve firing releases more NE into the synapse (15). However, if NET function does not proportionally increase with NE release, excess NE would accumulate in the neuroeffector junction, sustain the constrictive stimulation on the blood vessels and spillover into the circulation (13). NET deficient mice showed a significant higher BP than control animals (28), which further indicates the role of NET in hypertension. However, the molecular mechanisms underlying the regulation of NET in hypertension are poorly understood. It is expected that alterations in the function of NET could be a result of one or several changes, including changes in the amount of NET inserted into the plasma membrane, changes in the transport affinity or capacity, and alterations in NET gene/protein expression and regulation. Interestingly, ROS displayed inhibitory effects on both NET function and expression in vitro (41 ). The purposed of this thesis is to examine whether NADPH oxidase-derived ROS modulates NET expression and function in peripheral neurons. Sensory Neurons in Hypertension -- More than Just Baroreflex The role of sensory neurons in BP regulation is best recognized in the baroreflex response. The baroreceptor reflex is a key regulator of BP; baroreceptors in the carotid sinuses and aortic arch detect changes in BP and trigger reflex circulatory adjustments that buffer or oppose the change in pressure. 8 The central initiation of this reflex begins with baroreceptive afferent release of glutamate to activate second-order neurons in the nucleus tractus solitarii. The reflex circus then modulates the sympathetic outflow and thus regulates BP. It is suggested that essential hypertenSion is accompanied by modification of the arterial baroreceptor reflex, that is, a resetting of the range of action of the reflex toward higher blood pressure value (23). In fact, the abdominal circulation is sensitive to baroreceptor activation (24). However, not all sensory mediated reflexes on blood volume regulation are through baroreceptors. For example, intestinal distention can evoke an inhibitory junction potential (IJP) in the mesenteric blood vessels and this IJP mediates vasodilatation (42; 43). This peripheral reflex is mediated solely outside of the CNS and involves only pathways through peripheral sensory and sympathetic ganglia. In addition to its afferent properties, sensory neurons can synthesize and release vasoactive neuropeptide directly on the blood vessels and thus serve as an efferent mechanism. The vasodilatory neural transmitters released from the sensory fibers include calcitonin gene-related peptide (51), substance P (19), neurokinin A (14) and nitric oxide (58). Changes in expression or dynamics of these sensory neuropeptide have been associated with some types of hypertension animal models (27; 54; 56). The involvement of peripheral sensory neurons in BP regulation was best documented in salt-sensitive hypertension. Neonatal degeneration of 9 capsaicin-sensitive sensory nerves in rats leads to a significant increase in BP when high salt diet was given (54), suggesting that sensory innervation plays significant roles in antagonizing the development of salt-induced hypertension. The interaction between sensory nerves and the renin-angiotensin system or the sympathetic nerve activation may be involved in mediating this effect but detailed mechanisms are not fully understood (53). Oxidative stress can cause neuropathies in animal models of diabetes (52). During aging, the higher incidence of neuropathic pain in the elderly is suggestive of an association between progressive degeneration of primary sensory neurons and ROS generation (39). On the other hand, NADPH oxidase-derived ROS seem to have a beneficial effect on the maintenance of DRG neuron integrity and pain perception under normal physiological conditions (46). It is therefore reasonable to predict that DRG neuronal properties are under tight control of cellular redox homeostasis. However, the role of ROS in sensory cardiovascular regulation is very little studied. Role of NADPH Oxidase and Endothelin-1 in Hypertension ROS include superoxide anion (02"), hydrogen peroxide (H202), hydroxyl radicals ('OH) and peroxynitrite (ONOO’) (12). Increased levels of ROS are correlated with numerous cardiovascular diseases including hypertension (5). 10 Among several enzyme systems that catalyze ROS production, NADPH oxidase is a predominant source of 02“, the precursor of all other ROS, in hypertension. NADPH oxidase was first identified in phagocytes (neutrophils) (2), in which it plays a vital role in nonspecific hostdefense against pathogens by generating mill molar quantities of 02" during the respiratory burst (50). The NADPH oxidase enzyme system consists of two integral membrane proteins p22""°" and a NOX catalytic subunit (NOX1, gp91°“°*, NOX3, NOX4, or NOX5), and several cytoplasmic regulatory elements that include p47p“°", p40”"°", p67p“°" and a G protein (Rec-1). Activation of NADPH oxidase involves the translocation of regulatory elements from the cytoplasm to combine with catalytic subunits in the membrane (8) (Figure 2.1). In addition to phagocytes, NADPH oxidase is present in endothelial cells in blood vessels (3), vascular smooth muscle cells (34), kidney cortex (7) and nervous system (9; 29). Unlike those in neutrophils, the NADPH oxidase in these tissue make 02" in small amounts for purposes of signaling under physiological conditions (25). However, excessive 02" production will lead to a variety of intracellular signaling events that ultimately cause cell dysfunction (47). Endothelin-1 (ET-1) is a potent 21-amino acid vasoconstrictor peptide produced by endothelium and to a lesser extent, the nervous system (36). ET-1 expression in vasculature is increased in salt-dependent hypertensive animal models as well as in human hypertensive individuals (48). Although most studies 11 0000000000 000000.00 202" ._ V 1 202.- NADPH *Figure 2.1. Schematic diagram of NADPH oxidase structure. At rest, NADPH oxidase contains two membrane-bound subunits NOX and p22phox and several cytosolic subunits p47phox, p40phox, p67phox and Rac-GDP. upon activation, cytosolic subunits are recruited to the membrane and bind with the two membrane subuints. The assembly of the oxidase complex on the membrane initiates the production of 02" from O2 utilizing cytosolic NADPH as electron 'Images in this dissertation are presented in color. 12 to date have been focused on vascular ET-1, several lines of evidence suggest that ETs may function as neurotransmitters or neuromodulators within the nervous system (11). ET-1 can potentiate NE-induced cardiac contractile response by either facilitating NE release from the sympathetic nerve terminal or impairing NE re-uptake by the neuronal NET (1). ET also modulates NE release in the posterior hypothalamus, a sympathoexcitatory region in the central nervous system that is involved in BP regulation (11). ET-1 may be exerting its effects on the vasculature and nervous system by increasing the production of 02“, thereby altering the cellular redox environment and cell function. In the vasculature, ET-1 increases 02“ production via the activation of NADPH oxidase (37; 38; 55). The vascular NADPH oxidase activity and expression are increased in DOCA-salt hypertension (4), a salt—dependent hypertensive model associated with elevated ET-1 level (32; 33). In peripheral nervous system, 02" levels are increased in inferior mesenteric ganglia (IMG) of DOCA-salt hypertensive rats compared to normotensive rats (10). In cultured celiac ganglia (CG) neurons treated with ET-1, 02" production is significantly increased and this increase is blocked by NADPH oxidase inhibitor Apocynin (9), but not other oxidase inhibitors. Furthermore, NADPH oxidase enzymatic activity and ETB receptor expression are both increased in CG from DOCA-salt hypertension compared to controls, indicating correlated upregulation of NADPH oxidase and ET-1 signaling in prevertebral sympathetic ganglion neurons (9; 10). 13 On the other hand, both ET-1 and its receptors are localized to DRG sensory neurons (44; 45). However, less is known about its role in BP regulation. Summary Hypertension is a multi-factorial disease and is an independent risk factor for various other cardiovascular disorders. Scientists have been working hard for decades trying to unravel the mysteries behind its mechanisms. However, although extensive progresses have been made so far, the etiology of hypertension is still unknown. Most studies showed pathophysiological features found in either animal models or human hypertensive individuals that are “correlated” with the onset of hypertension, with few of them clarifying whether those are actually “causative factors” or merely pathological changes secondary to increased BP. A typical example is the role of free radicals in hypertension. Elevated ROS levels have been reported from various types of hypertension yet anti-oxidant supplement showed little effect in lowering the risk for hypertension (26). More studies are clearly needed to determine the exact roles of ROS molecules in BP regulation. In this thesis, experiments are designed to characterize the ROS-generating enzyme NADPH oxidase in peripheral sympathetic and sensory nervous system and its regulation in DOCA-salt hypertension, with the hope to provide possible 14 mechanisms related to elevated sympathetic activation and altered sensory properties in salt-sensitive hypertension. The functional effects of ROS in sympathetic neurons are studied in more detail under the hypothesis that ROS can affect catecholamine handling in sympathetic ganglion neurons via its modulation on NE reuptake by NET. 15 Reference List . Backs J, Haunstetter A, Gerber SH, Metz J, Borst MM, Strasser RH, Kubler W and Haass M. The neuronal norepinephrine transporter in experimental heart failure: evidence for a posttranscriptional downregulation. J Mol Cell Cardiol 33: 461-472, 2001. . Batot G, Martel C, Capdeville N, Wientjes F and Morel F. 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This suggested a role for neuronal NADPH oxidase in autonomic neurovascular control, but the expression and localization of NADPH oxidase in the peripheral neurons was not clear. The purpose of this study was to examine the mRNA expression and subcellular localization of NADPH oxidase in sympathetic and sensory ganglion neurons. Using reverse transcription-polymerase chain reaction, we determined that the mRNA of NADPH oxidase subunits NOX1, NOX2, NOX4, p22phox and p47°“°" were present in celiac gangia. The same subunits were also present in dorsal root ganglia with the exception that NOX4 levels were much lower. Immunohistochemical staining was performed to localize NADPH oxidase protein in sympathetic and sensory neurons by examining one of the membrane-bound subunit, p22”"°", and one of the cytosolic subunit, p47°“°". We found that in rat celiac ganglia and inferior phox mesenteric ganglia, there was intense immunostaining of p22 associated with 23 ganglionic neuron somata and intercellular nerve fibers with no staining in satellite cells. P22°“°" also was localized to a subpopulation of dorsal root ganglia neurons that contain calcitonin gene related peptide. In mesenteric arteries, p47°"°" and p22p"°" were colocalized with neuropeptide Y or calcitonin gene related peptide in perivascular nerve terminals. A similar pattern of nerve terminal staining of p47°"°" and p22p“°" also was found in cultured celiac ganglia neurons and one-week nerve growth factor -differentiated PC12 cells. These data demonstrate a previously uncharacterized localization of NADPH oxidase in prevertebral sympathetic ganglia and sensory ganglia. The presence of a O2"— generating enzyme in the close vicinity of the sites for neurotransmitter handling in these neurons may suggest novel reactive oxygen species-mediated mechanisms in peripheral sympathetic and sensory neurovascular control. Introduction Reactive oxygen species (ROS), such as superoxide (02") and hydrogen peroxide (H202) are signaling molecules which play important roles regulating cardiovascular function (14; 15). While first discovered in phagocytes, reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase is now thought to be a significant source of ROS in many cell types including smooth muscle cells (13; 41; 48), endothelial cells (38), fibroblasts (22) and neurons in the brain (11). 24 NADPH oxidase is a multi-subunit enzyme consisting of several cytosolic subunits (p47p"°", p67p"°", p40"“°" and Rae) and a membrane-associated cytochrome b558, which is composed of p22""°x and one NOX catalytic subunit. In phagocytes, enzyme activation requires the translocation of cytosolic subunits to the membrane where they associate with cytochrome b558 and facilitate the transfer of electrons from NADPH via FAD and two heme moieties to molecular oxygen, resulting in 02" formation. In non-phagocytic cells, intracellular pre-assembled NADPH oxidase may exist to facilitate 02" production in the cytoplasm (28; 55). Increased ROS production and NADPH oxidase activity are associated with cardiovascular dysfunction in hypertension (1 ), diabetes (17) and senescence (4). Studies of ROS in hypertension have focused primarily on vascular and endothelial ROS signaling (15). However, accumulating evidence indicates that peripheral neural components, including both sympathetic and sensory nerves, which innervate the splanchnic circulation, play a key role in regulating blood pressure, and that abnormalities in these neurons contribute to increased salt sensitivity and the development of hypertension (23; 33; 35; 49; 52). Enhanced NADPH oxidase activity in peripheral sympathetic neurons is associated with the onset of cardiovascular disorders (6; 32). This suggests a role of peripheral neuronal NADPH oxidase in the regulation of blood pressure. 