MSU LIBRARIES .—:-—. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped be10w. CENTRAL NERVOUS SYSTEM CONTRIBUTION TO PHYSIOLOGIC ACTIONS OF ANGIOTENSIN II By Cathy Ann Bruner A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1985 ABSTRACT CENTRAL NERVOUS SYSTEM CONTRIBUTION TO PHYSIOLOGIC ACTIONS OF ANGIOTENSIN II By Cathy Ann Bruner A central pressor effect of All has been postulated to contribute to the development of several models of experimental hypertension. Therefore, the effects of chronic selective stimulation of brain AII receptors were determined in rats and rabbits, and the effect of in- terruption of central AII mechanisms on several forms of hypertension was assessed. Chronic ivt AII infusion in rats and rabbits produced a hyperten- sive response which was enhanced by high sodium intake. Two mechanisms were identified that contribute to this form of hypertension. A small component appeared to be the result of leak of All from CSF into the periphery. The predominant mechanism supporting elevated arterial pressure was activation of the sympathetic nervous system as demon- strated by enhanced depressor responses to ganglionic and combined alpha- and beta-adrenergic blockade in rats receiving chronic ivt AII infusions. Furthermore, hypertension development was delayed by peri- pheral sympathectomy. Chronic ivt infusion of 1sar,8thr-angiotensin II (sarthran), a com- petitive AII receptor antagonist, was used to assess the role of central (ar‘nr 6’4 v‘ AHV a \d 3¥ nrzr .»‘, t, .Qr,‘ I' E‘CJ \- #6.. i.‘ 3 tPA ~ .- » . Cathy Ann Bruner AII pressor mechanisms in several forms of experimental hypertension. The development of DOC-salt hypertension and the established hyperten- sion of spontaneously hypertensive rats were unaffected by chronic ivt sarthran infusion. The hypertensive response to chronic iv AII infusion also was not blocked by ivt sarthran infusion. This latter observation suggested that chronic elevations in plasma AII produce hypertension by an action at brain sites that are relatively inaccessible to ivt sar- thran. Since the subfornical organ (SF0) and area postrema (AP) are two circumventricular organs that appear to have a CSF-brain barrier, the participation of these two structures in iv AII-induced hypertension was assessed. SFO ablation and knife cut of SFO efferent pathways did not attenuate chronic iv AII-induced hypertension. Furthermore, lesion of the median preoptic nucleus, an area through which all forebrain AII- sensitive pressor pathways are postulated to pass, did not prevent hypertension development. Preliminary results indicate that AP lesion may protect against this form of hypertension. Therefore, the AP may be the critical central site at which blood-borne AII acts to produce hypertension in the rat. To Greg, with many thanks ii AM a «d C» P. WI. T BID-‘4 uni-d ~9~ v ’r- .J a .. r. ‘4» . u r... m. .... I s a h fl. an‘ . ~ AU. Ann .1. J.“ Ps. P» h. ACKNOWLEDGEMENTS My deepest thanks go to my advisor, Dr. Gregory D. Fink, for creat- ing a stimulating laboratory environment and allowing an unusual amount of freedom in my research. I am deeply indebted to him for his ideas, support, encouragement, friendship, and for providing countless oppor- tunities to learn. I would like to thank Drs. T.M. Brody, G.D. Fink, G. Hatton, G.L. Gebber, and R.B. Stephenson for serving on my thesis committee. The work described in this dissertation could not have been per- formed without the excellent technical assistance of my friends and coworkers Mark Mann, Corie Pawloski, Doug Shinaver, Dave Walters and Jan Weaver. The work of Ben Kuslikis during his research rotation is gratefully acknowledged. Other investigators also have contributed importantly to this research. Specifically, I would like to thank Dr. Keith Demarest for measurement of tissue catecholamines, Dr. John Thorn- burg for measurement of plasma catecholamines, and Dr. Ian Reid for determination of plasma AII concentration. Studies involving SFO knife cuts, SFO ablation and median preoptic nucleus ablation were made possible by fruitful collaboration with Dr. Wally Lind and Dr. Michael Mangiapane. I also would like to thank the faculty of the Department of Pharma- cology and Toxicology for setting high standards of performance, and for iii the bread: Himgan S Lastl U’liiv’EPl n: the breadth and depth of the graduate education that I received while at Michigan State. Special thanks are due to Diane Hummel for her secre- tarial assistance over the past four years and for typing this disserta- tion. Lastly, I would like to acknowledge Leon and our families for their unwavering love and support. I couldn't begin to thank them enough. iv TABLE OF CONTENTS Page LIST OF TABLES --------------------------------------------------- ix LIST OF FIGURES -------------------------------------------------- x INTRODUCTION ----------------------------------------------------- l A. Peripheral Renin-Angiotensin System -------------------- l l. General ------------------------------------------- l 2. Physiological effects of blood-borne angiotensin II ------------------------------------------------ 2 3. Sodium-angiotensin interactions ------------------- 6 4 Evidence that blood-borne angiotensin II acts on the brain ----------------------------------------- 10 a. circumventricular organs --------------------- 10 b. pressor effects ------------------------------ ll c. Thirst --------------------------------------- 15 d. Vasopressin release -------------------------- l7 B. Brain Renin-Angiotensin System ------------------------- l7 l. Components present in brain ----------------------- l7 a. Angiotensinogen ------------------------------ l7 b. Renin ---------------------------------------- l8 c. Angiotensin converting enzyme ---------------- 18 d. Angiotensinase ------------------------------- l9 e. Angiotensins --------------------------------- 19 f. Angiotensin receptors ------------------------ 20 g. Angiotensinergic neurons --------------------- 23 2. Physiological effects of central AII administra- tI on— -------------------- . ------------------------- 24 a. Pressor effects ------------------------------ 24 b. Thirst and fluid/electrolyte effects --------- 27 C. Central Effects of Angiotensin All in Hypertension ----- 28 l. Brain lesion studies ------------------------------ 29 2. Central administration of inhibitors of the renin- angiotensin system -------------------------------- 32 TILE OF CE STITNE'TI C ”WINS F A. (1“ n. -n h \ TABLE OF CONTENTS (continued) STATEMENT OF PURPOSE --------------------------------------------- MATERIALS AND METHODS ............................................ A. General Methods ........................................ I. 2. 3 4. 5. 6 Animals ........................................... General surgical procedures ....................... Blood pressure measurement ........................ a. Direct catheterization: rats ---------------- b. Indirect blood pressure measurement ---------- 6- Direct measurement: rabbits ----------------- Implantation of intracerebroventricular cannulae-- Metabolic measurements ............................ Statistical analysis .............................. Experimental Protocols --------------------------------- 1. Chronic ivt AII infusion: rat -------------------- a. Sodium dependency ---------------------------- b. Plasma hormone levels ------------------------ c. Adrenalectomy -------------------------------- d. Acute blockade of vascular AII receptors and ganglionic blockade -------------------------- e. Acute blockade of vascular AVP receptors ----- f. Peripheral sympathectomy --------------------- 9. Verification of degree of sympathectomy ------ h. PharmaCOTOgic assessment of sympathetic tone- i. Interactions of AVP, sympathetic nervous sys- tem and peripheral renin-angiotensin system-- j. Spinal cord stimulation ---------------------- k. Acute ivt sarthran/chronic ivt AII ----------- l. Acute ivt sarthran/acute ivt AII ------------- m. Effect of iv saralasin on response to acute ivt AII -------------------------------------- Chronic ivt AII infusion in rabbits: sodium dependency ---------------------------------------- Chronic pharmacologic blockade of brain AII re- ceptors or brain converting enzyme ................ a. Chronic ivt saralasin ........................ 1) dose determination ---------------------- 2; effects in normal- and high-sodium rats- 3 chronic iv saralasin .................... vi Page 34 36 36 36 36 37 37 38 39 39 4O 4O 4] 52 55 55 55 56 57 TABLE OF CO ‘ if? t' ~. ‘EU‘IE is... -__l ___A .