ROLE OF CENTRAL EICOSANOIDS IN THE DEVELOPMENT OF ANGIOTENSIN II -SALT HYPERTENSION IN THE RAT By Ninitha-Margret -Julfiya Asirvatham -Jeyaraj A DISSERTATION Submitted to Michigan State University in parti al fulfillment of the requirements for the degree of Pharmacology and Toxicology Ð Doctor of Philosophy 2013 ABSTRACT ROLE OF CENTRAL EICOSANOIDS IN THE DEVELOPMENT OF ANGIOTENSIN II -SALT HYPERTENSION IN THE RAT By Ninitha-Margret -Julfiya Asirvatham -Jeyaraj Human essential hypertension (HTN) causes end organ damage, cardiovascular disease and premature death. While increased sympathetic nerve activity (SNA) is a principal risk factor for the development of HTN, the central mechanisms that drive high sympathetic outflow remain unclear . Increase d SNA and blood pressure (BP) can be triggered by high salt intake (2% NaCl ) and the hormone angiotensin II (AngII). Our lab has extensively studied a rat model of HTN caused by these two factors in combination (AngII-salt HTN), and have shown t hat SNA contributes to the HTN and block ing whole -body eicosanoid (prostaglandin, thromboxane, lipoxygenase) synthesis with cyclooxygenase (COX) inhibitors attenuated both the increased SNA and HTN development. My project was to determine how eicosanoid products contribute to AngII -salt HTN. In initial experiment s, I showed that whole-body COX inhibition failed to reverse established AngII -salt HTN in rats (similar to human hypertensives). In contrast, COX inhibition only duri ng the first several days of 14 -day AngII -salt treatment successfully prevented subsequent HTN development and sympathetic support of BP (i.e. neurogenic pressor activity). I concluded that COX products exert important physiological effects only during t he early phase . I next investigated the ro les of the two isoforms of COX and found that COX-1 specific products drive the development of AngII -salt HTN. Because most eicosanoid products in peripheral tissues (blood vessel, kidney) lower BP, I decided to test if central (i.e. brain) eicosanoids , acting early in the process of AngII -salt HTN development, cause a long -lived increase in SNA and BP. I chronically administered the COX inhibitor into the brain at a dose designed to block COX in the brain, and showed that this prevented increase in SNA and HTN development. I concluded that one or more COX -1 products in the brain contribute to HTN development. I also performed PCR and Western blot analysis of COX pathway -associa ted gene and protein expression in known cardio -regulatory regions of the brain. This revealed only modest changes for the most part, but I observed significant changes in the prostaglandin D2 (PGD2) pathway (downstream from COX) in the OVLT (cardio -regulatory brain region), choroid plexus (CP) a nd cerebrospinal fluid (CSF). Importantly, I showed increased lipocalin -prostaglandin D synthase ( L-PGDS ) expression in the CP and CSF , the main site s of L -PGDS synth esis and secretion, respectively. These findings were the first ever to implicate brain PG D2 in HTN. Thus, to investigate this finding in more detail, I measure brain levels of PGD2 with mass spectrometry and found high levels in CSF and rostral ventrolateral medulla (RVLM) of HTN rats . PGD2 in the brain binds mainly to the G -protein coupled re ceptor DP1R . Immunofluorescence staining revealed down -regulation of DP1R in the RVLM during the early phase of AngII -salt HTN; perhaps predictable in the presence of increased agonist concentration. Finally, blockade of L -PGDS prevented the increase in PG D2 levels in the RVLM during the early phase of AngII -salt treatment and attenuated subsequent HTN development. In conclusion, the results of my studies suggest a novel mechanism for neurogenic HTN development: PGD2 generated in the brain from L -PGDS acts on DP1R in the RVLM, which ultimately leads to increased SNA, neurogenic pressor activity and HTN. Copyright by NINITHA-MARGRET -JULFIYA ASIRVATHAM -JEYARAJ 2013 v ACKNOWLEDGEMENTS I dedicate this work to my mentor Dr. Gregory Fink who constantly guided me an d made this work possible. I thank him for his remarkable support, generosity, motivation, critical thinking and wisdom shown at every step of this project . His zeal for science and humble persona is incomparable and I am honored to have his guidance durin g my PhD and thank him for being my Òguru Ó and showing infinite patience. I extend my heartfelt thanks to my committee members Dr. P.S. Mohankumar, Dr. James Galligan, Dr. Carrie Northcott and Dr. Hui Xu for their constant guidance and support t hroughou t these years. They are like a family to me who constantly helping me to grow as a researcher by providing valuable suggestions and guidance when I needed their expert opinion. This work would not be possible without the generous help from Dr. Daniel Jon es, Dr . Ken neth Strauss, Robert B urnett and the RTSF staff at MSU . I am grateful for their guiding and assistance with the mass spectrometry work. I am also thankful to the histopathology lab members Kathy and Amy for their help with the immunofluorescence study, Dr. Melinda Frame for her guidanc e with the confocal microscopy and Dr. Patri ck Mueller for his technical expertise and collaboration . My work would not have bee n possible with out the constant support and words of advice from Dr. Sheba Moha nkumar to whom I am obliged. Furthermore, I specially vi thank my lab members Jeremiah T Phelps, Hannah Garver, Robert B urnett and Cris tiane P ereira for their good company and for making the lab a haven for me . I am also grateful to my friends Priya, Lakshmikripa, Haritha, Madhan, Nandakumar, Sarguru, Paulo, Roxanne, Natasha, Suga and Madhu , who were alwa ys there to support me and are the epitome of the quote, Ò a friend in need is a friend in deedÓ. Finally, I thank the almighty and his image in my life in the for m of my parents: Jeyaraj and Prema, who gave me their love, dedic ation, encouragement, and blessi ng when I needed it most. I thank both of my siblings: Sukanya who encouraged me to study Pharmacology and Richard s for his supports when I decided to leave my job and join a PhD program in the States . vii TABLE OF CONTENTS LIST OF TABLES. !!!!!!!!!!!!!!!!!!!!!!!!!!. !!... .xi LIST OF FIGURES . !!!. !!!!!!!!!!!!!!!!!!!!!!!!!.xii KEY TO ABBREVIATIONS !.. !!!!!!!!!!!!!!!!!!!!!!!..xiv CHAPTER 1 INTRODUCTION !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.1 1. Hypertension !!!!!!!!!!!!!!!!!!!!!!!.!!!!!!......2 1.1. Classification of hypertension !!!!!!!!!!!!!.!!!!!!.........2 1.2. Causes of hypertension !!!!!!!!!!!!!!!!!.!!!!...........3 1.3. Treatment of hypertension !!!!!!!!!!!!!!!!!!!!..!!..4 2. Neural regulation of blood pressure !!!!!!!!!!!!!!!!!.. !!!..5 2.1. Central n ervous system !!!!!!!!!!!!!!!!!!!!!!!!.6 2.2. Baroreceptor reflex !!!!!!!!!!!!!!!!!!!!!!!!!.....7 3. Sympathetic nervous system activity (SNA) in hypertension !!!!!!!.............9 3.1. Physiological mechanisms of increased sympathetic effects on BP !!!... !...9 3.2. What causes increased SNA and neurogenic activity in HTN ?.!!!.. !!!.10 4. Renin angiotensin aldosterone system !!!!!!!!!!!!!!!!!........11 5. Angiotensin II as a cause of incre ased SNA in hypertension !!!!!!!!.......15 6. Salt !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.........15 6.1. Salt sensitivity of blood pressure !!!!!!!!!!!!!!!!!!..!.16 6.2. Brain salt sensors and sympathetic nerve activity !!!!!!!!!!!......17 7. Angiotensin II -salt model of hypertension !!!!!!!!!!!!!!!!!!.18 8. Eicosanoids !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!...22 8.1. Synthesis, signaling and function of eicosanoids !!!!!!!!!!!.!...24 8.1.1. Prostaglandin E2 !!!!!!!!!!!!.. !!!!!!!!!!.. ...26 8.1.2. Prostaglandin D2 !!!!!!!!!!!!.. !!!!!!!!!!.. ..26 8.1.3. Prostagla ndin I2 !!!!!!!!!!!!.. !!!!!!!!.. .!!...27 8.1.4. Prostaglandin F2 !!!!!!!!!!!!.. !!!!!!!!... !!...27 8.1.5. Thromboxane !.. !!!!!!!!!!!!.. !!!!!!!!!!....28 8.1.6. Non-steroidal anti -inflammatory drugs !!!!!!!!... !!!!.!..28 8.2. Eicosanoids in the brain !!!!!!!!!!!!!!!!!!!!!!....30 8.2.1. Role of eicosanoids in hypertension !!!!!!!!!!!!!!.. ...33 8.2.2. Effect of eicosanoids in AngII HTN development !!!!!!!!!.. .34 8.2.3. Eicos anoids may increase BP and /or SNA by actions in the brain !.. ...35 8.2.4. Angiotensin II, neuroplasticity and brain eicosanoids !!!!!!!....37 9. Central hyp othesis !!!!!!!!... !!!!!!!!!!!!!.. !!!!!38 10. Significance !!!!!!!!!!!!!!!!!!!!!!!!!!!!........38 viii CHAPTER 2 MATERIALS AND METHODS !!!!!!!!!!!!!!!!... !!!!!.. !.40 1. Animals !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!. !.41 2. General anesthesia and post -operative analgesia !!!!!!!!!!!!..... !41 3. Radiotelemetry implantation .. !!!!!!!!!!!!!!!!!!!!!...!42 4. Mini-osmotic pu mp implantation !!!!!!!!!!!!!!!!!!!!!!42 5. Intracerebroventricu lar brain cannula implantation !!!!!!!!!!!!!. .. 44 6. Femoral catheterization !!!!!!!!!!!!!!!!!!!!!!!!!.. 44 7. Animal eutha nasia !!!!!!!!!!!!!!!!!!!!!!!!!!......45 8. Microdissection of rat brain.. !!!!!!!!!!!!!!!!!!!!!!!..45 9. Polymerase chain reaction (PCR) array and quantitative real time -PCR !!!!... 45 9.1. PCR array !!!!!!!!!!!!!!!!!!!!!!!!!!!...!.46 9.2. Quantitative RT -PCR. !!!!!!!!!!!!!!!... !!!!!!!!.47 10. Cerebrospinal fluid collection !!!!!!!!!!!!!!!!!!!!!... ....48 11. Western blotting !!!!!!!!!!!!!!!!!!!!!!!!!!!!..49 12. Cyclooxygenase activity assay . !!!!!!!!!!!!!!!!!. !!!.... 49 13. Blood collection and high perf ormance liquid chromatography !!!!!!!!50 14. Immunofluorescent sta ining and confocal m icro scopy !!!!!!!!!!!!.51 15. Confocal microscopy !!!!!!!!!!!!!!!!!!!!!!!!!!.. 52 16. Liquid chromatography and t andom mass spectrometry (LC -MS/MS) !!!!... ..52 16.1. Solid phase extraction of lipid med iators from brain punches !!!!!!!53 16.2. Liquid-liquid extraction of lipid mediators from cerebrospi nal fluid !!!!!55 16.3. Chromatograp hy !!!!!!!!!!!!!!!!!!!!!!!!!!55 16.4. Mass spectrometry !!!!!!!!!!!!!!!!!!!!!!!!!56 17. Statistical analys es .............................................................................................. ..57 CHAPTER 3 CYCLOOXYGENASE -1 AND NOT CYCLOOXYGENA SE-2 INHIBITION ATTENUATES ANGIOTENS IN II -SALT HYPERTENSION AN D NE UROGENIC PRESSOR ACTIVITY IN THE RAT !!!!!!!!!!!!!!!!!.. !!!!58 1. Introduction .............................................................................................................. 59 2. Experimental protocols !!!!!!!!!!!!!!!!!!!!!!!!!... 60 2.1. Selective COX -1 and COX-2 inhibition in chronic AngII -salt hypertensive rats !!!!!!!!. !!!!!!!!!!!!!!!!!!!!!!!....60 3. Resu lts!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.... 61 3.1. Effect of selective COX -1 and COX-2 inhibition on chronic AngII -salt HTN .!.62 4. Discussion !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.. 66 CHAPTER 4 TRANSCRIPTIONAL AND TRANSLATIONAL REGULATION OF CENTRAL ENZYMES AND RECEPTORS IN THE EICOSANOID SYNTHESIS PATHWAY DURINNG THE DEVELOPMENT OF ANGIOTENSIN II-SALT HYPERTENSION IN THE RAT !!!.. !!!!!!!!!!!!!!!!!!!!!!!!!!. !!!..71 1. Introduction !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.72 2. Experimental protocols !!!!!!!!!!!!!!!!!!!!!!!!!!.72 2.1. Non-selective COX -inhibition in AngII -salt treated rats with established HTN...72 ix 2.2. Non-selective COX -inhibition prior to and during early stages of AngII -salt HTN !!!!!!!!!!!!!!!!!!!!!!!!!!!73 2.3. Brain eicosanoids in the early stage of AngII -salt HTN !!!!!!!!!!74 2.3.1. Transcriptional and translational regulation of eicosanoid pathway related genes !!!!!!!!!!!!!!!!!!!!!!!!..74 2.3.2. PCR array !!!!!!!!!!!!!!!!!!!!!!!!!!74 2.3.3. Quantitative real time polymerase chain reaction (q RT -PCR)... !!...75 2.3.4. Western blot analysis !!!!!!!!!!!!!!!!!!!... !.75 3. Results .................................................................................................................... 75 3.1. Non-selective COX inhibition in AngII -salt established HTN rats !!!!!...75 3.2. Non-selective COX -inhibition prior to and during early st ages of AngII -salt HTN !!!!!!!!!!!!!!!!!!!!!!!!!!!.78 3.3. Gene array analysis of eicosanoid related gene expression in the early stage of AngII -salt HTN !!!!!!!!!!!!!!!!!!!!!!!..81 3.4. Quantitative RT-PCR !!!!!!!!!!!!!!!!!!!!!!!!..81 3.5. Western blot analysis !!!!!!!!!!!!!!!!!!!!!!!!..87 4. Discussion !!!!!!!!!!!!!!!!!!!!!!. !!!!!!!!....90 CHAPTER 5 CENTRAL INHIBITION OF CYCLOOXYGENASE AND LIPOCALIN PROSTAGLANDIN D SYNTHASE BLOCKS THE DEVELOPMENT OF ANGIOTENSIN II SALT HYPERTENSION IN THE RAT !!!!!!..!!!!!!94 1. Introduction !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!95 2. Experimental protocols !!!!!!!!!!!!!!!!!!!!!!!!!!96 2.1. Mean arterial pressure and cyclooxygenase activity in cardio -regulatory brain regions during the early stage of AngII -salt HTN development !!!!..96 2.2. Central cyclooxygenase inhibition in AngII -salt hypertensive rats !!!!!!96 2.3. Plasma ketorolac measurement using high performance liquid chromatography !!!!!!!!!!!!!!!!!!!!!!!!!!!97 2.4. Western blot analysis for lipocalin prostaglandin D synthase expression in the cerebrospinal fluid during early stages of AngII -salt HTN !!!!!!...97 2.5. Central and peripheral L -PGDS inhibition !!!!!!!!!!!!... !!!.98 3. Results !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!...98 3.1. Mean arterial pres sure and cyclooxygenase activity in cardio -regulatory brain regions during the early stage of AngII -salt HTN development !!!!.. 98 3.2. Brain cyclooxygenase inhibition in AngII -salt hypertensive rats !!!!!!.102 3.3. Plasma ketorolac measurement using high performance liquid chromatogra phy !!!!!!!!!!!!!!!!!!!!!!!!!!.. 104 3.4. Lipocalin -prostaglandin D synthase contents in CSF during the early stages of AngII -salt HTN in rats !!!!!!!!!!!!!!!!!!!...104 3.5. Central and peripheral L -PGDS inhi bition !!!!!!!!!!!!!!!..104 4. Discussion !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!...108 x CHAPTER 6 BRAIN PROSTAGLANDIN D2 AND EICOSANOID RECEPTOR CHARACTERIZATION DURING DEVELOPMENT OF ANGIOTENSIN II -SALT HYPERTENSION IN THE RAT !!!!!!!!!!!!!!!!!!!!!..!.114 1. Introductio n ............................................................................................................ 115 2. Experimental protocols .......................................................................................... 116 2.1. AngII -salt HTN and tissue collection for eicosanoids measurement !!!!.116 2.2. Ultra performace liquid chromatography -tandem mass spectrometry for measurement of AA and prostaglandins !!!!!!!!!!!!!!!...117 2.3. Immunofluorescence detection of DP1 receptor in early stages of AngII -salt HTN !!!!!!!!!!!!!!!!!!!!!!!!!!...117 3. Results .................................................................................................................. 118 3.1. Blood pressure and heart rate !!!!!!!!!!!!!!!!!!!!.118 3.2. Eicosanoids levels in cardio -regulatory brain regions during t he early stage of AngII -salt HTN development !!!!!!!!!!!!!!!!!!!...118 3.3. Immunofluorescence detection of DP1 receptor in early stages of AngII -salt HTN !!!!!!!!!!!!!!!!!!!!!!!!!!...121 4. Discussion ............................................................................................................. 125 CHAPTER 7 DISCUSSION ............................................................................................................ 130 7.1. Function of choroid plexus in angiotensin II -salt hypertension development !...133 7.2. Possible role of L -PGDS in the development of AngII -salt hypertension .............134 7.3. Possible role of PGD2 acting on DP1Rs in the development of AngII -salt hyperten sion !!!!!!!!!!!!!!!!!!!!!!!!..136 7.4. Importance of RVLM as the site of action of PGD2 to increase BP !!!!!...137 7.5. Future directions !!!!!!!!!!!!!!!!!!!!. !!!!!!!139 7.6. Overall significance, perspectives and therapeutic implications .!.. !!!!!..139 REFER ENCES !!!!!!!!!!!!!!!!!!!!!!!!!!!!!... 141 xi LIST OF TABLES Table 2-1 96-well custom PCR array template of eicosanoid related genes 46 Table 2-2 Arachidonic acid and prostanoi d mass to charge ratio, dwell time and voltage for mass spectr ometry analysis 57 Table 4-1 Phospholipase A2, cyclooxygenase -1, prostaglandin D synthase protein expression in 4 -day angiotensin II compared to vehicle treated rats 88 xii LIST OF FIGURES Figure 1 -1 Renin-angiotensin -aldosterone system 14 Figure 1-2 Brain sites potentially involved in changes in sympathetic nerve activity and blood pressu re in AngII -salt hypertension (AngII -salt HTN) 21 Figure 1-3 Eicos anoid biosynthesis and receptors 23 Figure 1-4 Prosta glandin synthesis and actions 29 Figure 1-5 Hypothesis and specific aim 39 Figure 2 -1 Osmotic mini pump 43 Figure 3-1 Effect of selective cyclooxygenase (COX) -1 inhibition on mean arterial pressure (MAP) 62 Figure 3-2 Effe ct of selective COX -2 inhibition on MAP 64 Figure 3-3 Depressor response to ganglionic blockade 10 days after AngII infusion in DMSO , SC560 and nimesulide (NM) treated rats 65 Figure 4-1 The effect of non -specific cyclooxyg enase inhibition on MAP and neurogenic pressor activity in established AngII -salt HTN rats 77 Figure 4-2 The effect of non -selective cyclooxygenase inhibition prior to AngII administration on AngII -salt HT N and neurogenic pressor activity 79 Figure 4-3 The effect of non -selective cyclooxygenase inhibition prior to and during the early phase of AngII administration on AngII -salt HT N and neurogenic pressor activity 80 Figure 4-4 Eicosanoid related gene expression in rats treated with Ang II or vehicle for 4 days 83 Figure 4-5 Phospholipase A2 and lipocalin prostaglandin D synthase gene expression using q -RT PCR in 4 -day Ang II or vehicle treated rats 86 Figure 4-6 Phospholipase A2, cyclooxygenase -1 and lipocalin prostaglandin xiii D synthase protein expression in 4 -day Ang II or vehicle treated rats 88 Figure 5-1 MAP and heart rate (HR) in AngII -salt HTN rats 99 Figure 5-2 Change in cyclooxygenase activity in cardio -regulatory brain regions on day 4 of AngII -salt HTN 101 Figure 5-3 Effect of chronic COX inhibition on MAP and neurogenic pressor activity 103 Figure 5-4 Lipocalin -prostaglandin D synthase expression in cerebrospinal fluid during early stages (A4) of Ang II-salt HTN development 105 Figure 5-5 The effect of chronic L -PGDS inhibition with AT56 106 Figure 6-1 Ultra performance liquid chromatography -tandem mass spectrometric analysis of lipid mediators 119 Figure 6-2 Representative chroma togram with PGD2 levels in RVLM 120 Figure 6-3 Double immunofluorescent stain ing for neuronal cell bod ies and the G-protein coupled receptor DP1R in early stages of HTN development 122 Figure 6-4 DP1 receptor expression in RVLM of 4-day AngII -salt HTN rats 123 Figure 6-5 DP1R expression in CP, SFO, PVN and OVLT of AngII treated rats 124 Figure 7-1 A hypothetical signaling pathway by which circulating AngII and high salt intake may cause increased splanchnic SNA and HTN 131 xiv KEY TO ABBREVIATIONS AA Arachidonic acid AngII Angiotensin II AP Area postrema AT1R Angiotensi n II receptor type 1 BP Blood pressure CG Celiac ganglion c-PGES Cytosolic prostaglandin E synthase cPLA2 Cytosolic phospholipase A2 CO Cumulus oophorus cell CP Choroid plexus CRTH2 Chemoattractant receptor homologous receptor molecule expressed on T helper 2 cells (or DP2) CSF Cerebrospinal fluid CVO Circumventricular organ DBP Diastolic blood pressure DP1 Prostanoid D receptor -1 DP2 Prostanoid D receptor -2 or (CRTH2) EP1-4 Receptor subtypes for prostaglandin E2 FP Prostaglandin F receptor H-PGDS Hematopoietic prostaglandin D synthase HR Heart rate HTN Hypertension xv ICV intracerebroventricular im intramuscular IML Intermediolateral cell column ip intraperiton eal L-PGDS Lipocalin type prostaglandin D synthase LOX Lipoxygenase LT Leukot riene LTR Leukotriene receptor MAP Mean arterial pressure mPGES -1 Microsomal prostaglandin E synthase -1 mPGES -2 Microsomal prostaglandin E synthase -2 NTS Nucleus tractus solitaries OVLT Organum vasculosum lamina terminalis PL Phospholipid PGD2 prost aglandin D2 PGE2 prostaglandin E2 PGF2 ! prostaglandin F2 ! PGFS Prostaglandin F synthase PGG2 Prostaglandin G2 PGH2 Prostaglandin H2 PGI2 prostaglandin I2 or prostacyclin PGIS Prostacyclin (Prostaglandin I2) synthase PGT Prostaglandin transporter xvi PVN Paraventric ular nucleus RAS Renin angiotensin system RVLM Rostral ventrolateral medulla SBP Systolic blood pressure sc subcutaneous SFO Subfornical organ SNA Sympathetic nerve activity SNS Sympathetic nervous system SSNA Splanchnic sympathetic n erve activity TP Thromboxane receptor Tx Thromboxane TS Thromboxane synthase VSMC Vascular smooth muscle cell 1 CHAPTER 1 INTRODUCTION 2 1. Hypertension High blood pressure, medically termed hypertension (HTN), is a principal risk factor for cardiovascular diseases. A recent report published in 2013 on the occasion of World Health Day by the World Health Organization (WHO) addresses HTN as Òa global public health problemÓ and a Ò silent killerÓ (1). One in three Americans adults over 18 years of age is hypertensive (196). HTN is a modifiable risk factor that predisposes to the development of cardiovascular and renal diseases; it is the major factor responsible for myocardial infarction ( 69%), stroke (77%) , and chronic heart failure ( 74%) (99). In the US and the world, healthcare costs due to the impact of HTN have surpassed billions when considering direct medical expenses and lost productivity (101). Pat ients who are clinically diagnosed with HTN are prescribed multiple drugs like diuretics, angiotensin receptor inhibitors, angiotensin converting enzyme inhibitors etc., to control their blood pressure (BP) and prevent multiple organ damage. Lifestyle modi fications suggested to reduce blood pressure include a nutritionally balanced diet low in sodium and fat content along with regular exercise. In particular, it has been suggested that lowering sodium intake population -wide from 3,300 mg to 2,300 mg per day could reduce the number of cases of high BP and save billions of dollars annually on health care (178). 1.1 . Classification of h ypertension A pressure of " 120 mmHg exerted against the walls of blood vessels when the left ventricle of the heart is contracting and " 80 mmHg when the heart is relaxing are typical systolic and diastolic blood pressures found in healthy young adults, respectively. 3 Therefore, adult humans with BP at or below 120/80 mmHg are considered ÒnormotensiveÓ (28). Individuals can exhibit different stages of HTN that are defined by the cardiovascular risk encountered with different levels of elevated BP. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure categorized HTN into prehypertension (120 -129/80-89 mmHg) , stage 1 hypertension ( 140-159/90-99 mmHg) and stage -2 hypertension ( # 160/ # 100 mmHg) (28). The stage of prehypertension is considered a grey area during HTN de velopment because the relative risk for cardiovascular disease development is low, but such patients have an increased chance of progressin g to true hypertension. C linical management of patients with prehypertension is controversial at the same time . 1.2 . Causes of hypertension Only 5 to 10 percent of patients have a clearly identifiable etiology for their HTN, such as pheochromocytoma, primary aldosteronism, CushingÕs syndrome, coarctation of aorta, or adrenal cortical tumors. Patients with a known cause for their HTN are generally curable once the underlying cause is removed and are said to have Òsecondary hypertensionÓ (87). The remaining 90 to 95 percent of patien ts have HTN with no single identifiable cause: this category is termed ÒessentialÓ or ÒprimaryÓ hypertension. The term ÒessentialÓ is a misnomer that was coined in the 1940s -1950s based on the mistaken belief that increased BP was essential to maintain ade quate tissue perfusion through an abnormally constricted arterial system. Modifiable risk 4 factors that increase the probability of developing essential HTN include increased body weight, excessive salt intake or alcohol consumption, environmental stresses, and physical inactivity (3). Non-modifiable risk factors include age, sex and genetics. Human hypertension is a polygenic trait with many gene variants contributing to a quantitative phenotype such as blood pressure. It is impossible to point at any one particular gene as the cause of HTN , instead it arises from the interaction of many genes with small effects (10). Therefore, identification of new genes requires novel stat istical approaches based on many thousands of patients (142). There are few outward symptoms of HTN, hence the designation Òsilent killerÓ. Severe HTN occasionally is often signaled by headache and/or vertigo. Stroke, left ventricular hypertrophy an d vascular disease leading to myocardial ischemia and congestive heart failure are frequent complications that account for much of the morbidity and mortality associated with HTN (82, 198). 1.3. Treatment of hypertension Numerous categories of hypertensive drugs are prescribed to patients including diuretics, beta -blockers, alpha -blockers, centrally acting sympathol ytics, vasodilators, calcium channel blockers, ACE inhibitors, angiotensin receptor blockers and renin, or a combination of these drugs (28). Most patients ultim ately need to take drugs from two or more of these classes to achieve adequate control of their HTN. Some individuals do 5 not reach therapeutic BP targets even when taking three or more drugs and are therefore described as having resistant hypertension. In recent years catheter -based renal sympathetic denervation has shown efficacy (50, 238) in lowering blood pressur e in some patients with resistant hypertension, emphasizing the important role of the sympathetic nervous system in the pathophysiology of HTN. 2. Neural regulation of blood pressure Hippocrates, ÒThe Father of MedicineÓ, over 2500 years ago appreciated the link between the mind and physical health. In recent decades, many details have been discovered about how the brain regulates BP (145, 244). Changing neuronal activity in a number of distinct Òcardio -regulatoryÓ neuronal regions of the brain controls BP by affecting cardiac function, blood vessel tone, hormone secretion, body fluid volume regulation and other factors (88). The primary sites in the brain containing neurons that regulate cardiovascular function are the spinal cord, brainstem, and hypothalamus. In addition, a network of peripheral afferent and efferent autonomic neurons comprises a short -term and long -term feedback control system that allows the brain to tightly regulate BP (36). BP control is mostly invested in the sympathetic nervous system (SNS), one of two major arms of the efferent autonomic nervous system (the other being the parasympathetic nervous system). Anatomically, short preganglionic neurons of the SNS originate from the thoracolumbar region of the spinal cord, in particular from the cell bodies of intermediolateral (IML) cell column. They send their axons to the paravertebral and prevertebral ganglia and adrenal medulla. Long postganglionic fibers project from the ga nglia to various effector organs , where they release the primary 6 sympathetic neurotransmitter norepinephrine (NE) along with numerous other con -transmitters. Widespread physiological responses to increased SNS activity (SNA) were initially characterized a s the Òfight or flight response.Ó With regard to BP regulation, sympathetic innervation of effector organs like the peripheral blood vessels, sinoatrial node and myocardium allows precise minute -to-minute control of BP via changes in arterial vasoconstrict ion, heart rate, and stroke volume (86, 88). While long-term control of BP was generally thought to be determin ed by the kidney pressure natriuresis mechanism (78, 87), it is now well accepted that the SNS plays a key rol e not only in short term but also in long -term control of BP (36, 50, 86, 128). For example, one autonomic pathway that operates over the long -term to control BP is renal SNA (49), which regulates renal vascular resistance, sodium and water excretion, and renin release. 2.1. Central nervous system As noted earlier, neural pathways originating in the brain maintain BP primarily by affecting SNA. Activity in these pathways is determined both by the intrinsic firing rates of neurons and by the concerted action of circulating hormones (e.g angiotensin II, leptin), chemicals (e.g. glucose), afferent neural inputs from the periphery (e.g. baroreceptors and chemoreceptors) and i nputs from other brain regions (e.g. cere bral cortex) on pathway neurons . 7 Brain regions known to be important in BP regulation that are considered important for signaling include the amygdala in the cerebral cortex, the paraventricular nucleus (PVN), preo ptic nuclei and dorsomedial nucleus in the hypothalamus, the A5 noradrenergic nuclei in the pons, the rostral ventrolateral medulla (RVLM) and nucleus of the solitary tract (NTS) in the brainstem, and circumventricular organs such as the hypothalamic subfo rnical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT) and the brainstem area postrema (AP) (59, 96, 208). Circumventricular organs (CVO) are the first brain structures to come into contact with peripheral hormonal signals that regulate SNA, like angiotensin II (176). Neuronal connections link these CVOs to other cardioregulatory regions involved in control of SNA and BP (217). 2.2. Baroreceptor reflex Although many factors influence SNA at rest and during physiolog ical stresses, the arterial baroreceptor reflex exerts overriding control of SNA over short time intervals (seconds to minutes). The brain receives continuous inputs from arterial baroreceptor neurons, i.e. neurons with cell bodies in the nodose and petros al ganglia and deformation -sensitive nerve endings situated in the carotid sinus and the aortic arch. Increased arterial pressure stretches the blood vessel wall and activates these nerve endings, which causes the baroreceptor neurons to discharge and inhi bit sympathetic vasomotor activity. Peripheral afferent fibers arising from the baroreceptor neurons synapse on first order neurons of the NTS located in medulla (brainstem ). These NTS neurons excite second -order neurons through glutamatergic synapses (140). The 8 glutamate sensitive NTS second -order neurons convey the baroreceptor signal to the caudal ventrolateral medulla (CVLM), which sen ds inhibitory signals to neurons within the RVLM (86, 186). RVLM neurons are tonically active and provide mos t of the ÒbasalÓ excitatory drive to sympathetic preganglionic neurons in the spinal cord. The reduced activity in RVLM neurons caused by baroreceptor activation causes a fall in sympathetic nerve discharge to the blood vessels and heart and a decline in b lood pressure (and heart rate) (186, 197). Aside from its role in the baroreceptor reflex, the RVLM serves as a Òfinal common pathwayÓ for most neural regulation of BP by causing sympathetically mediated changes in heart rate, stroke volume and systemic vascular resistance. This role of the RVLM became evident when researchers observed dose dependent reductions in SNA and BP when the inhibitory neurotransmitter gamma amino butyric acid ( GABA ) was microinjected into the RVLM in experimental animals (98, 182). Among other brain nuclei, the PVN is considered a particularly key integrative center for control of SNA and BP (125, 170). The PVN regulates SNA and BP by direct neuronal connections to: 1) the NTS and RVLM in the brainstem, and 2) spinal sympathetic pre -ganglionic neurons. The PVN receives input from many important integ rative centers of the hypothalamus (e.g. the SFO, median preoptic nucleus, arcuate nucleus, suprachiasmatic nucleus), pons (lateral parabrachial nucleus), and brainstem (NTS and RVLM) (187). Although the PVN is known to play an important role in stress, metabolism, growth, reproduction, immune, gastrointestinal, and renal function, recent 9 studies point to an important function of the PVN in regulating SNA and BP in hypertension as well (4, 31, 125, 148). 