25 Because of a short half life (1><10'6 see), the direct actions of 02" in the cell are confined to a limited region near the subcellular site of its production. In neurons, the major functional compartments—the cell body, dendrites, axons, and terminals— are separated by considerable distances; therefore it is important to evaluate the localization of NADPH oxidase in these compartments in order to fully understand the physiological consequences of 02" production. In particular, transmission at the neuro-vascular junctions modulates vascular tone, the 02“ produced by NADPH oxidase in the cell body would not be expected to diffuse to the terminals; if NADPH oxidase were to influence neurotransmitter dynamics it would have to produce 02" locally. Therefore, a systemic evaluation of the localization of NADPH oxidase in the peripheral sympathetic and sensory neurons is needed to address this issue. A series of experiments were designed to localize NADPH oxidase subunits, p22phox and p47p"°", to prevertebral sympathetic ganglia and sensory ganglia as well as perivascular nerve fibers and endings to assess their anatomical location. Our results showed that NADPH oxidase subunits were present in both the cell bodies and the nerve fibers of these neurons. The presence of NADPH oxidase subunits in the neurons innervating the splanchnic circulation may have important implications in the role of NADPH oxidase in blood pressure regulation and hypertension.- 26 Methods Tissue Harvest and Cell Culture All cell culture reagents are GIBCO® brand (lnvitrogen, Carlsbad, CA) unless otherwise noted. Primary sympathetic ganglion neg/ran culture Celiac ganglia (CG) from postnatal 3 to 5-day-old Sprague-Dawley (SD) rats were harvested and enzymatically dissociated (2.5mg/ml collagenase 10 minutes at 37°C followed by 2.5mg/ml trypsin 45min at 37°C). Freshly dissociated neurons were plated as a monolayer on cover glass in culture dishes double coated with 100pg/ml poly-D-lysine (Sigma-Aldrich, St. Louis, MO) and collagen. Cells were maintained in N2 medium (49% DMEM, 49% F-12 nutrient mixture, 0.5mg/ml bovine serum albumin, 2mM L-glutamine, 1% N2 supplement, 100ng/ml nerve growth factor 2.5 (Millipore, Billerica, MA), 0.7% B-27) supplemented with 1% fetal bovine serum at 37°C in a 5% CO2 humidified incubator. 0.24pg/ml 1B-arabinofuranosylcytosine (Ara-C) (Calbiochem, San Diego, CA) was added to N2 medium from the second day of culture to eliminate non-neuronal cell growth. Neurons were kept in culture for 7 days before immunostaining experiment to ensure full neurite outgrowth. PC-12 Cell Culture 27 PC-12 cells are derived from a rat catecholamine-secreting chromaffin tumor. They can differentiate into cells with a sympathetic neuronal phenotype after one week of NGF treatment (12). PC-12 cells were obtained from American Type Culture Collection, and maintained at 37°C in a 5% CO2 humidified incubator in RPMI 1640 medium supplemented with 10% heat inactivated horse serum, 5% fetal bovine serum, 100U/ml penicillin, 100pg/ml streptomycin and 0.25pglml Fungizone. To differentiate PC12 cells, 50ng/ml NGF 2.58 (Millipore) was added to the medium for 7 days. RNA Isolation RNA was extracted from cultures of CG neurons, cultures of PC-12 cells, rat dorsal root ganglia (DRG) (spinal level T13 - L2), rat aorta, and rat cerebral cortex by using the standard TRIzol procedure (Invitrogen). The concentration/purity/integrity of RNA was ascertained spectrophotometrically (A260/A230). To eliminate residual genomic DNA in the preparation, total RNA samples were treated with 10 U/pl RNase-free DNase I (Roche, Nutley, NJ) for 30 min at 37°C, and DNase I was inactivated by heating for 10 min at 75°C. Reverse Transcription - Polymerase Chain Reaction (RT-PCR) cDNA was synthesized from DNase-treated RNA using Superscript II mix (Invitrogen). The cDNA synthesized from 2.49 total RNA was used in subsequent 28 PCR. All primers were derived from the Rattus Norvegicus gene (National Center for Biotechnology Information GenBank). Primer sequences are shown in Table 3.1. PCR products were electrophoresed on a 2.0% agarose gel for 60 minutes at 9V/cm gel. Bands corresponding to PCR amplicons were stained by ethidium bromide and visualized by UV light. lmmunohistochemical Staining of Rat Inferior Mesenteric Ganglia and Cultured Celiac Ganglion Neurons and PC12 Cells All antibodies used in these experiments are listed in Table 3.2. Postnatal 7 to 10-day-old SD rats were euthanized with sodium pentobarbital (50mg/kg). The inferior mesenteric ganglia (IMG) were surgically removed and maintained in Hank’s Balanced Salt Solution (Invitrogen). The IMG were cleaned of surrounding connective tissue and blood vessels and the isolated ganglia were placed in fixative (4% paraformaldehyde, 0.1% Triton X-100 in Dulbecco’s phosphate buffered saline (DPBS)) for 30min at room temperature. Cultured cells were cleaned from culture medium by three washes in DPBS and then placed into fixative for 30min. Samples (ganglia or cultured cells) were then incubated in DPBS with blocking solution (5% goat serum, 3% BSA) for 1 hour at room temperature, followed by primary antibody incubation for overnight at 4°C. The next day, samples were washed in DPBS for three times and then incubated with corresponding secondary antibodies in a dark chamber at room temperature for 1 29 Table 3.1 Primer sequences for NADPH oxidase subunits NOX, NOX2, NOX4, p479“)( and p22phox and B-actin. Gene Sequence Amplicon NCBI Length accession (bp) Number NOX1 For:5' TGAACAACAGCACTCACCAATGCC 3' 245 AF152963 Rev:5' AGTTGTTGAACCAGGCAAAGGCAC 3' NOX2 For:5' GTGGAGTGGTGTGTGAATGC 3' 324 AF298656 Rev:5' TCCACGTACAATTCGCTCAG 3' NOX4 For:5' ACCAGATGTTGGGCCTAGGATTGT 3' 261 AY027537 Rev:5' AGTTCACTGAGAAGTTCAGGGCGT 3' p47phox For:5' GGCCAAAGATGGCAAGAATA 3' 221 AF260779 Rev:5’ TGTCAAGGGGCTCCAAATAG 3' p22phox For:5' TTGTTGCAGGAGTGCTCATC 3' 282 U18729 Rev:5' TAGGCTCAATGGGAGTCCAC 3' B-actin For:5' GGCTACAGCTTCACCACCAC 3' 500 V01 21 7 Rev:5' TACTCCTGCTTGCTGATCCAC 3' 3O Table 3.2 Antibodies for immunohistochemical staining. Primary antibodies Antigen Host species Dilution Source p47phox (R360) Rabbit 1:300 Dr. Mark T. Quinn (Montana State University) p47phox Rabbit 1:150 Santa Cruz Biotech., Inc., Santa Cruz, CA p22phox"(H44.1) Mouse 121000 Dr. Mark T. Quinn p22phox (R5554) Rabbit 1:300 Dr. Mark T. Quinn NeuN" Mouse 1:500 Millipore, Billerica, MA, SGII" Mouse 1:1000 Abcam Inc., Cambridge, MA TH" Mouse 1 :150 Calbiochem, La Jolla, CA NPY Goat 1:300 Santa Cruz Biotech., Inc., CGRP Sheep 1:1000 Abcam Inc., Cambridge, MA SGII = secretogranin Il; TH = tyrosine hydroxylase; NPY = Neuropeptide Y; CGRP = calcitonin gene-related peptide; "=monoclonal antibodies Secondary antibodies Target species Host species Conjugated to: Dilution Mouse Donkey FITC 1 :40 Rabbit Donkey Cy3 1 :200 Sheep Donkey Cy3 1 :200 Goat Donkey F lTC 1 1200 Mouse Donkey Cy3 1 :200 Rabbit Goat Alexa 488 1:500 Mouse Rabbit Cy3 1 :500 All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, except for goat anti-rabbit Alexa 488 was purchased from lnvitrogen, lnc., Carlsbad, CA 31 hour, followed by three washes in DPBS. Samples were mounted onto glass slides using Prolong Gold anti-fade reagent (lnvitrogen) for confocal laser scanning using Pascal (Zeiss, Thomwood, NY) or Fluoview (Olympus, Center Valley, PA). lmmunohistochemistry for Rat Celiac Ganglia and Dorsal Root Ganglia Ganglia were dissected from adult SD rats and fixed in 10% formalin for 2 hours then transferred to 70% ethanol for storage ranging from several hours to overnight. Tissue was processed using a vacuum infiltration tissue processor (Thermo Electron Excelsior) with decreasing concentrations of ethanol followed by xylene. Tissues were embedded in paraffin, sectioned on a rotary microtome into 5 pm sections, and mounted on to glass slides (Corning Glass). Heat induced epitope retrieval (HIER) was used. Samples were blocked for endogenous elements with hydrogen peroxide/methanol for 30 minutes then rinsed. Due to the use of HIER, an additional blocking step of avidin and biotin was used with a 15-minute incubation. Normal goat serum or donkey serum (1:28, Vector Laboratories, Burlingame, CA) was used as a protein block followed by incubation primary antibodies for 60-minutes. Incubation of biotinylated goat-anti-rabbit secondary antibody (1:200, Vector Laboratories) for 30 minutes was followed by a 15 minute incubation with Nova Red chromagen (Vector Laboratories). Slides were counterstained with Lerner 2 hematoxylin then dehydrated. Images were 32 collected using standard bright field microscopy (Olympus BX60 with SPOT Insight Digital Camera, Olympus America Inc. Center Valley, PA). A “no primary control” was run in parallel without addition of primary antibody to assess antibody specificity. For fluorescent staining, 'slides were incubated with primary antibodies followed by incubation in fluorophore-conjugated secondary antibodies. Images were collected using Fluoview confocal microscope (Olympus). immunostaining of Periarterial Nerve Fibers 8 week old SD rats were euthanized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). The mesentery was surgically removed and maintained in 0.1M phosphate-buffered saline (PBS). Mesenteric arteries were cleaned of adipose and connective tissue and cleared of blood via an intravascular PBS bolus. Tertiary branches were excised and isolated tissues were placed in Zamboni fixative (2% [vol/vol] formaldehyde and 0.2% [vol/vol] picric acid in 0.1M phosphate buffered saline, PBS) overnight (4 °C). The next day, the tissues were washed 3x with 0.1M PBS and then incubated in PBS with blocking serum (donkey) diluted in Triton X-100 (1.0 %) for 1 hour. Tissues were then co-incubated for 2 hours at 37 °C in diluted primary antibodies (in Triton-PBS). Next, tissues were washed 3x in 0.1M PBS buffer and then incubated for 1 hour in a dark, humidified chamber at room temperature in corresponding secondary antibodies. Vessels were then washed 3x with 0.1 M 33 PBS at 5-minute intervals and coverslipped with Prolong Gold anti-fade reagent for fluorescence confocal microscopy. Tissues were examined using a Leica TSL laser confocal microscope (Leica Microsystems Inc., Bannockburn, IL). Results Expression of NADPH Oxidase mRNA in CG, DRG and Differentiated PC-12 cells PCR amplicons of NADPH oxidase catalytic core subunits NOX1 and NOX2 were detected in aorta, brain, dissociated CG neurons, DRG and differentiated PC-12 cells (Figure. 3.1 A, B and C) at the expected sizes of 245 and 324 bp, respectively. By contrast, NOX4 was present in aorta and CG neurons but not in brain or PC-12 cells (Figure. 3.1 A and B). NOX4 was present in rat DRG at very low level (Figure. 3.10). Thus, NOX4 was the only homologue not found in all 4 cell types examined. PCR amplicons for NADPH oxidase regulatory subunits p47°“°" and p22phox were present in both rat CG and DRG (Figure 3.1 B and C). The sequenced PCR amplicons were aligned in GenBank. Greater than 99% of sequenced amplicons of p47°“°", p22""°", NOX2, NOX1, and NOX4 in CG neurons and PC-12 cells matched published sequences. 34 SIDbp—e bani ---- a b c p avb c_"_p __a_,b c p__ a b c p NOX1 NOX2 'NOX4 ' ' B-actin {2.1 5000p _. N 300bp _. :7: 200bp —* ' ' '§""""" an... - .1.-u. - ... _§__.P_,.-.___°.__ . P a b C P NTC No RT Fl ure .1. NADPH oxidase subunits In celiac an Iia CG dorsal root an Ii DRG and PC12 cells. PCR amplicons for catalytic core subunits NOX1, NOX2 were present on ethidium bromide-stained agarose gels from aorta (a), brain (b), CG (c) and PC12 cells (p) (A); NOX4 was found only in aorta and CG, not in brain or PC-12 cells (A). NOX4 was also found in DRG but at a very low level (C); NADPH oxidase regulatory subunits p47phox and p22phox were found in both CG (B) and DRG (C). B-actin was used as a loading control in all experiments. No-cDNA template control (NTC) and omission of the RT step (No RT) were both performed as a negative control. 35 Figure 3.1 continued 3 -- co “uh-fl Ohm 500bp —r- ri— 400bp —> -- - 300bp —> "' - 200bp —> " ' nox1 NOX4 I p22”? NTC 1 Ladder NOX2 p47""'°x B-actin No RT C DRG 400bp—> W- .. 300bp-—> W“ Q m zoom—p "" NOX1 NOX4 I p22”? NTC I Ladder phox NOX2 p47 B-actin No RT NADPH Oxidase Subunit p22""°" is Present in Neuronal Somata in Rat lMG. In whole mount rat IMG, p22phox immunoreactivity was found in ganglion neurons using two different antibodies targeting p22°“°". First, monoclonal mouse 36 anti-p22”"°" antibody (H44.1, see Table 3.2) staining showed that p22°“°" was localized to neuronal cell bodies in IMG with little staining in intemeuronal structures (Figure 3.2). Second, in double labeling staining, p22"“°" (R5552, see Table 3.2) immunoreactivity was localized in Neuronal Nuclei (NeuN)-positive ganglion neurons (Figure 3.3). Although in some species, NeuN does not stain sympathetic neurons (53); in rat IMG NeuN immureactivity was localized primarily in the nuclei of neurons with lighter staining in the cytoplasm. There was no NeuN immunoreactivity in non-neuronal cells. The use of NeuN as a sympathetic neuronal marker was further verified in cultured CG neurons (Figure 3.4) The staining pattern of NeuN in both IMG and CG are identical to those found in other types of neurons (54). The colocalization of p22phox with NeuN indicates the presence of NADPH oxidase in sympathetic ganglion neuronal cell bodies in rat IMG. 37 p229h°x p22phox \ Fl ure 3.2. Immunolocalization of 22 hox in rat Inferior meson erlc an llon IMG. IMG was dissected from 3-week old male Sprague-Dawley rats and fixed immediately. The ganglion was incubated with mouse monoclonal antibody against p22phox (H44.1) followed by Cy3 tagged secondary antibodies. P22phox was localized in the cytosol and plasma membrane of ganglion neurons (arrow). Images were taken under confocal laser scanning microscopy. 38 p22phox B Overlay was removed from 10 day- old Sprague- -Dawley rats and put in fixative immediately. The ganglion was incubated with (A)polyclona| rabbit anti-p22phox (R5554) and (B)monoclonal mouse anti-NeuN followed by secondary antibodies. (C)P22phox and NeuN immunoreactivity were found in the same neuron (arrow). Images were taken under confocal laser scanning microscopy. Scale bar is 20pm. 39 Figure 3.4 lmmunostaining of NeuN in Cultured Celiac Ganglia (CG) Neurons. Freshly dissociated CG neurons were dissected from 3 to 5-day old Sprague-Dawley rats and were kept in culture medium for 7 days before immunocytochemical staining. Cells were incubated with mouse monoclonal anti-NeuN at a 1:500 dilution overnight at 4°C followed by incubation with Cy3 conjugated goat anti-mouse antibody. NeuN staining was found presumably in the nuclei of the neurons with less staining in the cytoplasm. Scale bar is 50pm in the upper pannels and 20pm in the lower pannels. 