__4 TABLE OF CONTENTS (continued) A. Page b. Chronic ivt sarthran ------------------------- 57 1; dose determination: Sprague-Dawley ----- 57 2 effects in high-sodium rats ------------- 58 3) dose determination: spontaneously hypertensive rats ----------------------- 59 c. Chronic ivt teprotide ------------------------ 6O 1; dose determination: Sprague-Dawley ----- 60 2 dose determination: spontaneously hypertensive rats ----------------------- 61 d. Chronic ivt sarthran/chronic iv angiotensin II ------------------------------------------- 62 e. Chronic ivt sarthran/DOC-salt hypertension--- 63 f. Chronic ivt sarthran/spontaneously hyperten- sive rats ------------------------------------ 63 9. Chronic ivt teprotide/spontaneously hyperten- sive rats ------------------------------------ 64 4. Brain lesion studies: Chronic intravenous AII infusion ------------------------------------------ 65 a. Chronic intravenous AII infusion protocol---- 65 b. Subfornical organ lesion --------------------- 65 c. Knife cut of subfornical organ efferents ----- 67 d. Median preoptic nucleus lesion --------------- 67 RESULTS ---------------------------------------------------------- 69 Chronic ivt AII Infusion: Rat ------------------------- 69 l. Sodium dependency --------------------------------- 69 2. Plasma hormone levels ----------------------------- 78 3. Adrenalectomy ------------------------------------- 85 4. Acute blockade of vascular AII receptors and ganglionic blockade ------------------------------- 9O 5. Acute blockade of vascular AVP receptors ---------- 95 6. Peripheral sympathectomy -------------------------- 95 7. Verification of degree of sympathectomy ----------- 102 8. Pharmacologic assessment of sympathetic tone ------ 108 9. Interactions of AVP, sympathetic nervous system and peripheral renin-angiotensin system ----------- 113 10. Spinal cord stimulation --------------------------- 122 ll. Acute ivt sarthran/chronic ivt AII ---------------- 122 12. Acute ivt sarthran/acute ivt AII ------------------ 127 13. Effect of iv saralasin on response to acute ivt AII ----------------------------------------------- 127 vii TABLE OF C L.) TABLE OF CONTENTS (continued) Page 8. Chronic ivt AII Infusion in Rabbits: Sodium Dependency 127 C. Chronic Pharmacological Blockade of Brain AII Receptors or Brain Converting Enzyme ----------------------------- 148 1. Chronic ivt saralasin ----------------------------- 148 a. dose determination --------------------------- 148 b. Effects in normal- and high-sodium rats ------ 156 2. Chronic ivt sarthran ------------------------------ 165 a. Dose determination: Sprague-Dawley ---------- 165 b. Effects in high-sodium rats ------------------ 165 c. Dose determination: Spontaneously hyperten- sive rats ------------------------------------ 170 3. Chronic ivt teprotide ----------------------------- 170 a. Dose determination: Sprague-Dawley ---------- 170 b. Dose determination: Spontaneously hyperten- sive rats ------------------------------------ 176 4. Chronic ivt sarthran/chronic iv angiotensin II---- 176 5. Chronic ivt sarthran/DOC-salt hypertension -------- 179 6. Chronic ivt sarthran/spontaneously hypertensive rats ---------------------------------------------- 185 7. Chronic ivt teprotide/spontaneously hypertensive rats ---------------------------------------------- 185 D. Brain Lesion Studies ----------------------------------- 191 l. Subfornical organ lesion -------------------------- 191 2. Knife cut of subfornical organ efferents ---------- 191 3. Median preoptic nucleus lesion -------------------- 194 DISCUSSION ------------------------------------------------------- 200 A. Cardiovascular and Fluid/Electrolyte Response to Chronic ivt AII Infusion ------------------------------- 200 B. Physiologic Mechanisms Involved in the Hypertensive Response to Chronic ivt AII Infusion ------------------- 205 C. Chronic Interruption of Central AII Mechanisms in Several Forms of Experimental Hypertension ------------- 218 D. Central Site of Action of Blood-borne Angiotensin II--- 230 CONCLUSIONS ------------------------------------------------------ 237 BIBLIOGRAPHY ----------------------------------------------------- 240 viii .7 Table 10 LIST OF TABLES Page Plasma catecholamine levels before, during, and after ivt AII infusion --------------------------------------- 83 Plasma aldosterone levels before, during, and after ivt AII infusion ------------------------------------------- 84 Tissue norepinephrine and dopamine content in normal and sympathectomized rats ------------------------------ 109 Resting mean arterial pressure before various vaso- depressor interventions -------------------------------- 110 Effect of sequential administration of AVP antagonist and saralasin on blood pressure in sympathectomized rats receiving chronic ivt infusions of AII ------------ 121 Resting mean arterial pressure before and during chronic ivt saralasin infusion ------------------------- 149 Resting mean arterial pressure before, during, and after ivt sarthran or teprotide infusion in SHR -------- 173 Effect of chronic ivt sarthran infusion on DOC-salt hypertension ------------------------------------------- 184 Effect of AVP antagonist and hexamethonium on blood pressure in DOC-salt hypertensive rats ----------------- 186 Effect of chronic iv AII infusion on mean arterial pressure in rats with electrolytic ablation of the median preoptic nucleus -------------------------------- 199 ix C. - . 57“]. -1\..) d R) C3 Figure 10 11 LIST OF FIGURES Page Effect of chronic ivt AII infusion on cardiovascular and fluid/electrolyte parameters in the rat ------------ 70 Chronic ivt AII infusion in rats on restricted fluid intake ------------------------------------------------- 72 Cardiovascular and fluid/electrolyte responses to chronic ivt AII infusion (1 ug/hr) in rats maintained on normal and high sodium intake ----------------------- 74 Cardiovascular and fluid/electrolyte responses to chronic ivt AII infusion (6 ug/hr) in rats maintained on normal and high sodium intake ----------------------- 76 Cardiovascular response to chronic ivt AII infusion in rats used for plasma hormone determination ------------- 79 Effect of chronic ivt AII infusion on plasma electro- lytes, osmolality, hematocrit and body weight ---------- 81 Plasma AII concentration in rats receiving chronic ivt AII infusion ------------------------------------------- 86 Effect of adrenalectomy on the hypertensive response to chronic ivt AII infusion ---------------------------- 88 Cardiovascular and fluid/electrolyte response to chronic ivt All in rats that were subject to acute saralasin and hexamethonium treatment ------------------ 91 Change in mean arterial pressure and heart rate in re- sponse to acute iv saralasin infusion before, during, and after chronic ivt AII infusion --------------------- 93 Change in mean arterial pressure and heart rate in re- sponse to hexamethonium before, during, and after chronic ivt AII infusion ------------------------------- 96 22 LIST OF FIGURES (continued) Figure 12 13 14 15 16 17 18 19 20 21 22 23 Page Change in mean arterial pressure and heart rate in re- sponse to AVP antagonist before, during, and after chronic ivt AII infusion ------------------------------- 98 Efficacy of AVP antagonist in blocking responses to exogenous AVP infusion --------------------------------- 100 Effect of peripheral sympathectomy on hypertensive re- sponse to chronic ivt AII infusion --------------------- 103 Blood pressure and heart rate response to hexamethonium and phentolamine in normal and sympathectomized rats--- 105 Change in mean arterial pressure in response to elec- trical stimulation of sympathetic vasomotor outflow in normal and sympathectomized pithed rats ---------------- 107 Pharmacologic assessment of sympathetic tone in rats receiving chronic ivt infusions of AII ----------------- 111 Effect of sequential administration of AVP antagonist, saralasin, and phentolamine on blood pressure and heart rate in normal rats receiving chronic ivt infusions of All ---------------------------------------------------- 114 Effect of sequential administration of saralasin and AVP antagonist on blood pressure and heart rate in normal rats receiving chronic ivt infusions of AII ----- 117 Effect of sequential administration of AVP antagonist and saralasin on blood pressure and heart rate in