3. Sympathetic nervous system activity (SNA) in hypertension In varied experimental animal models such as spontaneously hypertensive rats (116), renovascular hypertension (21), angiotensin II hypertension (162), angiotensin II -salt hypertension (175, 268), Dahl salt sensitive rats (146) and mineralocorticoid hypertension (119), there is evidence for increased SNA . There also is compelling evidence for increased SNA in human hypertensive patients. For example , a substantial subset (around 50%) of essential hypertensive patients has elevated SNA and/or sympathetic support of BP (45, 48, 80, 81, 134, 205). This evidence comes from a variety of techniques for assessing SNA such as measurement of plasma norepinephrine or norepinephrine spillover, direct recording of single or multi -unit sympathetic nerve fiber firing rate , measurement of plasma catecholamine in hypertensive population the d epressor response to ganglionic blockers, and changes in blood pressure after surgical sympathectomy (191, 205). 3.1. Physiological mechanisms of increased sympathetic effects on BP Broadly speaking, sympathetic effects on blood pressu re, which in my dissertation I call neurogenic pressor activity, can be caused by two general sets of physiological effects classified as ÒprejunctionalÓ and ÒpostjunctionalÓ. Prejunctional mechanisms determine the concentrations of NE in the neuro -effecto r junctions of target tissues (blood vessels, heart, kidney), and include: 1) increased centrally -mediated sympathoneural or adrenal 10 medullary outflow (this is generally described as Òincreased SNA Ó), 2) increased ganglionic neurotransmission and 3) increa sed NE release or decreased uptake by sympathetic nerve terminals (often called Òincreased neurotransmissionÓ) (86). The postjunctional mechanisms are 1) vascular remodeling, 2) augmented vascula r, cardiac or renal adrenoreceptor -mediate d tissue responses, 3) altered cardiac or vascu lar ion channels, 4) abnormal intracellular signaling, 4) increased release of intracellular calcium from stores, or 5) decreased actions of physiological antagonists such as nitric oxide (86). The net effect of all these factors determines the degree to which the sympathetic nervous system determines blood pressure, i.e. neurogenic pressor activity. 3.2. What causes increased SNA and neurogenic pressor activity in HTN? A myriad of factors contribute to increased SNA or neurogenic pressor activity in human hypertension. Broadly these could be categorized into genetic factors and behavioral or environmental factors (48). The increase in SNA seen in many hypertensive patients is partly caused by genetic factors (56, 245). Another prime cause f or increased SNA is behavioral stress (47, 183). Obesity (6), insulin resistance (150) and inactivity (154) also are believed to enhance SNA in hypertensive patients . Finally, sleep deprivation (184), hypoxia (277) and inflammation (144) could account for increased SNA in human hypertension. From a physiological control system perspective, i ncrease d SNA could be a consequence of baroreceptor , chemoreceptor or metaboreceptor impairment (36, 37). Other physiological factors possibly responsible for increased SNA in hypertension include: increased plasma or tissue osmolal ity (173), elevated levels of circulating 11 hormones like angiotensin II (92, 193), and both inadequate or excess intake of salt (18, 108) . A major goal of my research project is to more fully elucidate specific brain signaling mechanisms that can cause increased SNA that result in increase BP . To this end, my experiments employed two factors that are well known to be import ant in the development of HTN, namely angiotensin II and high dietary salt intake. Thus, before proceeding further I will give brief overviews on the physiology of the renin -angiotensin -aldosterone system and salt, with specific emphasis on how they can af fect SNA. 4. Renin angiotensin aldosterone system The renin angiotensin aldosterone system (RAAS) producing the effector molecule angiotensin II (AngII) in response to physiological stimuli is a classic endocrine system (246). The main components of the classical renin angiotensin aldosterone system (Figure 1 -1) include: Renin: The juxtaglomerular (JG) cells that surround the renal afferent arterioles secrete renin. It is a protease enzyme synthesized first as an inactive precursor pro -renin. Angiotensinogen: It is a large protein with over 450 amino acids and is synthesized in the liver. The r enin angiotensi n system cascade starts with the release of enzyme renin from the kidney . Renin then acts on the precursor angiotensinogen in the blood to form AngI. 12 Angiotensin I: AngI is a decapeptide formed by renin activity . It has limited physiological activity, but is hydrolyzed by circulating and locally expressed angiotensin converting enzyme to the physiologically active AngII . Angiotensin converting enzyme (ACE): The enzyme ACE is mainly expressed in the lungs and to some extent in other tissues like vascular e ndothelial cells, kidneys , forebrain circumventricular organs and the adrenal (25). ACE catalyzes the conversion of angio tensin I into angiotensin II. ACE inhibitors are widely used as antihypertensive drugs. Angiotensin II (AngII): It is an octapeptide produced by the renin angiotensin pathway. Physiological effects of AngII include: potent vasoconstriction; stimulation of certain brain regions controlling SNA, thirst responses and vasopressin release; angiogenesis; release of aldosterone from the adrenal cortex; and direct actions on renal tubular cells to cause sodium absorption (68, 246). Angiotensin II receptors: Most established physiological effects of AngII are mediated through interaction with cell membrane re ceptors. The known AngII receptors are angiotensin type 1 receptor and angiotensin type 2 (AT1 and AT2) that belong to the superfamily of seven transmembrane spanning G -protein coupled receptors. AT1 mediates vasoconstriction, cardiac hypertrophy, fibrosis and inflammation. AngII -AT1 signaling trigger s aldosterone release and sodium retention (250). Action s of AngII at AT1 receptors are in some instances partially opposed by actions at AT2 receptors. AT2 receptor stimulation activates phospholipase A2, and nitric oxid e release and thereby produces anti-inflammatory, vasodilat ory and anti -proliferative effects (65, 230). AT1 receptor blockers are effective in lowering vascular tone and blood pressure along 13 with reduc ing catecholamine, vasopressin and aldosterone release (230). Aldosterone: The adrenal cortex is the site of synthesis of the steroid hormone aldosterone. Aldosterone acts primarily on the epithelium of the renal collecting tubules and distal tubules to cause an increase in th e reabsorption of sodium and secretion of potassium resulting in sodium conservation and enhanced excretion of the potassium ion (261). Angiotensin II stimulates secretion of aldosterone from adrenal cortex and aldosterone in turn acts on renal tubules to increases blood volume and blood pressure. The RAAS controls BP after being activated by one o r more physiological cues. For example, a reduction in renal perfusion pressure (or salt deprivation) stimulates the release of renin from the JG cells of the kidney into circulating blood. Under most circumstances, the rate -limiting step in the formation of AngII is the rate of release of renin from the kidney. In addition to reduced perfusion pressure and salt depletion, renin secretion is stimulated by activation of beta -1 adrenergic receptors on JG cells by norepinephrine released from renal sympathetic nerves (9). In addition to the classical renin -angiotensin system (RAS) , tissue specific RAS have been identified in brain, blood vessels, heart, adrenal gland, testes, ovaries, skin, adipose tissue and leukocytes (34). These tissue -specific RAS have roles like control of blo od flow, cell growth and repair, to name a few. It remains unclear how important tissue -specific RAS are to normal physiological function and the degree to which they are actually independent of the classical RAS (100, 258, 274). 14 Figure 1-1: Renin -angiotensin -aldosterone system . Circulating renin fro m kidney cleaves liver angiotensinogen to form angiotensin I (AngI). In the lung, angiotensin -conver ting enzyme (ACE) acts on AngI to form angiotensin II (AngII). AngII acts mainly on the angiotensin type 1 receptor (AT1) to cause vasoconstriction, sympath etic activation, and the secretion of vasopressin and aldosterone. 15 5. Angiotensin II as a cause of increased SNA in hypertension Abundant evidence supports the idea that AngII can increase SNA and/or sympathetic support of blood pressure by acting on both the brain and peripheral SNS (24, 85, 141, 268). With regard to brain mechanisms, pivotal work by Brody and colleagues revealed the critical role of brain CVOs in the AngII -induced HTN. Neurons in CVOs like the SFO and OVLT innervate neurons in the PVN t hat sends projections to the RVLM in the brain stem , and directly to sympathetic neurons in the spinal intermediolateral cell column (IML) , and thereby modulate sympathetic tone (185). Although there is support for the idea that AngII could be a player in the elevated SNA observed in some human hypertensives, that idea remains controversial (79, 141). This is likely due to: 1) important differences in the experimental animal models used to study the question; 2) the disparate approaches that have been used to quantify SNA in experimental animals and human patients; and 3) the often disregarded impact of other factors (e.g. salt intake) on the physiological actions of AngII (see below). 6. Salt Salt is regarded as an essential human commodity. If we take pages from the annals of history, we find many wars were fought to have control over salt. The Indian civil disobedience movement against the taxation of salt was a hallmark of the Indian Independence movement in 1930, where Gandhi led freedom fighters in what was called the ÒSalt March to DandiÓ to make salt from the sea. With modernization, the use of salt has been under scrutiny because its widespread use as a food preservative has produced higher levels in the body, with possible adverse consequences. The human 16 body i s 60% water, a handful of solutes, mainly sodium, potassium, calcium, magnesium, chloride, bicarbonate, phosphate , organic anions, and proteins. ÒSaltÓ is made up of sodium and chloride and is the major contributor to human extracellular and serum osmolali ty. In these fluids the concentration of sodium is tightly regulated in the range of 135 -145mnol/L and physiological disturbances occur if the concentration becomes higher or lower. In large population groups, average dietary salt intake has been shown to be an important risk factor for hypertension and related cardiovascular diseases. In contemporary western society, sodium intake is around 100 -200 mmol/day (240). Groups of individuals with intake of sodium below 50 to 100mmol/day have a lower incidence of cardiovascular diseases (42). Groups with higher daily intake than average are at increased risk for hy pertension and modest reduction in salt intake reduces blood pressure (97, 240). 6.1. Salt sensitivity of blo od pressure Despite the results from population studies cited above, individuals exhibit different BP responses to changes in salt intake. Some individuals respond to an increase in salt intake with a prompt rise in BP of over 10 mmHg and are called Òsalt -sensitive,Ó whereas others show no change in BP and are called Òsalt -resistantÓ. Some studies claim that as many as 50% of hypertensive subjects are salt -sensitive (55, 163, 252), although others report a lower fraction (61). Factors affecting salt sensitivity are race, birth weight, age, renal function and diabetes (77, 122, 251). The physiological mechanisms underlying salt sensitive hypertension are not fully known. Impaired renal sodium excretion has been hypothesized as one explanation (89-91), but some studies 17 find no difference in sodium handling between salt -sensitive and salt -resistant hypertensive humans and ins tead show critical differences in regulation of vascular tone between the two groups (20, 41). High salt in take decreases renin secretion (218), and the resulting decrease in circulating AngII is a major factor in facilitating renal salt excretion (90). Interestingly, however, this decrease in renin -secretion is blunted in some salt -sensitive hypertensive patients (16, 256, 266) and these same subject s exhibit higher SN A than salt -resistant hypertensives (266). What could account for the increased SNA in these individua ls? The answer may be that under certain conditions (especially inappropriately high RAS activity) the brain is capable of ÒsensingÓ high dietary salt intake and respond ing in a way that leads to elevated SNA and BP (6, 18, 136, 216, 217, 226). 6.2 Brain salt sensors and sympathetic nerve activity An organism tightly maintains the homeostasis of water and solute content in the body. In experimental animals, raising the sodium concentr ation in the cerebrospinal fluid by infusion of hypertonic saline into the lateral ventricles of the brain causes a rapid increase in BP and SNA (217). This effect is mediated throug h Òsalt sensorsÓ or sodium sensors in the brain (216). The central sodium sensing mechanism was proposed to be critical in salt sensitive hypertension because infusion of the sod ium channel blockers amiloride and benzamil into the cerebral ventricles successfully attenuated HTN development in salt -sensitive animal models (2, 169). The most sensitive osmoreceptors are localized in CVOs like the SFO, OVLT and AP. As noted earlier, these structures lack a blood brain barrier and therefore are exposed to alterations in 18 blood osmolality and hormone levels (e.g. AngII) (227). In response to increase salt and other signals that influence the SNS (like AngII), projections from the OVLT and SFO stimulate the median preoptic nucleus and thereby activate excitatory interneurons projecting to the PVN to cause sympathoexcitation as described earlier (18, 216). In summary, both circulating AngII and high salt intake salt can affect brain circuits that increase SNA and blood pressure vi a converging neural pathways that originate in the forebrain CVOs and ultimately include both the PVN and RVLM (176). This combined action may be operative in at least a subset of human patients with sa lt-sensitive essential hypertension. A primary goal of the research in this dissertation was to employ an experimental animal model of AngII -dependent, salt -sensitive hypertension to develop a more detailed understanding of the brain signaling mechanisms r esponsible for the increased SNA and BP initiated by salt and AngII. 7. Angiotensin II -salt model of hypertension Our lab and others have previously characterized in detail an experimental model of HTN (see (176) for more details) produced by chronically infusing exogenous AngII into male Sprague -Dawley rats ingesting a diet containing 2% NaCl by weight (normal rat chow contains 0.4% NaCl). The HTN is very likely caused by multiple mechanisms (includ ing actions of AngII on the vasculature and kidney), but King et al. (127) demonstrated that generalized sympathetic outflow (measured using both plasma NE concen trations and whole -body NE spillover) was greater in rats infused with AngII and fed a 2%NaCl diet (AngII -salt) than in rats receiving either AngII or salt treatment alone. 19 As noted above, high salt diet typically reduces RAS activity but the study by Kin g et al. supports the idea that one possible mechanism of salt -sensitive HTN is increased SNA caused by inappropriately high AngII levels during high salt intake. Sympathetic effects on BP in the AngII -salt model also was assessed by measuring neurogenic p ressor activity from the acute fall in BP after eliminating all postganglionic sympathetic activity with a ganglion block ing drug; the results strongly support a role for SNA in the development of AngII -salt HTN (126). Surprisingly, however, measurement of regional NE spillover and chronic, direct microneurographic recordings of SNA revealed no changes in either lumbar or renal SNA in rats with AngII -salt HTN (175, 268) and neither renal nor lumbar sympathectomy affected AngII -salt HTN development (175). On the other hand, splanchnic denervation significantly attenuated AngII -salt HT (128) suggesting that increased splanchnic SNA was the major player in the neurogenic component of HTN in the model. Furthermore, it appears that both increased splanchnic vascular resistance (133) and decreased vascular capacitance (126) mediate the effects of splanchnic sympathetic activity on AngII -salt HTN. Finally, centra l sodium sensors have an important part in AngII -salt HTN, since blockade of brain sodium channels with benzamil attenuates HTN development in the model (unpublished data) . An advantage of using the AngII -salt model to study sympathetic mechanisms in the development of hypertension is that there is a clear pre -hypertensive phase, followed by a short ÒdevelopmentalÓ phase, and finally an ÒestablishedÓ phase, all occurring over a period of less than two weeks. Furthermore, neurogenic pressor activity during each of these phases can be assessed in relatively undisturbed, conscious animals using 20 telemetric measurement of BP and a ganglion-blocking drug as described earlier . Importantly, b ased on such analyses, the mechanisms causing AngII -salt HTN appear to dif fer during the developmental and established phases of the model . During the first 3-5 days of AngII infusion (the early developmental phase), non -neurogenic mechanisms seem to have the largest role in increasing BP, whereas during the late developmental a nd established phases of hypertension, neurogenic mechanisms become predominant (133). Thus, studying the AngII -salt model potentially allows for a more clear se paration of cause and effect when seeking brain mechanisms that initiate changes in BP as opposed to those that occur in response to HTN development. A hypothetical scheme for sympathetically mediated increases in BP in Ang II-salt HTN is shown in Figure 1 -2. 21 Figure 1 -2: Brain sites potentially involved in changes in sympathetic nerve activity and blood pressure in AngII -salt hypertension (AngII -salt HTN) . Changes in osmolarity due to high salt diet, and/or peripheral AngII levels, are sensed by the SF O and OVLT that then signal mainly through the MnPO, PVN and RVLM to cause increased splanchnic sympathetic nerve activity. The resultant increase in total peripheral resistance and decrease in vascular capacitance cause HTN. Not shown in the figure is the potential for modulation of SNA by baroreceptors activated by increased BP. SFO -subfornical organ, OVLT -organum vasculosum lamina terminalis, MnPO -median preoptic nucleus, PVN -paraventricular nucleus, RVLM -rostral ventrolateral medulla. 22 8. Eicosanoids Eicos anoids are a class of lipid mediators that carry information from one cell to another (35). These cellular messengers have various physiological and pathophysiological roles in feve r, inflammation, pain, sleep, gastrointestinal function, bone remodeling, allergic asthma, luteolysis and parturition (35). The term ÒeicosaÓ in Greek means 20 as eicosanoids are de rived from polyunsaturated fatty acids containing 20 carbons (Figure 1-3). They play a vital role in host defense against adverse conditions, e.g. protection against bacterial pathogens. The eicos anoids are part of a family of biologically active lipids de rived from the action of cyclooxygenases (COX) or prostaglandin synthases upon the twenty -carbon essential fatty acids or eicosanoids. Prostanoids can be further subdivided into three main groups, the prostaglandins, prostacyclins and thromboxanes. Prostag landins (PGs) are the major eicosanoids studied in detail beginning with work by Von Euler in 1936, who first reported prostaglandins as vasodilators and muscle stimulating agents derived from the prostate glands of humans and experimental animals (243). PGs have been used clinically in patients with congenital ductus arteriosus to maintain duct patency until surgical correction (44). P Gs regulate gastric secretion, increase uterine contraction and cause labor induction (64). Receptors for PGs are a class of 7 -trans -membrane domain, G -protein coupled pro teins with strong intra -class structural similarities (29). 23 Figure 1 -3: Eicosanoid biosynthesis and receptors: Eicosanoids are formed by a sequence of enzyme reactions after cell activation. First, cy tosolic phospholipase A2 cleaves arachidonic acid (AA) from membrane phospholipids. AA is converted to prostaglandin H2 by the membrane bound cyclooxygenase (COX) enzyme and lipoxygenase converts AA to leukotrienes that act on the leukotriene ( LT) receptor . PGH2 is further isomerized by prostaglandin synthases (PGIS, microsomal PGES1/2, cytosolic PGES, and PGDS) and thr omboxane (TX) synthase to form PGI2, PGE2, PGF2 $ , P GD2 and TX, which act on their receptors IP, EP1 -4, FP, DP1, DP2 and TP. 24 8.1. Synthesis, signaling and function of eicosanoids Eicosanoids function as local hormones. They are not stored intracellul arly, but instead are synthesized (as illustrated in Figure 4) Òon demandÓ. Upon cell injury or other stimuli like binding of ligands such as bradykinin or angiotensin II, the rate -limiting enzyme phospholipase A2 translocates to the nuclear envelope, end oplasmic reticulum and Golgi apparatus to release arachidonic acid (AA) from membrane phospholipids (39). Among the PLA2 isoforms, Type IV cytosolic PLA2 (cPLA2) is the major player involved in eicosanoid synthesis because cells that do not contain this enzyme cannot synthesize eicosanoids. Oxygenation of AA by the rate -limiting enzyme COX (with two isoforms: COX -1 and COX -2) forms intermediate precursors viz., the pr ostaglandins PGG2 and PGH2 with a half -life of 3 minutes (35, 209). COX -1 and COX -2 are integral membran e proteins of the endoplasmic reticulum (ER) and nucleus. Subgroups of prostanoid synthases (acting on PGH2) and lipoxygenases (acting on AA directly) produce PGI2, PGE2, PGF2 $ , PGD2 and TxA2 and leukotrienes (LT). Products of this pathway have a very shor t half-life (20 -30 seconds) (35). The requirement for two distinct COX enzymes is not fully understood. C OX-1, a 70kD protein is found ubiquitously in all tissues and acts as a co nstitutively active enzyme (209). The other isoform , COX-2 (72kD) , produces prostaglandins in response to inflammatory stimul i. Separate genes encode the two forms of COX but the two forms of COX exhibit structural homology with almost identical catalyti c sites. Other differences between COX -1 and COX -2 include varied subcellular localization, substrate specificity and the manner in which they are coupled to upstream and downstream 25 enzymes (241). In 1994, Picott, Loll and Gravito established the three -dimensional structure of COXs (138). The small differences in the catalytic domains of these enzymes have been exploited for development of isoform specific inhibitor s. The COX -1 isoform in the endoplasmic reticulum generates PG when there are high levels of AA substrate available to act on. The PG product is then rel eased in an autocrine or paracrine manner to signal downstream through numerous cell -surface G -protein coupled receptors. The main role of the COX -1 products is to maintain homeostasis and hence the name Òhousekeeping protein.Ó COX -1 expression in kidney, stomach, vascular endothelium, and blood platelet support s the idea that it is expressed as a signaling mediator in tissues with specialized needs. In platelets the COX-1 product thromboxane is a potent vasoconstrictor and cause s platelet aggregation (209). COX-2 on the other hand is an inducible enzyme in most tissues . However the kidney and brain COX -2 is constitutively active. Stimuli like cytokines, growth factors and tumo r promoters , produced as part of an inflammatory response , lead to eicos anoid s ynthesis from even low concentrations of the substrate AA in cell types involved in inflammatory responses like macrophages and monocytes. The prostanoids then help resolve the inflammation. Listed below are some important eicosanoids (Figure 1 -4) along with their important biological roles. 26 8.1.1. Prostaglandin E2 : Prostaglandin E is synthesized from the precursor PGH2 by prostaglandin E synthases cytosolic -PGES (c -PGES) and microsomal -PGDS (m -PGES -1 and m -PGES -2). Cytosolic PGES is constitutively expr essed in various tissues, however microsomal PGDS is a perinuclear protein. PGE2 has a very short half -life of 30 seconds. PGE2 acts locally by binding to its receptors EP1, EP2, EP3 or EP4. EP1 is a Gq coupled receptor that increases IP3 by calcium signal ing. EP2 and EP4 are Gs coupled receptors that increase cAMP. The EP3 receptor is a Gi coupled receptor t hat decreases cAMP and increase calcium. PGE2 has proinflammatory effect s: for example, LPS -induced PGE2 production causes harmful effects on neurons a nd enhances pain sensations. On the other hand, PGE2 blocks LPS -induced cytokine synthesis and neuroinflammation providing evidence for an anti -inflammatory effect (194). 8.1.2. Prostaglandin D2 : PGD2 is the major prostaglandin synthesized in the brain. Its precursor PGH2 is acted upon by lipocalin type PGD2 (L -PGDS) or hematopoietic PGD2 (h -PGDS). L -PGDS is present in cells of a variety of tissues especially in the central nervous system. H -PGDS is predominant in the cytosol of immune and inflammatory cells. The CP, leptomeninges and oligodendrocytes synthesize L -PGDS in the CNS . It is unique in that it functions not only as a synthase to form PGD2 but also acts as a carrier for lipophilic molecules (235). L -PGDS is secreted into the cerebrospinal fluid and is also known as beta trace protein (172). Other sites of PGD2 synthesis are mast cells and leukocytes (dendritic cells and T helper 2 cells). PGD2 is metabolized into PGJ2 and 15d -PGJ2. PGJ2 is then converted into 15-deoxy-%12, 14 - 27 PGJ2 and %12-PGJ2. 15 -deoxy-%12, 14 -PGJ2 acts as a ligand for the nuclear receptor peroxisome proliferator -activated receptor gamma ( PPAR&) and inhibits nuclear factor kappa light chain enhancer of kappa light chain enhancer of B cells ( NFKB) (195). A proinflammatory effect of PGD2 is mediated through the Gs -protein coupled DP1 receptor that increases cAMP; and the DP2 receptor, a Gi coupled receptor that decreases cAMP and increases intracellular calcium (194). PGD2 is involved in type I acute allergic responses, pain perception and regulating physiological sl eep (95, 194, 233). 8.1.3. Prostaglandin I2 : PGI2 i s synthesized by prostacyclin synthase (PGIS) that is co-localized with COX -1 in the endoplasmic reticulum and constitutively expressed by endothelial cells (194). PGI2 is a potent vasodilator and inhibits platelet aggregation. PGI2 is metabolized by non -enzymatic hydrolysis to form the inactive product 6-keto -PGF 1$. PGI2 acts through Gs, Gi or Gq coupled receptors to decrease cAMP and increase IP3 signaling. R elevant IP receptors are localized in kidney, liver, lung, platelets, aorta and heart (210). 8.1.4. Prostaglandin F2": PGF synthase acts on PGH2 to form PGF2 $ . It is required for normal parturition (219) and plays an important role in ovulation and contraction of uterine s mooth muscle cells. The PGF2 $ receptor FP is a Gq coupled receptor and its deletion reduces blood pressure and atherosclerosis (270). 15 -keto -dihydro -PGF2 $ is 28 the ma in metabolic product of PGF2 $ and can be detected in the plasma and urine as an indicator of PGF2 $ produced in response to acute or chronic inflammation. 8.1.5. Thromboxane: Thromboxane A2 is synthesized by thromboxane synthase mainly in mast cells and ma crophages. Later TxA2 is converted into inactive TxB2. The actions of TxA2 are through the Gq, G12/13 or small G protein coupled receptor TP that increases cAMP, intracellular calcium and IP3 signaling. TxA2 promotes platelet adhesion and smooth muscle con traction. TP receptor deletion has been linked to lower blood pressure response but also bleeding defects (225). 8.1.6. Non-steroidal anti -inflammatory drugs Drugs that inhibit the synthesis of the eicos anoids are called non-steroidal anti -inflammatory drugs (NSAIDS) . In 1899 the first NSAID , aspirin , was introduced ; it revolutionized the drug industry by relieving patients of pain and inflammation (93). NSAIDs can be differentiated based on their ability to selectively inhibit COX -1 versus COX-2. The drugs that have much higher potency against COX -1 are so-called Ò COX-1 selectiveÓ inhibitors like naproxen and flurbiprofen. The selective COX-2 inhibi tors include celecoxib and rofecoxib . Aspirin , a Ònon-selective COX inhibitor Ó actually produces more COX -1 than COX -2 inhibition (Cox -2/COX-1 IC50 ratio of 4.3 , which is high and similar to COX-1 specific NSAIDS) (93, 248). 29 Figure 1-4: Prostaglandin synthesis and actions . Cytokines, growth factors or trauma triggers translocation of t ype IV cytosolic phospholipase (cPLA 2) and release of arachidonic acid from membrane lipids . M etabolism by COX -1 or COX -2 forms intermediate PG H2 that is converted to PGE 2, PGD 2, PGF 2$, PGI 2 (prostacyclin) and TxA2 (thromboxane). The synthesized prostaglandins then exert autocrine or paracrine actions by act ing on prostaglandin transporter (PGT) or other carrier s. The effector specific effect is caused by interaction with different receptors like EP1, EP 2, EP 3, E P4, DP1, DP2, FP, IP, TP. CO=cumulus oophorus cell. VSMC=vascular smooth muscle cell. [Adapted f rom C D Funk Science 2001; 294: 1871-1875]. 