4O NADPH Oxidase Subunits p22P"°' and p479” are Present in Rat cc Neurons. Fixed CG tissue was cut into 5pm sections for immunohistochemisty. As shown in Figure 3.5 A, immunoreactivity for p47p“°" was found in all neurons in CG. The staining was presumably in neuronal cell bodies with little staining in non-neuronal structures. Similar staining pattern was also observed with p22"“°" antibody (data not shown). Previously, in guinea pig prevertebral sympathetic ganglia, approximately 20% of all neurons contain immunoreactivity to neuropeptide Y (NPY) and have been speculated to be vasoconstrictor neurons (36). On the other hand, 18.9% of neurons in the IMG that innervate the inferior mesenteric artery are NPY-positive (2). In order to identify whether NADPH oxidase is present in vasomotor neurons in CG, double-labeling of p22°"°" and NPY was performed. In the rat CG, all neuron cell bodies examined were immunopositive for NPY and p22phox (Figure 3.5 B). Thus, the presence of both proteins was not limited to a subpopulation of neurons in rat CG. P22p"°" is localized in calcitonin gene-related peptide (CGRP) -positive neurons in DRG. CGRP is a vasodilatory neuropeptide that is released from the sensory nerve fibers (47). The synthesis of the peptide occurs in neuronal cell bodies of the DRG. The intensity of CGRP immunostaining in rat DRG varied depending on the 41 103m Figure 3.5. Immunolocalization of fiTghox and gZZghox in celiac ganglia (CG) neurons. CG were removed from adult SD rats and were fixed, embedded in paraffin and sectioned at 5pm for immunostaining. A) Polyclonal rabbit anti-p47phox (Santa Cruz Biotech) was used with NOVA RED chorrnagen such that p47phox immunoreactivity is shown in red. Images were captured using standard brightfield microscopy. P47phox immunoreactivity was found in nearly all neural cell bodies in CG although the intensity of staining varies across the tissue. This may suggest variable levels of NADPH oxidase expression among neurons in the same ganglia. Scale bar is 30pm in the upper left pannel and 10pm in the rest; B) CG sections were incubated with polyclonal rabbit anti-p22phox (R5554) and polyclonal goat anti-NPY followed by incubation with fluorophore-conjugated sencondary antibodies. Fluorescent images were taken using confocal laser scanning microscopy. Virtually all the neurons that contain NPY immunoreactivity showed positive staining for p22phox as well, indicating the colocalization of these two proteins in the same neurons in rat CG. Scale bar is 100pm in the upper pannel and 30pm in the lower pannel. 42 Figure 3.5 continued Neuropeptide Y Overlay NeuropeptIde Y Overlay 43 size of the neurons (Figure 3.6), in which neurons in smaller sizes showed higher immunoreactivity to CGRP (18). The distribution of p22""°x in DRG was identical to CGRP. Staining for p22phox was also found to be more concentrated in the cytoplasm of the neurons with smaller sizes, while staining was lighter and more diffuse in neurons with larger cell bodies. In addition to neurons, satellite cells that surround the neurons also showed immunoreactivity to both CGRP and p22p“°". These results indicate that the NADPH oxidase subunit p22”"°" and CGRP are colocalized in rat DRG neuronal cell bodies and some non-neuronal cells. P47""°" and p22””°" are present in the neurites of nerve growth factor (NGF)-differentiated PC12 cells and cultured CG Neurons. In order to examine the presence of NADPH oxidase in neuronal compartments outside of the cell bodies, I first did immunostaining of p47p“°" in NGF-differentiated PC12 cells. P47phox was present in PC-12 cell bodies, as well as the neurites that extended from the somata and was colocalized with secretogranin ll (SGII), a large dense core vesicle marker protein (9), on the cell membrane and the neurites (Figure 3.7 A-C). In dissociated CG neurons cultured 7 days, p479“)( and p229” were colocalized with SGII In both cell bodies and neurites (Figure 3.7 D-F and H-J). We also used NPY and tyrosine hydroxylase (TH) to label the nerve fibers in CG cultures. All NPY immunoreactive fibers were 44 “w I “(III I ll Figure 3.6. Immunolocalization of gZZghox ln dorsal root ganglia (DRG). DRG were removed from adult SD rats and were fixed, embedded in paraffin and sectioned at 5pm for immunostaining. DRG sections were incubated with polyclonal rabbit anti-p22phox (R5554) and polyclonal sheep anti-CGRP followed by incubation with fluorophore-conjugated sencondary antibodies. Fluorescent images were taken using confocal laser scanning microscopy. lmmunoreactivities for p22phox and CGRP are localized in the same DRG neurons. both of which are more intense in the neurons with smaller sizes. In addition to neurons, satellite cells surrounding the neurons are also positive for p22phox and CGRP. Scale bar is 100nm in the upper pannel, 30pm in the lower pannel. 45 7h x 947' O Secretogranin II Overlay and cultured celiac ganglia (CG) neurons. For immunostaining assay, PC12 cells were treated with NGF for 7 days to achieve neurite outgrowth. CG was removed from neonatal SD rats and ganglion neurons were freshly dissociated from the ganglia and were kept in culture medium for 7 days. Cells were incubated with polyclonal rabbit anti-p47phox (R360) or anti-p22phox (R5554) and monoclonal anti-secretogranin II, a marker for large dense core vesicles in sympathetic neuronal cell body and nerve endings. P47phox was co-labeled with secretogranin II in both cell bodies and neurites in PC12 cells (A-C) (Scale bar=20pm). P47phox and p22phox were co-localized with secretogranin II in CG neurons (D-F and H-J) (Scale bar=30pm). 46 Figure 3.7 continued Secretogranin ll Overlay 47 Figure 3.7 continued Secretogranin ll Overlay 48 positive for p22phox or p47°“°" (Figure 3.8), while TH immunoreactivity was found in some NADPH oxidase positive fibers but not others (Figure 3.9). The absence of TH in some of the nerve fibers from a sympathetic ganglion neuronal culture is unexpected. It is possible that some ganglia neurons developed non-adrenergic phenotype. For example, cholinergic property can be differentiated in sympathetic ganglia neurons under certain conditions (8). Further studies are needed to identify the TH-negative fibers in the culture. Nevertheless, these results suggest that in addition to cell bodies, NADPH oxidase is also present in nerve fibers. NADPH oxidase subunits, p479” and p22P”°" colocalize to NPY immunoreactive periarterial nerve fibers. In order to determine if NADPH oxidase subunits co-localize to sympathetic nerve fibers and endings, tertiary branches of mesenteric arteries were fixed and labeled with anti-NPY, as a marker for sympathetic perivascular nerves, and either anti-p47p“°" or anti-p22ph°". Upper rows in each panel are low power magnification images showing the meshwork pattern of nerve fibers innervating mesenteric arteries (40). Lower rows are high power magnifications, focusing on a single peri-vascular nerve bundle. P47p"°" and NPY were found in the same nerve fiber bundles (Figure 3.10A). lmmunostaining for p22°“°" was also found in some of the same nerve fibers as NPY, but not all of them. The merged images show that p22""°" and NPY co-Iocalize to some, but not all periarterial nerve fibers. 49 p47 ”h” Neuropeptide Y Overlay Neuropeptide Y Overlay Fi ure 3.8. 7 hox and 22 hox colocalize with neuro e tide Y in nerve fibers in cultured celiac ganglia (CG) neurons. CG were removed from neonatal SD rats and neurons were dissociated and kept in culture medium for 7 days before immunostaining. After fixation, cells were incubated with polyclonal rabbit anti-p47phox (R360) or anti-p22phox (R5552) and polyclonal goat anti-NPY. Most nerve fibers in CG culture showed immunoreactivity for both p47phox or p22phox and NPY. Scale bar = 20pm. 50 ‘ Tyrosine Hydroxylase ‘ Overlay l I l p22ph°" j Tyrosine Hydroxylase Overlay Figure 3.9. g47ghox and g229hox colocalize with tyrosine hydroxylase (TH) in nerve fibers in cultured celiac ganglia (CG) neurons. CG were removed from neonatal SD rats and neurons were dissociated and kept in culture medium for 7 days before immunostaining. After fixation, cells were incubated with polyclonal rabbit anti-p47phox (R360) or anti-p22phox (R5552) and monoclonal anti-TH. In some nerve finbers, NADPH oxidase and TH were found to be colocalized in the same fiber (Arrow a). However, some fibers showed only staining for TH not NADPH oxidase (Arrow b) or NADPH oxidase not TH (Arrow c). Scale bar = 20pm. 51 p47p"°" Neuropeptide Y Overlay p47ph°X Neuropeptide Y Overlay Fi ure 3.10. 7 hox and 22 hox colocalize with neuro e tide Y NPY in eriarterial nerve fibers in mesenteric arteries. Tertiary branches of mesenteric arteries from adult SD rats were fixed and labeled with anti-NPY. as a marker for sympathetic perivascular nerves, and either anti-p47phox (R360) or anti-p22phox (R5554). Upper rows in each panel are low power magnification images showing the meshwork pattern of nerve fibers innervating mesenteric arteries. Lower rows are high power magnifications, focusing on a single peri-vascular nerve bundle. A) P47phox and NPY were found in the same nerve fiber bundles although the localization of the staining within the nerve fiber was variable. NPY staining appears to be more vesicular, while p47phox staining is diffuse throughout the nerve fibers. B) lmmunostaining for p22phox was found in some of the same nerve fibers as NPY, but not all of them. The merged images show that p22phox and NPY co-Iocalize to some, but not all periarterial nerve fibers. Scale bar is 38.1 pm in upper panels, 9.53pm in lower panels. 52 Figure 3.10 continued Neuropeptide Y Overlay Neuropeptide Y Overlay 53 These results indicate that NADPH oxidase subunits are localized to sympathetic periarterial nerve fibers. NAPDH oxidase subunits p479"°" and p229” colocalize to CGRP immunoreactive periarterial nerve fibers. CGRP was used as a marker for sensory nerves on tertiary mesenteric arteries. Figure 3.11 A shows that p47""°x was colocalized with CGRP in the same nerve fibers. Images in Figure 3.11 B shows the localization of p22""°" and CGRP. P22""°x immunostaining co-localized with CGRP positive fibers (yellow), but was also found in non CGRP containing fibers. These fibers may be non-sensory source such as sympathetic nerve fibers. These results indicate that NADPH oxidase subunits are present in sensory periarterial nerve fibers. Discussion In this study, I have shown that NADPH oxidase was expressed in sympathetic and sensory ganglion neuronal somata and perivascular nerve fibers originated from these neurons. 1) NADPH oxidase subunits NOX1, NOX2, p22""°" and p47'Dhox mRNA were expressed in NGF-differentiated PC12 cells and CG and DRG neuron, with the exception that detectable level of NOX4 mRNA was only found in CG but not in PC12 cells or ORG; 2) p22""°" and p47phox expression were 54 p47 p"°‘ Overlay Overlay Figure 3.11. fimhox and Q222hox colocalize with CGRP in geriarterial nerve fibers in mesenteric arteries. Tertiary branches of mesenteric arteries from adult SD rats were fixed and labeled with anti-CGRP, as a marker for sensory perivascular nerves, and either anti-p47phox (R360) or anti-p22phox (R5554). Upper rows in each panel are low power magnification images showing the meshwork pattern of nerve fibers innervating mesenteric arteries. Lower rows are high power magnifications, focusing on a single peri-vascular nerve bundle. A) P47phox and CGRP were found in the same nerve fiber bundles. B) P22phox was found in CGRP immunoreactive nerve fibers. The p22phox positive fibers that lack immunoreactivity for CGRP may be sympathetic nerves. Scale bar is 38.1pm in upper panels. 9.53pm in lower panels. 55 Figure 3.11 continued Overlay Overlay ' associated with neuronal cell bodies in rat IMG, CG and DRG; 3) the protein expression of p22°“°" and p47"“°x were present in NGF-differentiated PC12 cells as well as primary CG and DRG cultured neurons across the cell bodies and the neurites; 4) p22”"°" and p47phox were present in perivascular sympathetic and sensory nerve fibers on mesenteric arteries. This is the first study to systemically evaluate the NADPH oxidase expression in peripheral sympathetic and sensory nervous system. In addition to the verification of the presence of NADPH oxidase in prevertebral sympathetic ganglion neurons and primary sensory neurons, the localization of NADPH oxidase subunits to perivascular nerve fibers innervating the splanchnic circulation is novel, and this may have important implications in blood pressure regulation and hypertension. The innervation of the mesenteric circulation consists of sympathetic neurons in celiac and mesenteric ganglia and sensory DRG neurons, respectively. The axons of the neurons travel to the mesenteric arteries and veins in the paravascular nerves, which divide in the adventitia of the blood vessels to form the perivascular nerve plexus(26; 27; 37). NADPH oxidase was previously shown to be localized to rat superior cervical ganglion (SCG) neurons and was suggested to be involved in regulating neuronal apoptosis (19; 46). However, since there is considerable heterogeneity in the morphological, neurochemical, and electrical properties of neurons from different sympathetic ganglia (21 ), the findings in SCG may not fully represent the case in other types of ganglia like. Therefore, a 57 complete understanding of the localization of NADPH oxidase in prevertebral sympathetic ganglia requires studies on other ganglia including CG and IMG. This study showed for the first time that p22p“°" and p47p“°" were present in rat CG and IMG neurons. The presence of NADPH oxidase in the neurons may provide novel mechanisms underlying the regulation of their activities. Evidence suggests that the neuronal properties, including firing rates and ion channel function, is indeed regulated by 02" in the brain cardiovascular center (45; 51). There is also evidence showing that ROS modulate cellular gene expression (14), this also can be applicable to neurons. For example, we found that NADPH oxidase co-localizes with NPY in nearly all neurons in rat CG. Transcription factors such as activator protein 1 (AP-1) can be induced by R08 (24). Meanwhile, the modulation of NPY gene expression involves the activation of AP-1 signaling (20). Therefore, the coexistence of NADPH oxidase and NPY in the same neurons indicates a possible interaction between these two proteins. Given that NADPH oxidase can be activated by environmental stimulants like angiotensin II (Ang II) (13) or endothelin-1 (ET-1) (29), it is reasonable to hypothesize a possible mechanism by which NPY can be regulated by Angll or ET-1 via NADPH oxidase. On the other hand, sensory neurons located in the DRG revealed positive p22phox staining in the same neurons that were also positive for CGRP, a vasoactive neural peptide found in vasomotor sensory neurons. The colocalization of these two proteins may also indicate their potential interaction in the sensory neurons. 