sym- pathectomized rats receiving chronic ivt infusions of AII ---------------------------------------------------- 119 Effect of chronic ivt AII infusion on pressor re- sponses to electrical stimulation of sympathetic vaso- motor outflow in pithed rats --------------------------- 123 Ability of acute ivt sarthran injection to reverse hypertension induced by chronic ivt AII infusion ------- 125 Ability of acute ivt sarthran injection to reverse the pressor effect of acute ivt AII injection ---------- 128 xi LIST C Figure 24 25 (.A.) A.) (U LIST OF FIGURES (continued) Figure 24 25 26 27 28 29 3O 31 32 33 34 Page Mean arterial pressure, heart rate, and body weight re- sponses to chronic ivt AII infusion in rabbits main- tained on low and high sodium intake ------------------- 130 Effect of chronic ivt AII infusion on plasma sodium and potassium concentrations and plasma osmolality in rab- bits maintained on low and high sodium intake ---------- 133 Water intake, urine output, and water balance in re- sponse to chronic ivt AII infusion in high-sodium rabbits ------------------------------------------------ 135 Water intake, urine output, and water balance in re- sponse to chronic ivt AII infusion in low-sodium rabbits ------------------------------------------------ 137 Ratio of water intake to food intake in rabbits main- tained on low and high sodium intake in response to chronic ivt AII infusion ------------------------------- 139 Effect of chronic ivt AII infusion on body fluid com- partment volumes and hematocrit in rabbits maintained on low and high sodium intake -------------------------- 142 Effect of chronic ivt AII infusion on sodium and po- tassium balance in rabbits maintained on low and high sodium intake ------------------------------------------ 144 Effect of chronic ivt AII infusion on creatinine clear- ance and blood urea nitrogen in rabbits maintained on low and high sodium intake ----------------------------- 146 Pressor responses to acute intravenous and intraven- tricular AII administration in rats before and after a 5-day sham period -------------------------------------- 150 Effect of continuous ivt infusion of saralasin (6 pg/ hr) on pressor responses to acute intravenous and in- traventricular AII administration ---------------------- 152 Effect of continuous ivt infusion of saralasin (12 ug/ hr) on pressor responses to acute intravenous and in- traventricular AII administration ---------------------- 154 xii LIST I PO 3 w of ‘E '1‘ 4.41 0% P k. l:* 4 HM‘: LIST OF FIGURES (continued) Figure 35 36 37 38 39 4O 41 42 43 44 45 46 47 Page Cardiovascular and fluid/electrolyte responses to chronic ivt saralasin infusion in normal sodium rats--- 157 Cardiovascular and fluid/electrolyte responses to chronic ivt saralasin infusion in high sodium rats ----- 159 Effect of chronic intravenous saralasin infusion (18 ug/hr) on cardiovascular and fluid/electrolyte para- meters ------------------------------------------------- 161 Comparison of cardiovascular and fluid/electrolyte responses to chronic ivt saralasin and AII ------------- 163 Effect of chronic ivt sarthran infusion on pressor responses to acute ivt and iv AII administration ------- 166 Cardiovascular and fluid/electrolyte responses to chronic ivt sarthran infusion (l ug/hr) in rats on high sodium intake ------------------------------------- 168 Effect of chronic ivt sarthran infusion on pressor re- sponses to acute ivt and iv All in SHR ----------------- 171 Effect of chronic ivt teprotide infusion on pressure responses to acute ivt and iv AII administration ------- 174 Effect of chronic ivt teprotide infusion on pressor responses to acute ivt and iv AII administration in SHR 177 Effect of chronic ivt sarthran infusion (l ug/hr) on cardiovascular and fluid/electrolyte responses to chronic iv AII infusion (10 ng/min) -------------------- 180 Effect of chronic ivt sarthran infusion (1 ug/hr) on cardiovascular and fluid/electrolyte responses to chronic iv AII infusion (20 ng/min) -------------------- 182 Tail cuff blood pressure and body weight responses of SHR to ivt sarthran infusion (1 ug/hr for 2 weeks followed by 6 ug/hr for 1 week) ------------------------ 187 Tail cuff blood pressure and body weight responses of SHR to ivt teprotide infusion (10 ug/hr for 1 week)---- 189 xiii LIST OF FIGURES (continued) Figure 48 49 50 Page Effect of chronic iv AII infusion on cardiovascular and fluid/electrolyte responses in rats with electrolytic ablation of the subfornical organ ---------------------- 192 Responses to chronic ivt AII infusion in rats with knife cuts of SEQ efferent pathways---------------¢---- 195 Arterial pressure and heart rate responses to acute in- travenous AII infusion in rats with knife cuts of SFO efferent pathways -------------------------------------- 197 xiv INTRODUCTION A. Peripheral Renin-Angiotensin System 1. General It is generally accepted that the peripheral renin-angiotensin system plays an important homeostatic role in the control of fluid and electrolyte balance and cardiovascular function. The first report that a factor of renal origin was capable of increasing arterial blood pres- sure (Tigerstedt and Bergman, 1898) described increases in blood pres— sure following intravenous injection of saline extracts of kidney in anesthetized rabbits. However, it was not until the pioneering work of Goldblatt gt_gl, (1934) and Pickering and Prinzmetal (1938) that the link between the kidney and high blood pressure was recognized. The critical finding of these studies was that reduction in renal blood flow can lead to a prolonged increase in arterial pressure. In the 1940's, two groups working independently (Braun-Menendezugt_al,, 1940; Page and Helmer, 1940) demonstrated that renin, the substance released from the kidney in response to a reduction in blood flow, is not pressor in itself. Rather, renin acts on a plasma substrate to produce a heat stable, short-acting vasoconstrictor substance, which is now known as angiotensin II. These initial observations have led to our current understanding of the functions of the renin-angiotensin system. synthes ized ce T971). tin; re recesto pressur thafiges If] is!” a 2 Renin is a proteolytic enzyme (Inagami and Murakami, 1977) synthesized and secreted by juxtaglomerular cells, which are special- ized cells in the wall of the afferent arteriole of the kidney (Cook, 1971). Davis and Freeman (1976) have classified the mechanisms regula- ting renin secretion as: a) intrarenal, including the renal vascular receptor in the afferent arteriole sensitive to changes in perfusion pressure and the macula densa receptor in the distal tubule sensitive to changes in tubular sodium and/or chloride load; b) sympathetic, in- cluding circulating catecholamines and the renal nerves, and c) humoral, including vasopressin, angiotensin II and prostaglandins. Stimuli such as a decrease in renal perfusion pressure, sodium depletion, or sympa- thetic activation cause the release of renin from the kidney. Renin catalyzes the conversion of angiotensinogen, a plasma globulin, to angiotensin I (AI), an inactive decapeptide. Converting enzyme, present in high concentration in lung, but also found in plasma and many other tissues (Erdos, 1975) acts to cleave a dipeptide fragment from the carboxyterminus of angiotensin I to form the biologically active octa- peptide, angiotensin II (AII). It also has been shown that a metabolite of angiotensin II, des-aspl-angiotensin II (angiotensin III; AIII) has considerable physiologic activity, especially with regards to stimula- tion of aldosterone secretion from the adrenal gland (Freeman gt_al,. 1976). 