30 8.2. Eicosanoids in the brain Next to adipocytes , the brain contains the hi ghest amount of lipids: around 36 -60% (221). Arachidonic acid (AA) and docasohexanoic acid (DHA) are the two main polyunsaturated fatty acids present in the brain , located in the sn -2 position of the phosphoglycerides of neural cell membrane s. Ac tivation of phospholipase A2 liberates AA from those phosphoglycerides. In the brain c PLA2 is expressed in astrocytes (220), endothelial cells of cerebral blood vessels (213), glial cells, pia matter and choroid plexus (135). Cyclooxygenases (COX -1 and COX -2) metabolize free AA to form eicos anoids by synthetic pathways similar to those described earlier in this chapter. COX-1 in the brain is expressed constitutively in hippocamp al CA3 and CA4 neurons, granular neuron s of the neocortical layer, microglial cells in the white and grey matter in all brain regions (267), spinally project ing PVN neurons (264) and SFO (22). On the other hand, the inducib le enzyme COX-2 is expressed in neurons in neo -cortices and allo-cort ices, hippocampus, amygdala, dendritic spine s involved in synaptic signaling , PVN neurons, locus coeruleus neurons and astrocytes surrounding the blood vessels (123). Both AA and DHA can be metabolized into leukotrienes and hydroxyl de rivatives by lipoxygenase, whereas AA can form lipoxins by the action of 5 -lipoxygenase (224). In the brain, 5 -lipoxygenase is expr essed in neurons and glial cell; however, the brain microvessel endothelium is devoid of 5 -LOX (272). COX-1 and COX -2 catalyze the formation of PGH2, the intermediate metabolite of AA. PGH2 is further transformed into PGE2, PGD2, PGI2 and TxA2 by various synthases in the brain . PGE2 is synthesized by three different PGE synthases viz., the constitutive 31 cytosolic prostagla ndin E synthase (cPGES) ; membrane bound mPGES -2 and the inducible mPGES -1. In rat brain, neurons and astrocytes of the ipsilateral cortex express mPGES -2 and SFO neurons express cPGES (22, 201). Rat and human brain L-PGDS is expressed in arachnoid trabecular cells (158), oligodendrocytes, leptomeninges and choroid plexus (232) and it synthesizes PGD 2, the most abundant eicosanoid in the brain . The isoform hematopoietic PGDS is widely distributed in the periphery, and to some extent in the brain in the microglial cells (160), T -helper2 cells, antigen presenting cells and the mast cells (117). The eicosanoid prostacyclin synthesizing enzyme prostacyclin (PGI2) -synthase is shown to be present both rat s and humans in the cortical neurons, purkinje cells of the cerebellum but not in the glial cells (152). In addition to th ese, PGI2 synthase was also localized in blood vessels, microglial cells, oligodendrocytes and hippocampal neuron (207). The thromboxane synthesizing enzyme thr omboxane synthase in the ovine brain is localized in the neuronal cell body and axons of NTS and RVLM along with ventricular ependymal cells, the cerebellar peduncle in the rostral pons and purkinje fibres (110). Together, the products of COX pathway called the eicosanoids take part in critical neural functions. For example , PGE2 has been linked to physiological long -term potentiation, spatial learning and synapt ic plasticity. Other critical roles of eicosanoid s in the brain include resolution of inflammation, anti -inflammatory and neuroprotective function s (224). PGD2 is one of the several known humoral sleep inducing factors: it stimulates DP1 receptors localized in the leptomeninges of the basal forebrain causing release of adenosine that in turn acts as a paracrine sleep promoting signaling molecule (109). A dose dependent increase in sleep time occurs with increase s in brain PGD2 32 levels. Studies w ith the PGD2 synthesis inhibitor selenium chloride, as well as tran sgenic and knockout mice, demonstrate the critical role of PGD2 as a sleep inducer (27, 94). When there is injury to brain tissue, free radicals (reactive oxygen species) cause damage by oxidation of lipids. Lipid peroxidation can result in neurodegenerative diseases (224). Eicosanoids take part in the process of neuroinflammation as seen in diseases like Alzheimers, multiple sclerosis and epilepsy. The p athogenesis of neuroinflammation is characteriz ed by neural injury that causes migration of microglia and adhesion of leukocytes to the site of i njury. Microglia produces cytokines like tumour necrosis factor -$ and inter leukin -1' along with chemokines and vascular cell adhesion molecule at the site of injury. These cytokines an d chemokines then increase cPLA2 activity (54). This c PLA2 activity is the source of increased release of pro -inflammatory eicosanoids . The importance of eicosanoids in neur odegenerative diseases is shown by the fact that long -term use of NSAIDs has been proven beneficial in delaying the onset and slowing th e progression of the disease (74). COX-2 mediated synaptic signaling further elucidates its role in inflammation especially in neurons (265). Effectiveness of COX inhibitors like aspirin and ibuprofen in reducing fever, sleep iness and anorexia (the Òsickness syndromeÓ) during systemic inflammation show that eicosanoids play a critical role during inflammation by signaling in the br ain (202). In rat models PGE2 is suggested to act on the EP3 receptors localized the median preoptic nucleus neurons to cause fever (167). 33 8.2.1. Role of eicosanoids in hypertension The eicosanoids have both pro -hypertensive and antihypertensive effects (115). These lipid mediators are produced and act in multiple tissues like kid ney, brain, blood vessels and immune system where they can influence blood pressure (168). Our labÕs interest in the role of eicosanoids in hypertension originated in part from data on human HTN patients taking NSAIDs: most commonly an increase in BP was observed (11, 26). This suggests that the net effect of COX products on BP in patients with HTN is depressor. Interestingly, however, the effects of COX inhibitors on BP seem to vary depending on the type of HTN studied. For example, in normotensive and hypertensive patients with a stimulated renin angiotensin system, COX inhibitors lower blood pressure (113) . NSAID treat ment also attenuates HTN development in human renovascular hypertension (111), and in renin -dependent animal models like two-kidney one -clip hypertension (214), AngII hypertension (168), and aortic coarctation induced hypertension (139). Therefore, it appears that the net effect of COX products in angiotensin -dependent HTN (unlike other forms) is to increase BP. Since a large fraction of human patients with HTN show a significant fall in BP when treated with renin -angiotensin system inhibitors (i.e. have angiotensin -dependent HTN), even when they have no evidence of an ÒactivatedÓ renin -angiotensin system, a better understanding of how COX products regulate BP in angiotensin -dependent HTN is needed. Some earlier work by others has address ed that topic, as described in t he next section. 34 8.2.2. Effect of eicosanoids on AngII HTN development Are eicosanoids one cause of AngII mediated HTN? COX products stimulate renin secretion, and COX inhibitors reduce plasma renin activity (62). This may explain why COX inhibitors blunt HTN development in some renin -dependent animal models like the aortic coarctation model (114), and two kidney one clip model (214), i.e. eicosanoid synthesis inhibition with NSAIDs likely lower BP by decreasing plasma renin activity. Aside from an action on renin secretion (and thus AngII formation) as a mechanism for eicosanoid effects, many studies have examined the role of eicosanoids in HTN caused by chronic administration of AngII itself (AngII HTN), thus bypassing any influence of renin. The most common approaches have been with the use of COX inhibitors (22, 57, 211) and COX knockout mice (22, 188, 260). T he studies have yielded contradictory conclusions, possibly because they employed different treatment regimens, treatment durations and methods of measuring blood pressure. Furthermore, the relative importance of COX -1 versus COX -2 in AngII HTN is disputed (22, 188, 211). Two main mechanisms have been proposed to explain pro -hypertensive effects of various eicosanoids in AngII HTN. Early work on this topic is well summarized by Nasjletti (119). He suggested that systemic and renal vasoconstrict ion induced by Tx A2 and PGH2 played a critical part in AngII - and AngII -salt HTN . Wilcox and colleagues (70, 124, 129) also found that TxA2 induced renal vasoconstriction played an important role in AngII HT N. Interestingly, COX blockade during AngII HTN in dogs impaired hypertension development, but actually augmented renal sodium retention (76), so COX products donÕt seem to exert a pro -hypertensive effect in AngII HTN by impairing renal 35 sodium excretion. Finally, a limited number of investigations have suggested a link between eicosanoids and increased SNA and neurogenic pressor activity in AngII HTN. The first support for this idea came from Luft and coworkers who reported an increase in directly recorded splanchnic SNA that was associated with increased eicos anoid synthesis in a chronic AngII HTN model in rats (143). Later, WilcoxÕs group (71) showed increased neurogenic pressor activity in rats made hypertensive by chronic infus ion of a TxA2/PGH2 receptor agonist. Neither of these studies provided clear evidence, however, on specific tissue targets where eicosanoids might act to increase sympathetic regulation of BP. 8.2.3. Eicosanoids may increase BP and/or SNA by actions in th e brain Studies with acute ICV administration of various eicosanoids into brain showed that PGE2 and PGD2 cause pressor response s that require sympathetic nervous system activation . Similarly, stimulation of TxA2/PGH2 receptors in the brain inc reases BP (70). These finding provide the basis for the idea that brain eicosanoids may regulate BP by affecting SNA. Interestingly, the pressor response to ICV PGE2 was abolis hed by lesion of the median eminence (an important link between SFO, OVLT and PVN) (104) suggesting th at Òsalt sensorsÓ may be involved in eicosanoid mediated increase s in sympathetic nerve activity and BP. Lesion s of both SFO and OVLT ha ve been found to reduce AngII-salt HTN in rats (30, 177). Very recently, Cao et al reported that COX -1 inhibition block s AngII HTN a nd reduces SNA in mice by reducing PGE2 formation and therefore activation of EP1 receptor s in the SFO (22). PGE2 induced ROS production (247) in the SFO was proposed to mediate the increase d SNA and BP in AngII HTN 36 (275). Collectively, these observations strongly suggest that angiotensin signaling in the brain may include recruitment of sympathoexcitatory and pro -hypertensive mechanisms mediated by COX-derived eicosanoids. Brain eicosanoids also activate SNA in other conditions like heart failure and stress, and studies on these conditions may provide insights into the specific brain pathways involved. For example, in a rat model of heart failure, PGE2 was reported to act directly on PVN neurons to increase SNA (269). Stress -related corticotropin r eleasing factor (CRF) signaling in the brain also brings out an intriguing connection between brain eicosanoids and SNA. Katafuchi et al has reported that ICV CRF or PGE2 increase splenic sympathetic nerve activity. The PGE2 mediated increase in SNA was bl ocked completely when the rats were pretreated with a CRF antagonist indicating a sequential signaling of CRF followed by PGE2 in causing pressor effect in rats (120). Furthermore, levels of CRF in the cerebrospinal fluid have been strongly correlated with those of L-PGDS in obese humans (43). A role for COX in sympatho excitatory CRF signaling in the brain was suggested by the finding of increased COX1 and COX2 expression in pre-sympathetic PVN neurons by Yamaguchi et al (264). These same neurons were found to activate sympathetic pre -ganglionic neurons specifically in the celiac and stellate ganglia (239). With the reports of success ful attenuation of AngII -salt HT N by celiac ganglionectomy, it is possible that the COX expressing PVN neurons play pivotal role in increased splanchnic sympathetic nerve activity in AngII -salt HT N (128). 37 8.2.4. Angiotensin II, neuroplasticity and brain eicosanoids The brain can exhibit long -term ÒadaptationÓ in response to even brief stimuli by Òexperience -dependent plasticity causing long -lasting, functional an d structural changes in brain circuits Ó (262). Interestingly, it has long been known that even a brief increase in circulating AngII levels can induce a long -term s ensitization to effects of AngII on the brain that are pro duced days or weeks later; for example, stimulation of salt appetite (199). Recently, Johnson and colleagues showed that transient short -term exposure to circ ulating AngII also could accentuate or ÒsensitizeÓ later (at least one week) development of AngII -HTN at least in part by brain mechanisms (262). Others also have shown that transient increases in circulating AngII can produce salt -sensitive hypertension later in life without the need for further exposure to AngII (107). Gabor and Leenen (69) recently summarized evidence that sympathoexcit ation in AngII HTN involves both very rapid and very slow changes in brain cardio -regulatory pathways. Therefore, it is of great interest that brain eicosanoids have been shown to participate in long-term adaptation of neuronal pathways as part of the ove rall Òstress responseÓ (67). 38 9. Central hypothesis Increased sympathetic activity and hypertension development caused by chronic infusion of angiotensin II in rats o n high dietary salt intake are mediated by eicosanoid signaling in specific cardio -regulatory brain regions (See Figure 1 -5) Specific Aim 1 : Test the hypothesis that AngII -salt HTN is associated with increased transcription of eicosanoid related genes in established cardio -regulatory brain regions during the early stages of HTN development. Specific Aim 2 : Test the hypothesis that COX products in the brain during the early stages of HTN development contribute to increased SNA and BP in AngII -salt HTN. Spec ific Aim 3 : Test the hypothesis that increased brain L -PGDS expression during early stages of HTN development contributes to elevated SNA and HTN. Specific Aim 4 : Test the hypothesis that PGD2 signaling through the DP1R causes increased SNA and BP in AngII -salt HTN. 10. Significance Increased SNA contributes to human hypertension . Understanding how specific eicosanoids act on the brain to increase SNA and BP would help fill a gap in our knowledge of how dysregulation of SNA occurs in hypertension. The pro posed work could help identify new therapeutic strategies for treating HT N and preventing cardiovascular disease. 39 Figure 1-5: Hypothesis and specific aim : Chronic infusion of angiotensin II in rats on high dietary salt intake increase SNA and hypertens ion by altering eicosanoid signaling in brain. Abbreviations and acronyms are described in the text. CG = celiac ganglion 40 CHAPTER 2 MATERIALS AND METHODS 41 1. Animals Male Sprague Dawley rats weighing 225 -275g were obtained from C harles River laboratories (Wilmington, MA) and housed in groups of 3 prior to experimentation. The rats were maintained in light - and temperature -controlled animal rooms and were allowed free access to food and water. The daytime light cycle was from 6:30a m to 6:30pm. After the surgical procedures, rats were housed individually in cages with cage tops containing high salt (2% NaCl) or normal chow diet (Research Diets, New Brunswick, NJ) that was provided throughout the experimental period along with ad -lib distilled water. All procedures were performed in compliance with the NIHÕs Laboratory Animal Care and Use guidelines and after approval from the Institutional Animal Care and Use Committee at Michigan State University. 2. General anesthesia and post -operative analgesia Mini-osmotic pump and telemeter implantation surgeries were performed under isoflurane anesthesia. The rat was placed in an anesthetic chamber and breathed a mixture of 2 to 4% isoflurane in oxygen at a flow rate of 2.0 L/min for induction . The rats were then placed in a surgical area with nose cone and heated pad and anesthesia was maintained with a mixture of 0.5 -2% isoflurane and oxygen at a flow rate of 2.0 L/min. Intracerebroventricular (ICV) cannula implantation surgery was performed using the injectable anesthetics ketamine (75mg/kg body weight) and xylazine (7.5mg/kg body weight), both administered intraperitoneally (ip) in combination . 42 The rats were given post -operative antibiotic and analgesics for a period of 24 -48 hours depending on the surgery. Enrofloxacin (Baytril, Bayer Healthcare, Kansas), intramuscularly ( im) at 5mg/kg body weight was used as the antibiotic. Carprofen (Rimadyl, Pfizer, NY), subcutaneously ( sc) at a dose of 5mg/kg body weight was used as for analgesi a. 3. Radiotelemetry implantation The surgical site was prepared using chlorhexidine scrub. Under general anesthesia as described above, a 2mm lateral midline incision on the thigh and the femoral artery was exposed. The tip of a radio -telemeter ( TA11PA -C40, Data Science International (DSI) , St Paul, MN) catheter was introduced (approximately 4cm) through the left femoral artery into the abdominal aorta just cranial to aortic bifurcation. The body of the transmitter was placed in a subcutaneous pocket along the caudo -ventral abdomen. This device recorded blood pressure and physical activity for 10 seconds at every 10 -minute interval. Afte r surgical recovery, 24hr/day data values were transmitted via radio signals from the animals to DSI plate receiver positioned under the animalsÕ cages and then to a computer data acquisition program (Dataquest ART 4.1, Data Sciences International, St. Paul, MN) for analysis. Variables recorded include; systolic, diastolic and mean (calculated) arterial pressure; heart rate (calcu lated); and activity level. 4. Mini-osmotic pump implantation Mini-osmotic pumps (2ML2 and 2004 models, Alzet, Cupertino, CA) (Figure 2 -1) were used for drug delivery in a chronic fashion with a constant rate of infusion. 43 Figure 2-1: Osmotic mini pump Osmotic pumps work on the principle of osmotic pressure difference between the inner chamber of the pump and the tissue where it is implanted. The pumps osmotic layer helps water to flux inside the pump through the semipermeable membrane. This flux causes the agent of interest (angiotensin II and other drugs) to be pumped out of the inner chamber at a constant and predetermined rate. The 2ML2 mini -osmotic pumps had a flow rate of 0.083333 ( l/min, and when filled with angiotensin II in powdered form disso lved in physiological saline (Sigma, St. Louis, MO) at appropriate concentrations produced a drug delivery rate of 150ng/kg body weight/minute. Mini -osmotic pumps filled with angiotensin II were incubated at 37 ¡C for 4 hours or overnight before implantatio n to get a constant release of the drug from the day of implantation. The surgery was performed under general anesthesia (as described above). After making an incision in the dorsal region of the neck, a small subcutaneous pocket was made on the back of th e rat to place the pump. The incision was closed with 5 -0 silk, non -absorbable sutures. 44 5. Intracerebroventricular brain cannula implantation After rats were anesthetized with ketamine and xylazine as described earlier, they were placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). An incision was made in the skin on top of the skull. Cotton swabs were used to clean the surgical site to expose bregma on the skull surface. The coordinates relative to bregma, anterioposterior = -0.8mm, m ediolateral = -1.5, and ventral = 4.2mm, were used to position the ICV cannula on the right side of the ratÕs skull. Stainless steel screws were attached to the skull and dental acrylic (Dentsply International inc., York, PA) was used to secure the cannula to the skull. Rats were housed in individual cages after surgery. At the end of the experiment, cannula placement was confirmed in each study. In case of using the brain for mRNA or western blot study, visual confirmation of the ICV site was done and in o ther cases either dye was injected or sections were stained to confirm the ICV cannula placement. 6. Femoral catheterization A polyurethane catheter (RFA -01, Strategic Applications Inc., Chicago, IL) was used for arterial catheterization. Under general a nesthesia with isoflurane, a midline incision on the thigh was made and the catheter tip was inserted into the abdominal aorta through the left femoral artery. The saline -filled catheter was connected to a Power Lab recorder (AD Instruments, Inc., Colorado Springs, CO) for blood pressure and heart rate measurements. 45 7. Animal euthanasia Rats were euthanized by injecting Fatal Plus¨ ( Pentabarbital, 390mg/kg, ip ) followed by decapitation using guillotine . Rongeurs were used to quickly remove the brain out carefully by cutting the occipital bone and the skull attachments. 8. Microdissection of rat brain Using a Microm HM525 cryostat maintained at -150C, 500 ( m thick serial brain sections were obtained. The sections were then transferred to a cold stage maint ained at the same temperature. The paraventricular nucleus (PVN), subfornical organ (SFO), organum vasculosum lamina terminalis (OVLT), rostral ventrolateral medulla (RVLM) and nucleus tractus solitarius (NTS) were microdissected using the Palkovit sÕ micr odissection technique (179) with a 500 ( m punch. A rat brain atlas (179) was used to identify and using a stainless steel needle of 1mm diameter, punch out the nuclei of interest. The isolated brain tissue was stored at -800C for future analyses. 9. Polymerase chain reaction ( PCR) array an d quantitative real time -PCR RNA was extracted from brain punches using a GenElute Mammalian Total RNA Miniprep Kit (Sigma Aldrich, St. Louis, MO USA). To evaluate the quality of extracted RNA, a Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, D E, USA) was used and samples with low quality RNA (assessed by OD 260/280 ratio outside the range of 1.8 and 2.1) were excluded from further analysis. Reverse transcription for cDNA synthesis was performed with an RT 2 first strand kit (SABiosciences, Fred erick, MD) using 100 ng of RNA from the samples. 46 9.1. PCR array: A custom Rat RT 2 profiler TM PCR array (SABiosciences, Frederick, MD) with 21 eicosanoid related gene primers (Table 2-1) in a 96 well plate format was used. The PCR reaction mixture for th e 96 well plates included 102 ( l diluted cDNA synthesis reaction, 1048 ( l RNA grade water, and 1150 ( l RT 2 SYBR Green master mix. PCR reactions were carried out on ABI 7500 Fast real time PCR system (Applied Biosystems, Carlsbad, CA) using an RT 2 SYBR Gre en master mix (SABiosciences, Frederick, MD). The PCR conditions included a holding stage (95 0C for 10 mins) and cycling stage (95 0C for 15 sec, 60 0C for 1 minute and 72 0C for 35 seconds) followed by a melt curve to confirm the specificity of the amplified products. To control for DNA contamination, no template control was included. The Ct values were normalized to beta actin . The house keeping genes in the array were Lactate dehydrogenase (ldha), Ribosomal protein13a (Rpl13a) and '-actin. The fold change was calculated by 2 %%CT method. Table 2 -1: 96 -well custom PCR array template for eicosanoid related genes Number Gene Gene symbol Gene RefSeq # 1 PG-endoperoxide synthase 1 (COX -1) Ptgs1 NM_017043 2 PG-endoperoxide syntha se 2 (COX -2) Ptgs2 NM_017232 3 Arachidonate 5 -lipoxygenase Alox5 NM_012822 4 Alox5 activating protein Alox5ap NM_017260 5 Arachidonate 12 -lipoxygenase Alox12 NM_001105798 6 Arachidonate 15 -lipoxygenase Alox15 NM_031010 7 Thromboxane A synthase 1 Tbxas 1 NM_012687 47 Table 2-1 (contÕd) 8 PGI2 (prostacyclin) synthase Ptgis NM_031557 9 PGE synthase 1(microsomal) Ptges1 NM_021583 10 PGE synthase 2 Ptges2 NM_001107832 11 PGE synthase 3 (cytosolic) Ptges3 NM_001130989 12 PGD2 synthase (brain) Ptgds NM_013015 13 PGD2 synthase 2 (hematopoietic) Ptgds2 NM_031644 14 Thromboxane A2 receptor Tbxa2r NM_017054 15 PGI2 (prostacyclin) receptor Ptgir NM_001077644 16 PGE receptor 1 (subtype EP1) Ptger1 NM_013100 17 PGE receptor 2 (subtype EP2) Ptger2 NM_031088 18 PGE receptor 3 (subtype EP3) Ptger3 NM_012704 19 PGE receptor 4 (subtype EP4) Ptger4 NM_032076 20 PGD receptor Ptgdr NM_001135164 21 PGD receptor -like Ptgdrl NM_001030643 22 Beta actin Actb NM_031144 23 Lactate dehydrogenase ldha NM_012583 24 Ribosomal protein13a Rpl13a NM_017008 9.2. Quantitative RT -PCR : To amplify and simultaneously quantify the DNA of interest we performed real time polymerase chain reaction or quantitative PCR reactions. It was carried out on ABI 7500 Fast real time PCR syste m (Applied Biosystems, Carlsbad, CA) using SYBR Green master mix (Cat. No. 330500, SABiosciences, Frederick, MD). The 48 PCR reaction mixture included 1 ( l cDNA, 10.5 ( l RNA grade water, 1 ( l primer and 12.5 ( l SYBR Green master mix. The PCR conditions were the following: holding stage (95 0C for 10 min) and cycling stage (95 0C for 15 sec, 60 0C for 1 minute and 72 0C for 35 seconds) followed by melt curve to confirm the specificity of the amplified products. 10. Cerebrospinal fluid collection The skin on the head was shaved and surgically prepared with 3 chlorhexadine scru bs and 3 alcohol scrub s. The surgical site was draped with sterile gauze and a small incision was made on the skin above the atlanto -occipital region. The head was placed in a stereotaxic apparat us and flexed downwards, so that it made a 45 ¡ angle to the ratÕs body. One end of a micro -hematocrit capillary tube was pulled with a pipette puller to get a pointed tip with 0.5mm diameter for insertion into the cisterna magna to collect the CSF. The ot her blunt end of the micro -hematocrit capillary tube was attached to a 7-10 cm length of Tygon¨ tubing. An 18G needle with a blunt tip was inserted into the other end of the Tygon¨ tubing and the needle was attached to a 1cc syringe. The plunger of the sy ringe was disconnected before cerebrospinal fluid ( CSF ) collection to allow its free flow. Once the spinotrapezius muscles on the ratÕs back region were separated from one other at the midline using cotton swabs, the landmark for CSF collection at the atla nto-occipital junction was seen. The pointed micro -hematocrit capillary tube was inserted into the atlanto -occipital junction and clear CSF (75 to 100 micro liters) flowing through the tube was collected in an Eppendorf¨ tube by either allowing it to flow freely or pulling the plunger if necessary. 49 11. Western blotting The brains removed from rats anesthetized with pentobarbital sodium (50mg/kg ip) were extracted and then frozen on dry ice. Cryostat sections (500µm) from PVN, SFO, NTS, RVLM, CP and OVLT we re made and punched using the Palkovits micro -dissection technique. Tissues were lysed using lysis buffer [0.5 mmol/l Tris )HCl (pH 6.8), 10% SDS, and 10% glycerol] with protease inhibitors (0.5 mmol/l PMSF, 10 µg/µl aprotinin, and 10 µg/µl leupeptin). Prot ein concentration was measured using the bicinchoninic acid protein assay (Sigma, St Louis, Mo), which was run on SDS -PAGE 10% gel or 12% gel and transferred to a nitrocellulose membrane and later blocked for 1 h with Odyssey ¨ Blocking Buffer. Blots prepar ed were then incubated overnight (4¡C) with primary antibody Anti -COX-1 (1:500; Millipore, Temecula, CA) or Anti -tubulin (1:2,000 dilution; Millipore; Temecula, CA) or Anti -PGDs (Cayman chemicals, Ann Arbor, MI) or anti-PLA2 (Novus biologicals, Littleton, CO). After rinsing the membrane 4 times for 5 minutes with tris buffered saline with tween -20 ( TBS -T) and a final rinse in TBS, the blots were incubated using a secondary antibody for 1 h at room temperature. An Odyssey ¨ imager was used to visualize the ba nds. For a positive control, one lane in the membrane had the purified form of the protein of interest. 12. Cyclooxygenase activity assay The bifunctional enzyme COX exhibits both cyclooxygenase activity and peroxidase activity. The COX activity leads to conversion of the substrate arachidonic acid into PGG2 (hydroperoxy endoperoxide), and the peroxidase component converts PGG2 into PGH2, a precursor for the production of various other prostanoids through reduction 50 reactions. To determine the activity of the enzyme (both COX1 and COX2) in brain tissue, we used a cyclooxygenase fluorescent activity assay (Cayman Chemicals, Ann Arbor, MI). The assay utilizes the peroxidase component of the enzyme. The compound ADHP (10 -acetyl -3, 7 -dihydroxyphenoxazine), when added to tissue containing PGG2, forms a fluorescent compound resoruffin. This fluorescent compound is analyzed with an excitation wavelength between 530 -540nm and an emission wavelength between 585-595nm. The test system included purified ovine COX -1 as a positive control. Sample preparation: The rat brains were snap frozen on dry ice and stored at -80¡C. 500 ( m brain sections were prepared using a cryostat at -15¡C and brain punches from SFO, OVLT, PVN), RVLM, NTS and the third and fourth ventricle CP were homogenized in 50 ( l of lysis buffer (0.1M Tris HCl, pH 7.5, containing 100mM PMSF, leupeptin and aprotinin). The homogenized tissue was centrifuged at 10,000g for 15 minutes at 4¡C. The supernatant was then collected and frozen at -80¡C before assay. 13. Blood collection and high performance liquid chromatography For blood collection the rats were anesthetized using 0.5ml of Fatal Plus¨ (pentobarbital , 390mg/kg ip). 2 -3ml of blood was collected using a 22gauge needle from the abdominal aorta. The blood w as collected into 2ml Eppendorf¨ tubes and centrifuged at 14000 rpm for 10 minutes (Spectrafuge 16M Microcentrifuge). Plasma was collected from the top layer and placed in fresh Eppendorf¨ tubes without disturbing the pellet. HPLC was performed using a Waters 510 HPLC pump (Millipore), Waters "717 Autosampler (Millipore) and Waters " 996 photodiode Array Detector. Ketorolac 51 tromethamine (Cayman chemicals, Ann Arbor, MI) standards were prepared by dissolving 1mg in 1ml of saline. The working standard solutions (62.5pg/ ( l, 125pg/ ( l, 250pg/ ( l, 500pg/ ( l) were prepared by dilution of the primary stock solution with the saline. The plasma containing the compound was separated using Luna 5u C18 (5 µm particle size, 250 mm length x 4.6 mm I.D.) reversed -phase columns (Phenomenex, Torrance, CA). A 50:50 (v/v) mixture of acetonitrile containing 0.065% triethylamine and 1.65% glacial acetic acid (pH 4.3) was used as the mobile phase. The optimum flow rate of 1.0 mL/min was used for separation and the column was maintaine d at 20 -22oC. The detection wavelength was 314nm. 14. Immunofluorescent staining and confocal microscopy Previously flash frozen slides containing 10 ( m thick sections of SFO, OVLT, PVN, RVLM and NTS were used for immunofluorescent staining by the MSU hist opathology laboratory. Samples were cryoprotected and sections were placed in -80 ¼C for storage until staining. Upon removal from -80¼C, slides were air dried overnight at room temperature and were placed in Tris buffered saline (pH 7.5) for 5 minutes f or pH adjustment. Heat Induced Antigen Retrieval (ScyTek Laboratories, Utah) was performed at pH 6.0 in a rice steamer for 10 minutes at 100 degrees followed by an additional 20 minutes at room temperature. Slides were then blocked in 3% hydrogen peroxide in TBS for 10 minutes at room temperature. Autofluorescence was blocked using ammonium hydroxide and ethanol for 60 minutes at room temperature. Following these pretreatments, slides were loaded on a Dako Autostainer. Slides were blocked for non-specific protein with Normal Donkey Serum (Jackson ImmunoResearch, PA) for 30 52 minutes. An antibody cocktail of mouse anti -DP1 at a dilution of 1:100 (Thermo Scientific, MA) and rabbit anti -FOX/NeuN at a dilution of 1:1000 (Abcam, Cambridge, MA) in Normal Antibody D iluent (NAD) (Scytek Ð Logan, UT) was incubated for 60 minutes at room temperature. Secondary antibody cocktail of Donkey anti -Mouse Dylight 647 @ 1:500 and Donkey anti -Rabbit Dylight 405 @ 1:100 was made in 2% normal rat serum (Jackson ImmunoResearch, PA) . Cover slips were placed on the slides after covering them with ProLong Antifade. 15. C onfocal microscopy: An Olympus FluoView 1000 laser scanning confocal microscope was used to take 20X and 40X magnified images of the 10 ( m thick coronal brain sections. Differential interference contrast images and overlay images were taken to localize the receptors on the neurons and ependymal cells. 16. Liquid chromatography and tandem mass spectrometry (LC -MS/MS) Mass spectrometry is an analytical technique that separ ates ionic species based on the electrical charge and atomic mass using magnetic and electrical fields. The main components of a mass spectrometer are the inlet for the sample, ion source, mass analyzer and an ion detector. After solid phase extraction the sample in the mass spectrometer is sprayed through a thin capillary in an electric field to create charged droplets. These droplets then pass through a heated inlet gas in the mass analyzer. Here they are separated according to their mass to charge (m/z) ratio. MS/MS is a two -stage system, where in the first stage an ion is preselected, and in the second stage, fragments induced by collision with an inert gas like argon or helium are analyzed. 