58 Noticeably, neurons have specific features that distinguish them from cells in other tissues, in which the chief functional compartments -the cell body, dendrites, axons, and terminals— are separated by considerable distance. Chemical/peptide transmission occurred at the neuro-vascular junctions directly determines the vascular tone. It is regulated at two levels: the electrical signals sent from the cell bodies and the local handling of neurotransmitters. In addition to its possible effects on neuronal firing as mentioned above, we would also like to know whether 02" can more directly regulate the neurotransmission at the nerve terminal. However, 02" is known to have an extremely short half-life and therefore, the direct actions of 02" in the cell is greatly confined within a certain region that is determined by the subcellular site of its production. It is very unlikely that the 02" produced up in the cell bodies can diffuse to the nerve terminals on the blood vessels. Most studies on the neuronal localization of NADPH oxidase to date have been limited to the cell bodies, with the exception that recent work from Picker’s group showed a dendrite-associated NADPH oxidase staining in rat medial nucleus tractus solitarius (10; 11). We are by far the first group to investigate the localization of neuronal NADPH oxidase outside neural somata in the peripheral. Our novel findings of NADPH oxidase in perivascular sympathetic and sensory nerves suggest a possible role of NADPH oxidase-derived 02" in the regulation of local neurotransmission. 59 The prototypical model of NADPH oxidase found in phagosome suggests that the catalytic core of the enzyme is a membrane-bound structure composed of a NOX subunit and p22p“°". Upon activation, it binds to NADPH and other cytosolic subunits at one side of the membrane while catalyzes the reduction of O2 to 02" to the other side (5). In addition to plasma membrane, several recent studies also suggested the presence of an intracellular vesicular membrane associated NADPH oxidase (30; 39). Based on these findings, we propose two possible locations of NADPH oxidase in the nerve fibers: the plasma membrane and the endosome/synaptic vesicle. The presence of these local NADPH oxidases could potentially have the following physiological significance. Take sympathetic nerve for example (Figure 3.12), it is suggested that nitric oxide (NO) can modulate sympathetic neurotransmission at the junction by reacting with norepinephrine (NE) resulting in its deactivation, thereby act as a protective effect against extra NE release from the sympathetic nerves (25). However, if there is a local O2"-producing site at the nerve terminal plasma membrane, like NADPH oxidase, the bioavailability of NO is then largely determined by the amount of 02“ on site because 02" deactivates NO (16; 43). Therefore, the activity of NADPH oxidase at the sympathetic nerve terminal can potentially have an impact on the amount of NE being released to the neurovascular junction and the downstream vascular tone. In addition to release, sympathetic nerve varicosities are also the sites for the synthesis, storage and reuptake of NE. Once synthesized in the axonal 60 Sympathetic Nerve Varicosity VMAT Tyr —§—>-—>———9 DA'" Blood Vessel Fl ure 3.12. Sch matlc dia ram of ro sed mechanisms for the function of NADPH gxidase at the mrivascular nerve termlngl. The cartoon shows neurovascular junction forms by a single sympathetic varioosity and a blood vessel. The presence of NADPH oxidase at the nerve terminal may be located at the plasma membrane or the membrane of the synaptic vesicles. NADPH oxidsae may have an impact on noerpinephrine handling via three proposed mechanisms: 1) decrease bioavailability of nitric oxide (NO), and thereby limit the deactivation of NE by NO resulting in higher junctional NE level; 2) interfere with the reuptake of NE back to the presynaptic terminal via the modulation of norepinephrine transporter (NET) expression or function; 3) generate 02—- into the synaptic vesicle so as to affect the loading of NE into the vesicle via vesicular monoamine transporter (VMAT). Tyr=tyrosine; DA=dopamine. 61 cytoplasm, dopamine is loaded into synaptic vesicles by vesicular monoamine transporter 2 (VMAT2) and be further catalyzed into NE. After being released, NE is quickly cleared from the neurovascular junction mainly via uptake 1 through norepinephrine transporter (NET) on the plasma membrane and be further reloaded into synaptic vesicles via VMAT2. The function of VMAT2 and NET therefore plays indispensible roles in NE handling in the sympathetic varicosities. Interestingly, there is evidence showing that both VMAT2 and NET may be affected by R08 although the mechanisms underlying these effects have not been elucidated (7; 34). If NADPH oxidase is indeed present at the plasma membrane or synaptic vesicular membrane level at the nerve terminal, it is possible that it can involve in regulating the function/expression of these transporters thereby modulate the NE handling. There are also non-sympathetic sources of NADPH oxidase in the nerve fibers surrounding the mesenteric arteries. The co-immunostaining with CGRP indicated its presence in sensory fibers, which is consistent with its staining in DRG neurons. Although the role of sensory nerves in blood pressure regulation is less defined as it is in sympathetic nerves, accumulating evidence has shown that sensory nerves play a counter-balancing role in preventing increases in blood pressure particularly in salt-induced hypertension (50), and there may be a ROS-associated mechanism as suggested by some recent studies including ours (3; 44). The presence of NADPH oxidase in the perivascular sensory fibers now 62 further provides insight into the role of locally produced 02". NO also was shown to modulate sensory neurotransmission at the neurovascular junction (42). Therefore, the mechanism we proposed above for the role of 02" in sympathetic neurotransmission may also apply to sensory nerves. Noticeably, the staining of NADPH oxidase was not yet limited to sympathetic and sensory nerve fibers. After both of them were depleted from the rats by celiac ganglionectomy (CGx), there was still substantial amount of remaining NADPH oxidase present in some nerve fibers (data not shown). These could either be some CGRP negative sensory fibers innervating the blood vessel without passing through the prevertebral ganglia which therefore cannot be abolished by CGx, or intestinofugal fibers which originate in the myenteric plexus, and terminate in prevertebral ganglia (31). More studies are needed to further identify the sources of these nerve fibers. In summary, we have demonstrated that NADPH oxidase is expressed in sympathetic and sensory neurons as well as their perivascular nerve fibers. The findings of localized 02" production at both the neuronal cell bodies and the prejunctional nerve terminals on the blood vessels are of great importance. 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Wolf HK, Buslei R, Schmidt-Kastner R, Schmidt-Kastner PK, Pietsch T, Wiestler OD and Blumcke I. NeuN: a useful neuronal marker for diagnostic histopathology. J Histochem Cytochem 44: 1167-1171, 1996. 54. Xu GY, Liu S, Hughes MG and McAdoo DJ. Glutamate-induced losses of oligodendrocytes and neurons and activation of caspase-3 in the rat spinal cord. Neuroscience 2008. 55. Yi XY, Li VX, Zhang F, YI F, Matson DR, Jiang MT and Li PL. Characteristics and actions of NAD(P)H oxidase on the sarcoplasmic reticulum of coronary artery smooth muscle. Am J Physiol Heart Circ Physiol 290: H1136-H1144, 2006. 70 CHAPTER FOUR Xian Cao; Xiaoling Dai; Lindsay M. Parker; David L. Kreulen. (2007). Differential Regulation of NADPH Oxidase in Sympathetic and Sensory Ganglia in Deoxycorticosterone Acetate — Salt Hypertension. Hypertension. 2007 Oct; 50(4):663-71 71 CHAPTER 4: DIFFERENTIAL REGULATION OF NADPH OXIDASE IN SYMPATHETIC AND SENSORY GANGLIA IN DOCA-SALT HYPERTENSION Abstract We demonstrated recently that superoxide anion levels are elevated in prevertebral sympathetic ganglia of deoxycorticosterone acetate—salt hypertensive rats and that this superoxide anion is generated by reduced nicotinamideadenine dinucleotide phosphate oxidase. In this study we compared the reduced nicotinamide-adenine dinucleotide phosphate oxidase enzyme system of dorsal root ganglion (DRG) and sympathetic celiac ganglion (CG) and its regulation in hypertension. The reduced nicotinamide-adenine dinucleotide phosphate oxidase activity of ganglion extracts was measured using fluorescence spectrometry of dihydroethidine; the activity in hypertensive dorsal root ganglion was 34% lower than in normotensive DRG. In contrast, activity was 79% higher in hypertensive CG than normotensive CG. mRNA for the oxidase subunits NOX1, NOX2, NOX4, p47°"°*, and p229“x were present in both CG and DRG; mRNA for NOX4 was significantly higher in CG than in DRG. The levels of mRNA and protein expression of the membrane-bound catalytic subunit p22”"°" and of the regulatory subunits p47phox and Rac-1 were measured in CG and DRG in normotensive and hypertensive rats. P22”"°"mRNA and protein expression was 72 greater in CG of hypertensive rats but not in DRG. Compared with normotensive controls, p47°“°" mRNA and protein, as well as Rec-1 protein, were significantly decreased in hypertensive DRG but not in CG. lmmunohistochemical staining of p47”"°" showed translocation from cytoplasm to membrane in hypertensive CG but not in hypertensive DRG. This suggests that reduced nicotinamide-adenine dinucleotide phosphate oxidase activation in sympathetic neurons and sensory neurons is regulated in opposite directions in hypertension. This differential regulation may contribute to unbalanced vasomotor control and enhanced vasoconstriction in the splanchnic circulation. Introduction The splanchnic circulation is of great importance in regulating systemic blood pressure. It receives approximately 60% of the cardiac output and contains about one third of the total blood volume (26). The splanchnic circulation is innervated by both the sympathetic division of the autonomic nervous system (prevertebral sympathetic ganglion neurons, including celiac ganglia [CGs], superior and inferior mesenteric ganglia) and by spinal sensory nerves (dorsal root ganglia neurons [DRGs]). Elevated sympathetic nervous system activation has been shown in. various types of hypertension (20; 36). In particular, sympathetic ganglionic blockade can reduce the arterial blood pressure increase in 73 deoxycorticosterone acetate (DOCA) -salt hypertension (16), indicating an important role of sympathetic ganglia in the development and maintenance of salt-induced hypertension. On the other hand, sensory nerves play a counter-regulatory role in preventing increases in blood pressure through either afferent baroreceptor-mediated mechanisms (8) or efferent release of vasodilatory neuropeptides, such as calcitonin gene—related peptide (CGRP) and substance P (SP) (15) (18). Altered synthesis or release of these vasodilator neuropeptides occurs in genetic and experimental hypertensive animal models (24; 40; 44). Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase is an enzyme that catalyzes the production of superoxide anion (02") from oxygen and NADPH, and is considered the predominant source of 02" in hypertension (45). It is a complex enzyme consisting of 2 membrane-bound components (p22°“°" and NOX) and 3 components in the cytosol (p47p"°" or NOXA1, p67phox or NOXO1 and p40"“°"), plus a GTPase (Rec-1 or Rac-2)(2). Activation of NADPH oxidase involves the translocation of regulatory elements from the cytoplasm to combine with catalytic subunits in the membrane (10). NADPH oxidase was first identified in phagocytes (4). It plays a vital role in nonspecific host defense against pathogens by generating large (millimolar) quantities of 02'“ during the respiratory burst (41). More recently, the presence of NADPH oxidase in non-phagocyte cell types has been well identified. This is especially true in cardiovascular system 74 related tissues such as vascular endothelium (23), vascular smooth muscle (27), kidney cortex (7), and nervous system (25). Unlike in neutrophils, the NADPH oxidase in these tissues makes 02" in small amounts for purposes of signaling under physiological conditions (22). However, excessive amounts of 02" production leads to a variety of intracellular signaling events that ultimately may cause dysfunction of the system (35).This brings more attention to the pathophysiological role of this enzyme system in the regulation of cardiovascular diseases, such as hypertension. Elevated NADPH oxidase-derived 02" production in the vasculature (6) and the sympathetic neurons (13), accompanied by enhanced endothelin-1 (ET-1) signaling (28) and increased sympathetic system activity (16) are characteristic of DOCA-salt hypertension. Studies using this hypertensive animal model have shown that elevated arterial ET-1 levels lead to enhanced vascular 02" production via the endothelinA (ETA) receptor/NADPH oxidase pathway (29), while in prevertebral sympathetic ganglia, 02" levels are increased due to enhanced activation of the ETB/NADPH oxidase pathway (13). Although sensory neurons are known to participate in innervating the vasculature, the regulation of NADPH oxidase activity in sensory neurons has not been investigated in DOCA-salt hypertension. Possible differential regulation of 02" in sympathetic and sensory ganglion neurons in cardiovascular diseases has been shown in apolipoprotein E 75 deficient mice, in which the levels of 02" is increased in sympathetic ganglia neurons but not in nodose sensory neurons (31 ). In this study, we measured the 02" levels and the expression of NADPH oxidase subunits in sympathetic ganglia (CG) and sensory ganglia (DRG) and compared the expression levels in both normotensive and hypertensive conditions. We tested the hypothesis that NADPH oxidase is regulated differentially in sympathetic and sensory ganglia in DOCA-salt hypertension, in which the enzyme system is upregulated