2. Physiological effects of blood-borne angiotensin II Angiotensin II is a potent vasoconstrictor, and the increase in blood pressure produced by acute intravenous infusions of AII is due in large part to its direct vascular constrictor action (DeBono et_al,, increas: vatisns by the a These 11 sub;*ess in dogs. that at dogs tne naive dc TnquiOr DTESSOr been 7.130 and Yu, SiOlLI-c VASCCORS 51011 in by SEVEr 3 1963; Regioli gt_al,, 1974). However, the magnitude of the sustained increase in blood pressure observed with chronic (days to weeks) ele- vations in plasma AII concentration is greater than can be accounted for by the acute vasoconstrictor action of AII alone (Bean gt_al,, 1979). These investigators found that chronic (2-week) infusion of acutely subpressor doses of AII produced a sustained increase in blood pressure in dogs. Measurement of plasma AII concentration in these dogs revealed that at any given plasma level of AII, blood pressure was higher in the dogs that had been receiving chronic intravenous AII infusion than in naive dogs in whom plasma AII was acutely raised with an intravenous AII infusion. Similar reports that prolonged infusions of acutely sub- pressor doses of AII produce sustained increases in blood pressure have been made by others in dogs (Cowley and McCaa, 1976), rabbits (Dickinson and Yu, 1967) and rats (Brown gt_al,, 1981). Thus, it appears that a slowly-developing pressor action of AII, distinct from its acute direct vasoconstrictor effect, plays an important role in maintaining hyperten- sion in response to chronic elevations of plasma AII. Despite efforts by several research groups to identify the mechanism by which chronic elevations in plasma AII produce hypertension, no consensus has been reached. Many of the known physiologic effects of All could potentially contribute to the hypertension produced by long-term intravenous AII infusions. For example, circulating All has been demonstrated to en- hance activity of the sympathetic nervous system at several levels of the neuraxis. A component of the pressor response to both acute (Lappe and Brody, 1984) and chronic (Sweet et_al,, 1971; Yu and Dickinson, 1971) infusion of AII into the cerebral circulation has been demon- strated to be the result of a central effect of AII to cause increased sympathetic outflow. Angiotensin II also can act at the level of the "I” "I 4 ganglia to increase sympathetic tone since AII has been shown to be a non-nicotinic ganglionic stimulant (Lewis and Reit, 1965, 1966). At the level of the sympathetic neuroeffector junction, All has been shown to enhance both the vasoconstrictor responses to nerve stimulation in isolated vascular bed preparations (Zimmerman, 1978; Campbell and Jackson, 1979) and the contractile response of vascular strips to field stimulation (Zimmerman, 1978). In addition, the constrictor effect of exogenous norepinephrine administration jn_yjyg_is enhanced by AII, but to a much lesser extent than the response to nerve stimulation (Zimmer- man, 1973). The AII-induced facilitation of peripheral adrenergic function has been attributed to enhanced transmitter release (Boke and Malik, 1983; Hughes and Roth, 1971), blockade of reuptake (Khairallah, 1972), and increased responsiveness of vascular smooth muscle to nor- epinephrine (Pals gt_al,, 1968; Zimmerman, 1978). Facilitation of cardiac sympathetic function has been forwarded as an explanation for the positive chronotropic and inotropic effects that have been ob- served with AII (Lokhandwala §t_al,, 1978). Alternatively, these effects may be due to direct stimulation of myocardial AII receptors (Baker gt_al,, 1984). One mechanism that has been postulated to contribute to the slowly-developing pressor response to AII is resetting of the baro- receptors (Cowley and DeClue, 1976). Indeed, it has been convincingly demonstrated that peripheral AII, by a central action, inhibits baro- receptor reflex function. Reflex increases in cardiac vagal activity (Lumbers gt_al,, 1979) and decreases in sympathetic activity (Stein §t_ al,, 1984; Guo and Abboud, 1984) for a given increment in arterial pressure are blunted by AII. A +1 rarsrui and v. Sustair afiltfev Cfifoni: and 015 VETCJS U TLET‘M 9;:AA "Cg: ‘ 9&3. 31, 1. \3 I: 5 Acute intravenous administration of angiotensin II has been reported to increase plasma vasopressin concentration in dogs (Bonjour and Malvin, 1970; Ramsay et_al,, 1978; Reid gt_al,, 1982). The direct vasoconstrictor effects of AVP could theoretically contribute to hyper- tension observed with chronic iv AII infusion. However, chronic infu- sion of the same dose of AII that produces acute increases in plasma AVP (20 ng/kg/min) although producing hypertension does not result in a sustained increase in plasma vasopressin (Cowley gt_al,, 1981). ACTH is another potential contributor to iv AII-induced hypertension, since chronic ACTH infusions will cause hypertension (Scoggins gt_al,, 1984), and plasma ACTH levels have been reported to increase with acute intra- venous AII infusions (Ramsay gt al,, 1978; Reid gt_a1,, 1982). However, the doses of AII needed to produce increases in plasma ACTH are rela- tively large in comparison to the doses needed to produce hypertension. Circulating angiotensin II functions to conserve sodium, in part by stimulation of aldosterone release from the adrenal glomerulosa (Laragh et_§l,, 1960; Fraser gt_al,, 1965) and in part by a direct renal effect to increase tubular sodium reabsorption (Hall §t_al,, 1980). Chronic aldosterone infusion has been demonstrated to produce hyperten- sion (Garwitz and Jones, 1982), therefore AII-induced aldosterone re- lease may be a mechanism by which chronic iv infusions of All produce hypertension. In experimental animals, short term (minutes to hours) intravenous infusions of All cause plasma levels of aldosterone to increase with a concomitant decrease in sodium excretion (Borresen gt_ 31,, 1982), however this increase in plasma aldosterone is not main- tained with longer term (days to weeks) All infusions (McCaa gt 21,, 1975; C at dose only tr. aldoste caronic in addi‘ Elevate: Chronic 6 1975; Cowley §t_al,, 1976). Since chronic intravenous infusion of A11, at doses that produce sustained elevations in arterial pressure, produce only transient increases in plasma aldosterone, it is unlikely that aldosterone is responsible for maintaining hypertension in response to chronic, low-level intravenous AII infusion. The hypertensive response to chronic intravenous AII infusion, in addition to being "slowly-developing" in nature, is sodium-sensitive. Elevated levels of sodium intake augment the hypertensive response to chronic iv infusions of All in the dog (Cowley and McCaa, 1976) and rat (Fink gt_gl,, 1982b). Not only are the mechanisms by which AII produces a slowly-developing pressor response as yet undefined, but the reason for the sodium-sensitivity of this response also is not yet known. Interactions of sodium and angiotensin have been quite extensively studied. What follows is a summary of the known interactions of sodium and AII, and how these may relate to the sodium-dependent hypertension produced by chronic iv AII infusions. 3. Sodium-Angiotensin Interactions Plasma renin activity and plasma AII levels are elevated during sodium depletion and depressed during sodium loading (Brown gt_ 21,, 1964; Laragh gt 31,, 1972). Dietary sodium intake, in addition to being a determinant of endogenous All levels, is known to modulate the pressor and aldosterone-stimulating effects of exogenous AII infusions. Changes in sodium intake have opposite effects on vascular and adrenal responsiveness to AII. For example, in subjects on low sodium, the pressor response to acute exogenous AII infusion is suppressed while the increase in plasma aldosterone is enhanced relative to normal sodium subjects (Shoback §t_al,, 1983). Efforts have been made to dissociate adreia the ac tr::i' u , e'ETl 1 d.“ ‘t.";c . ~\. In ’ I ‘e ‘ I h C'Qa.. PO‘ .~ I- ‘U:h ’ 7 the effects of prior receptor occupancy by endogenous All and other effects of sodium itself on tissue responsiveness to exogenous AII. In the case of the adrenal responses to AII, the elevated circulating levels of All seen in sodium deprivation have a trophic effect on the adrenal glomerulosa to increase the number of All receptors and increase the activity of enzymes in the aldosterone biosynthetic pathway. These trophic effects observed during sodium depletion can be only partially reproduced by All infusion. Thus, the adrenal responses to changes in sodium intake are due partially to changes in plasma All and also to an effect of sodium ion or some other factor that is altered with sodium intake (Aguilera and Catt, 1983). The vascular responses to exogenous All are suppressed in animals on a low sodium diet and enhanced by high sodium intake. Several jg_yjtrg_studies have examined the effects of sodium intake on responsiveness of isolated vascular strips to AII. In these studies, receptor occupancy by endogenous AII is eliminated as a factor. It has been shown that the contractile response to All is potentiated in iso- lated aortic strips from rabbits maintained on a high sodium intake when compared to aortae obtained from low-sodium animals (Strewler gt_al,, 1972). This change in responsiveness is specific for All and