53 Preparation of standards: All standards were obtained from Cay man Chemicals, Ann Arbor, MI. a) PGE2 MS standard (Catalog No.10007211): 1mg/ml DMSO b) PGD2 MS standard (Catalog No.10007202): 1mg/ml DMSO c) PGJ2 MS standard (Catalog No.10007233): 500 ( g/100 ( l methyl acetate (5mg/ml) d) %12-PGJ2 lipid maps MS standard ( Catalog No.10007234): 100 ( g/100 ( l methyl acetate (1mg/ml) e) 15 -deoxy-%12, 14 -PGJ2 lipid maps MS standard (Catalog No.10007235): 100 ( g/100 ( l methylacetate (1mg/ml) f) Arachidonic acid: Item # 90010 500mg/ml 16.1. Solid phase extraction of lipid mediators from brain punches Solid phase extraction (SPE) was performed using OASIS max ( Elution Plate 30 ( m (Waters Corporation, Milford, MA). The steps followed for SPE were first, s ample prep aration (total volume of 500 ( l). To the sample (brain punches from regio n of interest), 18 * water (245 ( l), 0.01% butylated hydroxytoluene to prevent oxidation (1 ( l of 1% BHT (25mg BHT from Cayman chemicals, Ann Arbor, MI in 1ml methanol) and Internal standard (4 ( l of 500pg/ ( l of PGD2 -d4) were added. Then 250 (l of 4% phosphoric acid (Sigma Aldrich, St. Louis, MO) (4% H3PO4=2g in 50ml water) was added to disrupt the protein binding and help flow through the SPE cartridge. The sample was homogenized (Omi TH int. Ð for 5 -10 seconds) and centrifuged at ~12000g for 10 min (Spectrafug e 16M -14000rpm). The supernatant was separated and the 54 volume was made to up to 750 ( l. The OASIS max ( Elution cartridge was conditioned with 100 ( l methanol and equilibrated with 100 ( l water. For loading, 750 ( l of sample was loaded to the cartridge and a f low rate of 1 drop/ sec was maintained. [Note: The flow through the liquid was collected the first time to perform extraction efficiency]. For the first wash, 200 ( l of 2% ammonium hydroxide (2ml NH4OH+100ml water) at pH 10 -11 was used for removal the salt and charge the acidic analytes and lock them in the cartridge. [Note: The flow through the liquid was collected the first time to perform extraction efficiency]. For the second wash, 200(l of 1ml 100% methanol was used to remove the basic and the neutral i nterference. [Note: The flow through the liquid was collected the first time to perform extraction efficiency]. For elution, 50 ( l of a mixture of 100% methanol and 2% formic acid was used for eluting the lipid prostaglandins and AA from the cartridge. (0.2 27ml of 88% formic acid + 10ml methanol) . The eluted solution was placed in a screw cap vial (Part # 5182 -0715, Agilent Technologies, Santa Clara, CA) with a vial insert (Part # 5183 -2085) and blue screw cap (Part # 5182 -0717). The vials were stored at -80¡C before LC -MS/MS analysis using Waters Xevo TQ -S UPLC MS/MS (Waters Corporation, Milford, MA). The experiments were performed using the biochemistry RTSF facility at Michigan State University. Extraction efficiency= Samples with standard mixed before S PE X 100 Samples with standards spiked post SPE 55 The loading waste, washes were dried in vacuum and reconstituted in 50 ( l acetonitrile/water (1:1, V/V) for analysis. This determined the loss of analytes and was performed du ring the first experimental run. 16.2. Liquid-liquid extraction of lipid mediators from cerebrospinal fluid Liquid-liquid extraction was used for processing cerebrospinal fluid (CSF). 100 ( l of CSF sample was added to 100 ( l of 0.15M EDTA, 80 ( l of 45nM pho sphoric acid and 20 ( l of methanol. The liquid -liquid extraction was performed as described previously (72) where samples were incubated with 600 ( l of ethyl acetate. After vortexing thoroughly, two layers were obtained. The top layer containing the analytes was separated and 600 ( l of ethyl acetate was added. After vortexing, the top layer was removed and subjected to a gentle stream of nitrogen at 45 ¡C. The residues w ere reconstituted with 50 ( l of acetonitrile/ water/formic acid (20:80:0.0025, v/v, pH 4.0) and centrifuged for 2 minutes at 10,000g. It was transferred to a glass vial and stored at -80¡C prior to injection into the Xevo TQ -S for analysis. 16.3. Chromatog raphy Mobile phase C consisted of 0.1% formic acid and mobile phase D consisted of 100% acetonitrile. Compounds were separated using Ascentis¨ Express C18 HPLC column (10cm x 2.1mm, 2.7 ( m) maintained at a temperature of 40 ¡C. Initial conditions for separat ion were 1% mobile phase C and 99% mobile phase D at a flow rate of 0.3ml/min. The proportion of mobile phase C gradually increased to 99% for 14 minutes at a flow rate of 0.3ml/min. The total run time was 14 minutes. 56 16.4. Mass spectrometry Mass spectrometry wa s performed using an Acquity UPLC system coupled to a Waters Xevo TQ -S mass spectrometer (Waters, Milford, MA) in a n electrospray negative ionization mode. An extract volume of 10 ( l was injected into the UPLC system and eluted with a gradient mixture of 0 .1% formic acid and 100% acetonitrile. The mass spectrometer was optimized following tuning in multiple reaction monitoring (MRM) mode with a capillary voltage of 3.0 V, cone voltage of 35.0 V, source offset of 50.0 V, analyser LM resolution 1 of 2.93, HM resolution 1 of 14.60, ion energy 1 of 0.6, LM resolution 2 of 2.79, HM resolution 2 of 14.90, ion energy 2 of 0.9, desolvation temperature of 500¡C, desolvation gas flow of 600L/Hr, cone gas flow of 150L/Hr and collision gas pressure of 7 bar and collisio n gas flow of 0.20mL/min. The mass to charge ratio (m/z) of parent and daughter ions of individual compounds with their dwell time, cone voltage and collision voltage were as shown in table 1. Data was acquired and analyzed using Target Lynxª Software. Ca libration curves were constructed by plotting peak area ratio (standard to internal standard) versus the nominal concentration and were fit using least -square regression with 1/x weighting. 57 Table 2-2: Arachidonic acid and prostanoid mass to charge ra tio, dwell time and voltage for mass spectrometry analysis Compound Parent (m/z) Daughter (m/z) Dwell (s) Cone (V) Collision (V) 1 AA 303.2 259 0.125 45 16 2 Prostaglandin J2 333.1 271.1 0.1 33 16 3 %12-PGJ2 333.1 189 0.1 21 16 4 15-deoxy-%12, 14 -PGJ2 315.2 271.1 0.1 39 10 5 Prostaglandin E2 351.2 271 0.125 55 16 6 prostaglandin D2 351.2 203.1 0.125 55 22 7 Prostaglandin D2 -d4 355.2 319 0.125 33 10 17. Statistical analys es Within group differen ces were assessed by a one -way repeated measures ANOVA with post -hoc multiple comparisons using DunnettÕs procedure (GraphPad Instat 3). Between group differences were assessed by a two -way mixed design ANOVA and post -hoc testing at each time point was per formed using BonferroniÕs procedure to correct for multiple comparisons (GraphPad Prism 4). For the PCR array between group differences were analyzed by one -sample t -tests with a hypothetical mean as 1. StudentÕs t -test was used to analyze the western blot data. A p -value of < 0.05 was considered significant. All results are presented as means ± standard error of the mean (SE). 58 CHAPTER 3 CYCLOOXYGENASE -1 AND NOT CYCLOOXYGENA SE-2 INHIBITION ATTENUATES ANGIOTENS IN II-SALT HYPERTENSION AN D NEUROGENIC PRE SSOR ACTIVITY IN THE RAT 59 1. Introduction Angiotensin II is involved in the etiology of systemic h ypertension through various mechanisms. These include direct contraction of blood vessels and subsequent remodeling of their structure (83), reductions in renal sodium excretion (174), release of aldosterone (253), and activation of the sympathe tic nervous system (SNS ) (32, 58). McGiff proposed the idea that the renin -angiotensin system influences eicosan oid formation and reported the release of prostaglandin -like substances during infusion of AngII (151). But are eicos anoids involved in AngII HTN? This question has been addressed in numerous previous studies through the use of COX inhibiting drugs (22, 57, 211) and COX knockout mice (22, 188, 260). However these efforts produced contradictory conclusions, possibly because the studies employed quite varied treatment regimens, treatment durations and methods of measuring blood pressure. Also, has noted earlier, eicos anoids cause both pro -hypertensive and antihypertensive effects by acting on the kidneys, blood vessels, endocrin e organs and brain. In addition , the relative importance of COX -1 versus COX -2 in AngII HTN is disputed (22, 188, 211). As reviewed in Chapter 1, s ympathoexcitation is an important cause of HTN during AngII infusion, particularly in the setting of high dietary salt intake (127, 203). Both older (115, 143) and recent (22) experiments indicate that the sympathetic nervous system is activated by eicos anoids in experimental AngII HTN and/or that eicos anoids are involved in mediating neurogenic hypertension. But the precise mechanisms by which eicosanoids cause sympathoexcitation remain to be fully elucidated. Therefore, in this study I used radiotelemetric methods and AngII -salt HTN protocol to determine the role 60 of eicos anoids in causing neurogenic HTN. In addition, pharmacological tools were used to determine the relative contribution of COX -1 and COX -2 to sympathetic control of BP in AngII -salt HTN. 2. Experimental Protocols 2.1. Selective COX -1 or COX-2 inhibition in chronic AngII -salt hypertensive rats: Radiotelemetery- implanted rats, fed a high salt diet were used in this experiment. Following a 5 -7 day of surgical recovery period and 3 days of baseline blood pressure recordings, DMSO (vehic le) or a selective COX -1 inhibitor SC560 (10mg/kg ip) or a selective COX -2 inhibitor nimesulide (10mg/kg ip) were injected once daily for the remainder of the study. The doses for COX -1 and COX -2 inhibitor s were chosen based on previous reports (84, 242) of the use of 10mg/kg ip dose, successfully mimicking the effects of the widely used COX inhibitor aspirin as well as the successful reduction of eico sanoid levels in various tissues. Both SC560 and nimesulide are reported to cross the blood brain barrier (215) and therefore could act both peripherally and centrally. After 4 days of COX inhibition or DMSO injection, AngII or physiological saline infusion was initiated using a miniosmotic pump (2ML2, Alzet, Cupertino, CA). AngII was infused at the rate of 150ng/kg/mi n sc, for 14 days. MAP and HR were measured for the entire duration of the experiment. Animals were subjected to ganglionic blockade with hexamethonium (Sigma, St. Louis, MO; 30mg/kg ip) 10 days after starting AngII administration to assess neurogenic pr essor activity (228). T he fall in MAP was recorded 15 minutes after injecting hexamethonium, and the magnitude of that fall was used as an estimate of neurogenic pressor activity . 61 3. Results 3.1. Effect of selective COX -1 and COX-2 inhibition on chronic AngII -salt HTN: COX-1 inhibition with SC560 (Figure 1): In vehicle (DMSO ) treated rats (n=7) fed a high salt diet, MAP was 105 ±2 mmHg on pretreatment day 3 and gradually increased to 135±8 mmHg on day 14 of infusion. In the SC-560 treated group (n=10) , during the pretreatment period or during treatment with SC560 alone MAP was not different from that of the vehicle treated group . During AngII administration , however, MAP rose to only 117± 1 mmHg 14 days after AngII infusion in SC560 treated rats , and was significantly lower than that of the control group on days 6 -14 of AngII in fusion. 62 Day 12345678910111213141516171819202122Mean Arterial Pressure (mmHg) 100120140160DMSO (n=7) SC 560 (n=10) Control SC 560 (10mg/kg/day) intraperitonially Angiotensin II (150ng/kg/min) sc ##p<0.05 Figure 3-1: Effect of selective cyclooxygenase ( COX)-1 inhibition on mean arterial pressure (MAP) . Response to AngII in rats fed a high salt diet. Rats were treated daily with the selective COX -1 inh ibitor SC560 or vehicle. # =p<0.05 SC560 vs. DMSO as vehicle . 63 COX-2 inhibition with nimesulide (Figure 2): Treatment with nimesulide alone did not affect MAP relative to the vehicle (DMSO ) ip treated control group. Both groups had similar MAP during the control period. The vehicle treated group (n=5) on a high salt diet had a baseline MAP of 102 ± 2 mmHg on pretreatment day 3, which was similar the drug treatment groupÕs (n=5) day 3 MAP of 101 ± 1 mmHg . From day 4 to 7, nimesulide treatment did not ca use any change in MAP compared to the vehicle treated rats. After starting the AngII infusion, MAP in the vehicle treated group increased to 1 26 ± 6 mmHg at the end of infusion period. In the nimesulide treat ed group, MAP increased to 135 ± 12 mmHg. Theref ore, AngII infusion caused the same ma gnitude of hypertension in vehicle and nimesulide treated rats. The data suggests that the COX -2 inhibitor causes a slight increase in blood pressure in AngII -salt HTN rats. Neurogenic pressor activity: The maximum fal ls in MAP after ganglionic blockade on day 10 of AngII infusion in vehicle treated rats and in rats subjected to selective COX inhibition are shown in Figure 3. In experiments with SC560 (Figure 3A) , ganglion blockade decreased MAP significantly more in c ontrol (39 ± 4 mmHg) versus drug -treated (27 ± 4 mmHg) rats. In experiments using nimesulide (Figure 3B) , the fall in MAP with ganglion blockade was similar in control (58 ± 9 mmHg) and drug treated (62 ± 12 mmHg) animals. 64 Day 12345678910111213141516171819202122Mean Arterial Pressure (mmHg) 80100120140160DMSO (n=5) Nimesulide (n=5) Control Nimesulide (10mg/kg/day) intraperitonially Angiotensin II (150ng/kg/min)sc Figure 3-2: Effect of selective COX -2 inhibition on MAP. Rats were treated with the selective COX -2 inhibitor nimesulide or vehicle and were infused with angiotensin II subcutaneously. 65 DMSO SC 560 Peak fall in MAP (mmHg) -60 -40 -20 0A# DMSO NMPeak fall in MAP (mmHg) -80 -60 -40 -20 0B Figure 3-3: Depressor response to ganglionic blockade 10 days after AngII infusion in DMSO, SC560 and nimesulide (NM) treated rats . Peak fall in MAP in response to hexameth onium administration (30mg/kg ip ) in SC560 (A) and NM (B) treated rats. # =p<0.05 SC560 vs. DMSO 66 4. Discussion Previous investigators have used both pharmacological and gene knockout approaches to determine if eicos anoids play a critical role in AngII mediated hypertension, but their results have led to conflicting c onclus ions (22, 188, 260). Eicos anoid products can exert both pro - and anti-hypertensive effects: thus, their net effect on blood pressure will depend on where increased eicos anoids formation occurs and on which specific products are released. Earlier studies showed that dietary salt intake is one factor that influences the contribut ion of eicos anoids to AngII dependent hypertension (115, 157). More recent studies by King found that COX inhibition with ketoprofen, a non -selective COX inhibitor, did not prevent hypertension in rats on a normal salt diet (0.4%NaCl). However, in rats that were on a high salt diet (2% NaCl), COX -inhibition prevented the later phase of AngII -salt hyperte nsion (beyond day 5 of AngII infusion) , but not the early rise in MAP (days 1 -4) of AngII infusion . There are multiple potential explanations for why eicos anoids could make a higher contribution to AngII hypertension under conditions of high salt diet. Ho wever my lab and others have shown that high salt intake amplifies the role of the sympathetic nervous system in AngII HTN (176, 203, 260). Furthermore, Kings work showed that selective removal of the splanchnic sympathetic innervation significantly attenuated chronic AngII -salt HTN (128) during the later phase of HTN development , indicating that specifically splanchnic SNS activity is important for HTN development in this model. Since direct recordings in conscious rats wi th chronic AngII HTN showed an increase in splanchnic SNS activity that was associated with a significant rise in urinary excretion of cyclooxygenase products (143), we hypothesized that COX -derived prostanoids could 67 increase blood pressure in the AngII -salt model by stimulating SNS activity . This i dea was further supported by the work that shows that ketoprofen treatment had a more prominent effect on AngII -salt hy pertension development during the later days of AngII infusion (8).It has been previously reported that neurogenic mechanism s appear to play a greater role in this hypertension model after 4 -5 days of AngII infusion (127, 128, 133). The increase in MAP on days 1 -4 appears to be caused by either the direct pressor actions of AngII or perhaps renal sodium and water retention. The increase in neurogenic pressor activity only during the l ater phase of AngII -salt HTN may be due to an ability of COX products to ÒprimeÓ subsequent changes in sympathetic regulation of BP. This idea will be addre ssed in more detail in Chapter 4 of my dissertation. Earlier, King et al tested the role of COX prod ucts in the neurogenic actions of AngII in two ways. First the SNS activity was assessed using measurement of whole body norepinephrine spillover in ketoprofen treated rats. A statistically significant 71% increase in whole body NE spillover on days 7 and 14 of AngII infusion was seen in rats on a high salt , but not low salt, diet (127). I n unpublished studies in rats treated with ketoprofen there was a much smalle r (though statistically significant) increase in whole body NE spillover on day 7 of AngII infusion (36% vs. control) and a statistically non -significant increase (14% vs. control) on day 14 of AngII infusion in high salt fed rats. Therefore, t he global SN S activation caused by AngII infusion in rats on high salt diet , as indicated by significant elevation in plasma NE and whole body NE s pillover (127) was largely, but not completely , prevented by non -selective COX inhibition. 68 Furthermore, in KingÕs studies measurement of neurogenic pressor activity (the acute contribution of the autonomic nervous system to arterial pressure) was used to test if eicos anoid-related S NS activity contributed to AngII -salt hypertension. Vehicle treated AngII -salt HTN rats exhibited a marked drop in MAP during ganglionic blockade; AngII -salt rats treated with ketoprofen showed a substantially smaller drop in pressure, although the differe nce was not statistic ally significant. This suggests that a eicos anoid-driven increase in SNS activity may be at least partially responsible for the hypertens ion in AngII -salt treated rats. As a first step in my study I asked whether eicos anoids involved in AngII -salt HTN were derived from the enzymatic isoforms COX -1 or COX -2. It is known that AngII exerts physiologically significant pro -inflammatory effects that contribute to numerous cardiovascular diseases (38, 57). AngII appears to produce inflammatory responses predominantly in a localized manner in target tissues such as blood vessels (38) kidney (254) and brain (276). COX-2 is the isoform most strongly implicated in generating inflammatory eicos anoids, so it is logical to assume that COX -2 the key player in AngII -salt HTN. Some published studies support this conclusion. For example , pretreatment with COX -2 inhibitors like refecoxib and nimesulide attenuated AngII -HTN in Sprague Dawley r ats (149, 190, 260). However, in contrast to this id ea, other studies indicate that COX -1 is the source of eicos anoids involved in AngII HTN (7). In a recent report, Cao and colleagues showed that AngII HTN was att enuated in COX -1-/- mice but not COX-2-/- mice (22). As noted earlier, t he contradictory reports from various laboratories could be attributed to the differences in animal models used, blood pressure 69 measurement methods like tail cuff plethysmography versus radiotelemetry, or the mode of drug administration. My studies indicate that the key eicos anoids involved in AngII -salt hypertension are derived from the COX -1 and not the COX -2 isoform: hypertension development was markedly and significantly attenuated by treatment with the COX -1 inhibitor SC560, but not by treatment with the COX -2 inhibitor nimesulide. However, with selective COX -1 inhibition, we did not see a s complete an attenuation of hypertension as we observed with the non -selective COX inhibitor ketoprofen. This may be due to 1) additional pharmacological actions of ketoprofen, or 2) involvement of COX -2 in the development of hypertension, although our re sults with the selective COX -2 inhibition do not support the latter idea. The doses of the inhibitors used to totally block COX enzyme could possibly fall on different stages of the ir dose response curve s. This differential COX inhibition with a particular dose of NSAID could explain the increase d attenuation of HTN with keto profen (2mg/kg, sc) compared to SC560 (10mg/kg, ip). It is further notable that COX -1 inhibition with SC560 significantly reduced neurogenic pressor activity in AngII -salt HTN rats. Th is result supports the hypothesis that eicos anoids increase arterial pressure in AngII -salt HTN in part by increasing SNS activity. In conclusion, this study suggests that in rats fed a high salt diet, chronic AngII infusion stimulates the formation of eicos anoids from COX -1 that activate the SNS and increase arterial pressure. This data does not allow us to establish where these eicos anoids are produced, or where they act, to increase SNS activity or arterial pressure. However, 70 central injections of eicos anoids can increase SNS activity (171) and arterial pressure (112), and a recent study in mice strongly implicated brain COX-1 activity in AngII HTN (22). Thus, studies described in my next chapter focus on exploring the role o f brain eicos anoids in AngII -salt hypertension. 71 CHAPTER 4 TRANSCRIPTIONAL AND TRANSLATIONAL REGULATION OF CENTRAL ENZYMES AND RECEPTORS IN THE EICOSANOID SYNTHESIS PATHWAY DURING THE DEVELOPMENT OF ANGIOTENSIN II -SALT HYPERTENSION IN THE RAT 72 1. Introduction Samuelsson first described the eicosanoids in the brain in 1964 (200) where these Òlocal hormonesÓ are known to act as mediators of inflammation , fever, feeding, sleep and other functions . Relatively little evidence has been published supporting a role for bra in eicosanoids in BP regulation, and I summarized that evidence in Section 8.2.3 of Chapter 1. As desc ribed earlier my dissertation, both other investigators and I found that treatment with COX inhibitors during AngII -salt HTN development d oes not affect BP during the early s tage (1 -4 days of AngII infusion) but significantly lowered BP during the later neurogenic phase of HTN (5 -14 days after AngII infusion). Therefore, here I hypothesize that 1) eicosanoids act early in HTN development to ÒprimeÓ a later engagement of neurog enic pressor mechanisms, and 2) early changes in the expression of COX pathway enzymes and /or receptors in the brain could be the cause of this ÒprimingÓ effect in AngII -salt HTN . To address these hypotheses I did experiments designed to 1) identify the cr itical time period during with eicosanoids operate to affect AngII -salt HTN development, and 2) analyze eicosanoid pathway gene and protein expression during that critical time window in specific brain nuclei known to regulate sympathetic activity and BP. 2. Experimental protocol s 2.1. Non-selective COX inhibition in AngII -salt treated rats with established HTN : In order to test the effect of NSAID treatment on rats with established AngII -salt HTN the following experiment was performed. R ats were allowed a 5 -day recovery period after telemeter surgery. After three days of control MAP and HR recordings the rats 73 were divided into two groups, both receiving AngII (Sigma) delivered at a constant rate of 150ng/kg/min using a mini -osmotic pump (2ML2, Alzet) impl anted subcutaneously, starting on day 3 until day 17 (figure 1). On days 9, 1 0 and 1 1 of the AngII infusion period (after HTN was well -established) , one group received once daily subcutaneous injections of either vehicle ( saline, n=5) or the potent non -sel ective COX inhibitor ketoprofen (2mg/kg, n=7). The subcutaneous AngII pumps were removed on day 1 4 of AngII infusion to allow reversal of the hypertension . Then starting 4 days later rats from one group (n=5) received once daily subcutaneous injections of saline vehicle for three consecutive days while the other group received ketoprofen (2mg/kg, n=7) . Hexamethonium (30mg/kg, IP) was given during the control period (C2), drug treatment period (D3) and the angiotensin treatment period (A10) of the protocol , respectively , to determine the effect of ketoprofen treatment on neurogenic pressor activity in established AngII -salt hypertensive rats. The magnitude of the acute (10 -30 minutes) fall in BP was used as an indicator of the degree of sympathetic regulat ion of blood pressure , as described in General Methods. 2.2. Non-selective COX inhibition prior to and during early stages of AngII -salt HTN: My next goal was to identify potentially critical time periods during which eicos anoids contribute to the develop ment of AngII -salt HTN. To achieve this end, I investigate d the effect of non -selective cyclooxygenase inhibition at two different time points during the development of AngII -salt HT. In the first set of experiments, after 3 control days of BP measurement either saline vehicle (n=3) or ketoprofen (2mg/kg, n=3) 74 was administered for 4 days prior to AngII infusion. H examethonium (30mg/kg, IP) was given once acutely 10 days after starting AngII to evaluate neurogenic pressor activity in the two groups of rats. In the second set of experiments, after 3 control days of BP measurement ketoprofen (2mg/kg, sc.) or saline was administered once daily for 4 days (prior to starting AngII infusion) and then once daily for an additional week (during the first 7 days of An gII infusion). Neurogenic pressor activity was measured on day 10 of AngII infusion, as described above. 2.3. Brain eicosanoids in the early stage of AngII -salt HTN 2.3.1. Transcriptional and translational regulation of e icosanoid pathway related genes: For performing PCR and western blot analysis of tissue punches collected from cardio -regulatory brain regions , a total of 22 radiotelemeter -implanted rats were used. After 3 days of control recording, AngII (150ng/kg/min, sc., n=12) or physiological saline (n=10) was delivered for 4 days using a mini-osmotic pump (Alzet, 2ML2). Then the rats were sacrificed, brains were collected, snap frozen in dry ice and kept at -80¡C until further analysis as described in Chapter 2. 2.3.2. PCR array : Expression level of 21 genes involved in eicosanoid signaling was measured in PVN, OVLT , SFO , CP, RVLM and NTS as described in Chapter 2. 75 2.3.3. Quantitative real t ime polymerase chain reaction (q RT-PCR) : To confirm L -PGDS expression in OVLT and to measure the cPLA2 gene expression in 4 day Ang II treated and vehicle treated rat brain s, punches were collected from PVN, SFO, OVLT, RVLM, NTS, third ventricle CP (3VCP) , fourth ventricle CP (4VCP) and the middle cerebral artery (MCA) (n=5/group) and q RT-PCR was performed as desc ribed in Chapter 2. 2.3.4. Western blot analysis : To analyze changes in eicosanoid pathway proteins during early development of HTN, the brain from 4 day AngII (n=7) or vehicle (n=5) treated rats were collected. Punches from PVN, SFO, OVLT, NTS, RVLM, 3VC P and 4VCP were analyzed as described in Chapter 2. 3. Results 3.1. Non-selective COX inhibition in established AngII -salt HTN rats: The baseline levels of MAP in both groups were identical from control day 1 to control day 3 . After starting AngII infusio n MAP increased from 101 ±1 mmHg on control day 3 to 125 ±7 mmHg on day 8 of AngII infusion in the vehicle group, and from 10 2 mmHg on day 3 to 12 2±3 mmHg on day 8 in the group that would later receive treatment with ketoprofen (Figure 4-1A). Rats were then given either saline vehicle or ketop rofen (2mg/kg/day, ip) for 3 consecutive days . MAP was not affected by ketoprofen treatment in these animals with established hypertension, being 126±8 mmHg in the vehicle group and 128 ±3 mmHg in the ketoprofen group, respectively , on the last day of treatment 76 (Figure 4-1A). Ganglion blockade with hexamethonium (10 days after AngII infusion ), on the second day of ketoprofen or vehicle treatment caused similar depressor responses in the vehicle treated (66 ±14 mmHg) and ket orprofen treated (55 ±8 mmHg) rats (Figure 4-1B). After the 14-day AngII infusion period the osmotic pumps were removed and MAP fell over several days to 10 3±1 mmHg and 103 ±2 mmHg in the vehicle and ketoprofen group s, respectively. At this point we again administered ketoprofen or vehicle for 3 days and saw no change in MAP in either group (Figure 4-1A). 77 Day 1234567891011121314151617181920212223242526Mean Arterial Pressure (mmHg) 80100120140160Vehicle (n=5) Ketoprofen (n=7) Angiotensin II 150 ng/kg/min,sc Control Ketoprofen scAngiotensin II control Ketoprofen scA vehicle ketoprofen Peak fall in MAP (mmHg) -100 -80 -60 -40 -20 0B Figure 4-1: The effect of non -specific cyclooxygenase inhibition on MAP and neurogenic pressor activity in established AngII -salt HTN rats . A) MAP in rats on 2% NaCl diet given a three -day treat ment of non-selective cyclooxygenase inhibitor or vehicle during and after infusion of AngII. B) Peak fall in MAP in response to ganglion blockade with hexamethonium during ketoprofen or vehicle treatment in rats with established HTN rats after 10 days of AngII infusion . 78 3.2. Non-selective COX inhibition prior to and during the early stages of AngII -salt HTN: Figure s 4-2 and 4-3 sho w data from rats treated with ketoprofen for different lengths of time prior to, or during the early days of, chronic AngII infusion. Figure 4-2A shows MAP in rats pretreated with vehicle (n=3) or ketoprofen (2mg/kg, sc, n=3) for 5 days prior to AngII infu sion. After 14 days of AngII infusion MAP rose to 147 ±2mmHg in vehicle treat ed rats and to only 129±8mmHg in ketoprofen treated rats, but there was no statistically significant difference in MAP between the two groups . Acute d epressor response s to hexameth onium on day 10 of AngII infusion were similar in vehicle ( -57±5mmHg) and ketoprofen ( -56±14mmHg) treated groups (Figure 4-2B). Figure 4-3 shows results from studies in which ketoprofen administration was started 4 days prior to beginning AngII infusion a nd then extended for 7 more days (after the start of AngII infusion). Another group of animals received only saline vehicle treatment over the same time course. MAP was similar in the two groups prior to AngII infusion, e.g. 100±3 mmHg on D1 in vehicle tre ated rats and 103 ±1 mmHg on D1 in ketoprofen treated animals prior to starting AngII infusion (Figure 4-3A). In rats treated with vehicle only, MAP increased markedly during AngII infusion and was 141±10 mmHg by the end of the infusion period . In rats tre ated with ketoprofen, however, MAP rose initially up to A4 (125 ±3) during AngII infusion but was only 109±7 mmHg by the end of the infusion period (significantly lower than MAP in vehicle treated animals). The depressor response (Figure 4-3B) was significa ntly less in ketoprofen treated rats ( -35±7 mmHg) than in vehicle treated rats ( -92±12 mmHg). 