in CG but not in DRG. Methods Animals All animal experiments were performed in accordance with the “Guide for the Care and Usage of Laboratory Animals” (National Research Council) and were approved by the Animal Use and Care Committee of Michigan State University. Adult male Sprague Dawley rats (250-300 9; Charles River Laboratories, Inc., Portage, MI) underwent uninephrectomy and subcutaneous implantation of DOCA (200 mg kg") under isofiurane anesthesia. Post-operatively, the rats were given drinking water containing 1% NaCI and 0.2%KCI (herein, the DOCA-salt treated group is referred to as hypertensive (HT)). Normotensive controls (NT) to the hypertensive rats were uninephrectomized but were not given DOCA implantation 76 or salt drink. Four weeks after surgery, the arterial blood pressure was measured using the tail cuff method. Rats with a mean systolic arterial pressure of > 150 mmHg were considered hypertensive (30). The mean systolic arterial pressure for the HT rats and NT rats were 206.3:506 mmHg and 119.7135 mmHg. respectively (p<0.05; n=20 in each group). Tissue Harvest Rats were sacrificed with a lethal dose of sodium pentobarbital (65 mg/kg ip); and the CG and DRG (spinal levels T13-L2) from HT and NT rats were removed and cleaned for further processing. Measurement of NADPH Oxidase Activity Activity of NADPH oxidase was measured using fluorescence spectrometry of dihydroethidine (DHE) in tissue homogenates of DRG and CG from NT rats and HT rats. DHE is oxidized to fluorescent ethidium by 02". Ethidium will intercalate with DNA to further amplify the fluorescent signal and The intensity of the fluorescent signal is proportional to 02" levels (5; 51).In a microtiter plate, freshly prepared DRG homogenates were incubated with DHE (10pmol/L), salmon testes DNA (0.5mg/mL, Stratagene, La Jolla, CA) and the substrate for NADPH oxidase, B-NADPH (0.1mmoI/L, Sigma, St. Louis, M0), for 30 minutes at 37°C in a dark chamber. Salmon testes DNA was added to bind to ethidium and consequently 77 stabilize ethidium fluorescence, thereby increasing the sensitivity of 02" measurement >40-fold (51). A parallel control group was analyzed in each run with no substrate added into the reaction. Ethidium-DNA fluorescence was measured at an excitation of 4853:40nm and an emission of 590135nm using a Biotek FL600 fluorescence plate reader (Bio-Tek Instruments, Inc., Winooski, VT). The enzyme activity was measured as total fluorescence units per minute per milligram tissue homogenate. Before statistical analysis, to eliminate the background fluorescence the no-substrate control readings were subtracted from the fluorescence readings of the wells with substrate. . NT rat ganglia were normalized to 100% in both CG and DRG independently. Experimental results are presented as the percent changes of fluorescence from NT to HT. Reverse Transcription - Polymerase Chain Reaction (RT-PCR) and Quantitative Real-time RT-PCR (qPCR) Fresh CG and DRG harvested from NT and HT rats were immediately placed in RNA/ater RNA stabilization Reagent (Qiagen, Valencia, CA). Total RNA was isolated from the ganglia using RNeasy Mini kit (Qiagen). cDNA was synthesized using Superscript II mix (Invitrogen, Carlsbad, CA). The cDNA synthesized from 2ug or 50ng total RNA was used in subsequent PCR or qPCR, respectively. All primers were derived from the Rattus Norvegicus gene (National Center for Biotechnology Information GenBank). Primer sequences are shown in Table 4.1. 78 Table 4.1 Primer sequences for NADPH oxidase subunits NOX1,NOX2, NOX4, p47phox and p22phox, B-actin and GAPDH Gene Sequence Amplicon NCBI accession Length (bp) Number NOX1 For:5' TGAACAACAGCACTCACCAATGCC 3' 245 AF152963 Rev:5' AGTTGTTGAACCAGGCAAAGGCAC 3’ NOX2 For:5' GTGGAGTGGTGTGTGAATGC 3' 324 AF298656 Rev:5' TCCACGTACAATTCGCTCAG 3' NOX4 For:5' ACCAGATGTTGGGCCTAGGATI'GT 3' 261 AY027537 Rev:5' AGTTCACTGAGAAGTTCAGGGCGT 3' NOX4 For:5' TCATGGATCTTTGCCTGGAGGGTT 3' 110 (qPCR) Rev:5' AGGTCTGTGGGAAATGAGCTTGGA 3' p47phox For:5’ GGCCAAAGATGGCAAGAATA 3' 221 AF260779 Rev:5’ TGTCAAGGGGCTCCAAATAG 3' p47phox For: 5’ AGGTTGGGTCCCTGCATCCTATTT 3’ 95 (qPCR) Rev: 5' TGGTTACATACGGTTCACCTGCGT 3' p22phox For:5' TTGTTGCAGGAGTGCTCATC 3' 282 U18729 Rev:5' TAGGCTCAATGGGAGTCCAC 3' p22phox For: 5' TGTTGCAGGAGTGCTCATCTGTCT 3' 150 (qPCR) Rev: 5’ AGGACAGCCCGGACGTAGTAAITI’ 3’ B-actin For:5' GGCTACAGCTTCACCACCAC 3' 500 vo1217 Rev:5' TACTCCTGCTTGCTGATCCAC 3' GAPDH For:5' ATCACTGCCACTCAGAAG 3' 317 NM017008 (qPCR) Rev:5' AAGTCACAGGAGACAACC 3' 79 PCR products were electrophoresed on a 2.0% agarose gel for 60 minutes at 9V/cm gel. Bands corresponding to PCR amplicons were stained by ethidium bromide and visualized by UV light. qPCR was performed using Mx3000P QPCR system (Stratagene). SYBR green was used as the fluorescence detector in the qPCR. Serial dilution was performed for each set of qPCR primers to determine its qPCR amplification efficiency (E) before the experimental run. A dissociation protocol (60-95 °C melt) was done at each end of the experiment to verify that only one amplicon was formed during the process of amplification. End point, used in qPCR quantification and Ct value, is defined as the PCR cycle number that crosses an arbitrarily placed signal threshold. The relative expression ratio of the target gene was calculated, based on its E and Ct difference (A) of sample versus control (ACtmmm. -samp.e) (Equation 1). Statistical analysis was performed by Pair Wise Fixed Reallocation Randomization Test© mtth/wwwgene-quantification.info) using Relative Expression Software Tool (REST) (34). . . _ ACt ACt EXPTGSSIOH Rat'o ' (Etarget) target(control — sample/(EGAP) GAP (control - sample) "‘ 1 Protein Isolation and Subcellular Fractionation CG and DRG homogenates were extracted on ice with lysis buffer (10mM HEPES, 150mM NaCI, 1mM EDTA, 0.5% Triton X-100, protease inhibitor cocktail (1:100, Sigma, St. Louis, MO) ). After homogenization the tissue lysates were 80 quickly centrifuged at 700xg for 5 min 4°C to pellet nucleic protein and any insoluble debris. The supernatant was saved for measurement of total protein. For subcellular fraction of CG protein, tissue was first harvested and homogenized in ice cold lysis buffer without Triton X-100. Tissue homogenates were then first centrifuged as described above, after which the supernatant were furthered centrifuged at 100,000 xg for 60 min at 4°C. The resulting supernatant was saved as cytosolic protein. The pellet was resuspended in lysis buffer and saved as plasma membrane rich protein. Protein quantification was performed using Bradford protein assay (Bio Rad Laboratories, Hercules, CA). Western Blotting Equal amounts of protein were separated by 7.5%-15% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane. After 2hrs of blocking in Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20, membranes were incubated overnight at 4°C with specific primary antibodies: p22”“°"(1:500, Santa Cruz Biotechnology, Santa Cruz, CA), p47p“°" (1 :1000, Santa Cruz) and Rec-1 (1:1000, Upstate, Charlottesville, VA). Following incubation in primary antibody, blots were washed and exposed to the HRP-linked secondary antibody for 1 hour at 4°C with rocking (anti-rabbit lgG, 1:2000; anti-mouse lgG, 1:5000; Santa Cruz). Finally, blots were washed and chemiluminescent detection of bands was performed (Pierce, Rockford, IL). To control for variation in protein 81 loading, membranes were stripped and reprobed with anti-d-actin (121000; Sigma) or with anti-Pan-Cadherin (1 :1000, Zymed Laboratories Inc, South San Francisco, CA), a plasma membrane protein marker. CG subcellular fraction western blot was performed on one membrane with protein from 5 ganglia. Scanned films were analyzed for band density and area using NIH Image software. lmmunohistochemistry Ganglia were dissected and fixed in 10% formalin for 2 hours then transferred to 70% ethanol for storage ranging from several hours to overnight. Tissue was processed using a vacuum infiltration tissue processor (Thermo Electron Excelsior) with decreasing concentrations of ethanol followed by xylene. Tissues were embedded in paraffin, sectioned on a rotary microtome into 5 pm sections, and mounted on to glass slides (Corning Glass). Heat induced epitope retrieval (HIER) was used. Samples were blocked for endogenous elements with hydrogen peroxide/methanol for 30 minutes then rinsed. Due to the use of HIER, an additional blocking step of avidin and biotin was used with a 15 minute incubation. Normal goat serum (1 :28, Vector Laboratories, Burlingame, CA) was used as a protein block followed by incubation in p47""°x primary antibody (1 :150, Santa Cruz) for 60-minutes. Incubation of biotinylated goat-anti-rabbit secondary antibody(12200, Vector Laboratories) for 30 minutes was followed by a 15 minute incubation with Nova Red chromagen (Vector Laboratories). Slides were 82 counterstained with Lerner 2 hematoxylin then dehydrated. Images were collected using standard brightfield microscopy (Olympus BX60 with SPOT Insight Digital Camera, Olympus America Inc. Center Valley, PA). A “no primary control” was run in parallel without addition of primary antibody to assess antibody specificity. Images were analyzed for staining density and area using NIH Image software. Data Analysis Data are presented as meantSE of the mean. Statistical significance of NADPH oxidase activity, western blotting, lmmunohistochemistry data were assessed by Student ttest using Prism 4.0 software (GraphPad Software, San Diego, CA). qPCR data statistical significance were assessed by Pair Wise Fixed Reallocation Randomization Test© using REST software. In both cases, p<0.05 indicates statistical significance. Results Expression of NADPH oxidase mRNA in normal rat DRG and CG PCR amplicons of NADPH oxidase subunits p47p“°", p22P“°*, NOX1, NOX2 and NOX4 were detected in RNA extracts of DRG and CG from normal rats that did not receive DOCA-salt treatments (Figure 4.1A). These amplicons were at the 83 A .. . DRG 500bp-—> - - 400bp—> w... 300bp—-> , - - zoom—p m '- NOX1 NOX4 l p22ph°xT NTC 1 Ladder h NOX2 p47p °" B-actin No RT — CG 500bp —> i— . 400bp —> --- 300bp _> - - 200bp —> ....., NOX1 NOX4 I p2zph°xt NTC 1 Ladder NOX2 p47ph°x [iv-actin No RT Figure 4.1. NADPH oxidase subunits are expressed in dorsal root ganglia (DRG) and celiac ganglia (CG). A) PCR amplicons for NOX1, NOX2, p47phox, p22phox and B-actin were present on ethidium bromide-stained agarose gels from DRG (top) and CG (bottom). PCR step with no cDNA template added (NTC) and reverse transcription step without adding the transcriptase enzyme (No RT) were performed as negative controls. B) qPCR results show NOX4 mRNA level is significantly lower in DRG then in CG in normal rats. The expression ratio of NOX4 in DRG vs. CG is 0.077 (n=7 normal rats). Relative expression value calculation and statistical analysis were performed by REST software. The randomisation test output from REST is listed in table format (bottom) attached to the bar graph. The significance (P<0.05) is indicated by " vs. CG. 84 Figure 4.1 continued CD § 9 N 7' .° N L Relative Expression Value (Normalized to GAPDH) O § NOX4 mRNA VIII/[Ill] DRG Pair Wise Fixed Reallocation Randomisation Test © Output Genes PCR Efficiencies Control (CG) Ct Means Sample (DRG) Ct Means Expression Ratio p-Value Randomisations GAPDH NOX4 1.983 2.043 18.926 30.239 17.830 32.786 0.077 0.001 2000 of 2000 done 85 expected sizes of 221bp, 282bp, 324bp, 245bp and 261bp, respectively. PCR products from CG ganglia were consistent with our previous findings in dissociated CG neurons (12). However, DRG NOX4 mRNA was barely detectable on the regular PCR gel compared to ECG. qPCR was then performed to determine the relative expression levels of NOX4 in normal DRG and CG. Results showed that the expression ratio of NOX4 in DRG and CG was 0.077 (Figure 4.1 B) (p<0.05 vs CG; n=7). NADPH oxidase activity in DRGs and CGs in NT and HT Rats Tissue homogenates of DRGs and CGs from NT and HT animals were incubated with the NADPH oxidase substrate B-NADPH, and the formation of 02" was detected in the reaction mixture. The NADPH oxidase activity of DRG homogenates from HT rats was 34% lower than the activity of homogenates from NT animals (Figure 4.2; P<0.05 versus NT; n=3). This result demonstrates that the NADPH oxidase enzymatic activity in tissue homogenates of HT DRGs is less than this activity in NT DRGs. Meanwhile, the NADPH oxidase activity in HT 063 is 78.6% higher than NT CGs (P<0.05 versus NT; n=6) (12). 86 g DRG 2 0 53 83150. 5. gawo- __ '5> 34‘ E35. 