is not seen with other vasoconstrictors such as norepinephrine. Therefore, even in the absence of the high circulating AII levels seen in sodium depletion, the vasculature from low-sodium animals is less responsive to AII. Aguilera and Catt (1981) have shown that sodium restriction de- creases, and sodium loading increases, the number of AII receptors in mesenteric artery. Sodium ion also has been shown to increase binding of 125I-AII to a particulate fraction of rat mesenteric artery (Wright m r+ In: em: End. ‘4‘ 8 gt_al,, 1982). The above studies suggest that receptor alterations may influence the changes in All pressor responses seen with varying sodium intakes. However, there is convincing evidence that receptor occupancy by endogenous AII is sufficient to explain the pressor responses to exogenous AII seen with varying sodium intakes. Cowley and Lohmeier (1978) have demonstrated in nephrectomized dogs that when plasma sodium concentration is maintained at 140, 146, and 156 meq/liter with body fluid volumes held constant, no change in the AII dose-pressure response is obtained. Similarly, changes in body fluid volumes reflecting those seen in sodium depletion and sodium loading had no effect on the pressor responses to acute iv AII infusion. These authors concluded that changes in sensitivity to exogenous AII infusion with sodium intake are not caused by sodium ion concentration or volume changes that accompany changes in sodium intake, but probably result from changes in the pre- vailing levels of endogenous AII, which alter the availability of receptor sites. This view is supported by results from other studies in which the diminished pressor responsiveness to exogenous AII infusion observed during sodium depletion can be restored to levels seen in "normal-sodium" subjects by pretreatment with converting enzyme inhi- bitors (Thurston and Laragh, 1975; Shoback et_al,, 1983). As described above, the mechanisms by which alterations in sodium intake effect pressor responsiveness to acute iv AII infusion have been extensively studied. However, it is not known why elevated levels of sodium intake augment the hypertensive response to chronic iv infusions of AII in the dog (Cowley and McCaa, 1976) and rat (Fink gt_ al,, 1982b). The degree to which vascular receptors are occupied by endogenous All is an unlikely factor since chronic iv AII infusions will 9 produce sustained hypertension at doses that acutely are subpressor (Bean gt_al,, 1979). In addition, we are unable to lower blood pressure with acute AII antagonist infusions in rats made hypertensive by chronic iv AII infusion (unpublished observation). This result argues against a sodium-sensitive direct vasoconstrictor effect of AII in mediating the hypertensive response to chronic iv AII. A pivotal role for the kidney in determining the sodium sensiti- vity of the response to chronic iv AII infusion has been suggested (Cowley and McCaa, 1976; DeClue gt_al,, 1978; Hall gt_al,, 1980). These authors postulate that in an animal on normal sodium intake, the direct renal sodium retaining effect of All can be offset by other homeostatic mechanisms, such as suppression of renal nerve activity and plasma aldosterone. However, in an animal on high sodium intake, these mecha- nisms are already maximally suppressed. Therefore, they suggested that elevation of plasma AII in high-sodium animals will cause sodium and water retention, leading to volume expansion. As a means of maintaining sodium and water balance, arterial pressure rises until it reaches a level at which the sodium and water retaining effects of AII are com- pensated for by a "pressure natriuresis/diuresis" phenomenon. Evidence from our laboratory and others does not support this view. In the rat there is no evidence for sodium retention since urinary electrolyte excretion (unpublished observations) and sodium balance (Brown gt_al,, 1981) remain unchanged during chronic iv AII infusion. Water balance also is unaltered by chronic iv AII infusion in the rat (unpublished observations). Although a transient (l-day) sodium retention is ob- served in the rabbit and dog in response to chronic iv AII, no change in body fluid volumes is observed. Therefore, although AII undoubtedly has direct little infusi blair Scri‘: Tithe arts. 5&1 10 direct renal sodium-sparing effects, these effects probably contribute little to the sodium-dependent hypertensive response to chronic iv AII infusion. It has been demonstrated, however, that an action of AII on the brain, rather than the kidney, is important for the development of hypertension in response to chronic iv AII infusion. Electrolytic ablation of periventricular structures in the anterior hypothalamus (AV3V region) in rats largely prevents the chronic hypertension seen in response to elevated plasma levels of All (Fink et_al,, 1982b). There is abundant evidence that blood-borne AII acts on the brain (as reviewed in the next section). Some of the experiments de- scribed in this thesis examine the premise that a central effect of AII, rather than a renal effect, is responsible for chronic elevations in arterial pressure seen with intravenous infusions of this hormone. 4. Evidence that blood-borne angiotensin II acts on the brain a. Circumventricular organs A number of the physiological effects of circulating All can be wholly or partially attributed to an action of AII on the brain. Since All is a polar peptide, it would not be expected to cross the blood-brain barrier readily. The initial studies that were performed in an effort to examine the degree to which circulating AII had access to brain tissue involved the peripheral administration of radiolabelled All and subsequent quantitation of the label in CSF or brain tissue (Volicer and Loew, 1971; Johnson and Epstein, 1975). These investigators found that radiolabel did appear in CSF and brain tissue after intravenous AII administration, however, biochemical identification of the label as intact All was not performed. A subsequent study by Schelling gt_al, 11 (1976) employed polyacrylamide gel electrophoresis to separate All and metabolites. It was demonstrated that after intravenous administration of 3 H-AII, the small amount of label that did appear in CSF was not intact AII, but rather a breakdown product. It is now well accepted that blood-borne AII cannot cross the blood-brain barrier, except in situations where large increases in blood pressure disrupt integrity of the barrier (Johansson gt_al,, 1970). However, circulating AII does have access to brain tissue at sites that lack a functional blood-brain barrier. These sites have been collectively termed the circumventricu- lar organs (CVO's) and they are characterized anatomically by the ab- sence of capillary endothelial tight junctions. Instead, the fene- strated capillary endothelium in these areas permits the passage of blood-borne substances that would not normally cross the blood-brain barrier into brain parenchyma (Broadwell and Brightman, 1976). The circumventricular organs include the area postrema, pineal gland, subcomissural organ, subfornical organ, organum vasculosum of the lamina terminalis, and median eminence. Autoradiographic evidence has shown that blood-borne AII binds specifically within the CVO's and is excluded from the rest of the brain (van Houten gt_al,, 1980). It now appears that three of the circumventricular organs, namely the area postrema (AP), subfornical organ (SF0) and organum vasculosum of the lamina terminalis (OVLT) are important brain sites at which blood-borne AII acts to produce central effects. b. Pressor effects The first demonstration that blood-borne AII could act directly on the brain to produce an increase in arterial pressure was erse 33‘} res: 12 reported by Bickerton and Buckley (1961). They used a dog cross-circu- lation preparation in which the head of a recipient dog was vascularly isolated from the trunk to demonstrate that AII, when delivered into the cerebral circulation, could cause an increase in blood pressure that was independent of direct vascular constriction. Experiments in conscious rabbits (Dickinson et_a1,, 1965) showed that infusion of All into a vertebral artery caused a pressor response of greater magnitude than that seen when the same dose was infused intravenously. Similar find- ings have been made in anesthetized dogs (Ferrario gt_al,, 1970). The pressor activity of intravertebral AII infusion is associated with increased splanchnic preganglionic