79 Day 123456789101112131415161718192021Mean Arterial Pressure (mmHg) 80100120140160180200vehicle (5 days) (n=3) ketoprofen (5 days) (n=3) Angiotensin II (150ng/kg/min) sc Control Ketoprofen 2mg/kg sc A Vehicle Ketoprofen Peak fall in MAP (mmHg) -100 -80 -60 -40 -20 0B Figure 4-2: The effect of non -selective cyclooxygenase inhibition prior to AngII admini stration on AngII -salt HT N and neurogenic pressor activity . A) Mean arterial pressure in rats treated with cyclooxygenase inhibitor in one group and vehicle in another group for 5 days prior to making them h ypertensive with AngII infusion. B) Peak fall in MAP in response to ganglion blockade after 10 days of AngII infusion. # = (p<0.05) vs. control rats. 80 Day 123456789101112131415161718192021Mean Arterial Pressure (mmHg) 80100120140160180200Vehicle (11 days) (n=3) Ketoprofen (11days) (n=3) #p<0.05 Angiotensin II (150ng/kg/min) sc Ketoprofen (2mg/kg) sc Control #AVehicle Ketoprofen Peak fall in MAP (mmHg) -100 -80 -60 -40 -20 0B# Figure 4-3: The effect of non -selective cyclooxygenase inhibition prior to and during the early phase of AngII administration on AngII -salt HT N and neurogenic pressor activity . A) MAP in rats after non-selective cyclooxygenase inhibition or vehicle treatment for 5 days prior to and first 7 days of AngII infusion . B) Peak fall in MAP in response to ganglion blockade with hexamethonium on day 10 (A10) . # = (p<0.05) vs. control rats. 81 3.3. Gene array analysis of eicosanoid related gene expression in the brain of rats in the early stage of AngII -salt HT N: PCR array analysis was used to mea sure transcriptional regulation of genes in brain samples obtained from rats on day 4 of AngII -salt administration. MAP on day 4 was 105±2 mmHg in control rats and 125±5 mmHg in AngII -salt treated rat s. The PCR data are shown in Figure 4-4. Between group differences were analyzed by one -sample t -tests with a hypothetical mean as 1. AngII treated rats showed a significant up -regulation of Arachidonate 5 -lipoxygenase (Alox5) (2.0 fold), PGD synthase 2 -hematopoitic (Ptgds2) (1.6 fold) and down -regulation of PG-endoperoxide synthase 1 (Ptgs1) (0.9 fold) and prostacyclin synthase (0.6 fold), in the RVLM (n=4) . In the OVLT (n=2), lipocalin type prostaglandin D synthase or PGD2 synthase (brain) (ptgds) (4.3 fold) expression was increa sed, whereas COX1, PGH synthas e (0.2 fold) and PGD receptor (0.6 fold) expression were significantly down regulated. Eicosanoid gene expression was unaffected in the PVN, (n=5) , SFO (n=5) , NTS (n=5) and CP lining the third (n=5) and fourth ventricle (n=5) . The p value of mRNA fold chan ge for PGD2 synthase (brain) in CP of third ventricle was 0.0648. 3.4. Quantitative RT-PCR : Q RT-PCR was performed in order to confirm the result of gene expression changes found in OVLT with PCR array. In rats (n=5) treated with AngII for 4 days, there was a significant 3 -fold increase in L -PGD -synthase gene expression in the OVLT compared to vehicle treated rats. 82 Q RT-PCR was also performed for another rate limiting enzyme in eicosanoid synthesis, i.e. cPLA2, since it was inadvertently not included in the customized eicosanoid pathway PCR array we used. The enzyme cPLA2 expression was significantly higher in OVLT (2.2 -fold, n=5), PVN (2.4 -fold, n=5), NTS ( 9-fold, n=5) and MCA ( 22-fold, n=5) of AngII treated rats compared to vehicle treated rats. There were no change s in cPLA2 expression in RVLM or SF O. 83 Eicosanoid related genes Ptgs1 Ptgs2 Alox5 Alox5ap Alox12 Alox15 Tbxas1Ptgis Ptges1 Ptges2 Ptges3 Ptgds Ptgds2 Tbxa2rPtgir Ptger1 Ptger2 Ptger3 Ptger4 Ptgdr Ptgdrl mRNA fold change 0246Angiotensin II (n=5) A. PVN Eicosanoid related genes Ptgs1 Ptgs2 Alox5 Alox5ap Alox12 Alox15 Tbxas1 Ptgis Ptges1 Ptges2 Ptges3 Ptgds Ptgds2 Tbxa2r Ptgir Ptger1 Ptger2 Ptger3 Ptger4 Ptgdr Ptgdrl mRNA fold change 0246Angiotensin II (n=5) B. SFO Figure 4-4: Eicosanoid related gene expression in rats treated with Ang II or vehicle for 4 days. The mRNA fold ch ange of eicosanoid related gene s in samples collected on day 4 from A ) PVN, B) SFO, C ) OVLT, D ) RVLM, E) NTS, F) MCA, G ) CP-third ventricle (3VCP) and H ) CP-fourth ventricle (4VCP) . # S ignificant difference (p<0.05) upregulation of eicosanoid genes in AngI I treated rats vs. vehicle treated rats. # Significant d ownregulation of eicosanoid genes a fter AngII infusion compared to vehicle treated rats. Between group difference was analyzed by one -sample t -test with a hypothetical mean of 1. 84 Figure 4 -4 (ContÕd) Eicosanoid related genes Ptgs1 Ptgs2 Alox5 Alox5ap Alox12 Alox15 Tbxas1Ptgis Ptges1 Ptges2 Ptges3 Ptgds Ptgds2 Tbxa2rPtgir Ptger1 Ptger2 Ptger3 Ptger4 Ptgdr Ptgdrl mRNA fold change 0246Angiotensin II (n=2) *#*C. OVLT Eicosanoid related genes Ptgs1 Ptgs2 Alox5 Alox5ap Alox12 Alox15 Tbxas1Ptgis Ptges1 Ptges2 Ptges3 Ptgds Ptgds2 Tbxa2rPtgir Ptger1 Ptger2 Ptger3 Ptger4 Ptgdr Ptgdrl mRNA fold change 0246Angiotensin II (n=4) *##*D. RVLM Eicosanoid related genes Ptgs1 Ptgs2 Alox5 Alox5ap Alox12 Alox15 Tbxas1Ptgis Ptges1 Ptges2 Ptges3 Ptgds Ptgds2 Tbxa2rPtgir Ptger1 Ptger2 Ptger3 Ptger4 Ptgdr Ptgdrl mRNA fold change 0246Angiotensin II (n=5) * E. NTS 85 Figure 4 -4 (ContÕd) Eicosanoid related genes Ptgs1 Ptgs2 Alox5 Alox5ap Alox12 Alox15 Tbxas1 Ptgis Ptges1 Ptges2 Ptges3 Ptgds Ptgds2 Tbxa2r Ptgir Ptger1 Ptger2 Ptger3 Ptger4 Ptgdr Ptgdrl mRNA fold change 0246Angiotensin II (n=5) F. MCA Eicosanoid related genes Ptgs1 Ptgs2 Alox5 Alox5ap Alox12 Alox15 Tbxas1Ptgis Ptges1 Ptges2 Ptges3 Ptgds Ptgds2 Tbxa2rPtgir Ptger1 Ptger2 Ptger3 Ptger4 Ptgdr Ptgdrl mRNA fold change 0246Angiotensin II (n=5) G. 3VCP Eicosanoid related genes Ptgs1 Ptgs2 Alox5 Alox5ap Alox12 Alox15 Tbxas1Ptgis Ptges1 Ptges2 Ptges3 Ptgds Ptgds2 Tbxa2rPtgir Ptger1 Ptger2 Ptger3 Ptger4 Ptgdr Ptgdrl mRNA fold change 0246Angiotensin II (n=5) H. 4VCP 86 PVNNTS SFORVLM MCA 3V4VOVLT mRNA fold change 010203040Angiotensin II (n=5) ####A. PLA2 mRNA fold change 0246Control (n=5) Angiotensin II (n=5) #B. Ptgds Figure 4-5: Phospholipase A2 and lipocalin prostaglandin D s ynthase gene expression using q -RT PCR in 4-day Ang II or vehicle treated rats . (A) PLA2 gene expression in PVN, NTS, SFO, RVLM, MCA, th ird ventricle CP (3V) , fourth ventricle CP (4V) and OVLT. (B) Lipocalin prostaglandin D synthase gene expression in 4 day AngII treated rats in OVLT . # = p<0.05 , significant overexpression after 4 days of AngII infusion compared to vehicle treatment in rat s. Between group difference was analyzed by one-sample t -test with a hypothetical mean of 1. 87 3.5. Western blot analysis : Western blot analysis showed a significant overexpression of cPLA2 protein in the MCA and L-PGDS protein in the 3VCP (Figure 4-6). There w ere no change s in protein levels of COX-1 in AngII treated rats in MCA, SFO, OVLT, PVN or RVLM (Table 4-1). 88 % PLA2 expression 0204060vehicle (n=7) AngII (n=5) Beta actin cPLA2 #A. MCA % COX-1 expression 020406080100vehicle (n=3) AngII (n=5) Beta actin COX-1 B. MCA % L-Ptgds expression 0.0 0.5 1.0 1.5 2.0 vehicle (n=6) AngII (n=4) Beta tubulin L-PGDS C. 3VCP # Figure 4-6: Phospholipase A2, cyclooxygenase -1 and lipocalin prostaglandin D synthase protein expression in 4-day Ang II or vehicle treated rats . A) cPLA2 protein expression in 4 day AngII treated ra ts in middle cerebral artery (veh; n=7, AngI I; n=5) . B) COX-1 protein expression in 4 day AngII treated ra ts in middle cerebral artery (veh; n=3, AngII; n=5) . C) Lipocalin type prostaglandin D synthase protein expression in 4 day AngII treated rats in CP in third ventricle (veh; n=6, AngII; n=4) . # Overexpression of proteins (p<0.05) in AngII infusion day 4 compared to control rats. BBeta tubulin 89 Table 4-1: P hospholipase A2, cyclooxygenase -1, prostaglandin D synthase protein expression in 4 -day angiotensin II compared to vehicle treated rats . Western blot analysis of protein of interest in samples from a) PVN, b) SFO , c) OVLT , d) RVLM , e) NTS, f) MCA, g) CP-third ventricle (3VCP) and h) CP-fourth ventricle (4VCP) is represent ed as the % protein expression over control. * Overexpression of proteins (p<0.05) in AngII infusion day 4 compared to control rats. $ Lower expression of proteins after AngII infusion compared to control rats on day 4 of HT N.Enzyme Groups % Protein expression over control (Beta actin for MCA and Beta tubulin for brain regions) MCA OVLT PVN SFO RVLM NTS 3VCP 4VCP PLA2 Control 6.6±2.4 3.3±0.7 4.9±1.1 3.3±0.5 2.1±0.2 4.0±0.2 4.2±0.9 20±3 AngII 36.4±8.2! 2.2±0.8 4.2±0.4$ 4.2±0.5 2.2±0.4 3.3±0.2 5.4±1.1 27±5 COX-1 Control 62.5±15.4 0.077±0.02 0.075±0.01 0.17±0.05 0.08±0.07 0.01±0.03 1.04±0.2 4.8±2 AngII 4.7±9.2 0.078±0.02 0.085±0.01 0.16±0.1 0.07±0.05 0.06±0.03 0.8±0.1 2.6±0.5 L-PGDS Control - 0.4±0.03 0.2±0.08 0.5±0.2 0.02±0.003 - 0.5±0.2 1.1±05 AngII - 0.2±0.03 0.3±0.04 0.6±0.1 0.01 - 1.1±0.2! 0.3±0.2 90 4. Discussion The main findin gs of this study are : 1) acute COX inhibition does not reverse established Ang II-sal t HTN; 2) transient COX inhibition during the first phase of AngII -salt HTN development impairs the ultimate development of HT N in the second (neurogenic) phase ; 3) AngII -salt HT N is associated with early transcriptional regulation of eicosanoid pathways , especially in the OVLT and the cerebral vasculature ; and 4) altered expression of cPLA2 and L -PGDS proteins in the cerebral vessels and brain nuclei occur during the early phase of AngII -salt HT N. In previous stud ies in our lab (discussed in Chapter 3) continuous treatment with ketoprofen attenuated AngII -salt HT N development, but only du ring the second phase of the HTN (8). Since this late r phase of HTN is caused mainly by increased neurogenic pressor activity, we reasoned that eicosanoid p roducts might drive that increased neurogenic pressor ac tivity; and our results using ganglion blockade supported that idea. However, previous studies in experimental animals and humans indicate that NSAIDs like ketoprofen generally donÕt reduce BP in established HTN (see Chapter 1). Therefore, I wanted to test whether COX activity was required to maintain elevated SNA in the later phase of AngII -salt HTN. The results were clear: once HTN was established, blocking COX activity had no effect on BP or SNA (assessed from neurogenic pressor activity). I then went on to do experiments designed to determine when eicosanoids did act to increase SNA during AngII -salt HTN. I found that COX inhibition limited to the first week (early phase) of AngII -salt administration significantly attenuated later HTN development. Additi onally, neurogenic pressor activity in short -term ketoprofen treated rats was lower than their untreated counterparts on day 10 of AngII. Collectively these 91 data indicate that COX products act during the early phase of HTN development to somehow increase S NA during the later phase. How might this delayed effect of eicosanoids be produced? There is convincing evidence that short -term exposure to AngII can cause long -lasting changes in the brain pathways by which AngII regulate s SNA and BP. One manifestation of this phenomenon is the ability of short -term increases in circulating AngII to ÒsensitizeÓ responses to subsequent increases in circulating AngII occurring even weeks later (263). The mechanisms responsible for this ÒprimingÓ action of AngII have not been identified, but could be similar to those responsible for the known influence of brain AngII and its metabolites on learning and memory (259). My data suggest that COX inhibitors may prevent this priming action in AngII -salt HTN. Interestingly, a n analogous effect of COX inhibitors has been demonstrated in AlzheimerÕs disease (AD). Chronic COX -1 inhibition effectively delays the development of cognitive and other defects in AD, but has no benefit once the disease is established (106). COX-derived eicosanoids in the brain are able to induce long -lasting neuroplastic changes in numerous neuronal populations and pathways that after a period of time do not require continued e icosanoid action (12, 27). This phenomenon could explain the timing of the protective actions of COX inhibitors in both AD and HTN. Eicosanoids could regulate sympathetic activity and blood pressure in AngII -salt HT in a variety of ways, but my studies focused on a possible role of eicosanoids in the brain. Eicosanoid products are produced throughout the brain in r esponse to numerous physiological and pathophysiological stimuli , and are synthesized in various brain 92 regions by the pathways described in chapter 1 (Figure 1 -3). Because eicosanoid products can act in the brain to increase SNA and BP, and also cause long-lasting adaptive changes in brain regulatory pathways, I tested the hypothesis that discrete transcriptional and translational products of the brain eicosanoid pathway would be activated during the early development of AngII -salt HTN. Four days after st arting AngII -salt treatment in rats eating a high salt diet , I found an increase in mRNA levels of cPLA2 in some cardio -regulatory brain regions and cerebral arteries . Since cPLA2 protein also w as overexpressed in cerebral arteries , this could produce acce lerated conversion of membrane phospholipid s into AA, the major substrate for production of eicosanoids. AngII is known to stimulate membrane phospholipid metabolism in other tissues (153), and activate the AA cascade in the cerebral microvasculature (213). The possible importance of cPLA2 will be addressed further in Ch apter 6. Downstream to cPLA2, however, there was no change in the transcript levels of COX -1, another key enzyme in eicosanoid synthesis. This latter finding is consistent with the fact that COX -1 is generally a constitutively active enzyme in the brain an d elsewhere (241). Expression of genes in the COX and lipoxygenase enzymatic pathway s was evaluated to help identify specific eicosanoids that might act as brain mediators of elevated sympathetic activity and BP. Overall, other than cPLA2, very few changes in eicosanoid gene expression were found early in AngII -salt HTN, including genes for synthetic enzymes and known receptors. This could be taken as evid ence against an important 93 role for brain eicosanoids in AngII -salt HTN, but also could indicate that the relevant eicosanoids generated by AngII -salt are part of a constitutively active pathway involved in normal brain physiology. A notable and surprising exception was my finding that transcript ion of L -PGDS, which is found primarily in the brain in meningioepithelial cells and oligodendrocytes (232), was strongly upregulated in the OVLT, a circumventricular organ that comes in contact wit h circulating angiotensin II through a Ò leaky Ó blood brain barrier. Because the leptomeninges, arachnoid trabecular cells and CP epithelial cells are the primary source s of L -PGDS production and secretion into the cerebrospinal fluid (14), the implications of expression of the L-PGDS gene in the OVLT are not clear. Nevertheless, when c ombined with the facts that 1) the L-PGDS product PGD2 is the most abundant eicosanoid in the brain , and 2) PGD2 injected into the brain causes a sympathe tically mediated increase in BP (60), the dramatic increase in L -PGDS gene expression (along with cPLA2) led me to further inves tigate a possible role of brain eicosanoids, and especially L -PGDS, in the development of AngII -salt HTN. Those studies are described in Chapters 5 and 6. 94 CHAPTER 5 CENTRAL INHIBITION OF CYCLOOXYGENASE AND LIPOCALIN PROSTAGLANDIN D SYNTHASE BLOCKS THE DEVELOPMENT OF ANGIOTENSIN II -SALT HYPERTENSION IN THE RAT 95 1. Introduction In previous Chapters , I showed evidence that cyclooxygenase products play a key role in the developmental stages of a neurogenic model of AngII -salt HTN (Figure 4-3). Specifically, administering either a non -selective, or a specific COX1, cyclooxygenase inhibitor during HTN development significantly attenuated the increase in blood pressure and also reduced neurogenic pressor activity (determined as the acute depressor response to ganglion blockade) . Although many other previous reports implicate eicos anoids in angiotensin -dependent hypertension (155-157, 255), most of those reports suggest that the main tissue targets are blood vessels or the kidney. However, there is some evidence that COX1 in the brain participates in a sympathoexcitatory and pressor pathway (22, 23). So as part of my project I performed an analysis of eicosanoid pathway gene expression in cardioregulator y brain regions during the early phase of AngII -salt HTN . The analysis reveal ed no changes in COX1 or COX2 expression in the brain, but did highlight enzymes both upstream (cPLA2) and downstream ( L-PGDS ) of COX as potential regulated elements of the eicosa noid pathway during AngII -salt HTN development (Chapter 4). In the current study I tested the hypothesis that downstream products of cyclooxygenase activity in the brain contribute to AngII -salt HTN and that one mechanism for this effect is increased synt hesis and release of L -PGDS. To that end, I did the following experiments: 1) measured brain COX activity, and CSF L-PGDS protein content, during the early phase of AngII -salt HTN development; and 2) tested the effect of selective brain blockade of COX activity , and LPGDS activity , on AngII -salt HTN. 96 2. Experimental protocol s 2.1. Mean arterial pressure and cyclooxygenase activity in cardio -regulatory brain regions during the early stage s of AngII -salt HTN development : Rats were given high salt diet and either AngII (n=5) or saline vehicle (n=5) was infused subcutaneously for 4 days. B P was measured using a radio -telemeter implanted into the abdominal aorta through the femoral artery. On the fourth day of AngII infusion, the rats were euthanized and brain s were collected, snap frozen on dry ice and stored at -80¡C. Using a cryostat maintained at -15¡C, 500 ! m thick coronal brain sections were prepared and brain punches from SFO, OVLT , PVN and RVLM were obtained. COX activity was measured in the punches a s described in Chapter 2. 2.2. Brain cyclooxygenase inhibition in AngII -salt hypertensive rats : To verify the involvement of brain COX pathway s in HTN development, we used our standard AngII -salt HTN protocol. After three days of baseline control MAP record ing (days C1, C2 and C3), a brain infusion cannula attached to an osmotic pump was implanted into the lateral ventricle (co -ordinates; -0.8mm AP, -1.5mm ML and 4.2mm DV). A mini-osmotic pump (Alzet, model 2004) deliver ed ketorolac at different doses (33 , 3 .3. or 0.6 nmol/h) or vehicle (DMSO ) intracerebroventricularly (ICV) to different groups of rats throughout the Òdrug -aloneÓ control period (days D1, D2, D3 and D4) and the remainder of the experimental protocol. After the 4-day drug -alone period , mini-osm otic pumps (Alzet, model 2ML2) for sc AngII infusion (150 ng/kg/min) were implanted (days A1 -A14) . The control group received saline vehicle infusion . On days C2, D2 and A10, n eurogenic pressor activity was determined in rats receiving ICV 97 ketorolac at 33 n mol/h and in vehicle treated rats, using the acute fall in blood pressure after injection of the ganglionic blocker hexamethonium (30mg/kg, ip). 2.3. Plasma ketorolac measurement using high performance liquid chromatography : To determine an ICV infusion rate of ketorolac that would inhibit COX in the brain but not in peripheral tissues , we measured the drug concentration in blood during ICV infusion at three rates: 0 .6nmol/h, 3.3nmol/h and 33nmol/h . Ketorolac was infused into the lateral ventricles of mal e Sprague Dawley rats using brain infusion cannula attached to an osmotic pump (Alzet , model 2004 ). After one week of infusion, blood was drawn from the abdominal aorta of anesthetized rats and plasma was collected . Ketorolac concentrations in plasma were measured as described in Chapter 2. Plasma concentrations were compared to the published IC50 of ketorolac, namely 0.27 ! M or 100ng/ml in plasma (33). 2.4. Weste rn blot analysis for lipocalin prostaglandin D synthase expression in the cerebrospinal fluid during early stages of AngII -salt HTN : Rats were made hypertensive using our standard AngII -salt HTN protocol (n=5 /group ), and CSF was collected from both hyperte nsive and normotensive control rats on day 4 (A4) of AngII or vehicle infusion respectively. CSF was collected and assayed for L -PGDS protein concentration as described in Chapter 2. 98 2.5. Central and peripheral L -PGDS inhibition : AT56, a highly select ive inhibitor of L -PGDS enzymatic activity (63), was used to test the role of L-PGDS in the development of AngII -salt HTN. Osmotic mini -pumps ( Alzet, Model 200 4) delivered AT56 centrally as described earlier (5.8nmol/h, ICV, n=7) or peripherally (5.8nmol/h, sc, n=4). Th e delivery rate of AT56 was chosen based on an amount reported earlier to produce effective L -PGDS inhibition (16). After three days of baseline control MAP recording (C1, C2 and C3) and 5 days of AT56 drug treatment alone (D1, D2, D3, D4 and D5 ) that continued for the rest of the protocol, AngII (150ng/kg /min sc ) was infused for an additional 14 days (A1-A14, n=7/group). Neurogenic pressor activi ty was evaluated (as described earlier) on day 2 (C2), day 6 (D3 ) and day 17 (A10) of the protocol. 3. Results 3.1. Mean arterial pressure and cyclooxygenase activity in cardio -regulatory brain regions during the early stages of AngII -salt HTN development : MAP and HR were not different betwee n the groups during the control period. The AngII treated rats had a significantly higher MAP on A4 compared to vehicle infus ed rats (Figure 5 -1A). In contrast t here was no significant effect of AngII infusion on HR compared to the controls (Figure 5 -1B). 99 Day C1C2C3A1A2A3A4Mean Arterial Pressure (mmHg) 80100120140160Angiotensin II (n=5) Vehicle (n=3) Angiotensin II 150 ng/kg/min, sc Control #A Day C1C2C3A1A2A3A4Heart Rate (beats/min) 350400450500550Angiotensin II (n=5) Vehicle (n=3) Angiotensin II 150 ng/kg/min, sc Control B Figure 5 -1: MAP and heart rate (HR) in AngII -salt HTN rats . Mean arterial pressure (A) and heart rate (B) in high salt (2% NaCl) fed rats before and after they were infused with AngII (n=5) or vehicle control (n=5) . # = p<0.05 AngII versus vehicle treatment . 100 Total COX activity was measured in two hypothalamic CVOs (SFO and OVLT) and two brain regions known to regulate sympathetic activity (PVN and RVLM). T here w ere no significant difference s in COX activity on day A4 between vehicle treated and AngII trea ted rats in the RVLM (Figure 5 -2A), SFO (Figure 5 -2B) or OVLT (Figure 5 -2C). However, total COX activity was significantly higher in the PVN of AngII -salt HTN rats compared to vehicle treated rats (Figure 5 -2D). 101 Arbitrary fluorescent unit 020406080Vehicle (n=4) AngII (n=5) A. RVLM 0100020003000400050006000vehicle (n=7) AngII (n=5) Arbitrary fluorescent unit B. SFO 02000400060008000100001200014000vehicle (n=2) AngII (n=4) C. OVLT Arbitrary fluorescent unit 02004006008001000vehicle (n=5) AngII (n=4) Arbitrary fluorescent unit D. PVN # Figure 5-2: Change in cyclooxygenase activity in cardio -regulatory brain regions on day 4 of AngII -salt HTN. RVLM (Veh; n=4, AngII; n=5) (A), SFO (Veh; n=7, AngII; n=5) (B), OVLT (Veh; n=2, AngII; n=4) (C) and PVN (Veh; n=5, AngII; n=4) (D) . # =p<0.05 versus cont rol. 102 3.2. Brain cyclooxygenase inhibition in AngII -salt hypertensive rats : During the control period (C1, C2 and C3), baseline MAP was not differen t between the groups (Figure 5 -3A). MAP also was not affected by any ICV infusion rate of ketorolac or veh icle administration (D1, D2, D3 and D4). In the control group (ICV vehicle with sc vehicle) , MAP did not change from day A1 (105 ± 2mmHg) through day A14 (103 ± 2mmHg). In the AngII infused rats receiving ICV vehicle, MAP increased significantly from 110 ± 3mmHg on day A1 to 140 ± 10mmHg on day A14 (p<0.05). In the groups that received ICV ketorolac alone (33nmol/h or 0.6nmol/h) , MAP did not change throughout the study. In the AngII -infused rats receiving the two higher rates of ICV ketorolac (3.3 and 33nmo l/h), MAP rose for the first few days then declined to values not different from those seen in the control groups, i.e . ICV ketorolac with sc vehicle and ICV vehicle with sc vehicle. In rats receiving the lowest rate of ICV ketorolac infusion (0.6nmol/h) , MAP increased less during AngII infusion than in the control group, but the difference was not statistically significant. There were no significant differences in HR between th e groups at any time (figure not shown) . Finally, there w ere no difference s in neurogenic pressor activity (figure 5 -3B) between the 7 groups during the control period (day C2) or during drug control period ( day D2). However, after 10 days of AngII infusion ( day A10), the hexamethonium-mediated fall in MAP (mmHg ± SE) in the vehicle treated AngII -salt rats was 78 ± 10 mmHg. In comparison, the fall was 48 ± 5mmHg (p<0.05), 45 ± 9mmHg (p<0.05), and 57± 9mmHg (p>0.05) in AngII -salt rats treated with 33, 3.3 and 0.6mmol/h ICV ketorolac respectively that was not significantly different from the fall in MAP in rats not receiving AngII: ICV vehicle, 47 ± 1 mmHg; ICV ketorolac (33nmol/h), 43 ± 8mmHg ; and ICV ketorolac (0.6nmol/h), 36 ± 4mmHg . 103 Day C1C2C3D1D2D3D4A1A2A3A4A5A6A7A8A9A10 A11 A12 A13 A14 Mean Arterial Pressure (mmHg) 100120140160180200Veh ICV+Veh sc (n=4) Veh ICV+AngII sc (n=6) Keto(33nmol/h) ICV+Veh sc (n=4) Keto(33nmol/h) ICV+AngII sc (n=6) Keto(3.3nmol/h) ICV+AngII sc (n=5) Keto(0.6nmol/h) ICV+Veh sc (n=4) Keto(0.6nmol/h) ICV+AngII sc (n=9) Control Ketorolac ( 33nmol/h,3.3nmol/h or 0.6nmol/h) ICV Angiotensin II (150ng/kg/min) sc #*A Figure 5 -3: Effect of chronic COX inhibition on MAP and neurogenic pressor activity. MAP (A) and neurogenic pressor activity (B) in the rats on 2% s alt diet and infused with AngI I. # = p<0.05 keto 33nmlo/h + AngII vs veh + AngII, *=p<0.05 keto 3.3nmol/h + AngII vs. veh + AngII and $=p<0.05 all three control groups (veh + veh, keto 33nmol/h + veh, keto 0.6nmol/h + veh) vs. veh + AngII. Peak fall in MAP (mmHg) -120-100-80-60-40-200# $*Veh scKetorolac icv AngII sc-+-+---++++++--Veh icv+33330.63.30.6-------B 104 3.3. Plasma ketorolac measurement using high performance liquid chromatography : Three infusion rates of ketorolac, 33nmol/h (n=4), 3.3nmol/h (n= 5) and 0.6nmol/h (n=9) , were stud ied after measuring their MAP (Figure 5 -3A). After 1 4 days of ICV infusion in these rats the plasma concentration of ketorolac were 173±30ng/ml, 45±6ng/ml and undetectable, respectively in comparison to the concentration (100ng/ml) of ketorolac req uired to inhibit COX activity by 50% (IC50) in plasma (33). 3.4. Lipocalin-prostaglandin D synthase content in CSF during the early stages of AngII -salt HTN in r ats : L-PGDS protein content in CSF was significantly higher in the AngII -salt HTN rats compared to the vehicle treated rats (Figure 5-4). 3.5. Central and peripheral L -PGDS inhibition : MAP was not affected by either ICV or sc administration of AT56 alon e compared to vehicle treated rats (Figure 5-5A). During the first two days of AngII infusion, MAP rose similarly in all three groups of rats. By day A14, however, MAP rose to a significantly higher level in vehicle treated rats ( 124 ± 5 mmHg) compared to r ats given AT56 either ICV ( 105 ±5 mmHg) or sc ( 108 ± 1 mmHg). There were no significant differences in HR between the groups at any time (Figure 5 -5B). Neurogenic pressor activity (Figure 5-5C) was not different between the groups on day C3 or day D3, but wa s significantly lower in both AT56 treated groups compared to the control rats (p<0.05) on day A10 . 105 % Relative intensity (L-PTGDS/albumin) 05101520Control (n=5) Angiotensin II (n=5) #Albumin (66kD) L-PGDS (26kD) + control Veh AngII Figure 5 -4: Lipocalin -prostaglandin D synthase expression in cerebrospinal fluid during early stages (A4) of Ang II-salt HTN development. A representative western blot is shown in the upper panel. In the lower panel, the L-PGDS and albumin bands were scanned and the intensity of L -PGDS was normalized to the band intensity value of albumin . Data are expressed as relat ive intensity and # =p<0.05 AngII versus vehicle treated rats (n=5 in each group) . 