50- E .2 ‘D -- c g t it": NT g E 1? C . NT HT Ethidium Fluorescence Increase, ('56) Vs control $ Figure 4.2: NADPH oxidase actile in DRG and CG. NADPH oxidase activity is lower in DRG from DOCA-salt hypertensive (HT) rats than from normotensive (NT) control rats, but is higher in HT CG than NT CG. B-NADPH was used as NADPH oxidase‘substrate. Results represent the percent changes of dihydroethidine (DHE) fluorescence intensity in the ganglia homogenate from no-substrate controls to substrate-treated groups in both HT and NT rats. The NADPH oxidase activity of DRG homogenates from HT rats was 34% lower than from NT animals (n=3) (top); meanwhile, the NADPH oxidase in HT CG is 78.6% higher than NT CG (n=6) (bottom). The significance (P<0.05) is indicated by * vs. NT. 87 NADPH Oxidase Subunit mRNA Levels in DRGs and 063 in NT and HT Rats P22""°" and p47°“°" mRNA were both present in DRGs and 065 as shown above, and these subunits aregcritical in mediating the NADPH oxidase enzyme activity (1; 2) . We, therefore, compared the levels of p22phox and p47p“°" mRNA in RNA extracts of DRGs and CGs from NT and HT rats using qPCR. The mRNA level of p22""°x in CG was significantly greater in HT animals compared with NT by the factor 1.776 (P<0.05 versus NT rats; n=7 NT rats; n=6 HT rats), whereas its level was unchanged in DRG (Figure 4.3A and B). On the other hand, p47p“°" mRNA was significantly lower in HT DRGs compared with NT DRGs. The relative expression ratio of p47°“°" mRNA in HT DRGs to NT DRGs is 0.379 (P<0.05 versus NT rats; n=7 NT rats; n=5 HT rats), whereas there was no significant difference between p47p“°" mRNA in NT C68 and HT CGs (Figure 4.30 and D). NADPH Oxidase Subunit Protein Expression Levels in C63 and DRGs in NT and HT Rats In addition to p22°“°" and p47p“°", we also measured Rec-1 protein expression levels in the ganglia in NT and HT rats, because the protein expression of this regulatory factor has been associated with NADPH oxidase activity in the nervous system (49). The protein expression of p22p"°", p47p"°", and Rae-1 in CGs and DRGs was examined by Western blotting analysis. The 88 A DRG p22|"""x ‘1' '? p ‘1' Relative Expression Ratio (Normalized to GAPDH) P o .i NT HT Pair Wise Fixed Reallocation Randomisation Test © Output Genes GAPDH p22p"°°‘ PCR Efficiencies 1.983 2.107 Control (NT) Ct Means 17.711 22.014 Sample (HT) Ct Means 17.600 21.896 Expression Ratio 1.012 p-Value 0.955 Randomisations 2000 of 2000 done Figure 4.3: gZZghox mRNA level Is higher In CG In HT animals comgare to NT controls, and fiTghox mRNA is lower in HT DRG than In NT DRG. Panel A and B show the mRNA level of p22phox in CG was significantly greater in HT animals compared with NT by the factor 1.776 (n=7 NT rats, n=6 HT rats), while its level was unchanged in DRG. Panel C and D show p47phox mRNA was significantly lower in HT DRG compared to NT DRG. The relative expression ratio of p47phox mRNA in HT DRG to NT DRG is 0.379 (n=7 NT rats, n=5 HT rats), while there was no significant difference between p47phox mRNA in NT CG and HT CG. All qPCR data is normalized to GAPDH. Results are shown in table form from REST software analysis output (bottom), and in graphical form (top). The significance (P<0.05) is indicated by *vs. NT. 89 Figure 4.3 continued B cc pzzphm‘ go T go ‘3 ‘1' ? p ‘1' Relative Expression Ratio (Normalized to GAPDH) * P O «i NT HT I Pair Wise Fixed Reallocation Randomisation Test 69 Output Genes GAPDH p22""°x PCR Efficiencies 1.983 2.107 Control (NT) Ct Means 21.044 23.697 Sample (HT) Ct Means 22.200 23.988 Expression Ratio 1.776 I p-Value 0.028 I Randomisations 2000 of 2000 done 90 Figure 4.3 continued C DRG p479“ g A 1.00- .5 g 0.75- i : .... a 3 "' o i. a . NT Pair Wise Fixed Reallocation Randomisation Test © Output Genes GAPDH p47°“°°‘ PCR Efficiencies 1.983 2.038 Control (NT) Ct Means 18.559 27.264 Sample (HT) Ct Means 18.554 28.622 Expression Ratio 0.379 p-Value 0.048 Randomisations 2000 of 2000 done 91 Figure 4.3 continued D cc p479“x .2 .. 20- aé ,, C 1. .2 3 § 2 .. 1 o- 3.3; f‘z’ E 0.54 ‘5 3 '6 z. ‘1 o.c . NT Pair Wise Fixed Reallocation Randomisation Test (6) Output Genes GAPDH p47""°x PCR Efficiencies 1.983 2.038 Control (NT) Ct Means 21.434 28.100 Sample (HT) Ct Means 22.765 29.083 Expression Ratio 1.235 p-Value 0.546 Randomisations 2000 of 2000 done 92 expression of p22"“°" was greater in HT CGs than in NT CGs (P<0.05 versus NT rats; n=6), and this paralleled its greater mRNA levels shown above. Similar to its unchanged mRNA levels in DRGs, the p22”"°" protein expression was not significantly different between NT and HT rats (Figure 4.4A). In 065, there was no difference between NT and HT rats in the amounts of p47""°x and Rac-1 in total protein fractions. Meanwhile, HT DRGs showed a different pattern in the expression of these 2 subunits. There was a profound downregulation of p47"hox (P<0.05 versus NT rats; n=4; Figure 4.43), as well as a significant decrease in Rec-1 expression (P<0.05 versus NT rats; n=3; Figure 4.40) in total protein preparation. We also analyzed the p47""°x expression in CGs and DRGs with immunohistochemistry. In both CGs and DRGs there was intense staining associated with the neuron cell bodies with little or no staining of intercellular elements. Compared with NT CGs, there was a significant redistribution of p47phox to the plasma membrane of neurons in the HT CGs (P<0.05 versus NT rats; n=7 neurons in NT CGs; n=14 neurons in HT CGs; Figure 4.5A and B). We observed a similar redistribution pattern in Western blotting of CG subcellular fractions; in HT CGs there was lower expression of p47°“°" in cytosolic fractions accompanied by greater p47""°x expression in membrane fractions (Figure 4.50). On the other hand, immunohistochemical staining of DRGs showed that the total 93 A :ggion- DRG :5 E :5 7.5- 3:3 5.0- 8§ 5'; u- §~ M, l ' l “ NT p22phox . -‘ ‘ v.3. ci-actin-—> - - EE‘M- CG g? 7.5- a 3:8 £3 5"” QE 2.5- %5 o.c [ . 1 NT p22 phOL’ dank-"=4" - oc-actin—> «was: .m. Figure 4.4: Western blot data from ganglia homggenate reveals that pZthox, gTQhox and Rac-1 are present In DRG and CG, and are differentially regulated in HT and NT animals. Panel A shows that there is no significant difference in the amount of p22phox protein expression in NT DRG versus HT DRG (top), while it is higher in HT CG than in NT CG (bottom) (n=6). Representative blots are shown below each figure. Panel B shows p47phox protein is significantly decreased in HT DRG compare to NT DRG (n=4) but is not different between NT and HT CG. Panel C shows the protein expression of Rec-1 is lower in HT DRG than in NT DRG (n=3), and there is no significant difference between Rec-1 protein levels in NT and HT CG. All data are normalized to B-actin before statistical analysis. The significance (P<0.05) is indicated by *vs. NT. 94 Figure 4.4 continued DRG _l ms m. s o 1 1 o o .ezors 8 82.2.32. :5 558.28 cas§< B p47ph°-°-‘—> N- a a a menus.» 1 1 o o .5898 2 3.38.62. :5... 3553.230 E9532 m 7 M a-actin—> - - 95 Figure 4.4 continued H m G D C III! t—ll T N N rlll hmsose»» eases... » 2 z 1 1 o o 2 2 1 1 o o .58.: 8 63.3562. 4 cm .588 3 233.5%. .m “=5 Euchzccco >333: W m 2.5 E85263 2232 .0. R w W C 96 Figure 4.5 fi7phox protein Is redistributed In the CG neprong in HT animals compared to NT controls. A) lmmunohistochemistry reveals p47phox protein localization in CG neurons (shown in red). Left three panels are CG from NT rats and right three panels are HT CG (top to bottom: 20x magnification, 100x oil objective, no primary antibody controls). Representative images show p47phox protein is present in most cells within the ganglia. The membrane localization of p47phox is significantly higher in HT CG than in NT CG (arrow), indicating a translocation of this protein from the cytoplasm to the plasma membrane. Scale bars are 50pm. Plots of the density measurements are shown in panel B): In HT CG there was a significant increase in plasma membrane density (*p<0.05 vs NT; n=7 neurons in NT CG, n=14 neurons in HT CG) , but no change in total (plasma membrane + cytoplasm) staining density; C) Western blot performed on CG subcellular fraction protein shows that in cytosol fraction, NT CG has higher p47phox protein expression than HT CG. However, in membrane-rich fraction, HT CG has more p47phox than NT CG. Membranes are stripped and re-probed with Pan-cadherin, a plasma membrane marker, indicating membrane-rich protein preparation (n=5, pooled ganglia). 97 98 Figure 4.5 continued B Plasma Membrane p47phox 90- * so- 704 604 50- 40.. _—_. 30- 20- 10- Arbitrary Intensity Units Total p47ph°x 60- 50- 4o- 30- 20- 10-I Arbitrary Intensity Units NT 99 Figure 4.5 continued C M i" ‘ 7* 'Q ‘ -' VT'"‘~""‘!‘ -. P47ph°"-) i- - m-I-I M” ! Pan-Cadherin —) ~ ~ NT HT NT HT Cytosol Membrane 100 p47""°x staining was decreased in HT DRGs as compared with NT DRGs (P<0.05 versus NT rats; n=61 neurons in NT DRGs; n=91 neurons in HT DRGs; Figure 4.6). This is consistent with our Western blotting data in which there was decreased p47phox protein expression in HT DRGs (see Figure 4.4). However, there was no p47phox redistribution from cytosol to membrane in HT DRGs. This suggests that the translocation of p47p"°" from the cytoplasm to the plasma membrane may contribute to the elevated NADPH oxidase activity in HT CGs, whereas in HT DRGs the lack of this translocation, as well as the decreased expression of total p47°"°" protein, could contribute to the lower activity level of the enzyme. Discussion In this study, we have shown for the first time that, in DOCA-salt hypertension, NADPH oxidase— derived reactive oxygen species production is regulated in opposite directions in sympathetic ganglion neurons and in primary sensory neurons. Whereas 02“ production and NADPH oxidase activity are increased in sympathetic ganglia in HT rats (12; 13), they are decreased in DRGs. The expression of NOX4 is much higher in CGs than in DRGs. Furthermore, p22°“°" is increased in HT CGs, whereas p47°“°" and Rec-1 are 101 In. ‘ 1‘ I. ‘ 1 J u' 'I ~' .9 " , § 3 e a ‘ , ’ ~ I.» b v e ‘- . Q ’5. I 3‘“ [I Q- a I . r {it Nf - i" ‘l _’ HT y j 50 um ‘ 50 W“ .l . "3 . ‘1 NT 5‘. . . , . .5097! ”T sb'E'm r - . HT , NT -— 5am 50 pm «,. ,a Figure 4.6: lmmunohistochemistgy reveals fiTphox localization in DRG neurons. A)Left three panels are DRG sections from NT rats and right three panels are from HT rats (top to bottom: 20x magnification, 100x oil objective, no primary antibody controls). The micrographs show p47phox staining (red) throughout the cytoplasm of the ganglion cell bodies with limited membrane localization in both NT and HT DRG. Scale bars are 50pm. Plots of the density measurements are shown in panel B): The total amount of p47phox is significantly lower in HT DRG than in NT. (*p<0.05 vs NT; n=61 neurons in NT DRG, n=91 neurons in HT DRG). 102 Figure 4.6 continued B Plasma Membrane p47ph°X £50- _l_ 540- E 230- 3 520- Z‘ a .510- E < c . NT DRGp47ph°x 350- E r;- :40- E." 230- 3 520- E' 2 :10- E < c NT Total HT Total 103 decreased in HT DRGs. Finally, p47ph°" is translocated from the cytoplasm to the plasma membrane in HT CGs but not in DRGs. NADPH oxidase activity can be determined by 2 major factors: the capability of different NOX isoforms to catalyze electron transfer reactions and/or the availability of the cytosolic regulatory subunits. The expression pattern and level of the core protein, as well as the regulatory subunits, can affect the enzyme activity level. First, the differential regulation of NADPH oxidase activity in CGs and DRGs in hypertension may be because of their differences in the expression of NOX isoforms. The formation of the catalytic core of NADPH oxidase between either one of the NOX isoforms and p22""°" is essential for the production of 02" (39). However, whereas the activation of catalytic complexes made with NOX1/NOX2 and p22""°" requires the addition of cytosolic regulatory subunits, such as p47phox or the GTPase Rac (3), NOX4-p22p“°" produces 02“ constitutively without combining with other subunits (32). Different from the other 2 isoforms, the expression of NOX4 is much higher in CGs than in DRGs. It is then conceivable that, because a large part of the oxidase in CGs where NOX4 expression is high contains only NOX4-p22°h°", the p22""°" increase that we observed in HT CGs may be responsible for the elevated oxidase activity even if the expression levels of regulatory subunits p47IDhox and Rae-1 were unchanged. Second, differences in the availability of regulatory subunits can affect NADPH oxidase activity. For example, in NOX1- or NOX2-based NADPH 104 oxidase, 02" generation is regulated by the concentration of p47p“°" and Rec-1 (9; 11; 14),and inhibition of p47°“°" or Rac-1 expression can result in a decrease in 02“ production (38; 49). Therefore, the decreased expression of p47°"°" and Rec-1 in HT DRGs, where the NOX1 and NOX2 dominate, is likely to result in lower oxidase activity. Moreover, NADPH oxidase activation involves the translocation of regulatory subunits from the cytoplasm to combine with catalytic core in the membrane. The redistribution of regulatory subunits can be another indicator for oxidase activity level. There is increased membrane-bound p47phox in HT CG but not in HT DRG, indicating that the translocation of p47p“°" may contribute to enhanced oxidase activity in HT CG, whereas the lack of this translocation accompanied by decreased total p47p“°" expression may explain the attenuated oxidase activity in HT DRG. In hypertension, enhanced NADPH oxidase activity and expression occur in various tissue types, including vasculature (29; 37), kidney (7), and the nervous system (12; 50).There is a positive correlation between reactive oxygen species levels in the nervous system and sympathetic neuronal activity in hypertension. For example, removal of extracellular 02" or reactive nitrogen species within the rostral ventrolateral medulla by microinjection of superoxide dismutase reduces sympathetic nervous system activity in animals subjected to oxidative stress (46); also, intravenous administration of the superoxide dismutase mimetic Tempol lowers mean blood pressure and renal sympathetic nervous system activity in the 105 DOCA-salt hypertensive model (43). Activation of ETB receptors increases 02" production in prevertebral sympathetic ganglia both in vitro(13)and in vivo (17). In these ganglia, ETB receptor expression and NADPH oxidase-derived 02" generation are elevated in DOCA-salt hypertension (13). Because DOCA-salt hypertension is characterized by sympathetic hyperactivation, elevated 02" levels in sympathetic ganglia may directly or indirectly contribute to the hypertension. The relationship of changes in reactive oxygen species levels in sensory neurons to blood pressure regulation is not known but could be related to interactions between sensory neurons and sympathetic ganglionic neurons (26; 48) or of sensory nerves directly with the vasculature. In salt-sensitive hypertension, synthesis and release of vasoactive neuropeptides from sensory ganglia innervating the splanchnic circulation are increased (40; 42),and this may play a role in blood pressure regulation (33),but it is not known whether these are related to the observed decreases in the activity of NADPH oxidase. One of the important findings of the present study is that NADPH oxidase activity is decreased in extracts of spinal sensory ganglia in hypertension; this is in contrast to sympathetic ganglia, where it is increased in hypertension. Both types of ganglia are made up of neurons and satellite cells, but in both types, the presence of the enzyme appears limited to the neurons. Dorsal root ganglia are a mixture of neurons with different functional and neurochemical properties, and only a subset of the neurons innervates the vasculature and release 106 neuropeptides. Sensory nerve fibers that innervate the systemic blood vessels contain the vasodilatory neuropeptides calcitonin gene-related peptide and substance P (19),and subsets of dorsal root ganglion neurons are labeled with calcitonin gene-related peptide (33%) substance P (23%) (21) or NO synthase (12%) (47). Thus, if decreased NADPH oxidase activity in dorsal root ganglia is associated with changes in activity of the peptide-containing vascular neurons, it is possible that these changes could contribute to hypertension. The splanchnic vasculature is innervated by sympathetic nerves, which are vasoconstrictor, and by sensory nerves, which are vasodilator. The NADPH oxidase system that is responsible for generation of 02" is regulated differently in these 2 types of nerves in DOCA-salt hypertension. We suggest that 02" overproduction evoked by the increased NADPH oxidase in sympathetic ganglia may play a role in the increased neurogenic vasoconstriction. Decreased oxidase activity in sensory ganglia may also enhance blood vessel tone or it may be a response to increased blood pressure. 