activity and is prevented by adrener- gic blockade or spinal section at C2 (Ferrario et_al,, 1972). These experiments indicate that in the dog and rabbit, blood-borne AII acts at a site within the field of vertebral circulation to cause a pressor response mediated by an increase in vasomotor tone. The AP is the only CVO that is perfused by the vertebral arteries, suggesting that blood- borne AII acts at the AP in these species to produce a pressor response. Indeed, it has been shown that electrolytic ablation of the AP in the dog (Joy and Lowe, 1970) and rabbit (Yu and Dickinson, 1971) reduces the pressor effect of intravertebral and intravenous (Ferrario et_al,, 1979) All administration. In contrast to these species, the pressor response to intravertebral AII infusion in the rat is not different than the response to intravenous infusion, and AP lesion does not alter the pressor response to acute intravenous AII (Haywood gt 21,, 1980). However, in the rat All is a more potent pressor agent when given into the carotid circulation than when given intravenously (Fink et_a1,. (I. (‘9' l3 1980a). Thus, a more rostral site appears to be important for the central pressor effect of AII in this species. The two major candidates for the forebrain site at which blood-borne AII acts to produce a central pressor effect are the SF0 and periventricular structures within the anterior ventral third ventricular region (AV3V). Electrolytic ablation of the SFO has been shown to reduce the pressor response to systemic AII administration (Mangiapane and Simpson, 1980). In addition, a knife cut that interrupts ventrally- directed SFO efferent pathways attenuates the pressor response to acute intravenous AII infusion in rats (Lind et_al,, 1983). Thus, AII-recep- tive elements within the SFO appear to be sensitive to blood-borne AII and contribute to the pressor response to systemic AII. In addition to the SFO, structures within the AV3V region also appear to mediate a central pressor effect of blood-borne AII in the rat. The AV3V region encompasses the OVLT, the subcomissural por- tion of the median preoptic nucleus (MnPO), the periventricular preoptic nucleus and the medial part of the medial preoptic area. It has been demonstrated that the augmented pressor response to All seen with carotid 35, aortic AII infusion is abolished in rats with electrolytic lesion of the AV3V (Fink et_al,, 1980a). Furthermore, the pressor response to acute intravenous AII infusion is significantly decreased in rats with AV3V lesions (Buggy gt_al,, 1977), consistent with the idea that AV3V lesion eliminates that component of the iv AII pressor re- sponse that is central in origin. Intracerebroventricular administra- tion of All receptor antagonists also produce similar decrements in the acute iv AII pressor response in the rat (Brody et_al,, 1978). In addition, the sodium-sensitive hypertensive response to chronic intr vent cent site (Phi lati tral ts": ‘- ”UCTI l4 intravenous infusions of acutely subpressor doses of AII is largely pre- vented in rats with AV3V lesions, suggesting that the "slowly develop- ing" pressor response to iv AII is mediated by an action of All on the brain (Fink gt_gl,, 1982). These observations suggest that the CVO within the AV3V region, namely the OVLT, may be responsible for the centrally-mediated pressor effect of blood-borne AII. However, other sites within the AV3V region that are sensitive to AII such as the MnPO (Phillips gt_al,, 1979a) may be the critical site of action for circu- 1ating AII. Alternatively, the ability of AV3V lesion to abolish a cen- trally-mediated AII pressor effect in the rat may be due to disruption of known SFO efferent pathways that project ventrally along the lamina terminalis and terminate, among other areas, in the median preoptic nucleus and OVLT (Miselis, 1981; Saper and Levisohn, 1983). The importance of the ventral MnPO in mediating central AII pressor responses has recently been examined. Injections of lido- caine into the MnPO attenuate the pressor response to intravenous AII, but these same injection sites are not responsive to local microinjec- tion of All (O'Neill and Brody, 1984). This observation, coupled with the evidence that an anterior hypothalamic knife cut that interrupts efferents from SF0 and OVLT that course through the MnPO also decreases the pressor response to iv AII (Hartle and Brody, 1984), suggest that efferents from forebrain AII sensitive systems converge in the MnPO. Since the ventral MnPO is within the area of AV3V lesion, some effects of AV3V destruction may be due specifically to interruption of pathways in the MnPO. Wit: TAT; 15 c. Thirst An elevation in plasma AII levels is a potent stimulus for drinking behavior. Several experimental manipulations that increase plasma AII concentrations including isoproterenol treatment, caval ligation, and subcutaneous polyethylene glycol (extracellular thirst challenge) result in increased water intake (Leenen gt_a1,, 1974; John- son g1_a1,, 1981). In addition, subcutaneous or intravenous administra- tion of AII itself will cause drinking in virtually all mammalian species studied including the rat (Buggy and Johnson, 1977; Hsiao gt 21,, 1977; Eng and Miselis, 1981; Lind and Johnson, 1982) and dog (Fitz- simons g1_§1,, 1978; Thrasher gt_a1,, 1982a). Chronic intravenous AII infusion (10 days) has been reported to produce a sustained increase in water intake in the dog (Trippodo gt_a1,, 1976), whereas comparable infusions in the rat fail to alter water intake (Brown gt_a1,, 1981). Although there is evidence that angiotensin is a physiological dipsogen in situations such as water deprivation (Barney gt_a1,, 1983), this point is still one of considerable debate (Stricker, 1978; Mann gt_a1,, 1980; Johnson g1_g1,, 1981). Blood-borne AII has been proposed to elicit thirst by a direct effect on the brain rather than by altering nervous input from peripheral receptors. One compelling argument that AII acts on the brain to stimulate thirst is that AII is 1,000 times more potent as a dipsogen when delivered directly into the brain as compared to intra- venous administration (Epstein gt_a1,, 1970). Furthermore, thirst induced by peripheral All is attenuated by lower doses of AII antago- nists when given centrally than peripherally (Johnson and Schwob, 1975). In an effort to localize the specific brain area responsible for ' h E351 FIG r! .\ \. d1r l6 angiotensin-induced drinking, Johnson and Epstein (1975) mapped cannula sites that were effective in eliciting drinking to central injections of AII. They found that cannula sites bordering the cerebral ventricles or trajectories that passed through the ventricles were the most sensitive sites. Using a method of regional ventricular obstruction with cold cream plugs, it was found that intracerebroventricular (ivt) injections of A11 must have access to the AV3V region in order to be dipsogenic (Hoffman and Phillips, 1976a). However, these same plugs were without effect on drinking elicited by systemic AII administration (Johnson and Buggy, 1976). Lesions of the AV3V region, however, abolish drinking to subcutaneous AII injections (Buggy and Johnson, 1977), thereby impli- cating structures in this area in the dipsogenic response to peripheral AII. Lesions of the MnPO also attenuate the drinking response to subcu- taneous AII injections (Mangiapane et_a1,, 1983). In addition to the drinking behavior provoked by blood-borne AII, chronic intravenous AII infusion also has been reported to induce sodium appetite (Findlay and Epstein, 1980). There is evidence that the SFO also is important in mediating the dipsogenic effects of blood-borne AII. Injections of All directly into the SFO are more effective in inducing drinking than injections into surrounding brain areas (Simpson and Routtenberg, 1973; Mangiapane and Simpson, 1980a). SFO lesions reduce drinking to intra- venous AII in both the rat (Simpson and Routtenberg, 1975) and dog (Thrasher et_a1,, 1982), and transection of SFO efferent projections also attenuates angiotensin-induced drinking (Eng and Miselis, 1981; Lind and Johnson, 1982). Integrity of the SFO (or its efferent pathways) 17 and structures within the AV3V region appear to be necessary for drink- ing induced by blood-borne AII. d. Vasopressin release Although the cell bodies of magnocellular neurons in the supraoptic and paraventricular nuclei are sensitive to AII (Gregg and Malvin, 1978; Sladek and Joynt, 1979), blood-borne AII does not have direct access to these regions. However, the SFO has known neural connections to the paraventricular nucleus (Miselis, 1981). The re— lease of vasopressin in response to intravenous AII has been found to be attenuated in rats with knife cuts of SFO efferent pathways (Knepel 22_ 21,, 1980) or SFO lesions (Mangiapane gt_21,, 1984). Structures within the AV3V region also have been implicated in angiotensin-stimulated vasopressin release (Bealer 22_21,, 1979). B. Brain Renin-Aggiotensin System l. Components1present in brain The actions of angiotensin on the brain are of interest not only because many of the effects of blood-borne AII can be partially or entirely attributed to a central action, but because all the components necessary for the generation of AII are present in the brain (see Phillips, 1978; Ganong, 1984 for reviews). a. Angiotensinogen Angiotensinogen (or renin substrate) has been found in brain tissue (Ganten 21_21,, 1971; Printz and Lewicki, 1977; Lewicki 22_ 21,, 1978) and in CSF (Schelling 21 21,, 1980). The CSF angiotensinogen concentration is lower than that of plasma, but when expressed per mg in; :ic P- ‘d \ him 8 C h u 0 Q .. «.1... r . 