106 Day C1C2C3D1D2D3D4D5A1A2A3A4A5A6A7A8A9A10 A11 A12 A13 A14 Mean Arterial Pressure (mmHg) 100120140160vehicle ICV + AngII (n=7) AT 56 ICV + AngII (n=7) AT 56 sc + AngII (n=4) Control AT 56 (5.8nmol/h) ICV or sc Angiotensin II (150ng/kg/min) sc #*A Figure 5 -5: The effect of chronic L-PGDS inhibition with AT56 . Effect on the mean arterial pressure (A), heart rate (B) and depres sor response to ganglionic blockade (C) in rats infused with AngII and fed a high salt diet. # = p<0.05 Vehicle ICV + AngII sc versus AT56 ICV/ AngII sc treated rats. * = p<0.05 Vehicle ICV/AngII versus AT56 sc + AngII sc treated rats. 107 Figure 5 -5 (Cont Õd) Day C1C2C3D1D2D3D4D5A1A2A3A4A5A6A7A8A9A10 A11 A12 A13 A14 Heart Rate (beats/min) 360380400420440460480500vehicle ICV+AngII (n=7) AT56 ICV+AngII (n=7) AT56 sc+AngII (n=4) Control AT56 (5.8nmol/h) ICV or sc Angiotensin II (150ng/kg/min) sc B Peak fall in MAP (mmHg) -100 -80 -60 -40 -20 0veh ICV + AngII sc (n=7) AT56 ICV + AngII sc (n=7) AT56 sc + AngII sc (n=4) *p<0.05 vs veh ICV+AngII sc #p<0.05 vs veh ICV+AngII sc #*C 108 4. Discussion Cao et al (22) showed that COX -1 activity specifically in the brain i s critical for AngII -mediated HTN in mice. In previous studies we found evidence that COX -1 activity is vital for increased neurogenic pressor activity, increased SNA and HTN development in an AngII -salt model in rats (8). AngII -salt HTN in rats is characterized by two stages, an early non -neurogenic phase (1 -5 days), and a second neurogenic phase (6 -14 days) (28, 31). Interest ingly, I demonstrated that inhibiting COX activity only during the early phase (first 3 -5 days of a 14 day infusion ) of AngII administration i s sufficient to attenuate subsequent neurogenic HTN development ( Chapter 4, Figure 4-3). In an attempt to localize the tissue(s) where COX products influenced neurogenic hypertension development, I investigated COX gene expression (Chapter 4, Figure 4-4 and Figure 4 -5) and protein expression (Chapter 4, Figure 4-6 and Table 4-1) in various brain regions and peripheral tissues during this early phase of the rat model, but observed no significant changes . Therefore, in the first part of the investigation described in this Chapter 1 measured the direct enzymatic activity of COX in our AngII -salt HTN model. Circumventric ular organs with a deficient blood brain barrier like the SFO and OVLT contain AT1 receptors and facilitate signaling in the brain in response to circulating AngII (17, 180). Critical AngII signaling through central circuit s beginning at either the SFO or OVLT and passing through the hypothalamic PVN and brainstem RVLM has been reported in AngII HTN models both i n the absence (118, 249) and presence (137, 176, 177) of high salt intake. Therefore, I measured COX enzymatic activity in these 109 regions during the early phase of AngII -salt HTN. I found an increase in COX activity only in the PVN. It is not clear if this increase occurred in neurons or other cells in the PVN. Generally COX -1 in the brain is found primarily in microglia and is expressed constitutively (192). There is evidence though that COX -1 enzymatic activity in glial cells can be directly stimulated via G -protein based signaling pathways (13, 223). In addition, however, COX-1 expression is increased in spinally projecting PVN neurons during both stress (103) and corticotropin releasing factor (206) induced sympathetic activation. PGE2 is a major product of COX -1 and acute injection of PGE2 into the PVN causes elevated SNA and BP in rats (120, 153). Collectively these results sugges t that during the early phase of AngII -salt HTN increased COX -1 activity could generate PGE2 or other products in the PVN and thereby increase SNA and BP. But because no direct links between circulating AngII, COX activity in the PVN, SNA and blood pressure have been reported, we perfo rmed additional experiments exploring the role of brain COX in HTN development. In my previous studies on COX activity in AngII -salt HTN, I used systemic administration of COX inhibitors and thus could not rule out that possibility that the antihypertensi ve effects I found were due at least in part to blocking the formation of eicosanoids in the vasculature, kidney or other peripheral organs. In order to determine whether COX activity specifically in the brain is required for the development of AngII -salt HTN, I tested the effect of selective COX inhibition in the brain using chronic ICV infusion of the COX1/COX2 inhibitor ketorolac . I chose ketorolac because of its relatively high water solubility, making it easier to deliver high concentrations of drug in to the CSF. Chronic 110 central COX inhibition with ICV ketorolac had no effect on BP or HR in normotensive rats, but attenuated HTN development and reduced neurogenic pressor activity in the second phase of AngII -salt HTN. I infuse d different doses of ketorol ac ICV and measure d plasma ketorolac concentration in an attempt to insure maximal inhibition of COX in the brain with minimum inhibition of peripheral COX (due to diffusion of ketorolac out of the brain into the systemic circulation). Although all ICV inf usion rates of ketorolac successfully ameliorated AngII -salt HTN , itÕs important to note that rates of 3.3 and 0.6 nmol/h produced plasma concentrations much lower than the reported IC50 required for inhibition of COX (100 ng/ml). This suggests that these infusion rates inhibited COX only in the brain and not in peripheral tissues. Although a limitation to this study is that I did not directly confirm COX inhibition in the brain of ketorolac treated rats, the results still strongly suggest that COX-derived eicos anoids in the brain are necessary for the increase in neurogenic pressor activity and BP in AngII -salt HTN. I previously reported increased expression of L -PGDS , an enzyme downstream from COX in the eicosanoid pathway, in the CP during the early st age of AngII -salt HTN (Figure 4 -6C). L -PGDS is synthesized by CP lining the ventricle s of brain and is secreted i nto the cerebrospinal fluid (232, 235). COX converts arachidonic acid to PGH2 and L -PGDS catalyzes the conversion of PGH2 to PGD2 ; this occurs predominantly in the brain. Apart from PGD2 synthesis, L -PGDS acts as a transpor ter of lipophilic molecules (235, 257), and thereby is part of the superfamily of secretory proteins called lipocalins. Brain L -PGDS is i mportant in sleep induction (46, 132, 233, 236), temperatu re regulation (118, 164) and inflammation (47, 56, 150), but has not 111 previously been linked to BP regula tion. However, acute ICV injection of PGD2 causes a modest, sympathet ically mediated increase in BP (76, 112, 118). Therefore, I performed two studies to test the hypothesis that brain L -PGDS contributes to the development of AngII -salt HTN. First I measured L -PGDS in the CSF of rats during the early phase of AngII -salt HTN, and second I explored the effect s of chronically blocking L-PGDS in the AngII -salt mod el using chronic ICV and sc infusion of the drug AT56, a highly selective L-PGDS blocker. In the first experiment, consistent with my hypothesis, I found significantly elevated CSF content of L -PGDS protein in rats with early phase AngII -salt HTN. Relatively little is known about the factors regulating L -PGDS synthesis and secretion into CSF (71), and there are no published data at all on AngII, although AT1 receptors are found on the CP and leptomeninges (33, 67). One possibility is that AngII -salt could work by increasing oxidative stress in the CP, since the latter has been shown to drive L -PGDS expression (273). In the second experiment, I found that both ICV and sc administration of the L -PGDS inhibitor AT56 markedly reduced BP and neurogenic pressor activity during the late (neurogenic) phase of AngII -salt HTN. Un fortunately, limited data were available to guide my selection of dose for AT56, so I canÕt be certain how completely L -PGDS activity in the brain was inhibited. Likewise I cannot rule out that the effects observed were due to blockade of peripheral L -PGD S. However, PGD2 is a vasodilator in the systemic circulation and inhibition of peripheral L -PGDS would be expected to cause 112 vasoconstriction and increased BP (121, 271). Despite these caveats, when considered with my other findings, the results from this experiment support the idea that increased brain L -PGDS activity is important in the development of AngII -salt HTN. My data here in rats provide strong support for the recent findings of Cao et al. in mice that COX1 in the brain plays an important role in AngII mediated HTN. A particularly notable similarity is that both our results and those of Cao et al sho w that brain COX activity is important only during the early stage of HTN development . As noted in Chapter 4, this suggest s that COX -derived eicosanoids are ÒprimingÓ a later increase in neurogenic pressor activity and HTN rather than causing them directly . There are, however, also some important differences between our findings and those of Cao et al. Their data in mice indicate the COX -1 activity and eicosanoid generation are increased only in the SFO and not in other brain regions; we found COX activity to be increased in the PVN but not in the SFO. Their data support PGE2 acting on the EP1 receptor as the main downstream signaling mechanism causing HTN; we did not specifically investigate PGE2 or its receptors, but our results implicate (for the first ti me) an alternative downstream eicosanoid pathway enzyme (L -PGDS) and product (PGD2) in AngII -salt HTN. These disparities could reflect differences in species, AngII infusion rates, and/or salt intake between our studies and require further investigation. In conclusio n, the results presented in this chapter provide insight into the central signaling mechanism s driving neurogenic AngII -salt HTN. We found an increase in COX activity in the PVN during early HTN development stages with no change in COX 113 expressi on indicating the possibility of more conversion of substrate into eicos anoid products through increase s in COX activity. The importance of the COX pathway is further supported by successful attenuation of HTN development with brain -selective COX inhibitio n. D ownstream of COX, L-PGDS was discovered to be critical in increasing neurogenic pressor activity and BP during AngII -salt HTN. The final studies in my dissertation were performed to explore in more detail how L -PGDS contributes to AngII -salt HTN and ar e described in Chapter 6. 114 CHAPTER 6 BRAIN PROSTAGLANDIN D2 AND EICOS ANOID RECEPTOR CHARACTERIZATION DURING DEVELOPMENT OF ANGIOTENSIN II -SALT HYPERTENSION IN THE RAT 115 1. Introduction In the central nervous system L-PGDS is predominantly synthesized in the leptomeninges and CP and later, secreted into the CSF (222, 237). Urade et al reported dominant mRNA expression of L -PGDS in leptomeninges, CP and oligodendrocytes of adult rat brain (237). L -PGDS (also known as beta trace protein i n humans) is the second most abundant protein (next only to albumin) that is secreted into the CSF (105). The two important functions of L -PGDS are: synthesis of PGD2, a lipid mediator that regulates physiological processes like sleep (234), pain (118) and body temperature (13); and a transporter of lipophilic molecules viz., bilirubin, biliverdin, and retinol. Due to this transport property L -PGDS is considered a member of t he lipocalin family of lipid transporters (232). Inhibiting L -PGDS enzymatic activity results in low brain PGD2 levels and suppression of sleep (189, 234). The CSF concentration of PGD2 in rats fluctuates in a circadian fashion: from 903 ± 162 pg/ml at night to 503 ± 7 8 pg/ml during the day (222). PGD2 affects sleep by acting on D -type eicos anoid receptors ( DP1R ) (189) on arachnoid trabecular cells on the ventral surface of rostral forebrain (158). PGD2 also binds to DP -2 receptors (DP2 R), otherwise known as chemo -attractant receptor homologue molecule expressed on T helper 2 cells (CRTH2). DP2R are mainly localized on type 2 lymphocytes, basophils and eosinophils (206). Recently, however, DP2R on neurons have been shown to mediate communication between microglia and neurons in the spinal cord (118). 116 A role for brain L -PGDS or PGD2 in the pathogenesis of hypertension has not been previously reported, although, Hirawa et al (103) found higher levels of L -PGDS in the plasma and urine of patients with essential hypertension. As described in earlier chapters of my dissertation, I found increased transcript levels of L -PGDS in the OVLT and CP, and increase d L-PGDS protein expression in the CSF , during the developmental stage of our AngII -salt HTN rat model. Furthermore, I showed that inhibition of L -PGDS in the brain significantly attenuates AngII -salt HTN and the associated increase in neurogenic pressor activity. Those results, however, do not establish whether the enzymatic or transport function of L -PGDS is most important for HTN develop ment. Because most known physiological effects of L -PGDS in the brain are mediated by PGD2, I hypothesized that PGD2 levels in the brain increase during the development of AngII -salt HTN. DP1R s are expressed in the arachnoid and CP (158) but expression in brain cardio -regulatory regions has not been reported. Therefore I performed experiments designed to localize DP1R s in circumventricular organs (SFO, OVLT), and other BP regulating brain regions like the PVN and RVLM, and examine changes in their expression during the development of AngII -salt HTN. 2. Experimental protocol s 2.1. AngII -salt HTN and tissue collection for eicosanoid measurement: Tissues were obtained from high salt (HS) (2%NaCl) fed rats that were infused with AngII ( 150 ng/kg/min, sc; n=5) or saline (n=5) for 4 days. B P was measured using a radio -telemeter catheter implanted into the abdominal aorta through the femoral artery. On the fourth day of AngII infusion (A4), CSF from cisterna magna of these rats was 117 collected using the protocol that is described in the methods section in Chapter 2. The rats were then euthanized and brains were collected. Brains were snap frozen on dry ice and stored at -80¡C. Using a cryostat maintained at -15¡C, 500 ! m thick coronal brain sections were prepared and brain punches from SFO , OVLT , PVN and RVLM were obtained. 2.2. Ultra -performance liquid chromatography -tandem mass spectrometry for measurement of AA and pr ostaglandins : The substrate for prostaglandin synthesis (AA) and the resultant eicos anoids, in particular PGD2 and its metabolites PGJ2, delta -12-PGJ2 and PGE2 levels were measured in brain punches and CS using ultra high performance liquid chromatography coupled with mass spectrometry as described in Chapter 2. 2.3. Immunofluorescence detection of DP1 receptor in early stages of AngII -salt HTN: Two groups of rats (AngII infused or vehicle infused) were prepared u sing our standard AngII -salt HTN protocol as described in Chapter 2, but I did not measure BP in these animals. After four days of AngII (n=5) or vehicle (n=5) infusion, brain tissue was collected . Using a microtome, 10 ! m thick sections cor onal brain sections were prepared. After immunofluorescent staining, the neurons and DP1 receptor expression were imaged in different regions like SFO, OVLT, PVN, RVLM and CP using confocal microscopy as described in Chapter 2. 118 3. Results 3.1. Blood pressure and heart rate : The blood pressure in high salt fed ra ts after 4 -days of AngII infusion was consistently higher compared to vehicle infused control rats (data not shown) . 3.2. Eicos anoid levels in cardio -regulatory brain regions during the early stage of AngII -salt HTN development : During the early stage of hypertension development there were higher levels of AA in the CP lining the third ventricle in HTN versus control rats (Figure 6 -1A). PGD2 was significantly higher in CP (Figure 6 -1B), CSF (Figure 6 -1C) and RVLM (Figure 6 -2A, 2B, 2C). No differences were found in other brain regions. There also were no differences in the levels of PGE2, or the PGD2 metabolites PGJ2, delta -12-PGJ2 and 15 -deoxy-PGJ2 in the various brain regions or CSF of AngII -salt HTN rats compared to the control rats. 119 VehicleAngII01!1062!1063!1064!1065!106AA(pg/region)*Choroid plexusA VehicleAngII0300060009000120001500018000PGD2(pg/region)*Choroid plexusB VehicleAngII0100200300400500PGD2(pg/ml)*Cerebrospinal fluidC Figure 6 -1: Ultra perform ance liquid chromatography -tandem mass s pectrometric analysis of lipid mediators . Measured AA (A) and PGD2 (B) levels in the CP lining the third ventricle and PGD2 levels in the cerebrospinal fluid (C) . t test *=p<0.05 vs. vehic le. (n=5 in each group). 120 VehicleAngII050001000015000PGD2(pg/region)*C Figure 6-2: Representative chromatogram with PGD2 levels in RVLM. With a retention time of 4.06 minutes and m/z of 351.2 !203.1, PGD2 peaks were detected in the RVLM of vehicle treated (A) and angiotensin II treated (B) rats which was higher in the RVLM (C). t test *=p<0.05 vs. vehicle. (n=5 in vehicle treated rats and n=4 in AngII -salt treated rats) . 121 3.3. Immunofluorescence detection of DP1 receptor in early stage s of AngII -salt HTN: Control rats showed significant expression of DP1R in the RVLM (Figure 6 -3). In AngII infused rats there was a decrease in DP1R expression that was statistically significant (Figure 6 -4). Another area that expressed DP1R was CP (Figure 6 -5A) but there was no difference in the expression in AngII infused rats (data not shown). In the cardio -regulatory regions of the brain like SFO (Figure 6 -5B), OVLT (Figure 6 -5C) and PVN (Figure 6 -5D) there was no detectable DP1R expression in control or AngII treated rats. 122 Figure 6-3: Double immunofluo rescent s taining for neuronal cell bod ies and the G-protein coupled receptor DP1R in early stages of HTN development. Arrow s point at the neurons and the DP1 receptors with NeuN and DP1 receptor staining respectively. . The DP1R in the RVLM of 4 -day vehicl e treated rats as shown in the top panel, were localized on the NeuN immunoreactive neurons. 4 days of angiotensin II treatment decreased the DP1R expression on the NeuN immunoreactive RVLM neurons (as shown in the bottom panel). Scale bar=30 ! m. [For inter pretation of t he references to color in this and all other figures, the reader is referred to the electronic version of this dissertation] 123 Figure 6-4: DP1 receptor expression in RVLM of 4 -day AngII -salt HTN rats. DP1R expressi on was lower in the RVLM neurons of 4 -day angiotensin II treated rats compared to the vehicle treated rats. Paired t test # =p<0.05 vs. vehicle. 124 Figure 6 -5: DP1R expression in CP, SFO, PVN and OVLT of AngII treated rats. CP lining the brain ventricles expressed DP1R as shown in the DIC image of AngII HTN rat Scale bar=3 0!m in a 40X image (A) and the expression was not different from the control rats (fig not shown). DP1R were not seen in SFO (B), PVN (C) or OVLT (D) of AngII or ve hicle (fig not shown) treated rats. Scale bar=6 0!m in a 20X image. !! 125 Discussion There are two main findings of this study. First, during the early stage of development of AngII -salt HTN there is an increase in PGD2 levels in the CP and CSF: brain sites where the enzyme L -PGDS is synthesized and secreted, respectively. Surprisingly, PGD2 concentrations also were increased in the RVLM, but not in the SFO, OVLT or PVN. Second, I found that DP1R s are expressed on neurons in the RVLM, but not in the SFO, OVLT or PVN; and that their expression is decreased during the development of AngII -salt HTN. These findings suggest the existence of a heretofore -unknown brain signaling mechanism -involving PGD2 that may contribute to the pathophysiology of HTN. In my experi ments, AngII -salt HTN was associated with an early increase in AA levels in the CP (Figure 6 -1A), suggesting increased activ ity of cPLA2. Combined with my observation that neither cPLA2 gene expression nor protein concentration was increased in the CP at t he same time period during AngII -salt HTN development (Chapter 4) , I conclude that cPLA2 is fun ctioning here as a constitutive . Similar to L-PGDS, cPLA2 in the brain is found predominantly in the CP, oligodendrocytes and astrocytes (135), but its role in brain physiology remains largely unknown (53). Although AngII is a potent stimulus for membrane phospholipid metabolism in other tissues (153), and activates the AA cascade (including PGD2 synthesis) in the cerebral microva sculature (213), this is the first report of increased choroid AA production in AngII HTN. As noted earlier, t he CP is the predominant site of synthesis of PGD2 and the CSF acts as a reservoir of secreted PGD2 (14, 222, 237). I found increased PGD2 126 levels in the CP and CSF in AngII -salt treated rats compared to control rats, indicating increased synthesis and secretion into CSF . I speculate that a coordinated increase in AA levels and L -PGDS enzymatic activity in the CP is required to generate elevated levels of PGD2 in response to AngII -salt treatment. Most importantly, when combined with my data showing that AngII -salt HTN is attenuated in animals receiving an inhibitor of L -PGDS (AT56) in the brain (Chapter 5 ), the current results support the conclusion that PGD2 signaling in the brain is a critical part of AngII -salt HTN development. This raises the question of where and how PGD2 acts in the brain to facilitate HTN development. Current thinking is that PGD2 initiates signaling in the brain by accessing target tissues both from the CSF (sleep) and from local tissue synthesis (from L -PGDS) (232), but details on the la tter are sparse. Administering PGD2 into the subarachnoid space of rats was shown to elicit powerful neuronal activation in brain regions involved in sleep regulation (e.g. ventrolateral preoptic area) and modest activation of the PVN, but to have no effec t on neurons in the RVLM (204). As noted in Chapter 1, injection of PGD2 into the brain causes an acute, sympathetically mediated increase in BP. To my knowle dge, however, no one has previously measured PGD2 concentrations in the CVOs, or cardioregulatory regions like the PVN or RVLM, in experimental models of HTN. A new finding of my study was increased PGD2 levels in the RVLM (but not in SFO, OVLT or PVN) dur ing AngII -salt HTN development (Figure 6 -2C). This observation led me to conclude that PGD2 could act in the RVLM to increase SNA and neurogenic pressor activity in AngII -salt HTN . 127 As an aside, Cao et al revealed elevated COX1 -derived PGE2 levels in the SFO during the early stages of AngII HTN in mice (22). I also found a small increase in PGE2 levels in the SFO early in AngII -salt HTN, but this difference was not statistically significant. Disparity between my results and those of Cao et al may be due to use of different infusions rates of AngII or to species differences. I also measured the PGD2 metabolites PGJ2 and delta -12-PGJ2 in the CP, CSF and cardio -regula tory brain regions. No change s in the levels of these metabolites were observed in AngII -salt HTN rats . This suggest s that conversion of PGD2 into these metabolites does not play a role in mediating HTN development. Another metabolite of PGD2 is 15deoxy de lta-PGJ2. It acts as a ligand for the transcription factor peroxisome proliferator activated receptor gamma and could inhibit activation of RAS, sympathetic outflow and brain inflammation, especially in PVN (271). However, my assay was unable to detect 15deoxy delta -PGJ2 in any of the samples, so IÕm unable to make any conclusions about its role in AngII -salt HTN. The next question I addressed was how PGD2 might alt er SNA and neurogenic pressor activity in AngII -salt HTN. As reviewed earlier, PGD2 binds to two different receptors, DP1R and DP2R (166). I investigated the DP 1 receptor s because it has been linked to most actions of PGD2 in the brain, especially sleep. DP1R are localized in arachnoid trabecular cells of the rostral forebrain and CP (158). There is also evidence of localization of DP1R mRNA in the CP and leptomeninges of rat brain (257). But to my 128 knowledge no one has rep orted DP1R in cardioregulatory brain regions. While confirming sites identified in earlier reports, my study shows for the first time localization of DP1R on neurons of the RVLM. Combined with my finding that PGD2 concentrations are elevated in the RVLM ea rly during AngII -salt HTN development, these results support the hypothesis that PGD2 acting at DP1R on RVLM neurons contributes to AngII -salt HTN. My additional observation that DP1R in the RVLM were downregulated with AngII treatment (Figure 6 -4) is cons istent with an AngII -salt mediated increase in PGD2 signaling providing the feedback stimulus for receptor downregulation. How might AngII and salt cause an increase PGD2 in the RVLM? One possibility is that AngII -salt treatment could increase PGD2 levels in the CSF (via the pathway outlined earlier) and PGD2 could then diffuse into the RVLM. This raises the question though of why I did not observe elevated PGD2 levels in other brain regions. Alternatively, AngII -salt stimulated neuronal signals from CVOs to the RVLM could increase the activity of L-PGDS and generation of PGD2 within the RVLM. However, I did not observe an increase in L -PGDS gene expression in the RVLM of rats early in AngII -salt HTN (Chapter 4 , Figure 4 -4D). T hat does not rule out the poss ibility of changes in enzymatic activity. Finally, AngII could act on cerebral blood vessels coursing through the RVLM to release PGD2, which could then diffuse into the brain parenchyma. Again, however, this raises the question of why increases in PGD2 w ere not seen in other vascularized brain regions. 129 How might PGD2 act in the RVLM to increase neurogenic pressor activity and cause HTN? As described in Chapter4, m y hypothesis is that eicosanoid products are acting in the brain not to directly increase S NA, but rather to produce slowly developing, adaptive changes in brain circuits that modulate SNA over the long -term (i.e. ÒprimeÓ later sympathoexcitation). Therefore, presumably the same cellular mechanisms proposed to be responsible for eicosanoid -mediated neuroplasticity (27) and neuronal adaptive responses (66) in other neural responses (st ress, learning) could operate in the RVLM. A more detailed understanding at the cellular level of how long -term, adaptive changes occur in neurons in cardioregulatory brain regions is emerging ( 26). Finally, even though there is abundant evidence linking increased neuronal activity in the RVLM to th e pathophysiology of AngII HTN (132), Pedrino and colleagues (181) recently reported that increased resting sympathoexcitatory discharge from RVLM neurons is not required for the maintenance of AngII -salt HTN in anesthetized rats. Thus, I need to consider the possibility that PGD2 may not influence AngII -salt HTN by actions in the RVLM. The data in this study reveal a novel association between PGD2 signaling via the DP1R in the brain and the development of AngII -salt hypertension. More detailed studies are required to determine if this information can be used to develop new, practical therapies for treating HTN or other conditions involving increased SNA. 130 CHAPTER 7 DISCUSSION 131 As reviewed in Chapter 1, eicosanoid s have long been implicated in the pathogenesis of hypertension but actions of eicosanoids on the cardioregulatory regions of the brain have received relatively little attention. Studies from Wilcox and colleagues suggested that the most important brain e icosanoids involved in AngII -dependent HTN are thromboxanes acting on thromboxane (TP) receptors (70, 255). Mor e recently Cao et al (4) proposed a very different scheme linking brain eicosanoids to the development of HTN based on experiments in mice. They suggested that during the early phase of HTN development, AngI I increased PGE2 generation in the SFO, which then acted on EP -1 receptors to increase BP and neurogenic pressor activity. My work reveals a completely new paradigm for understanding how eicosanoids act in the brain during AngII HTN development. In partic ular, I propose a previously unsuspected role for L -PGDS and PGD2. Figure 7 -1 is a summary of my data and a hypothetical scheme based on those data to explain how exposure to AngII and high salt diet might produce HTN. Briefly, I propose that in rats fed a high salt diet, peripheral AngII acts on br ain AT-1 receptor s to cause direct sympathoexcitation via established pathways originating in the OVLT and SFO. In addition, however, Ang II acts on other AT-1 receptors to initiate eicosanoid -based signaling. In cerebral vessels and CP, cPLA2 is stimulated to produce AA, which is the n converted by COX1 to PGH2. L -PGDS is induced in the CP and secreted into the CSF, and as a result converts PGH2 to PGD2 in both CP and CSF. Possibly by diffusion from the CSF, PGD2 l evels also increase in the RVLM. 132 Figure 7 -1: A hypothetical signaling pathway by which circulating AngII and high salt intake may cause increased splanchnic SNA and HTN. Acronyms are as defined in the text. CG = celiac ganglion 133 PGD2 binds to DP 1R in the RVLM and causes a slowly developing change in either neuronal excitability or synaptic function that ultimately produces higher splanchnic SNA and HTN. 7.1. Function of choroid plexus in angiotensin II -salt hypertension development One unique as pect of my hypothesis is the postulated role of the CP in HTN development. In the central nervous system, CP lines the lateral ventricles, third ventricles and fourth ventricles. CP is made up of a monolayer of ependymal cells that are the choroid epitheli al cells with basal lamina, large central spherical nucleus and villi on the luminal surface. It plays an important role in the synthesis of CSF (19). Importantly , AT -1 receptors are found in the CP (73, 229) and previous work has suggested that the CP is a target o f AngII signaling in HTN (52) but m ainly to affect the ability of the CP to regulate CSF sodium concentration (5). On the other hand, my data indicate that the CP is a source of eicosanoids that regu late SNA. Binding of AngII to AT -1 receptors increases intracellular calcium and thereby causes cPLA2 to translocate to the intracellular endoplasmic reticulum and nuclear membranes (14). The 85kD enzyme cPLA2 is present in different organs including bra in and is localized in platelets, macrophages, neutrophils, endothelial cells and vascular smooth muscle cells (131). Cytosolic PLA2 mRNA is reported in pia mater both on the brain surface as well as in the inner core of the CP (135). In response to injury there is an increase in cPLA2 expression in the astrocytes that act as neuron support cells providing cell -to-cell communication in the brain (220). Phosphorylation regulates the 134 catalytic activity of cPLA2 at the phospholipid membrane (39). One caveat to my observation of increased cPLA2 activity in AngII -salt HTN is the lack of data on the levels of phosphorylated cPLA2 in hypertensive rats compared to the controls. However, increased production of AA in the CP after AngII administration is strong evidence of increased functional cPLA2 activity there, either in the vasculature or other cell types. Arachidonic acid release from the membrane phospholipids (PL) by the action of cPLA2 is acted upon by the two isozymes of cyclooxygenase viz., COX -1 and COX -2, local ized in the endoplasmic reticulum (ER) and nuclear envelope, respectively (75). The positioning of COX -1 near cPLA2 in the ER could provide ready access to free AA in the CP for eicosanoid synthesis. Since I observed a COX -1 dependent increase in BP in my model, it is logical to hypothesize that COX -1 mediates the synthesis of downstream eicosanoid products in the brain near the site of AA release --the choroid ple xus--during early stages of AngII -salt HTN. My data indicate that this likely does not require additional COX protein to be produced. 7.2. Possible role of L -PGDS in the development of AngII -salt hypertension CP is the major site of L -PGDS synthesis (15). The CP promptly responds to peripheral inflammatory stimuli with induction of genes like interleu kin 1 and tumor necrosis factor ", but L -PGDS (normally constitutive) also is strongly induced (147). The mRNA levels of L -PGDS are increased in the CP after 6, 12 and 24 hours, but return t o baseline after 72 hours (147). This could be why I did not see increased L -PGDS transcript levels in 135 the CP in 4 -day AngII salt treated rats. L -PGDS protein expression on the other hand was higher in the CP lining the third ventricle when compared to the control rats suggesting a differential transcriptional and translational regulation of L -PGDS in the CP during early developmental stages of AngII -salt HTN. L-PGDS in the brain is localized in subcellular structures like rough endoplasmic reticulum, Golgi apparatus, arachnoid trabecular cells and oligodendroglial cells (231). Interestingly, similar to the close association of cPLA2 with COXs, L -PGDS is also co -localized with COXs in choroid plexus, leptomeninges and perivascular micr oglial cells (14). Given the increase in L -PGDS expression in the CP (Figure 4 -6C) along with increased PGD2 levels in CP (Figure 6 -1B) and CSF (Figure 6 -1C), I postulate that synthesis of PGD2 in CP and CSF is due to increased L -PGDS activity during early development of AngII -salt HTN. This implies that the mechanism by which the competitive inhibitor of L -PGDS, AT56, reduces both PGD2 levels (Figure 6 -3) in t he brain and AngII -salt HTN development is via reduced PGD2 formation f rom L -PGDS in the CP . A limitation of my study is that I did not measure PGD2 levels in the CP or CSF in AT56 treated rats (I measured them only in the RVLM). The importance of L -PGDS i n AngII -salt HTN development is bolstered by the observed disparity in the regulation of L -PGDS in males compared to the female rats. Kittikulsuth and co -workers (130) recently report ed that female rats are protected against development of AngII -salt hypertension compared to male rats. This protection is likely caused by gonadal sex hormone estrogen (E2). The L -PGDS promoter contains E2 binding sites (40) and E2 decreases L -PGDS m -RNA levels in the brain (161). It is therefore tempting to speculate that regulation of L -PGDS by E2 could be the cause of the relative resistance of female rats to AngII -salt HTN. 136 7.3. Possible role of PGD2 acting on DP1Rs in the development of AngII -salt hypertension The main function of L -PGDS is synthesis of PGD2 from the precursor PGH2. PGD2 is the most abundant prostaglandin in the brain (234). It is present in CSF and rapidly transported out through a variety of lipid an d organic anion transporters (222). In AngII -salt HTN rats I found increased PGD2 levels in the CP, CSF and RVLM. So does PGD2 signaling in the brain alter blood pressure? As reviewed earlier, most PGD2 actions in the brain are mediated via DP1Rs. Because I found DP1R expression in the RVLM (Figure 6 -4), I predicted that paracrine signaling by PGD2 through DP1Rs in the RVLM could mediate AngII -salt HTN devel opment. Some support for this idea came from my finding that DP1Rs are downregulated in the RVLM during the early stage of AngII -salt HTN (perhaps as a response to increased local agonist concentration). Two important caveats to this idea, however, are t hat: 1) I did not demonstrate that PGD2 in the brain can by itself cause neurogenic HTN; and 2) I did not show that blocking DP1Rs in the RVLM attenuates development of AngII -salt HTN. Although not included in my dissertation, I did two preliminary studi es in an attempt to address both of these concerns. First, I infused PGD2 ICV for 14 days into rats on high salt diet and measured BP via telemetry. I expected to see a gradual rise in BP and neurogenic pressor activity after a delay of 3 -5 days, but inste ad there were no changes in either variable when compared to vehicle -infused control rats. Very surprisingly though, when I measured the concentration of PGD2 in the CSF of these rats at the end of the study I found equally low levels of PGD2 in the two gr oups of rats. Thus, it is likely 137 that this experiment was not a valid test of my original hypothesis. Second, I induced AngII -salt HTN with our standard protocol in rats treated ICV with either vehicle or the DP1R antagonist BW A868C . I hypothesized that t he DP1R antagonist would block the development of HTN in a manner similar to what I observed with ketorolac and AT56. Instead the rats receiving the DP1R antagonist actually became more hypertensive than the vehicle treated rats, and exhibited greater neur ogenic pressor activity. The results of this study certainly do not support my hypothesis concerning DP1R in AngII -salt HTN. However, I did not do experiments to confirm that the infusion rate of BW A868C I used was in fact effective in blocking DP1R in th e RVLM, so these results also are not definitive. In light of this uncertainty about the role of the DP1R in AngII -salt HTN, an alternative explanation may be an action of PGD2 on the other main receptor subtype, DP2R, also known as chemoattractant recepto r homologous molecule expressed on T helper cells (CRTH2). DP2 Rs are present at low levels in the brain and are predominantly localized on T helper 2 -lymphocytes, eosinophils and basophils (102). However recently it was shown that PGD2 derived from COX -1 in microglial cells could affect neuronal function by actions on DP2R, at least in the spinal cord (118). Therefore, a role for DP2Rs in PGD2 signaling during development of AngII -salt HTN is possible and could be tested with a pharmacological DP2R antagonist or with DP2R knockout approaches. 