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Zou AP, Li N and Cowley AW, Jr. Production and actions of superoxide in the renal medulla. Hypertension 37: 547-553, 2001. 113 CHAPTER 5: ENDOTHELIN-1 TRANSIENTLY DOWNREGULATES NOREPINEPHRINE TRANSPORTER VIA THE ACTIVATION or P22P”°"- CONTAINING NADPH OXIDASE IN PC12 CELLS Abstract Dysfunction of norepinephrine (NE) reuptake via norepinephrine transporter (NET) in the sympathetic nerve endings contributes to the elevated sympathetic drive to the vasculature in hypertension. In deoxycorticosterone acetate (DOCA)-salt hypertension, superoxide anion (02") levels are increased in sympathetic ganglion neurons; this increase is mediated in part by endothelin-1 (ET-1) which activates NADPH oxidase. The purpose of this study was to determine whether NET is regulated by NADPH oxidase-derived Oz" in response to ET-1 In sympathetic neuronal cell line PC12 cells. It was hypothesized that ET- 1 can downregulate NET; and this effect can be diminished by knockdown of p22°“°", an indispensable component of NADPH oxidase. PC 12 cells were treated with ET-1 (100nM) from 30 minutes to 24 hours. Compared to no treatment control, intracellular 02" levels measure by the intensity of dihydroethidium fluorescence were elevated by 360.7% after 1 hour of ET-1 incubation (n=3; p<0.05), while NET mRNA was decreased and reached its minimum at 2 hours (39.6% decrease, n=8; p<0.01). Apocynin (100pM) or actinomycin D (5p.M) pretreatment abolished NET mRNA decrease in response to ET-1, indicating a role of NADPH oxidase in transcriptional downregulation of 114 NET. Two lines of short hairpin RNA (shRNA) stably transfected PC12 cells, shRNA-p22 #1 and shRNA-p22 #2, were generated by transfecting undifferentiated PC12 cells with two shRNA-constructs targeting distinct parts in the p22""°" sequence. As compared to normal PC12 cells, p22""°" mRNA levels were decreased in shRNA-p22 #1 and shRNA-p22 #2 by 45.9% and 79.1%, respectively. P22°“°" immunoreactivity was also significantly diminished in shRNA-p22 #1 (40.2% decrease) and shRNA-p22 #2 (46.3% decrease). In contrast to normal PC12 cells or scramble control, neither 02“ production nor NET mRNA expression was significantly changed by ET-1 in shRNA-p22 cells. The results indicate that 1) ET-1 down-regulates NET transcription in PC12 cells; 2) the effects of ET-1 on 02" production and NET mRNA expression are p22""°"- dependent. These suggest that NADPH oxidase-derived reactive oxygen species as a result of enhanced ET-1 signaling in sympathetic neurons may contribute to NET dysfunction in hypertension. Introduction Endothelin-1 (ET-1), a 21-amino-acid peptide originally isolated from the supernatant of cultured endothelial cells from blood vessels, is the most extensively studied endothelin isoform in the cardiovascular system(45; 50). In addition to endothelial cells, ET-1 is also produced by other tissue including kidney and neurons (13; 23; 36). In the central nervous system, ET-1 is present in cerebral perivascular nerves and sympathetic and sensory ganglia neurons (39). The ET-1 mRNA and protein levels from these neurons were shown to be 115 higher in spontaneously hypertensive rats than in normal Wistar Kyoto rats(42), which suggests that neuronal ET-1 is important in hypertension. In the peripheral nervous system, our lab reported that endothelin B receptor (ETB), one of the ET- 1 binding receptors, is upregulated in prevertebral sympathetic ganglia from deoxycorticosterone acetate (DOCA)-salt hypertensive rats as compared to normotensive controls. This also indicates enhanced neuronal ET-1 signaling in hypertension (12). On the other hand, relatively little is known about the functional consequences of elevated ET-1 signaling in neurons. Cao et al is among the few studies who reported that ET-1 has a neuro-excitatory effect on vasomotor neurons in the rat brain (9). However, the mechanisms underlying this effect are not fully understood. Since elevated catecholamine levels were reported in patients with primary hypertension more than thirty years ago (14), sympathetic neuronal norepinephrine (NE) handling in hypertension has been intensively studied. Higher neuronal firing activity (26) and impaired nerve terminal NE reuptake via norepinephrine transporter (NET) (19) were reported from hypertensive patients, both of which can contribute to increased junctional NE and NE spillover in hypertension. Interestingly, recent studies showed that ET-1 regulates NE release and reuptake via the ET receptors located on the cardiac sympathetic nerve terminals (4; 30). This suggests the potential effect of ET-1 on sympathetic neuronal activity through the regulation of NE handling. Our lab has previously reported that ET-1 induced superoxide anion (02") production” through the activation of ETB/NADPH oxidase pathway in primary celiac ganglionic neurons and rat pheochromocytoma PC12 cells in vitro(12). Lau 116 et al used systemic administration of a selective ETB agonist, sarafotoxin 60 ($60), and observed a similar elevation of 02" levels in sympathetic ganglion in vivo, although the source of 02" was not specified in that study(37). More recently the same group reported that chronic activation of ETB in rats causes , sustained hypertension (21), which may be associated with the increased ganglionic 02". Meanwhile, the involvement of 02" in the regulation of NET was first suggested by Mao et al who showed that in PC12 cells NET activity and protein expression were inhibited when cellular oxidative stress level was increased(41). These studies together implicate a potential functional link among ET-1, 02" and NET in sympathetic neurons. NADPH oxidase is a multi-subunit enzyme system that contains. two membrane-bound catalytic subunits, NOX and p22""°", and several cytosolic regulatory subunits, namely p47°h°", p40°"°", p67p“°" and Rec (3). We have previously shown that NADPH oxidase activity was increased in sympathetic ganglion in DOCA-salt hypertension (11) and this elevated enzyme activity was accompanied by an upregulation of p22""°x expression (10). In this study, we sought to investigate the effect of ET-1 on neuronal NET. In addition to primary celiac ganglion neurons, we also used PC12 cells, which endogenously express ETB and p22”“°"-containing NADPH oxidase (11; 12) as well as the machinery for synthesis, release and reuptake of NE(15), as a model to further study the NADPH oxidase-mediated part of ET-1’s effect on NET by genetically manipulating the expression of p22""°x in PC12 cells using short interference RNA technique (22).We tested the hypothesis that in sympathetic neurons, ET-1 117 can downregulate NET; and this effect can be diminished by knockdown of NADPH oxidase subunit p22°“°". Methods Cell Culture The rat pheochromocytoma PC12 cell line was obtained from American Type Culture Collection (ATCC), and maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat inactivated horse serum, 5% fetal bovine serum, 100UIml penicillin, 100ug/ml streptomycin, and 0.25uglml Funizone in a humidified incubator at 37 °C and 5% 002 atmosphere. Medium was changed every other day and the cells were passaged every 7 days. All experiments were performed using cells under passage 15th to avoid cell line drift. For drug treatment, cells were plated as a monolayer on multi-well cell culture plates coated with 100ug/ml poly-D-lysine (Sigma-Aldrich, St. Louis, MO) one day prior to the experiment. All drugs were prepared as stock solutions in DMSO or H20. Aliquots were stored at -80°C and protected from light before use: ET-1 (Bachem Americas, Inc., Torrance, CA), 80788 (Sigma), BQ610 (Sigma), apocynin (Sigma), actinomycin D (Sigma). RNA Isolation and Reverse Transcription-Quantitative Polymorease Chain Reaction (R T-qPCR) Total RNA was isolated from the cells using RNeasy Mini kit (Qiagen, Valencia, CA). cDNA was synthesized using Superscript II mix (lnvitrogen). 118 qPCR analysis was then performed using Mx3000P QPCR system (Stratagene, La Jolla, CA). SYBR green was used as the fluorescence detector in the qPCR reaction (Applied Biosystems, Foster City, CA). All primers were derived from the Rattus Norvegicus gene (National Center for Biotechnology Information GenBank). NET fonivard: 5’-GCC TGA TGG TCG TTA TCG TT-3’; reverse: 5’- CAT GAA CCA GGA GCA CAA AG-3’. GAPDH forward: 5’-GGA GTC TAC TGG CGT CTT CAC-3’; reverse: 5’-GGT TCA CAC CCA TCA CAA AC-3'. Design of shRNA Insert Target sites for rat p22"“°" were selected following the guidelines for effective shRNAs (18). Two complementary DNA oligonucleotides incorporating each chosen target site, a loop sequence, and the corresponding reverse complement of the target site were designed. Xho I and Xba I overhangs were added at the end of the two oligonucleotides, respectively. The transcriptional termination signal for 5 T's was added at the 3' end of the inverted repeat (Figure 5.1A). The pair of oligonucleotides was then annealed and ready to be ligated into the plasmid vector. A scramble sequence with no known targeting sites in the rat genome was selected as control. Target sequences for p22"“°" and scramble control sequence are listed in Table 1. ShRNA Expressing Cassette Construction Vector-based short hairpin RNA (shRNA) expressing cassette was constructed by cloning the p22""°x shRNA insert into linearized pSuppressorNeo plasmid with neomycin/kanamycin selection cites and Sal I and Xba I cloning 119 A shRNA-p22 #1 Xhol Overhang Loop Stop 5’ TCGA GACGCTTCACGCAGTGGTA GAGTACTG TACCACTGCGI‘GAAGCGTC TTTTT 3’ 3‘ CTGCGAAGTGCGTCACCAT CTCATGAC ATGGTGACGCACTTCGCAG AAAAA GATC 5' Xbal Overhang shRNA-p22 #2 Xhol Overhang Loop Stop 5' TCGA GCTTCACGCAGTGGTACTTTG TTCAAGAGA CAAAGTACCACTGCGTGAAGC TTTTT 3' 3' CGAAGTGCGTCACCATGAAAC AAGTTCTCT GTTTCATGGTGACGCACTTCG AAAAA GATC 5' Xbal Overhang Scramble Ctl Xhol Overhang Loop Stop 5’ TCGA TTTCAGCACGTATATGGTCGT GAGTACTG ACGACCATATACGTGCTGAAA TTTTT 3’ 3' AAAGTCGTGCATATACCAGCA CTCATGAC TGCTGGTATATGCACGACTTT AAAAA GATC 5‘ Xbal Overhang B Sal | GACGCUUCACGCAGUGGUA’G \u ,..- .. Xbal ShRNA'922#1 llllllllil|||||||l| ' cucccmcucccucrxccrxm 4‘ U6 Promoter (RU/C GCAG 66 AC G UIUHQA u u uuu ’ ‘ . IMG-800 8““sz lllllllllllllllllllll i \ 3416 bps GCGUCACCAUGAAAC \A G A/G NeoIKan / \ \ G/A\G \_ .,» S bl Ctl UUUCAGCACGUAUAUGGUCGU’ b ‘w’ _E!.§E_‘E__ lllllllllllllllllllll A AAAGUCGUGCAUAUACCAGCA \ , G\U/C Fl ure 5.1. Plasma-based ex ression of shRNA- 22. A) Deoxyoligonucleotides used in the construction of shRNA-p22 #1, shRNA-p22 #2 and scramble control are shown as they would be paired after anneling. The color-coded labels above and below the oligonucleotides indicate the source or the function of the nucleotides in the indicated regions. B) Left: plasmid map of pSuppressorNeo plasmid with neomycin/kanamycin selection cites and Sal l and Xba I cloning sites from lmgenex Corp. shRNA constructs insertion site is marked in red. Right: the predicted stem-loop structure of shRNA-p22 and scramble ctl expressed from the plasmid. 120 Table 5.1 Target sequences for NADPH oxidase subunit p22phox for shRNA constructs. Sequence shRNA-p22 #1 5’- GACGCTTCACGCAGTGGTA -3’ shRNA-p22 #2 5’- GCTTCACGCAGTGGTACTTTG -3’ Scramble Ctl 5’-TTTCAGCACGTATATGGTCGT -3’ 121 sites (lmgenex, San Diego, CA) (Figure 5.1 B). Plasmids were transformed into One Shot® Chemically competent E. coli (Invitrogen). Cells were plated on LB plates containing 100ug/ml kanamycin (Sigma). Kanamycin resistant colonies were selected and amplified in LB medium and plasmids were isolated and purified using PureYieldT'lI Plasmid Midiprep System (Promega, Madison, WI). The resulting plasmid was sequenced to ensure that the insert was present and correct. The predicted shRNA expressed by this construct is shown in Figure 5.13. Transfection and selection of stably transfected PC12 cells An early passage of PC12 cells were plated as monolayer on 6-well cell culture plate pretreated with poly-D-lysine one day prior to the transfection. Cells (1 x 106) were transfected with 4 ug of plasmid constructs containing either p22”“°" shRNA sequences or scramble sequence as described above using Lipofectamine 2000 (Invitrogen). One day after transfection the cells were replated at a lower density and selected with 400 pg/ml G418 (Sigma) for 35 days until discrete colonies were formed. Individual colonies were isolated, grown up and maintained in RPMI medium containing 200pg/ml G418. Colonies with effective p22”“°" knockdown were defined as shRNA-p22 #1 or #2 and were used in later experiments. Immunocytochemistry of p22""°" in PC12 Cells Normal and transfected PC12 cells were plated onto poly-D-lysine coated cover glasses and were maintained in culture medium for one day before 122 immunostaining. The cells were cleaned from culture medium by three washes in Dulbecco’s phosphate buffered saline (DPBS) and then placed into fixative (4% paraformaldehyde, 0.1%triton-X100 in DPBS) for 30min at 4°C. Cells were then Incubated in DPBS with blocking solution (5% goat serum, 3% bovine serum albumin (BSA)) for 1 hour at room temperature followed by incubation with mouse monoclonal p22°“°" antibody (H44.1, kindly provided by Dr. Mark Quinn) at a 1:500 dilution in DPBS with 1%BSA for overnight at 4°C. The next day, samples were washed in DPBS for three times and then incubated with Cy3 conjugated secondary antibody in a dark chamber at room temperature for 1 hour followed by three washes In DPBS. Samples were mounted onto glass slides using Prolong Gold anti-fade reagent (lnvitrogen) for confocal microscopy using Pascal (Zeiss, Thomwood, NY). In order to compare immunostaining intensity across samples on different slides, samples in the same comparing group were prepared on the same day and the images were taken under the same microscopy parameters. Images were analyzed using Image J software (NIH). Measurement of 02" Level Intracellular 02" levels were examined by measuring fluorescence signal