11 T,» 6 DO A wb .T. e .3 P» (L\ 18 protein, CSF has about 3 times as much angiotensinogen as plasma. The highest tissue concentrations are found in the AP, OVLT, periventricular region of the thalamus and hypothalamus and the median eminence. Printz 21_21, (1980) also have provided evidence that CSF angiotensinogen is not derived from peripheral sources, but originates in the brain. Manipulations of plasma angiotensinogen are not always reflected in similar changes in brain angiotensinogen (Gregory gt_21,, 1982), suggest- ing independent regulation of plasma and brain levels. b. Renin Renin-like activity has been demonstrated in dog, rat and human brain tissue (Fischer-Ferrario 22_21,, 1971; Daul 21_21,, 1975) and this activity persists after nephrectomy (Ganten 22_21,, 1971), suggesting that brain renin is not of peripheral origin. Renin has been reported to be undectable in CSF (Schelling 22_21,, 1980), or present in extremely low amounts (Brosnihan 22_21,, 1982). Initial studies demon- strated that the pH optimum for brain renin-like activity (4.5-5.5) was lower than that for renal renin (5.0-6.0), and most of the renin-like activity observed in brain was attributed to cathepsin D and similar acid proteases (Day and Reid, 1976). However, chromatographic separa- tion of brain renin and cathepsin 0 activities has been achieved (Hirose 21_21,, 1978). Independent regulation of plasma and brain renin has been demonstrated in sodium-depleted dogs, where plasma renin increases concomitantly with either no change or decreases in regional brain renin (Brosnihan 22_21,, 1982). c. Angiotensin converting enzyme Converting enzyme (when measured by hydrolysis of the substrate hip-his-leu) is found both in CSF (Schelling 22_21,, 1980) and l9 brain tissue (Yang and Neff, 1972). Immunocytochemical localization of converting enzyme has shown that the enzyme is highly concentrated in the brush border of the choroid plexus (Igli 22_21,, 1977). d. Angiotensinase Peptidases that metabolize AII into smaller fragments are collectively termed angiotensinase. Angiotensinase activity is not present in CSF, but AII is degraded during jg_yjyg_ventriculo-cisternal perfusion, presumably by contact with tissue angiotensinase. Circumven- tricular organs (including the subfornical organ, median eminence, subcomissural organ and area postrema) have particularly high angioten- sinase activity (Schelling 22_21,, 1980), and it has been suggested that angiotensinase activity is intimately associated with angiotensin re- ceptors (Abhold 22_21,, 1984). e. Angiotensins Both angiotensin I and II have been found in CSF (Schell- ing 21.21,, 1980; Husain 22_21,, 1983), and immunoreactivity for an AII- like material has been detected in brain tissue (Phillips 22_21,, 1980; Quinlan and Phillips, 1981; Phillips 22_21,, 1981; Simonnet 22_21,, 1984). Since available antibodies for AII cross react with the 2-8 (AIII), 3-8 and 4-8 fragments of AII (Hutchinson 21_21,, 1978), some studies have undertaken electrophoretic or chromatographic separation of these peptide fragments to determine the relative amounts of AII and metabolites in biological samples. Using these techniques, some in- vestigators have concluded that AII measured in human CSF may be an artifact (Semple 21_21,, 1980), while others have been able to separate and measure AI, AII, and A111 in rat brain tissue and CSF (Hermann 22 not tion the int. Till no or 3-1.") tr 20 21,, 1982; Ganten gt_21,, 1983). Electrophoretic separation of immu- noreactive angiotensin from canine CSF has shown that A111 is the major angiotensin from this source (Hutchinson 22_21,, 1978). The concentra- tion of immunoreactive AII in the CSF has been found to increase during the development of 2-kidney Goldblatt hypertension in dogs (Suzuki_gg 21,, 1983). The CSF immunoreactive AII found in this study probably was not of peripheral origin since Nicholls (1980) has shown that manipula- tions that cause changes in the level of plasma immunoreactive AII in the dog (hemorrhage, furosemide, B-adrenergic blockade, and saline infusion) do not result in any changes in CSF immunoreactive AII. In this study it was also demonstrated that AIII, when infused intravenous- ly, does not enter the CSF. f. Angiotensin receptors Specific, saturable and reversible binding of 125I-AII to various regions of rat brain has been demonstrated (Sirett 22_21,, 1977; Harding 21 21,, 1981). These jg_yj§gg_binding studies have shown that high concentrations of All binding sites are present in area postrema, septum, superior colliculi, midbrain, thalamus and hypothala- mus. The technique of 12_!1222_receptor autoradiography has been used to further localize AII receptors to the lateral septal nucleus (Healy and Printz, 1984), superior colliculus, lateral olfactory tract, para- ventricular and periventricular nuclei, OVLT, median preoptic nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, and area postrema (Gehlert 22_21,, 1984a,b; Mendelsohn 22_21,, 1984). Angiotensin II has been demonstrated to bind specifically to circumven- tricular organs (OVLT, SFO, AP, median eminence) after intravenous injection (van Houten 22_21,, 1980). AII binding sites also were found us .I A.\v 21 in the OVLT after intraventricular AII injection by a fluorescent micro- scopic technique (Landas 22_21,, 1980). It is believed that brain AII binding sites are physiologically relevant for several reasons: 1) microiontophoresis of All in areas of dense AII binding, such as OVLT (Knowles and Phillips, 1980) and SFO (Phillips and Felix, 1976) cause neuronal excitation; 2) microinjection of All into these regions results in physiologic responses, such as increases in blood pressure (Mangia- pane and Simpson, 1980a), and the binding affinity of All analogs to brain membrane preparations correlates well with their physiological potency (Mann 22_21,, 1981). Factors that regulate brain AII receptor number and/or affinity, or the activity of the brain renin-angiotensin system as a whole are not well defined. The regulation of brain AII receptors in response to sodium intake has been studied. One report (Mann 22_21,, 1980a) has shown that low sodium intake results in a decreased number of AII binding sites in the hypothalamus-thalamus-septum-midbrain (HTSM) region in rats, and that sodium restriction also blunts the dipsogenic and pressor responses to acute ivt AII injection. However, others (Speth gt 21,, 1984) have found no alterations in brain AII receptors with sodium intake. Functional studies of brain AII receptor sensitivity have shown that the dipsogenic (Kapsha 22_21,, 1979) and pressor (Brosnihan 22_21,, 1979; Eguchi and Bravo, 1984) actions of acute ivt AII administration appear to be unaltered by dietary sodium intake. However, the hyperten- sive response to chronic ivt AII injection in the dog is sodium—sensi- tive (Buckley 22_21,, 1981), suggesting that AII and sodium may interact at the level of the central nervous system to produce chronic increases 22 in arterial pressure. A central angiotensin-sodium interaction has been demonstrated in the goat, where the pressor, dipsogenic, antidiuretic, natriuretic and plasma renin suppressing activities of acute third ventricular infusions of AII or hypertonic NaCl are potentiated when the All is given in a hypertonic NaCl solution (Anderson 22_21,, 1971, 1972; Eriksson 21_21,, 1976). Sodium 222.