7.4. Importance of RVLM as the site of action of PGD2 to increase BP Potentiated RVLM neuronal activity is believed to cause both experimental hypertension and human essential hypertension (132). As noted by Mueller (165): Ò The increased 138 output of the RVLM in hypertension is likely dependent on increased excitatory input from other brain regio ns, as well as an increase in sensitivity to excitatory input . Interestingly, the increase in sensitivity to excitation may occur together or separately from the increased tonic excitatory input depending on the model or risk factor studied. Ó In this dis sertation I propose that in AngII -salt HTN there may be both increased excitatory input from the OVLT and SFO , and increased sensitivity to excitatory input in the RVLM; and that the latter is caused in part by PGD2 derived mainly from the CP. Neurons expressing the Gs subtype DP1 receptors are cyclic AMP dependent. When stimulated, DP1 -Rs could activate protein kinase A and i ncrease intracellular calcium. An i ncrease in calcium is known to activate the calmodulin -mediated myosin light chain kinase (CAM kin ase II) that has been associated with memory formation in male mice (159). CAM kinase II expressed by the dendritic spine has been shown to modulate neural plas ticity (212). The differential ex pression of CAM kinase II with PGD2 signaling has not been reported but there could be a modulation of synaptic transmission through CAM kinase II that could cause enhanced synaptic transmission during HTN development. Mueller also highlights the evidence that increased sensitivity of RVLM neurons to excitatory inputs is the result of slowly developing but persistent structural and/or functional changes in synaptic transmission within the RVLM (i.e. neuroplasticity) (165). This is consistent with my finding, and that of Cao et al (22), that increased production of eicosanoid products is essential for the development but not the maintenance of AngII -salt HTN, suggesting that eicosanoids work by gradually changing neuronal sensitivity rather than by direct actions on neuronal activity. 139 7.5 Future directions The data presented in my dissertation support sequential roles for cPLA2, COX -1, L -PGDS and PGD2 in potentiating the development of An gII-salt HT. However, it does not exclude the possibility that the critical actions of L -PGDS in neurogenic HTN are independent of PGD2 formation. Therefore, more experiments need to be performed to confirm a cause -and-effect relationship between increased brain PGD2 and neurogenic HTN. Specifically, it needs to be determined whether the main func tion of L -PGDS is to act as a lipid carrier or to synthesize PGD2. This could be investigated by additional studies on the ability of PGD2 itself to cause chronic HTN, and by further investigation of DP1R and DPR2 antagonists or knockout animals. The contribution of PGD2 signaling at the molecular level in different cell types like neurons, astrocytes and microglia also needs to be further explored in order to devel op a complete understanding of how PGD2 in the brain affects SNA and BP. Finally, much additional work is necessary to confirm that the RVLM is the principal site of action of COX products in mediating neurogenic HTN. This would likely require tissue -speci fic genetic targeting strategies because of the difficulty of chronically and specifically accessing the RVLM with drugs in conscious animals. 7.6. Overall significance, perspectives and therapeutic implications In light of suggested roles for brain L -PGD S in sleep (189), obesity (43) and insulin resistance (51), and the known associations of these conditions with sympathetic overactivity and essential HTN, my findings reveal an up -to-now unknown signaling pathway in the brain that c ould explain why these physiological and pathological 140 conditions often occur together. Nevertheless, with regards to HTN it will be challenging to capitalize on my findings to generate novel therapies. This is because the new mechanism I discovered appears to operate only during the initiation of HTN and is not required once HTN is established. Current therapy of HTN is focused only on lowering BP once the condition is established. On the other hand, itÕs possible that a more refined understanding of how L -PGDS or PGD2 affects brain pathways regulating SNA could lead to strategies for reversing those changes and thereby modulating SNA for therapeutic benefit. 141 REFERENCES 142 REFERENCES 1. A global brief on hypertension silent killer, global public health crisis. World Health Day 2013, Geneva, World Health Organization 2013. 2. Abrams JM, and Osborn JW . A role for benzamil -sensitive proteins of the central nervous system in the pathogenesis of salt -dependent hypertension. Clinical and experimental pharmacology & physiology 35: 687-694, 2008. 3. Agarwal A, Williams GH, and Fisher ND . Genetics of human hypertension. Trends in endocrinology and metabolism: TEM 16: 127-133, 2005. 4. Allen AM . Inhibition of the hy pothalamic paraventricular nucleus in spontaneously hypertensive rats dramatically reduces sympathetic vasomotor tone. Hypertension 39: 275-280, 2002. 5. Amin MS, Reza E, Wang H, and Leenen FH . Sodium transport in the choroid plexus and salt -sensitive hype rtension. Hypertension 54: 860-867, 2009. 6. Ando K, and Fujita M . Reactive oxygen species and the central nervous system in salt -sensitive hypertension: possible relationship with obesity -induced hypertension. Clinical and experimental pharmacology & phys iology 39: 111-116, 2012. 7. Araujo M, and Welch WJ . Tubuloglomerular feedback is decreased in COX -1 knockout mice after chronic angiotensin II infusion. American journal of physiology Renal physiology 298: F1059-1063, 2010. 8. Asirvatham -Jeyaraj N, King A J, Northcott CA, Madan S, and Fink GD . Cyclooxygenase -1 inhibition attenuates angiotensin II -salt hypertension and neurogenic pressor activity in the rat. American journal of physiology Heart and circulatory physiology 2013. 9. Atlas SA . The renin-angiotensin aldosterone system: pathophysiological role and pharmacologic inhibition. Journal of managed care pharmacy : JMCP 13: 9-20, 2007. 10. Barlassina C, Lanzani C, Manunta P, and Bianchi G . Genetics of essential hypertension: from families to genes. Journal of the American Society of Nephrology : JASN 13 Suppl 3: S155 -164, 2002. 143 11. Barnes JN, Casey DP, Hines CN, Nicholson WT, and Joyner MJ . Cyclooxygenase inhibition augments central blood pressure and aortic wave reflection in aging humans. American journal of physiology Heart and circulatory physiology 302: H2629-2634, 2012. 12. Bazan NG . Lipid signaling in neural plasticity, brain repair, and neuroprotection. Molecular neurobiology 32: 89-103, 2005. 13. Bergmann BM, Landis CA, Zenko CE, and Rechtschaffen A . Sleep deprivation in the rat: XVII. Effect of aspirin on elevated body temperature. Sleep 16: 221-225, 1993. 14. Beuckmann CT, Lazarus M, Gerashchenko D, Mizoguchi A, Nomura S, Mohri I, Uesugi A, Kaneko T, Mizuno N, Hayaishi O, and Urade Y . Cellular loca lization of lipocalin -type prostaglandin D synthase (beta -trace) in the central nervous system of the adult rat. The Journal of comparative neurology 428: 62 -78, 2000. 15. Blodorn B, Mader M, Urade Y, Hayaishi O, Felgenhauer K, and Bruck W . Choroid plexus: the major site of mRNA expression for the beta -trace protein (prostaglandin D synthase) in human brain. Neuroscience letters 209: 117-120, 1996. 16. Borghi C, Boschi S, Costa FV, and Ambrosioni E . Factors associated with acute salt -sensitivity in borderli ne hypertensive patients. Clinical and experimental hypertension Part A, Theory and practice 14: 837-851, 1992. 17. Braga VA, Medeiros IA, Ribeiro TP, Franca -Silva MS, Botelho -Ono MS, and Guimaraes DD . Angiotensin -II-induced reactive oxygen species along t he SFO -PVN-RVLM pathway: implications in neurogenic hypertension. Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica [et al] 44: 871-876, 2011. 18. Brooks VL, Hay wood JR, and Johnson AK . Translation of salt retention to central activation of the sympathetic nervous system in hypertension. Clinical and experimental pharmacology & physiology 32: 426-432, 2005. 19. Brown PD, Davies SL, Speake T, and Millar ID . Molecul ar mechanisms of cerebrospinal fluid production. Neuroscience 129: 957-970, 2004. 144 20. Campese VM, Karubian F, Chervu I, Parise M, Sarkies N, and Bigazzi R . Pressor reactivity to norepinephrine and angiotensin in salt -sensitive hypertensive patients. Hypert ension 21: 301-307, 1993. 21. Campos RR, Oliveira -Sales EB, Nishi EE, Boim MA, Dolnikoff MS, and Bergamaschi CT . The role of oxidative stress in renovascular hypertension. Clinical and experimental pharmacology & physiology 38: 144-152, 2011. 22. Cao X, Pe terson JR, Wang G, Anrather J, Young CN, Guruju MR, Burmeister MA, Iadecola C, and Davisson RL . Angiotensin II -dependent hypertension requires cyclooxygenase 1 -derived prostaglandin E2 and EP1 receptor signaling in the subfornical organ of the brain. Hyper tension 59: 869-876, 2012. 23. Capone C, Faraco G, Anrather J, Zhou P, and Iadecola C . Cyclooxygenase 1 -derived prostaglandin E2 and EP1 receptors are required for the cerebrovascular dysfunction induced by angiotensin II. Hypertension 55: 911-917, 2010. 24. Capone C, Faraco G, Peterson JR, Coleman C, Anrather J, Milner TA, Pickel VM, Davisson RL, and Iadecola C . Central cardiovascular circuits contribute to the neurovascular dysfunction in angiotensin II hypertension. The Journal of neuroscience : the official journal of the Society for Neuroscience 32: 4878-4886, 2012. 25. Chai SY, Zhuo J, and Mendelsohn FA . Localization of components of the renin -angiotensin system and site of action of inhibitors. Arzneimittel -Forschung 43: 214 -221, 1993. 26. Chan CC, Re id CM, Aw TJ, Liew D, Haas SJ, and Krum H . Do COX -2 inhibitors raise blood pressure more than nonselective NSAIDs and placebo? An updated meta-analysis. Journal of hypertension 27: 2332-2341, 2009. 27. Chen C, and Bazan NG . Lipid signaling: sleep, synaptic plasticity, and neuroprotection. Prostaglandins & other lipid mediators 77: 65-76, 2005. 28. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL, Jr., Jones DW, Materson BJ, Oparil S, Wright JT, Jr., and Roccella EJ . The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure: the JNC 7 report. JAMA : the journal of the American Medical Association 289: 2560-2572, 2003. 145 29. Coleman RA, Smith WL, and Narumiya S . International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacological reviews 46: 205-229, 1994. 30. Collister JP, Olson MK, Nahey DB, Vieira AA, and Osborn JW . OVLT lesion decreases basal arterial pressure and the chronic hypertensive response to AngII in rats on a high-salt diet. Physiological Reports 1: 2013. 31. Coote JH . A role for the paraventricular nucleus of the hypothalamus in the autonomic control of heart and kidne y. Experimental physiology 90: 169-173, 2005. 32. Cox BF, and Bishop VS . Neural and humoral mechanisms of angiotensin -dependent hypertension. The American journal of physiology 261: H1284-1291, 1991. 33. Cryer B, and Feldman M . Cyclooxygenase -1 and cycloox ygenase -2 selectivity of widely used nonsteroidal anti -inflammatory drugs. The American journal of medicine 104: 413-421, 1998. 34. Cuadra AE, Shan Z, Sumners C, and Raizada MK . A current view of brain renin -angiotensin system: Is the (pro)renin receptor t he missing link? Pharmacology & therapeutics 125: 27-38, 2010. 35. Curtis -Prior P . The eicosanoids. 3 -13, 2004. 36. Dampney RA, Coleman MJ, Fontes MA, Hirooka Y, Horiuchi J, Li YW, Polson JW, Potts PD, and Tagawa T . Central mechanisms underlying short - and long-term regulation of the cardiovascular system. Clinical and experimental pharmacology & physiology 29: 261-268, 2002. 37. Dauphinot V, Kossovsky MP, Gueyffier F, Pichot V, Gosse P, Roche F, and Barthelemy JC . Impaired baroreflex sensitivity and the ri sks of new -onset ambulatory hypertension, in an elderly population -based study. International journal of cardiology 168: 4010-4014, 2013. 38. Davis PA, Mussap M, Pagnin E, Bertipaglia L, Savica V, Semplicini A, and Calo LA . Early markers of inflammation in a high angiotensin II state --results of studies in Bartter's/Gitelman's syndromes. Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 21: 1697-1701, 2006. 146 39. Dennis EA, Cao J, Hsu YH, Magrioti V, and Kokotos G . Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chemical reviews 111: 6130-6185, 2011. 40. Devidze N, Fujimori K, Ura de Y, Pfaff DW, and Mong JA . Estradiol regulation of lipocalin -type prostaglandin D synthase promoter activity: evidence for direct and indirect mechanisms. Neuroscience letters 474: 17-21, 2010. 41. Draaijer P, Kool MJ, Maessen JM, van Bortel LM, de Leeuw PW, van Hooff JP, and Leunissen KM . Vascular distensibility and compliance in salt -sensitive and salt -resistant borderline hypertension. Journal of hypertension 11: 1199-1207, 1993. 42. Eaton SB, and Konner M . Paleolithic nutrition. A consideration of its nature and current implications. The New England journal of medicine 312: 283-289, 1985. 43. Elias E, Benrick A, Behre CJ, Ekman R, Zetterberg H, Stenlof K, and Wallenius V . Central nervous system lipocalin -type prostaglandin D2 -synthase is correlated wit h orexigenic neuropeptides, visceral adiposity and markers of the hypothalamic-pituitary -adrenal axis in obese humans. Journal of neuroendocrinology 23: 501-507, 2011. 44. Elliott RB, Starling MB, and Neutze JM . Medical manipulation of the ductus arteriosu s. Lancet 1: 140-142, 1975. 45. Esler M . The sympathetic nervous system through the ages: from Thomas Willis to resistant hypertension. Experimental physiology 96: 611-622, 2011. 46. Esler M . The sympathetic system and hypertension. American journal of hyp ertension 13: 99S-105S, 2000. 47. Esler M, Julius S, Zweifler A, Randall O, Harburg E, Gardiner H, and DeQuattro V . Mild high -renin essential hypertension. Neurogenic human hypertension? The New England journal of medicine 296: 405-411, 1977. 48. Esler M, and Kaye D . Sympathetic nervous system activation in essential hypertension, cardiac failure and psychosomatic heart disease. Journal of cardiovascular pharmacology 35: S1 -7, 2000. 147 49. Esler MD, Krum H, Schlaich M, Schmieder RE, Bohm M, and Sobotka PA . Renal sympathetic denervation for treatment of drug -resistant hypertension: one -year results from the Symplicity HTN -2 randomized, controlled trial. Circulation 126: 2976 -2982, 2012. 50. Esler MD, Krum H, Sobotka PA, Schlaich MP, Schmieder RE, and Bohm M . Renal sympathetic denervation in patients with treatment -resistant hypertension (The Symplicity HTN -2 Trial): a randomised controlled trial. Lancet 376: 1903-1909, 2010. 51. Evans JF, Islam S, Urade Y, Eguchi N, and Ragolia L . The lipocalin -type prostaglandin D2 synthase knockout mouse model of insulin resistance and obesity demonstrates early hypothalamic -pituitary -adrenal axis hyperactivity. The Journal of endocrinology 216: 169-180, 2013. 52. Faraci FM, Baumbach GL, and Heistad DD . Cerebral circulation: hum oral regulation and effects of chronic hypertension. Journal of the American Society of Nephrology : JASN 1: 53-57, 1990. 53. Farooqui AA, and Horrocks LA . Brain phospholipases A2: a perspective on the history. Prostaglandins, leukotrienes, and essential f atty acids 71: 161-169, 2004. 54. Farooqui AA, Horrocks LA, and Farooqui T . Modulation of inflammation in brain: a matter of fat. Journal of neurochemistry 101: 577-599, 2007. 55. Felder RA, White MJ, Williams SM, and Jose PA . Diagnostic tools for hyperten sion and salt sensitivity testing. Current opinion in nephrology and hypertension 22: 65-76, 2013. 56. Ferrier C, Cox H, and Esler M . Elevated total body noradrenaline spillover in normotensive members of hypertensive families. Clin Sci (Lond) 84: 225-230, 1993. 57. Fierro-Carrion GA, and Ram CV . Nonsteroidal anti -inflammatory drugs (NSAIDs) and blood pressure. The American journal of cardiology 80: 775-776, 1997. 58. Fink GD . Long -term sympatho -excitatory effect of angiotensin II: a mechanism of spontaneou s and renovascular hypertension. Clinical and experimental pharmacology & physiology 24: 91-95, 1997. 148 59. Fink GD, Haywood JR, Bryan WJ, Packwood W, and Brody MJ . Central site for pressor action of blood -borne angiotensin in rat. The American journal of ph ysiology 239: R358-361, 1980. 60. Forstermann U, Heldt R, and Hertting G . Studies on the mechanism of central cardiovascular and temperature responses to prostaglandin D2. Prostaglandins, leukotrienes, and medicine 18: 301-308, 1985. 61. Frohlich ED, and V aragic J . Sodium directly impairs target organ function in hypertension. Current opinion in cardiology 20: 424-429, 2005. 62. Frolich JC, Hollifield JW, Dormois JC, Frolich BL, Seyberth H, Michelakis AM, and Oates JA . Suppression of plasma renin activity b y indomethacin in man. Circulation research 39: 447-452, 1976. 63. Fujimori K, Watanabe M, Urade Y, and Ishikawa K . Increased production of lipocalin -type prostaglandin D synthase in leptomeningeal cells through contact with astrocytes. Neuroscience letter s 423: 133-137, 2007. 64. Funk CD . Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 294: 1871-1875, 2001. 65. Funke-Kaiser H, Reinemund J, Steckelings UM, and Unger T . Adapter proteins and promoter regulation of the angiotensin AT2 receptor --implications for cardiac pathophysiology. Journal of the renin -angiotensin -aldosterone system : JRAAS 11: 7-17, 2010. 66. Furuyashiki T, and Narumiya S . Roles of prostaglandin E receptors in stress responses. Current opinion in pharmacology 9: 31-38, 2009. 67. Furuyashiki T, and Narumiya S . Stress responses: the contribution of prostaglandin E(2) and its receptors. Nature reviews Endocrinology 7: 163-175, 2011. 68. Fyhrquist F, and Saijonmaa O . Renin -angiotensin system revisited. Journal of intern al medicine 264: 224-236, 2008. 69. Gabor A, and Leenen FH . Central neuromodulatory pathways regulating sympathetic activity in hypertension. J Appl Physiol (1985) 113: 1294-1303, 2012. 149 70. Gao H, Peng B, Welch WJ, and Wilcox CS . Central thromboxane recept ors: mRNA expression and mediation of pressor responses. The American journal of physiology 272: R1493-1500, 1997. 71. Gao H, Welch WJ, DiBona GF, and Wilcox CS . Sympathetic nervous system and hypertension during prolonged TxA2/PGH2 receptor activation in rats. The American journal of physiology 273: H734-739, 1997. 72. Gao W, Schmidtko A, Wobst I, Lu R, Angioni C, and Geisslinger G . Prostaglandin D2 produced by hematopoietic prostaglandin D synthase contributes to LPS -induced fever. Journal of physiology a nd pharmacology : an official journal of the Polish Physiological Society 60: 145-150, 2009. 73. Gehlert DR, Gackenheimer SL, and Schober DA . Autoradiographic localization of subtypes of angiotensin II antagonist binding in the rat brain. Neuroscience 44: 501-514, 1991. 74. Giovannini MG, Scali C, Prosperi C, Bellucci A, Pepeu G, and Casamenti F . Experimental brain inflammation and neurodegeneration as model of Alzheimer's disease: protective effects of selective COX -2 inhibitors. International journal of immunopathology and pharmacology 16: 31-40, 2003. 75. Goetzl EJ, An S, and Smith WL . Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases. FASEB journal : official publication of the Federation of American So cieties for Experimental Biology 9: 1051-1058, 1995. 76. Gonzalez JD, Llinas MT, Moreno C, Rodriguez F, and Salazar FJ . Renal effects of prolonged cyclooxygenase inhibition when angiotensin II levels are elevated. Journal of cardiovascular pharmacology 36: 236-241, 2000. 77. Gonzalez -Albarran O, Ruilope LM, Villa E, and Garcia Robles R . Salt sensitivity: concept and pathogenesis. Diabetes research and clinical practice 39 Suppl: S15 -26, 1998. 78. Grassi G . Assessment of sympathetic cardiovascular drive in h uman hypertension: achievements and perspectives. Hypertension 54: 690-697, 2009. 150 79. Grassi G . Renin -angiotensin -sympathetic crosstalks in hypertension: reappraising the relevance of peripheral interactions. Journal of hypertension 19: 1713 -1716, 2001. 80. Grassi G . Role of the sympathetic nervous system in human hypertension. Journal of hypertension 16: 1979-1987, 1998. 81. Grassi G, and Esler M . How to assess sympathetic activity in humans. Journal of hypertension 17: 719-734, 1999. 82. Grassi G, Quarti -Trevano F, Dell'oro R, and Mancia G . Essential hypertension and the sympathetic nervous system. Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology 29 Suppl 1: S33 -36, 2008. 83. Griffin SA, Brown WC, MacPherson F, McGrath JC, Wilson VG, Korsgaard N, Mulvany MJ, and Lever AF . Angiotensin II causes vascular hypertrophy in part by a non-pressor mechanism. Hypertension 17: 626-635, 1991. 84. Gu B, Desjardins P, and Butterworth RF . S elective increase of neuronal cyclooxygenase -2 (COX -2) expression in vulnerable brain regions of rats with experimental Wernicke's encephalopathy: effect of nimesulide. Metabolic brain disease 23: 175-187, 2008. 85. Guild SJ, McBryde FD, Malpas SC, and Bar rett CJ . High dietary salt and angiotensin II chronically increase renal sympathetic nerve activity: a direct telemetric study. Hypertension 59: 614-620, 2012. 86. Guyenet PG . The sympathetic control of blood pressure. Nature reviews Neuroscience 7: 335-346, 2006. 87. Guyton AC, Coleman TG, Cowley AV, Jr., Scheel KW, Manning RD, Jr., and Norman RA, Jr. Arterial pressure regulation. Overriding dominance of the kidneys in long-term regulation and in hypertension. The American journal of medicine 52: 584 -594, 1972. 88. Hall JE. Guyton and Hall Textbook of Medical Physiology . 2011. 151 89. Hall JE, Guyton AC, and Brands MW . Pressure -volume regulation in hypertension. Kidney international Supplement 55: S35 -41, 1996. 90. Hall JE, Guyton AC, and Mizelle HL . Role of th e renin -angiotensin system in control of sodium excretion and arterial pressure. Acta physiologica Scandinavica Supplementum 591: 48-62, 1990. 91. Hall JE, Mizelle HL, Hildebrandt DA, and Brands MW . Abnormal pressure natriuresis. A cause or a consequence o f hypertension? Hypertension 15: 547 -559, 1990. 92. Harlan SM, and Rahmouni K . PI3K signaling: A key pathway in the control of sympathetic traffic and arterial pressure by leptin. Molecular metabolism 2: 69-73, 2013. 93. Hart FD, and Huskisson EC . Non -ster oidal anti -inflammatory drugs. Current status and rational therapeutic use. Drugs 27: 232-255, 1984. 94. Hayaishi O . Molecular genetic studies on sleep -wake regulation, with special emphasis on the prostaglandin D(2) system. J Appl Physiol (1985) 92: 863-868, 2002. 95. Hayaishi O, Matsumura H, and Urade Y . Prostaglandin D synthase is the key enzyme in the promotion of physiological sleep. Journal of lipid mediators 6: 429 -431, 1993. 96. Haywood JR, Fink GD, Buggy J, Phillips MI, and Brody MJ . The area postr ema plays no role in the pressor action of angiotensin in the rat. The American journal of physiology 239: H108-113, 1980. 97. He FJ, Li J, and Macgregor GA . Effect of longer -term modest salt reduction on blood pressure. The Cochrane database of systematic reviews 4: CD004937, 2013. 98. Heesch CM, Laiprasert JD, and Kvochina L . RVLM glycine receptors mediate GABAA and GABAB)independent sympathoinhibition from CVLM in rats. Brain research 1125: 46-59, 2006. 99. Heidenreich PA, Trogdon JG, Khavjou OA, Butler J, Dracup K, Ezekowitz MD, Finkelstein EA, Hong Y, Johnston SC, Khera A, Lloyd -Jones DM, Nelson SA, Nichol G, Orenstein D, Wilson PW, and Woo YJ . Forecasting the future of 152 cardiovascular disease in the United States: a policy statement from the American He art Association. Circulation 123: 933-944, 2011. 100. Herichova I, and Szantoova K . Renin -angiotensin system: upgrade of recent knowledge and perspectives. Endocrine regulations 47: 39-52, 2013. 101. Hing E, Hall MJ, Ashman JJ, and Xu J . National Hospital Ambulatory Medical Care Survey: 2007 outpatient department summary. National health statistics reports 1-32, 2010. 102. Hirai H, Tanaka K, Yoshie O, Ogawa K, Kenmotsu K, Takamori Y, Ichimasa M, Sugamura K, Nakamura M, Takano S, and Nagata K . Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven -transmembrane receptor CRTH2. The Journal of experimental medicine 193: 255 -261, 2001. 103. Hirawa N, Uehara Y, Yamakado M, Toya Y, Gomi T, Ikeda T, Eguchi Y, Takagi M, Oda H, Seiki K, Urade Y, and Umemura S . Lipocalin -type prostaglandin d synthase in essential hypertension. Hypertension 39: 449-454, 2002. 104. Hoffman WE, and Schmid PG . Cardiovascular and antidiuretic effects of central prostaglandin E2. The Jo urnal of physiology 288: 159-169, 1979. 105. Hoffmann A, Conradt HS, Gross G, Nimtz M, Lottspeich F, and Wurster U . Purification and chemical characterization of beta -trace protein from human cerebrospinal fluid: its identification as prostaglandin D synth ase. Journal of neurochemistry 61: 451-456, 1993. 106. Hoozemans JJ, Rozemuller JM, van Haastert ES, Veerhuis R, and Eikelenboom P . Cyclooxygenase -1 and -2 in the different stages of Alzheimer's disease pathology. Current pharmaceutical design 14: 1419-1427, 2008. 107. Howard LL, Patterson ME, Mullins JJ, and Mitchell KD . Salt -sensitive hypertension develops after transient induction of ANG II -dependent hypertension in Cyp1a1-Ren2 transgenic rats. American journal of physiology Renal physiology 288: F810-815, 2005. 108. Huang BS, Amin MS, and Leenen FH . The central role of the brain in salt -sensitive hypertension. Current opinion in cardiology 21: 295-304, 2006. 153 109. Huang ZL, Urade Y, and Hayaishi O . Prostaglandins and adenosine in the regulation of sleep a nd wakefulness. Current opinion in pharmacology 7: 33-38, 2007. 110. Husted D, Upshaw J, Gridley KE, and Wood CE . Cellular localization of thromboxane synthase in ovine spinal cord and hindbrain. Brain research 971: 107 -115, 2003. 111. Imanishi M, Kawamura M, Akabane S, Matsushima Y, Kuramochi M, Ito K, Ohta M, Kimura K, Takamiya M, and Omae T . Aspirin lowers blood pressure in patients with renovascular hypertension. Hypertension 14: 461-468, 1989. 112. Inoue M, Crofton JT, and Share L . Interactions between the brain renin -angiotensin system and brain prostanoids in the control of vasopressin secretion. Experimental brain research Experimentelle Hirnforschung Experimentation cerebrale 83: 131-136, 1990. 113. Izzo JL, Sica DA, and Black HR . Hypertension prime r. 94 -96, 2008. 114. Jackson EK, Oates JA, and Branch RA . Indomethacin decreases arterial blood pressure and plasma renin activity in rats with aortic ligation. Circulation research 49: 180-185, 1981. 115. Jia Z, Zhang A, Zhang H, Dong Z, and Yang T . Delet ion of microsomal prostaglandin E synthase -1 increases sensitivity to salt loading and angiotensin II infusion. Circulation research 99: 1243-1251, 2006. 116. Judy WV, Watanabe AM, Henry DP, Besch HR, Jr., Murphy WR, and Hockel GM. Sympathetic nerve activi ty: role in regulation of blood pressure in the spontaenously hypertensive rat. Circulation research 38: 21-29, 1976. 117. Kanaoka Y, and Urade Y . Hematopoietic prostaglandin D synthase. Prostaglandins, leukotrienes, and essential fatty acids 69: 163-167, 2003. 118. Kanda H, Kobayashi K, Yamanaka H, and Noguchi K . COX -1-dependent prostaglandin D2 in microglia contributes to neuropathic pain via DP2 receptor in spinal neurons. Glia 61: 943-956, 2013. 154 119. Kandlikar SS, and Fink GD . Splanchnic sympathetic ner ves in the development of mild DOCA -salt hypertension. American journal of physiology Heart and circulatory physiology 301: H1965-1973, 2011. 120. Katafuchi T, Ichijo T, and Hori T . Sequential relationship between actions of CRF and PGE2 in the brain on sp lenic sympathetic nerve activity in rats. Journal of the autonomic nervous system 67: 200-206, 1997. 121. Katsumata Y, Shinmura K, Sugiura Y, Tohyama S, Matsuhashi T, Ito H, Yan X, Ito K, Yuasa S, Ieda M, Urade Y, Suematsu M, Fukuda K, and Sano M . Endogeno us Prostaglandin D2 and Its Metabolites Protect the Heart Against Ischemia -Reperfusion Injury by Activating Nrf2. Hypertension 2013. 122. Katsuya T, Ishikawa K, Sugimoto K, Rakugi H, and Ogihara T . Salt sensitivity of Japanese from the viewpoint of gene po lymorphism. Hypertension research : official journal of the Japanese Society of Hypertension 26: 521-525, 2003. 123. Kaufmann WE, Worley PF, Pegg J, Bremer M, and Isakson P . COX -2, a synaptically induced enzyme, is expressed by excitatory neurons at postsy naptic sites in rat cerebral cortex. Proceedings of the National Academy of Sciences of the United States of America 93: 2317-2321, 1996. 124. Kawada N, Dennehy K, Solis G, Modlinger P, Hamel R, Kawada JT, Aslam S, Moriyama T, Imai E, Welch WJ, and Wilcox CS. TP receptors regulate renal hemodynamics during angiotensin II slow pressor response. American journal of physiology Renal physiology 287: F753-759, 2004. 125. Kenney MJ, Weiss ML, and Haywood JR . The paraventricular nucleus: an important component of the central neurocircuitry regulating sympathetic nerve outflow. Acta physiologica Scandinavica 177: 7-15, 2003. 