intensity resulting from intracellular probe oxidization. Normal or transfected PC12 cells were plated on poly-D-lysine coated cover glasses one day prior to the experiment. Cells were pretreated with ET-1 (100nM) for 1 hour, and then loaded with the dihydroethidine (DHE) (10pM) (Invitrogen) and incubated at 37°C for 45 minutes before measuring fluorescence (excitation: 514nm; emission: 560nm) with confocal microscopy (Zeiss). DHE was used as an intracellular 02" 123 probe. Upon its reaction with 02", DHE is oxidized to fluorescent ethidium. Ethidium will then intercalate with DNA in the nucleus to further amplify the red fluorescent signal and the intensity of the fluorescent signal is proportional to 02" levels (5; 53). Confocal images consisting of a 0.36pm optical slice through the approximate center of cells were captured and analyzed using Image J software (NIH). Data Analysis Data are presented as meantSE of the mean. Statistical significance of NADPH oxidase activity, lmmunohistochemistry data were assessed by Student t test using Prism 4.0 software (GraphPad Software, San Diego, CA). qPCR data statistical significance were assessed by Pair Wise Fixed Reallocation Randomization Test© using REST software. In both cases, p<0.05 indicates statistical significance. Results E T-1 Induced Transient Decrease in Norepinephrine Transporter mRNA Levels in PC12 Cells When 100nM ET-1 was applied to PC12 cells, there was a 39.6% decrease in NET mRNA expression at 2 hours. The relative expression ratio of NET mRNA at 2 hours to control is 0.6 $0.1 (n=8; p<0.01). After the transient 124 decrease, the levels of NET mRNA recovered to normal levels at 12 and 24 hours of ET-1 incubation (Figure 5.2). Apocynin Abolished NET mRNA decrease following E T-1 Treatment To determine whether the transient decrease of NET mRNA in response to ET-1 was mediated through NADPH oxidase-derived 02", apocynin (4-hydroxy- 3methoxy-acetophenone) was used as an NADPH oxidase inhibitor (49). Cells were incubated with apocynin (100pM) for 1 hour before the addition of ET-1 (100nM). Pretreatment of PC12 cells with apocynin completely abolished the decrease of NET mRNA in 2-hour ET-1 treated groups (Figure 5.3) (n=6; p=0.36). This indicates that ET-1 may induce NET mRNA decrease via the activation of NADPH oxidase. Actinomycin D prevented the decrease of NET mRNA in response to ET-1 NADPH oxidase derived 02" was shown to reduce mRNA stability of certain proteins (52). Actinomycin D was used in this study to block transcription in PC12 cells in order to test whether the reduction of NET mRNA in response to ET-1 was due to a decrease in NET mRNA stability. Normal PC12 cells were pre- treated with 5 pM actinomycin D 1 hour prior to the addition of ET-1 (100nM). Total RNA was collected for RT-qPCR for NET. Figure 5.4 shows that no NET mRNA decrease was found in cells treated with actinomycin D and ET-1. These results indicated that ET-1 reduces NET mRNA expression via the modification of NET transcription rather than its mRNA stability. 125 " N NET mRNA Relative Expression Value to Control (Normalized to GAPDH) P r‘ ‘I' T P o I 1 I I T 0 30min 2hr 12hr 24hr ET-1 [100nM] Figure 5.2. ET-1 Induces a transient decrease of NET mRNA expgpsslon In PC12 cells. Undifferentiated PC12 cells were incubated with ET-1(100nM) for 30 minutes, 2 hours, 12 hours or 24 hours. Cells were collected at the end of each time point. Total RNA was isolated for the purpose of RT-qPCR for NET mRNA measurement. Relative expression value to calibration (control) was calculated in MxProT" QPCR Software (Stratagene). All data points were normalized to their own GAPDH levels. The amplification efficiency of NET (99.1%) and GAPDH (95.0%) primers in PC12 cells were identified using serial dilutions. As compared to no treatment control, cells treated with ET-1 for 2 hours showed a significant decrease in NET mRNA levels. This decrease was recoverd to normal levels after 12 hours or 24 hours treatment. Data are expressed as the mean relative expression value :tSEM (n=8 cell preparations). * indicates significance by a paired Student’s ttest (p<0.01) at 2hr versus 0. 126 .5} 5 1.5- D Control ‘3 f - ET-1(100nM) : g - Apocynin(100uM) + ET-1(100nM) 3 (imam z > O ‘5 5% B it ,g z e g 0.5- % .. O ‘I‘. a .5 o.c A! O a: Fl ure .3. A c nin a Iishe the decrea of NET mRNA in res nse to ET-1 In PC12 cells. PC12 cells were either not treated or treated with ET-1 (100nM) for 2 hours. ET-1-treated cells were preincubated with apocynin (100uM) or vehicle 1 hour before the addition of ET-1. Total RNA was harvested from the cells for RT-qPCR analysis for NET mRNA. Relative expression ratio calculation and statistical analysis were performed by Pair Wise Fixed Reallocation Randomization Test© (http://www.gene-quantification.info) using Relative Expression Software Tool (REST) {Pfaffl, 2002 6906 lid}. All data points were normalized to their own GAPDH levels. As compared to control, cells treated with ET-1 showed a significant decrease in NET mRNA expression while this decrease was absent in cells pretreated apocynin. Data are expressed as the mean relative expression value :tSEM (n=6 cell preparations). * indicates significance p<0.01 at ET-1 versus Control. 127 1 .51 .d O l (Normalized to GAPDH) 9 0| P o Control Actinomycin D + ET-1 NET mRNA Relative Expression Value to Control Figure 5.4. Actinomycin D abolishg NET mRNA decrease in resmnse to ET-1 In PC12 cells. Normal PC12 cells were pretreated with actinomycin D (5pM) for 1 hour followed by ET-1 (100nM) incubation for 2 hours. Total RNA was harvested for RT-qPCR analysis for NET mRNA. Relative expression ratio calculation and statistical analysis were performed by Pair Wise Fixed Reallocation Randomization Test° (http ..I/www gene-quantification. info) using REST software. All data points were normalized to their own GAPDH levels. No significant difference of NET mRNA expression was found in treated cells as compared to controls. Data are expressed as the mean relative expression value :tSEM (n=9 cell preparations). 128 P22‘”""’t knockdown in PC12 cells by RNA interference PC12 cells transfected by plasmids containing shRNA sequences (Seq #1 or Seq #2, see Table 5.2) against p22""‘°x were selected for the subsequent experiments. They were defined as shRNA-p22 #1 and shRNA-p22 #2, respectively. PC12 cells transfected by scramble sequence-containing plasmid were defined as shRNA-scramble. RT-qPCR results revealed that the levels of p22""°x mRNA were significantly decreased in both shRNA-p22 #1 (45.916.3% knockdown) and shRNA-p22 #2 (79.1110.0% knockdown) (Figure 5.5A). The p22"“°" mRNA level was not affected by scramble sequence transfection. Off- target effects of RNAi was evaluated by measuring the mRNA level of signal transducers and activator of transcription protein-1 (STAT-1) because there is evidence of sequence-independent off target effects via interferon response that signals through STAT (31). Figure 553 shows that STAT-1 mRNA expression was not significantly different between transfected cells and control normal PC12 cells. This indicates the signaling pathway through STAT-1 was not activated by shRNA transfection. P22”"°" protein levels in shRNA-p22 cells were measured by immunocytochemistry. The staining of p22”"°" in normal PC12 cells was located in both the plasma membrane and the cytoplasm (Figure 5.6A). Using the same microscopy setting (gain and offset), shRNA-p22 cells revealed lower immunostaining intensity as compared to normal PC12 cells or scramble control cells (Figure 5.6). The decreases of mean fluorescent intensity in shRNA-p22 #1 129 '2' E 8 A 2-5‘ a Normal P012 3 E E ShRNA-p22 #1 2.0% g .3 g a: shRNA-p22 #2 «I E > O 15., a Scramble Ctl : H 6 .2 "g ‘5. 8 ._ 1.04 _ R 2 5 °' 3 g 0.5- ...... g V o.c , - - - - - - :_-:-:-:-:-:- .2 o n: Fl ure 5.5. P22 hox mRNA ex ression level Is su ressed b RNA Interference In P012 cells. P012 cells stably transfected by two shRNA sequences targeting p22phox were selected and defined as shRNA-p22 #1 and shRNA-p22 #2. Control cells were stably transfected by a scramble sequence with no known target in the rat genome and was defined as scramble ctl. Total RNA was harvested for RT-qPCR analysis for p22phox and STAT-1 mRNA. Relative expression ratio calculation and statistical analysis were performed by Pair Wise Fixed Reallocation Randomization Test© (http:/Iwww.gene-quantification.info) using REST software. All data points were normalized to their own GAPDH levels. A) As compared to normal P012 cells, shRNA-p22 #1 and shRNA-p22 #2 both have significant decreased levels of p22phox expression. The knockdown level of p22phox mRNA were 45.9% and 79.1%, respectively. Meanwhile, scramble ctl cells have a similar level of p22phox expression with normal cells. B) As compared to normal P012 cells, STAT-1 mRNA levels were not significantly different in shRNA-p22 #1, shRNA-p22 #2 and scramble control cells. Data are expressed as the mean relative expression value :I:SEM (n=6 cell preparations). * indicates significance p<0.01 at shRNA-p22 #1 vs Normal; # indicates significance p<0.01 at shRNA-p22 #2 vs Normal. 130 Figure 5.5 continued 2 m w. u .n.n.n...n.n.u.n.n.u.n.u. m n n. c . P n... m... b ..... mm m ............ m .h. m. a mu. .m M .m. c. 2 1 1 o 0 £23 2 33.2...on .9550 3 o2a> co_mmo.axm ozusom 530203.“. 932$. 3.23% 133 and #2 were 40.211.7% and 46.3:t1.6%, respectively. These results indicate the protein expression of p22°“°" was diminished in shRNA transfected cells. 02" production induced by E T-1 was attenuated in shRNA-p22 P012 cells ET-1 increases intracellular 02" production by NADPH oxidase in PC12 cells via the activation of ETB receptor (12). To test the function of NADPH oxidase in shRNA-p22 cells, we measured 02" production in response to ET-1. In normal PC12 cells and scramble control cells, 02" production was increased by 360.7:60.7% and 106.5:t14.1% by ET-1, respectively (Figure 5.7). However, no significant difference in DHE fluorescence was found in shRNA-p22 #1 or shRNA-p22 #3 cells treated with or without ET-1. These results showed that 02" production in response to ET-1 treatment was diminished in shRNA-p22 cells but not scramble control cells. This indicates that ET-1 induces 02" production in PC12 cells via NADPH oxidase. The difference in baseline 02" was unexpected. More studies are needed to evaluate the source of that 02". E T-1 showed no effect on NET mRNA expression in shRNA-p22 cells ET-1 (100nM) was applied to normal PC12 cells, shRNA-p22 #1, #2 and scramble control cells for 2 hours. NET mRNA levels were significantly 134 Figure 5.7. ET-1-incduced 02- production in PC12 cells is afienuatg ln ghRNAgZZ glia. A) Representative confocal fluorescent images of normal PC12 cells, shRNA-p22 #1 and #2 cells and scramble control cells incubated with DHE. Red fluorescence indicates the presence of 02--. In each group, control cells without ET-1 treatment are listed in the left row. ET-1 treated cells are listed in the right row. B) and C) Relative DHE fluorescence is analyzed using Image J software. Results are expressed as mean tSEM. (n=3 cell preparations for each group). The statistical significance (p<0.05) is indicated by *vs control. 135 Normal PC12 shRNA-x3221“ shRNA-p22 32 Scramble Ctl Control wormed BO. . .. ET»1 Control Control shRNA-p22 #2 Control 136 Figure 5.7 continued Normal PC12 Control q - u 6 4 2 3 £5.80 2 35.2.on c:_o> accomchoai min. eggs—om B ShRNA-p22 #1 1 .3550 8 85.9:on o:_m> acooocLoEu NE 3323. 137 Figure 5.7 continued ET-1 2 I # a 2 e m ... , c // I a m s m .. C 7/ .... g.“ n... g... ..u. as. 3 1n 2 2 1 1 0 0 cob—.00 on ce~=oEozv cob—30 3 33:2on c:_u> E3332". mro 9:53". o:_a> accomchoau. min 9:501 C 138 decreased in normal PC12 as well as scramble control cells (Figure 5.8). No change in NET mRNA was found in shRNA-p22 #1 or #2 cells. These data, together with the apocynin results, further indicate that the effect of ET-1 on NET mRNA in PC12 cellswas mediated via p22°“°"-containing NADPH oxidase. The reduction in NET mRNA expression is an effect of 02“ production on a side effect of the process of shRNA transfection. Discussion In this study, we examined the effects of ET-1 on NET mRNA expression. We found that ET-1 induced a transient decrease in NET mRNA levels and this decrease was mediated through an NADPH oxidase-dependent pathway. The findings from this study implicated a potential role of ET-1 induced 02" in the regulation of catecholamine handling in neuronal cells. This may suggest novel approach in understanding the role of sympathetic nervous system in blood pressure regulation in hypertension. NET belongs to a family of sodium- and chloride-coupled transporters. It is a 617 amino acid protein with 12 a-helical transmembrane domains that are interrupted by alternating intra- and extracellular loops. lts major function is to presynaptically terminate NE signaling at the neuroeffector junction (2) and therefore serves as an indispensible determinant of sympathetic innervation. There is in fact evidence for an impaired NE clearance by NET in hypertensive patients (19), suggesting that altered NET function might contribute to the elevated sympathetic tone in hypertension. NET was previously shown to be 139 fg Normal PC12 C O .. o f 1.5 2 o .. s % z N G 1.0“ n: > 0 E = “ |_ .2}; g g? 0.54 3 3 o E .5 o.c . .3 Control a: g Scramble Ctl C O .- 0 f 1.5 8 o 3 2 gfiotm > o a: c ,., 5.2 g '“ fig 05 2 ,_ g ' 1 3' a ‘3 a .. .. .5 0.0- % Control ET-1 n: Fl ure 5.8. NET mRNA is reduced b ET-1 in normal PC12 cells but not shRNA- 22 cell . Normal PC12 cells, shRNA-p22 #1, #2 and scramble control cells were either treated with ET-1 (100nM) or vehicle for 2 hours. Total RNA was harvested for RT-qPCR analysis for NET mRNA. Relative expression ratio calculation and statistical analysis were performed by Pair Wise Fixed Reallocation Randomization Test© (httpzllwww.gene-quantification.info) using REST software. All data points were normalized to their own GAPDH levels. ET-1 treatment elicited a significant reduction in NET mRNA expression in normal PC12 cells and scramble control cells (A). No change in NET mRNA in response to ET-1 was found in shRNA-p22 #1 and shRNA-p22 #2 cells (B). Data are expressed as the mean relative expression value tSEM (n=6 cell preparations). * indicates significance p<0.05 vs control. 140 Figure 5.8 continued shRNA-p22 #1 5- .0 5 1 1 0. 0. £920 3 35.2:on .9580 on c:_n> co_mmoaxm 9333. .853..an o>=o_cm