126. King AJ, and Fink GD . Chronic low -dose angiotensin II infusion increases venomotor tone by neurogenic mechanisms. Hypertension 48: 927-933, 2006. 127. King AJ, Novotny M, Swain GM, and Fink GD . Whole body norepinephrine kinetics in ANG II -salt hypertension in the rat. American journal of physiology Regulatory, integrative and comparative physiology 294: R1262-1267, 2008. 128. King AJ, Osborn JW, and Fink GD . Splanchnic circulation is a critical neural target in angiotensin II salt hypertension in rats. Hypertension 50: 547-556, 2007. 155 129. Kitiyakara C, Welch WJ, Verbalis JG, and Wilcox CS . Role of thromboxane receptors in the dipsogenic respo nse to central angiotensin II. American journal of physiology Regulatory, integrative and comparative physiology 282: R865-869, 2002. 130. Kittikulsuth W, Looney SW, and Pollock DM . Endothelin ET(B) receptors contribute to sex differences in blood pressure elevation in angiotensin II hypertensive rats on a high -salt diet. Clinical and experimental pharmacology & physiology 40: 362 -370, 2013. 131. Kramer RM, and Sharp JD . Structure, function and regulation of Ca2+ -sensitive cytosolic phospholipase A2 (cPLA2) . FEBS letters 410: 49-53, 1997. 132. Kumagai H, Oshima N, Matsuura T, Iigaya K, Imai M, Onimaru H, Sakata K, Osaka M, Onami T, Takimoto C, Kamayachi T, Itoh H, and Saruta T . Importance of rostral ventrolateral medulla neurons in determining efferent sympa thetic nerve activity and blood pressure. Hypertension research : official journal of the Japanese Society of Hypertension 35: 132-141, 2012. 133. Kuroki MT, Guzman PA, Fink GD, and Osborn JW . Time -dependent changes in autonomic control of splanchnic vascu lar resistance and heart rate in ANG II -salt hypertension. American journal of physiology Heart and circulatory physiology 302: H763-769, 2012. 134. Lambert E, Dawood T, Schlaich M, Straznicky N, Esler M, and Lambert G . Single -unit sympathetic discharge pa ttern in pathological conditions associated with elevated cardiovascular risk. Clinical and experimental pharmacology & physiology 35: 503-507, 2008. 135. Lautens LL, Chiou XG, Sharp JD, Young WS, 3rd, Sprague DL, Ross LS, and Felder CC . Cytosolic phosphol ipase A2 (cPLA2) distribution in murine brain and functional studies indicate that cPLA2 does not participate in muscarinic receptor -mediated signaling in neurons. Brain research 809: 18-30, 1998. 136. Leenen FH, Ruzicka M, and Huang BS . The brain and salt -sensitive hypertension. Current hypertension reports 4: 129-135, 2002. 137. Liang X, Wu L, Hand T, and Andreasson K . Prostaglandin D2 mediates neuronal protection via the DP1 receptor. Journal of neurochemistry 92: 477-486, 2005. 156 138. Lim SM, Chen D, Teo H, Roos A, Jansson AE, Nyman T, Tresaugues L, Pervushin K, and Nordlund P . Structural and dynamic insights into substrate binding and catalysis of human lipocalin prostaglandin D synthase. Journal of lipid research 54: 1630-1643, 2013. 139. Lin L, Mistry M , Stier CT, Jr., and Nasjletti A . Role of prostanoids in renin -dependent and renin -independent hypertension. Hypertension 17: 517-525, 1991. 140. Llewellyn -Smith AIJ . Central Regulation of Autonomic Functions. 2011, p. 23 -40. 141. Lohmeier TE . Angiotensin II infusion model of hypertension: is there an important sympathetic component? Hypertension 59: 539-541, 2012. 142. Luft FC . Geneticism of essential hypertension. Hypertension 43: 1155 -1159, 2004. 143. Luft FC, Wilcox CS, Unger T, Kuhn R, Demmert G, Rohme iss P, Ganten D, and Sterzel RB . Angiotensin -induced hypertension in the rat. Sympathetic nerve activity and prostaglandins. Hypertension 14: 396-403, 1989. 144. MacNeil BJ, Jansen AH, Janz LJ, Greenberg AH, and Nance DM . Peripheral endotoxin increases spl enic sympathetic nerve activity via central prostaglandin synthesis. The American journal of physiology 273: R609-614, 1997. 145. Malpas SC . Sympathetic nervous system overactivity and its role in the development of cardiovascular disease. Physiological re views 90: 513-557, 2010. 146. Mark AL . Sympathetic neural contribution to salt -induced hypertension in Dahl rats. Hypertension 17: I86-90, 1991. 147. Marques F, Sousa JC, Correia -Neves M, Oliveira P, Sousa N, and Palha JA . The choroid plexus response to pe ripheral inflammatory stimulus. Neuroscience 144: 424-430, 2007. 148. Martin DS, Haywood JR, and Thornhill JA . Stimulation of the hypothalamic paraventricular nucleus causes systemic venoconstriction. Brain research 604: 318 -324, 1993. 157 149. Martinez -Revell es S, Avendano MS, Garcia -Redondo AB, Alvarez Y, Aguado A, Perez -Giron JV, Garcia -Redondo L, Esteban V, Redondo JM, Alonso MJ, Briones AM, and Salaices M . Reciprocal relationship between reactive oxygen species and cyclooxygenase -2 and vascular dysfunction in hypertension. Antioxidants & redox signaling 18: 51-65. 150. Masuo K, Rakugi H, Ogihara T, Esler MD, and Lambert GW . Cardiovascular and renal complications of type 2 diabetes in obesity: role of sympathetic nerve activity and insulin resistance. Curren t diabetes reviews 6: 58-67, 2010. 151. McGiff JC, Crowshaw K, Terragno NA, and Lonigro AJ . Release of a prostaglandin -like substance into renal venous blood in response to angiotensin II. Circulation research 27: 121-130, 1970. 152. Mehl M, Bidmon HJ, Hil big H, Zilles K, Dringen R, and Ullrich V . Prostacyclin synthase is localized in rat, bovine and human neuronal brain cells. Neuroscience letters 271: 187-190, 1999. 153. Mehta PK, and Griendling KK . Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. American journal of physiology Cell physiology 292: C82-97, 2007. 154. Miki K, and Yoshimoto M . Sympathetic nerve activity during sleep, exercise, and mental stress. Autonomic neuroscience : basic & clinical 174: 15-20, 2013. 155. Mistry M, Muirhead EE, Yamaguchi Y, and Nasjletti A . Renal function in rats with angiotensin II -salt -induced hypertension: effect of thromboxane synthesis inhibition and receptor blockade. Journal of hypertension 8: 75-83, 1990. 156. Mis try M, and Nasjletti A . Contrasting effect of thromboxane synthase inhibitors and a thromboxane receptor antagonist on the development of angiotensin II -salt -induced hypertension in rats. The Journal of pharmacology and experimental therapeutics 253: 90-94, 1990. 157. Mistry M, and Nasjletti A . Role of pressor prostanoids in rats with angiotensin II-salt -induced hypertension. Hypertension 11: 758-762, 1988. 158. Mizoguchi A, Eguchi N, Kimura K, Kiyohara Y, Qu WM, Huang ZL, Mochizuki T, Lazarus M, Kobayashi T, Kaneko T, Narumiya S, Urade Y, and Hayaishi O . Dominant localization of prostaglandin D receptors on arachnoid trabecular 158 cells in mouse basal forebrain and their involvement in the regulation of non -rapid eye movement sleep. Proceedings of the National Academy of Sciences of the United States of America 98: 11674-11679, 2001. 159. Mizuno K, Ris L, Sanchez -Capelo A, Godaux E, and Giese KP . Ca2+/calmodulin kinase kinase alpha is dispensable for brain development but is required for distinct memories in ma le, though not in female, mice. Molecular and cellular biology 26: 9094-9104, 2006. 160. Mohri I, Eguchi N, Suzuki K, Urade Y, and Taniike M . Hematopoietic prostaglandin D synthase is expressed in microglia in the developing postnatal mouse brain. Glia 42: 263-274, 2003. 161. Mong JA, Devidze N, Frail DE, O'Connor LT, Samuel M, Choleris E, Ogawa S, and Pfaff DW . Estradiol differentially regulates lipocalin -type prostaglandin D synthase transcript levels in the rodent brain: Evidence from high -density oligonucleotide arrays and in situ hybridization. Proceedings of the National Academy of Sciences of the United States of America 100: 318-323, 2003. 162. Moretti JL, Burke SL, Davern PJ, Evans RG, Lambert GW, and Head GA . Renal sympathetic activation from long -term low -dose angiotensin II infusion in rabbits. Journal of hypertension 30: 551-560, 2012. 163. Morimoto A, Uzu T, Fujii T, Nishimura M, Kuroda S, Nakamura S, Inenaga T, and Kimura G . Sodium sensitivity and cardiovascular events in patients with essentia l hypertension. Lancet 350: 1734-1737, 1997. 164. Morimoto S, Sasaki S, Itoh H, Nakata T, Takeda K, Nakagawa M, Furuya S, Naruse S, Fukuyama R, and Fushiki S . Sympathetic activation and contribution of genetic factors in hypertension with neurovascular com pression of the rostral ventrolateral medulla. Journal of hypertension 17: 1577-1582, 1999. 165. Mueller PJ . Physical (in)activity -dependent alterations at the rostral ventrolateral medulla: influence on sympathetic nervous system regulation. American jour nal of physiology Regulatory, integrative and comparative physiology 298: R1468-1474, 2010. 166. Nagata K, and Hirai H . The second PGD(2) receptor CRTH2: structure, properties, and functions in leukocytes. Prostaglandins, leukotrienes, and essential fatty acids 69: 169-177, 2003. 159 167. Nakamura K . Central circuitries for body temperature regulation and fever. American journal of physiology Regulatory, integrative and comparative physiology 301: R1207-1228, 2011. 168. Nasjletti A . Arthur C. Corcoran Memorial Lecture. The role of eicosanoids in angiotensin -dependent hypertension. Hypertension 31: 194-200, 1998. 169. Nishimura M, Ohtsuka K, Nanbu A, Takahashi H, and Yoshimura M . Benzamil blockade of brain Na+ channels averts Na(+) -induced hypertension in rats. The American journal of physiology 274: R635-644, 1998. 170. Northcott CA, Watts S, Chen Y, Morris M, Chen A, and Haywood JR . Adenoviral inhibition of AT1a receptors in the paraventricular nucleus inhibits acute increases in mean arterial blood pressure in the rat. American journal of physiology Regulatory, integrative and comparative physiology 299: R1202-1211, 2010. 171. Okuno T, Lindheimer MD, and Oparil S . Central effects of prostaglandin E2 on blood pressure and plasma renin activity in rats. Role of th e sympathoadrenal system and vasopressin. Hypertension 4: 809-816, 1982. 172. Olsson JE . Human beta -trace in normal and pathological CNS tissues, genital organs and body fluids. Advances in experimental medicine and biology 433: 351 -354, 1997. 173. Orlov S N, and Mongin AA . Salt -sensing mechanisms in blood pressure regulation and hypertension. American journal of physiology Heart and circulatory physiology 293: H2039-2053, 2007. 174. Osborn JL . Relation between sodium intake, renal function, and the regulati on of arterial pressure. Hypertension 17: I91-96, 1991. 175. Osborn JW, and Fink GD . Region -specific changes in sympathetic nerve activity in angiotensin II -salt hypertension in the rat. Experimental physiology 95: 61 -68, 2010. 176. Osborn JW, Fink GD, Sve d AF, Toney GM, and Raizada MK . Circulating angiotensin II and dietary salt: converging signals for neurogenic hypertension. Current hypertension reports 9: 228-235, 2007. 160 177. Osborn JW, Hendel MD, Collister JP, Ariza -Guzman PA, and Fink GD . The role of t he subfornical organ in angiotensin II -salt hypertension in the rat. Experimental physiology 97: 80-88, 2012. 178. Palar K, and Sturm R . Potential societal savings from reduced sodium consumption in the U.S. adult population. American journal of health pro motion : AJHP 24: 49-57, 2009. 179. Palkovits M . Isolated removal of hypothalamic or other brain nuclei of the rat. Brain research 59: 449-450, 1973. 180. Paton JF, Wang S, Polson JW, and Kasparov S . Signalling across the blood brain barrier by angiotensin II: novel implications for neurogenic hypertension. J Mol Med (Berl) 86: 705-710, 2008. 181. Pedrino GR, Calderon AS, Andrade MA, Cravo SL, and Toney GM . Discharge of Rvlm Vasomotor Neurons Is Not Increased in Anesthetized Angiotensin Ii - Salt Hypertensi ve Rats. American journal of physiology Heart and circulatory physiology 2013. 182. Peng JF, Wu ZT, Wang YK, Yuan WJ, Sun T, Ni X, Su DF, Wang W, Xu MJ, and Wang WZ . GABAergic mechanism in the rostral ventrolateral medulla contributes to the hypotension of moxonidine. Cardiovascular research 89: 473-481, 2011. 183. Perini C, Muller FB, Rauchfleisch U, Battegay R, and Buhler FR . Hyperadrenergic borderline hypertension is characterized by suppressed aggression. Journal of cardiovascular pharmacology 8 Suppl 5 : S53 -56, 1986. 184. Perry JC, Bergamaschi CT, Campos RR, Andersen ML, Montano N, Casarini DE, and Tufik S . Sympathetic and angiotensinergic responses mediated by paradoxical sleep loss in rats. Journal of the renin -angiotensin -aldosterone system : JRAAS 12: 146-152, 2011. 185. Porter JP, and Brody MJ . Neural projections from paraventricular nucleus that subserve vasomotor functions. The American journal of physiology 248: R271 -281, 1985. 186. Potas JR, and Dampney RA . Sympathoinhibitory pathway from caudal midline medulla to RVLM is independent of baroreceptor reflex pathway. American journal of physiology Regulatory, integrative and comparative physiology 284: R1071-1078, 2003. 161 187. Pyner S . Neurochemistry of the paraventricular nucleus of the hypothalamus : implications for cardiovascular regulation. Journal of chemical neuroanatomy 38: 197 -208, 2009. 188. Qi Z, Hao CM, Langenbach RI, Breyer RM, Redha R, Morrow JD, and Breyer MD. Opposite effects of cyclooxygenase -1 and -2 activity on the pressor response t o angiotensin II. The Journal of clinical investigation 110: 61-69, 2002. 189. Qu WM, Huang ZL, Xu XH, Aritake K, Eguchi N, Nambu F, Narumiya S, Urade Y, and Hayaishi O . Lipocalin -type prostaglandin D synthase produces prostaglandin D2 involved in regulati on of physiological sleep. Proceedings of the National Academy of Sciences of the United States of America 103: 17949 -17954, 2006. 190. Quilley J . COX -2 and angiotensin II -induced hypertension and oxidative stress. American journal of hypertension 24: 1188. 191. Rahn KH, Barenbrock M, and Hausberg M . The sympathetic nervous system in the pathogenesis of hypertension. Journal of hypertension Supplement : official journal of the International Society of Hypertension 17: S11 -14, 1999. 192. Ram A, Pandey HP, Ma tsumura H, Kasahara -Orita K, Nakajima T, Takahata R, Satoh S, Terao A, and Hayaishi O . CSF levels of prostaglandins, especially the level of prostaglandin D2, are correlated with increasing propensity towards sleep in rats. Brain research 751: 81-89, 1997. 193. Ramchandra R, Yao ST, and May CN . Organ selective regulation of sympathetic outflow by the brain Angiotensin system. Current hypertension reports 15: 401-408, 2013. 194. Ricciotti E, and FitzGerald GA . Prostaglandins and inflammation. Arteriosclerosi s, thrombosis, and vascular biology 31: 986-1000, 2011. 195. Ricote M, Li AC, Willson TM, Kelly CJ, and Glass CK . The peroxisome proliferator -activated receptor -gamma is a negative regulator of macrophage activation. Nature 391: 79-82, 1998. 196. Roger VL, Go AS, Lloyd -Jones DM, Benjamin EJ, Berry JD, Borden WB, Bravata DM, Dai S, Ford ES, Fox CS, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, 162 Makuc DM, Marcus GM, Marelli A, Matchar DB, Moy CS, Mozaffarian D, Mussolino ME, Nichol G, Paynter NP, Soliman EZ, Sorlie PD, Sotoodehnia N, Turan TN, Virani SS, Wong ND, Woo D, and Turner MB . Heart disease and stroke statistics --2012 update: a report from the American Heart Association. Circul ation 125: e2-e220, 2012. 197. Ross CA, Ruggiero DA, Park DH, Joh TH, Sved AF, Fernandez -Pardal J, Saavedra JM, and Reis DJ . Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C 1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin. The Journal of neuroscience : the official journal of the Society for Neuroscience 4: 474 -494, 1984. 198. Rushmer RF. Cardiovascular dynamics . p. 177-187. 199. Sakai RR, Fine WB, Epstein AN, and Frankmann SP . Salt appetite is enhanced by one prior episode of sodium depletion in the rat. Behavioral neuroscience 101: 724-731, 1987. 200. Samuelsson B . Identification of a Smooth Muscle -Stimulating Factor in Bovine Brain. Prostaglandins and Related Factors 25. Biochimica et biophysica acta 84: 218 -219, 1964. 201. Samuelsson B, Morgenstern R, and Jakobsson PJ . Membrane prostaglandin E synthase -1: a novel therapeutic target. Pharmacological reviews 59: 207-224, 2007. 202. Saper CB, Romanovsky AA, and Scammell TE . Neural circuitry engaged by prostaglandins during the sickness syndrome. Nature neuroscience 15: 1088 -1095, 2012. 203. Sato Y, Ogata E, and Fujita T . Role of chloride in angiotensin II -induced salt -sensitive hy pertension. Hypertension 18: 622-629, 1991. 204. Scammell T, Gerashchenko D, Urade Y, Onoe H, Saper C, and Hayaishi O . Activation of ventrolateral preoptic neurons by the somnogen prostaglandin D2. Proceedings of the National Academy of Sciences of the Uni ted States of America 95: 7754-7759, 1998. 205. Schlaich MP, Lambert E, Kaye DM, Krozowski Z, Campbell DJ, Lambert G, Hastings J, Aggarwal A, and Esler MD . Sympathetic augmentation in hypertension: 163 role of nerve firing, norepinephrine reuptake, and Angiote nsin neuromodulation. Hypertension 43: 169-175, 2004. 206. Schuligoi R, Sturm E, Luschnig P, Konya V, Philipose S, Sedej M, Waldhoer M, Peskar BA, and Heinemann A . CRTH2 and D -type prostanoid receptor antagonists as novel therapeutic agents for inflammator y diseases. Pharmacology 85: 372 -382, 2010. 207. Siegle I, Klein T, Zou MH, Fritz P, and Komhoff M . Distribution and cellular localization of prostacyclin synthase in human brain. The journal of histochemistry and cytochemistry : official journal of the Hi stochemistry Society 48: 631-641, 2000. 208. Simpson JB . The circumventricular organs and the central actions of angiotensin. Neuroendocrinology 32: 248-256, 1981. 209. Smith WL, DeWitt DL, and Garavito RM . Cyclooxygenases: structural, cellular, and molecu lar biology. Annual review of biochemistry 69: 145-182, 2000. 210. Smyth EM, and FitzGerald GA . Human prostacyclin receptor. Vitamins and hormones 65: 149-165, 2002. 211. Smyth EM, Grosser T, Wang M, Yu Y, and FitzGerald GA . Prostanoids in health and disea se. Journal of lipid research 50 Suppl: S423 -428, 2009. 212. Soderling TR . CaM -kinases: modulators of synaptic plasticity. Current opinion in neurobiology 10: 375-380, 2000. 213. Spatz M, Stanimirovic D, Bacic F, Uematsu S, and McCarron RM . Vasoconstrictiv e peptides induce endothelin -1 and prostanoids in human cerebromicrovascular endothelium. The American journal of physiology 266: C654 -660, 1994. 214. Stahl RA, Helmchen U, Paravicini M, Ritter LJ, and Schollmeyer P . Glomerular prostaglandin formation in t wo-kidney, one -clip hypertensive rats. The American journal of physiology 247: F975-981, 1984. 215. Steiner AA, Li S, Llanos QJ, and Blatteis CM . Differential inhibition by nimesulide of the early and late phases of intravenous - and intracerebroventricular -LPS -induced fever in guinea pigs. Neuroimmunomodulation 9: 263-275, 2001. 164 216. Stocker SD, Madden CJ, and Sved AF . Excess dietary salt intake alters the excitability of central sympathetic networks. Physiology & behavior 100: 519-524, 2010. 217. Stocker S D, Osborn JL, and Carmichael SP . Forebrain osmotic regulation of the sympathetic nervous system. Clinical and experimental pharmacology & physiology 35: 695-700, 2008. 218. Stocker SD, Smith CA, Kimbrough CM, Stricker EM, and Sved AF . Elevated dietary salt suppresses renin secretion but not thirst evoked by arterial hypotension in rats. American journal of physiology Regulatory, integrative and comparative physiology 284: R1521-1528, 2003. 219. Sugimoto Y, Yamasaki A, Segi E, Tsuboi K, Aze Y, Nishimura T, O ida H, Yoshida N, Tanaka T, Katsuyama M, Hasumoto K, Murata T, Hirata M, Ushikubi F, Negishi M, Ichikawa A, and Narumiya S . Failure of parturition in mice lacking the prostaglandin F receptor. Science 277: 681-683, 1997. 220. Sun GY, Xu J, Jensen MD, Yu S, Wood WG, Gonzalez FA, Simonyi A, Sun AY, and Weisman GA . Phospholipase A2 in astrocytes: responses to oxidative stress, inflammation, and G protein -coupled receptor agonists. Molecular neurobiology 31: 27 -41, 2005. 221. Svennerholm L . Distribution and fat ty acid composition of phosphoglycerides in normal human brain. Journal of lipid research 9: 570-579, 1968. 222. Tachikawa M, Tsuji K, Yokoyama R, Higuchi T, Ozeki G, Yashiki A, Akanuma S, Hayashi K, Nishiura A, and Hosoya K . A clearance system for prostag landin D2, a sleep -promoting factor, in cerebrospinal fluid: role of the blood -cerebrospinal barrier transporters. The Journal of pharmacology and experimental therapeutics 343: 608-616, 2012. 223. Takahata R, Matsumura H, Kantha SS, Kubo E, Kawase K, Saka i T, and Hayaishi O . Intravenous administration of inorganic selenium compounds, inhibitors of prostaglandin D synthase, inhibits sleep in freely moving rats. Brain research 623: 65 -71, 1993. 224. Tassoni D, Kaur G, Weisinger RS, and Sinclair AJ . The role of eicosanoids in the brain. Asia Pacific journal of clinical nutrition 17 Suppl 1: 220 -228, 2008. 165 225. Thomas DW, Mannon RB, Mannon PJ, Latour A, Oliver JA, Hoffman M, Smithies O, Koller BH, and Coffman TM . Coagulation defects and altered hemodynamic resp onses in mice lacking receptors for thromboxane A2. The Journal of clinical investigation 102: 1994-2001, 1998. 226. Toney GM, Chen QH, Cato MJ, and Stocker SD . Central osmotic regulation of sympathetic nerve activity. Acta physiologica Scandinavica 177: 43-55, 2003. 227. Toney GM, and Stocker SD . Hyperosmotic activation of CNS sympathetic drive: implications for cardiovascular disease. The Journal of physiology 588: 3375 -3384, 2010. 228. Touw KB, Haywood JR, Shaffer RA, and Brody MJ . Contribution of the sympathetic nervous system to vascular resistance in conscious young and adult spontaneously hypertensive rats. Hypertension 2: 408-418, 1980. 229. Tsutsumi K, and Saavedra JM . Characterization and development of angiotensin II receptor subtypes (AT1 and AT2 ) in rat brain. The American journal of physiology 261: R209-216, 1991. 230. Unger T, Paulis L, and Sica DA . Therapeutic perspectives in hypertension: novel means for renin -angiotensin -aldosterone system modulation and emerging device -based approaches. Eur opean heart journal 32: 2739-2747, 2011. 231. Urade Y EN, Hayaishi O. . Lipocalin -Type Prostaglandin D Synthase as an Enzymic Lipocalin. In Madame Curie Bioscience Database [Internet]: Austin (TX): Landes Bioscience 2000. 232. Urade Y, and Hayaishi O . Bioc hemical, structural, genetic, physiological, and pathophysiological features of lipocalin -type prostaglandin D synthase. Biochimica et biophysica acta 1482: 259-271, 2000. 233. Urade Y, and Hayaishi O . Prostaglandin D2 and sleep regulation. Biochimica et biophysica acta 1436: 606-615, 1999. 234. Urade Y, and Hayaishi O . Prostaglandin D2 and sleep/wake regulation. Sleep medicine reviews 15: 411-418, 2011. 166 235. Urade Y, and Hayaishi O . Prostaglandin D synthase: structure and function. Vitamins and hormones 58: 89-120, 2000. 236. Urade Y, Hayaishi O, Matsumura H, and Watanabe K . Molecular mechanism of sleep regulation by prostaglandin D2. Journal of lipid mediators and cell signalling 14: 71-82, 1996. 237. Urade Y, Kitahama K, Ohishi H, Kaneko T, Mizuno N, and Hayaishi O . Dominant expression of mRNA for prostaglandin D synthase in leptomeninges, choroid plexus, and oligodendrocytes of the adult rat brain. Proceedings of the National Academy of Sciences of the United States of America 90: 9070-9074, 1993. 238. Urban D, Ewen S, Ukena C, Linz D, Bohm M, and Mahfoud F . Treating resistant hypertension: role of renal denervation. Integrated blood pressure control 6: 119-128, 2013. 239. Usui D, Yamaguchi -Shima N, Okada S, Shimizu T, Wakiguchi H, and Yokotani K . Selectiv e activation of the sympathetic ganglia by centrally administered corticotropin -releasing factor in rats. Autonomic neuroscience : basic & clinical 146: 111-114, 2009. 240. Van Vliet BN, and Montani JP . The time course of salt -induced hypertension, and why it matters. Int J Obes (Lond) 32 Suppl 6: S35 -47, 2008. 241. Vane JR, Bakhle YS, and Botting RM . Cyclooxygenases 1 and 2. Annual review of pharmacology and toxicology 38: 97-120, 1998. 242. von der Weid PY, Hollenberg MD, Fiorucci S, and Wallace JL . Aspir in-triggered, cyclooxygenase -2-dependent lipoxin synthesis modulates vascular tone. Circulation 110: 1320-1325, 2004. 243. von Euler US . On the specific vaso -dilating and plain muscle stimulating substances from accessory genital glands in man and certain animals (prostaglandin and vesiglandin). The Journal of physiology 88: 213-234, 1936. 244. Vongpatanasin W, Kario K, Atlas SA, and Victor RG . Central sympatholytic drugs. J Clin Hypertens (Greenwich) 13: 658-661, 2011. 167 245. Wallin BG, Kunimoto MM, and Sell gren J . Possible genetic influence on the strength of human muscle nerve sympathetic activity at rest. Hypertension 22: 282 -284, 1993. 246. Walmor C. DeMello, and Frohlich ED . Renin Angiotensin System and Cardiovascular Disease . 2009. 247. Wang G, Sarkar P , Peterson JR, Anrather J, Pierce JP, Moore JM, Feng J, Zhou P, Milner TA, Pickel VM, Iadecola C, and Davisson RL . COX -1-derived PGE2 and PGE2 type 1 receptors are vital for angiotensin -II-induced formation of reactive oxygen species and Ca2+ influx in the subfornical organ. American journal of physiology Heart and circulatory physiology 2013. 248. Warner TD, Giuliano F, Vojnovic I, Bukasa A, Mitchell JA, and Vane JR . Nonsteroid drug selectivities for cyclo -oxygenase -1 rather than cyclo -oxygenase -2 are asso ciated with human gastrointestinal toxicity: a full in vitro analysis. Proceedings of the National Academy of Sciences of the United States of America 96: 7563 -7568, 1999. 249. Watanabe K, Urade Y, Mader M, Murphy C, and Hayaishi O . Identification of beta-trace as prostaglandin D synthase. Biochemical and biophysical research communications 203: 1110-1116, 1994. 250. Weber KT, and Brilla CG . Pathological hypertrophy and cardiac interstitium. Fibrosis and renin -angiotensin -aldosterone system. Circulation 83: 1849-1865, 1991. 251. Weinberger MH . Hypertension in African Americans: the role of sodium chloride and extracellular fluid volume. Seminars in nephrology 16: 110-116, 1996. 252. Weinberger MH, Fineberg NS, Fineberg SE, and Weinberger M . Salt sensitivity, pulse pressure, and death in normal and hypertensive humans. Hypertension 37: 429-432, 2001. 253. Weir MR, and Dzau VJ . The renin -angiotensin -aldosterone system: a specific target for hypertension management. American journal of hypertension 12: 205S -213S, 1999. 254. Wilcox CS . Reactive oxygen species: roles in blood pressure and kidney function. Current hypertension reports 4: 160-166, 2002. 168 255. Wilcox CS, and Welch WJ . Thromboxane synthase and TP receptor mRNA in rat kidney and brain: effects of salt in take and ANG II. American journal of physiology Renal physiology 284: F525-531, 2003. 256. Williams GH, and Hollenberg NK . Sodium -sensitive essential hypertension: emerging insights into an old entity. Journal of the American College of Nutrition 8: 490 -494, 1989. 257. Wright DH, Nantel F, Metters KM, and Ford -Hutchinson AW . A novel biological role for prostaglandin D2 is suggested by distribution studies of the rat DP prostanoid receptor. European journal of pharmacology 377: 101-115, 1999. 258. Wright JW, and Harding JW . Brain renin -angiotensin --a new look at an old system. Progress in neurobiology 95: 49-67, 2011. 259. Wright JW, Yamamoto BJ, and Harding JW . Angiotensin receptor subtype mediated physiologies and behaviors: new discoveries and clinical tar gets. Progress in neurobiology 84: 157-181, 2008. 260. Wu R, Duchemin S, Laplante MA, De Champlain J, and Girouard H . Cyclo -oxygenase -2 knockout genotype in mice is associated with blunted angiotensin II -induced oxidative stress and hypertension. American journal of hypertension 24: 1239 -1244, 2011. 261. Xanthakis V, and Vasan RS . Aldosterone and the risk of hypertension. Current hypertension reports 15: 102-107, 2013. 262. Xue B, Beltz TG, Yu Y, Guo F, Gomez -Sanchez CE, Hay M, and Johnson AK . Central inter actions of aldosterone and angiotensin II in aldosterone - and angiotensin II -induced hypertension. American journal of physiology Heart and circulatory physiology 300: H555-564, 2011. 263. Xue B, Zhang Z, Johnson RF, and Johnson AK . Sensitization of slow p ressor angiotensin II (Ang II) -initiated hypertension: induction of sensitization by prior Ang II treatment. Hypertension 59: 459-466, 2012. 264. Yamaguchi N, and Okada S . Cyclooxygenase -1 and -2 in spinally projecting neurons are involved in CRF -induced s ympathetic activation. Autonomic neuroscience : basic & clinical 151: 82-89, 2009. 169 265. Yang H, and Chen C . Cyclooxygenase -2 in synaptic signaling. Current pharmaceutical design 14: 1443-1451, 2008. 266. Yatabe MS, Yatabe J, Yoneda M, Watanabe T, Otsuki M, Felder RA, Jose PA, and Sanada H . Salt sensitivity is associated with insulin resistance, sympathetic overactivity, and decreased suppression of circulating renin activity in lean patients with essential hypertension. The American journal of clinical nutr ition 92: 77-82, 2010. 267. Yermakova AV, Rollins J, Callahan LM, Rogers J, and O'Banion MK . Cyclooxygenase -1 in human Alzheimer and control brain: quantitative analysis of expression by microglia and CA3 hippocampal neurons. Journal of neuropathology and experimental neurology 58: 1135-1146, 1999. 268. Yoshimoto M, Miki K, Fink GD, King A, and Osborn JW . Chronic angiotensin II infusion causes differential responses in regional sympathetic nerve activity in rats. Hypertension 55: 644-651, 2010. 269. Yu Y, K ang YM, Zhang ZH, Wei SG, Chu Y, Weiss RM, and Felder RB . Increased cyclooxygenase -2 expression in hypothalamic paraventricular nucleus in rats with heart failure: role of nuclear factor kappaB. Hypertension 49: 511-518, 2007. 270. Yu Y, Lucitt MB, Stubbe J, Cheng Y, Friis UG, Hansen PB, Jensen BL, Smyth EM, and FitzGerald GA . Prostaglandin F2alpha elevates blood pressure and promotes atherosclerosis. Proceedings of the National Academy of Sciences of the United States of America 106: 7985-7990, 2009. 271. Yu Y, Zhang ZH, Wei SG, Weiss RM, and Felder RB . Peroxisome proliferator -activated receptor -gamma regulates inflammation and renin -angiotensin system activity in the hypothalamic paraventricular nucleus and ameliorates peripheral manifestations of heart fa ilure. Hypertension 59: 477-484, 2012. 272. Zhang L, Zhang WP, Hu H, Wang ML, Sheng WW, Yao HT, Ding W, Chen Z, and Wei EQ . Expression patterns of 5 -lipoxygenase in human brain with traumatic injury and astrocytoma. Neuropathology : official journal of the Japanese Society of Neuropathology 26: 99-106, 2006. 273. Zhang W, and Mifflin S . Plasticity of GABAergic mechanisms within the nucleus of the solitary tract in hypertension. Hypertension 55: 201-206, 2010. 170 274. Zhuo JL, and Li XC . New insights and perspe ctives on intrarenal renin -angiotensin system: focus on intracrine/intracellular angiotensin II. Peptides 32: 1551 -1565, 2011. 275. Zimmerman MC, Lazartigues E, Lang JA, Sinnayah P, Ahmad IM, Spitz DR, and Davisson RL . Superoxide mediates the actions of an giotensin II in the central nervous system. Circulation research 91: 1038-1045, 2002. 276. Zimmerman MC, Lazartigues E, Sharma RV, and Davisson RL . Hypertension caused by angiotensin II infusion involves increased superoxide production in the central nervo us system. Circulation research 95: 210-216, 2004. 277. Zoccal DB, Paton JF, and Machado BH . Do changes in the coupling between respiratory and sympathetic activities contribute to neurogenic hypertension? Clinical and experimental pharmacology & physiolog y 36: 1188-1196, 2009.