§2_appears to be important in this response since hypertonic solutions of non-electrolytes do not have the same effect. In contrast, Buggy gt_21, (1979) have shown that the dipsogenic effect of ivt infusions of hypertonic NaCl and AII in the rat are not synergistic, but additive, and furthermore that hypertonic sucrose solutions are as effective as hypertonic NaCl in eliciting blood pressure increases. Regardless of whether the effects of AII and sodium are additive or synergistic, it does appear that sodium can augment the central effects of AII. Although adrenal and vascular AII receptors regulate in response to the elevated plasma AII levels seen in sodium restriction, it is not yet well established whether brain AII receptors are altered in response to changes in endogenous levels of the peptide. During dehydration, a condition in which plasma AII is elevated, AII receptors in the SFO "up-regulate" as reflected by an increase in the number of AII binding sites (Israel 22_21,, 1984). However, chronic ivt AII infusion has no effect on All binding site number or affinity in the hypothalamus-thalamus-septum-midbrain region (Singh 22_21,, 1984). At this point it is still uncertain to what extent the central interactions of AII and sodium contribute to the pressor actions of central or peripheral AII administration in animals on varying dietary sodium intakes. Elle 23 g. Angiotensinergic neurons Receptor binding studies have localized AII receptors both in sites that would be expected to be exposed to blood-borne AII, and also in sites that are within the blood-brain barrier, and thus would not be expected to "see" circulating AII. The functional impor- tance of All receptors that are far removed from the ventricular system and inside the blood-brain barrier is not known. However, accumulating evidence suggests that these receptors may indicate the presence of neuronal systems that use angiotensin as a transmitter. It has been demonstrated that All can be synthesized 22_2932_in brain cells in tissue culture (Fishman 22_21,, 1981; Raizada 22_21,, 1984; Weyhenmeyer 21_21,, 1984), and that AII can be released from these cells upon chemical depolarization with KCl (Meyer 21_21,, 1984). Immunohisto- chemical studies have localized angiotensin peptides in particular cell groups and fiber systems in the central nervous system (Fuxe 22_21,, 1976; Changaris 22_21,, 1978; Kilcoyne 21_21,, 1981; Phillips 22_21,, 1981; Weyhenmeyer and Phillips, 1982; Lind gt_21,, 1984a). Of parti- cular interest in these studies is that All immunoreactivity is present in areas that are known to interact with blood-borne AII. The central part of the SFO contains an AII-immunoreactive terminal field which appears to arise from cells in the lateral hypothalamus, zona incerta and nucleus reuniens (Lind 22_21,, 1984). Cell bodies that stain for AII-like peptides are present in the peripheral part of the SFO. Many of the areas that receive SFO efferents are sensitive to AII, and some of these, including the paraventricular nucleus and median preoptic nucleus, receive AII-containing input from the SFO (Lind gt_21,, 1984a). 24 Among other CVO's, the OVLT was found to contain a plexus of fibers and varicosities that stain for AII. The AP contains AII-stained fibers and adjacent parts of the nucleus of the solitary tract contain immuno- stained cell bodies. 2. Physiological effects of central AII administration a. Pressor effects One method that has been commonly employed to study the central actions of A11 is to administer the peptide into the CSF. This route of administration presumably simulates endogenous brain production of AII. Acute or chronic administration of AII into the cerebral ventricles produces a pressor response (Severs 22_21,, 1970; DiNicolan- tonio 22_21,, 1982; Fink 22_21,, 1982; Fisher and Brown, 1984). Two mechanisms have been proposed to contribute to the pressor response seen with acute ivt injections of AII: the release of pressor quantities of vasopressin (AVP) and activation of the sympathetic nervous system. Acute ivt AII injections have been observed to produce antidiuresis (Severs 21_21,, 1971; Hoffman 22_21,, 1979), an indication of AVP re- lease. In addition, in virtually every study in which it has been measured, plasma AVP increases in response to acute ivt AII (Keil 22 21,, 1975; Haack and Mohring, 1978; Malayan 22_21,, 1979; Scholkens 22_ 21,, 1982; Fisher and Brown, 1984). Furthermore, the following obser- vations indicate that this increase in plasma AVP exerts an important pressor effect in the rat: 1) hypophysectomy, supraoptic nucleus lesion or median eminence lesion decreases the pressor response to acute ivt AII (Severs 22_21,, 1970; Hoffman 22_21,, 1979), 2) pretreatment with an AVP antibody (Haack and Mohring, 1978; Hoffman 22_21,, 1979) or 25 with a specific antagonist of the vascular AVP receptor (Unger 22_21,, 1981; Fisher and Brown, 1984) diminishes the magnitude of acute ivt AII- induced pressor responses and 3) Brattleboro rats, genetically deficient in AVP, exhibit markedly reduced pressor responses to central AII when compared to Long-Evans control rats (Hutchinson 21_21,, 1976; Haack and Mohring, 1978). In contrast to the elevated plasma AVP observed after acute ivt AII injection, plasma AVP is not elevated in response to chronic ivt AII infusion in rats or rabbits (Sterling 22_21,, 1980; Fink 22_21,, 1982a). In the dog, AVP appears to contribute less to the ivt AII-induced pressor response than in the rat. Although plasma AVP increases in the dog after acute ivt AII, hypophysectomy does not alter the magnitude of the pressor response (Malayan 22_21,, 1979). Activation of the sympathetic nervous system also appears to be involved in the pressor response to acute ivt AII, although the evidence for this is somewhat controversial. Some investigators report an increase in plasma catecholamines after ivt AII injection (Mann e_i:_ 21,, 1982; Scholkens 21_21,, 1982) while others report no change (Fisher and Brown, 1984). Ganglionic blockade does not decrease the magnitude of the pressor response (Severs 22 21,, 1970; Fisher and Brown, 1984), while a-adrenergic blockade has been reported to either decrease (Severs 22_21,, 1970; Unger 22_21,, 1981) or have no effect on (Mann gt_21,, 1982) the magnitude of the acute ivt AII-induced pressor response. Peripheral sympathectomy with 6-hydroxydopamine does not alter the magnitude of the pressor response to acute ivt All but prolongs the latency to peak blood pressure (Falcon 21_21,, 1978). Although the above evidence is equivocal, it is known that the reduced pressor response to acute ivt AII seen when the AVP system is blocked by either 26 hypophysectomy or AVP antagonists can be totally abolished in the pre- sence of ganglionic blockade (Severs 22_21,, 1970), suggesting that activation of the sympathetic nervous system does comprise a component of the total pressor response to acute ivt AII. The mechanisms involved in maintaining elevated arterial pressure in response to chronic ivt AII infusions have been less well studied. Chronic ivt administration of A11 in both rabbits (Fink 22 21,, 1982a) and dogs (Jandhyala 22_21,, 1979) produced persistent hyper- tension that does not appear to be mediated by elevated sympathetic vasomotor tone. In both studies, pressor responses to exogenous nor- epinephrine were enhanced in the hypertensive animals, and in the dogs an increased responsiveness to All also was observed. These results are in contrast to those obtained in rabbits and dogs with chronic intra- vertebral AII infusion (Dickinson and Yu, 1967; Sweet gt_21,, 1971). Chronic intravertebral AII infusion, at doses that are ineffective when given intravenously, causes hypertension that can be demonstrated to be mediated by the sympathetic nervous system. The apparent difference in the mechanisms supporting hypertension when A11 is delivered to the brain by the two routes may reflect differences in the receptor sites reached from blood and from CSF. The area postrema is believed to be the site at which vertebral artery infusions of All act (Joy and Lowe, 1970; Yu and Dickinson, 1971). In contrast, AII in the CSF probably interacts with structures in the AV3V region to produce both acute (Buggy and Johnson, 1977; Fink and Bryan, 1980) and chronic (Fink _e_t_ 21,, 1983) increases in blood pressure. Although chronic ivt AII infu- sion has been performed in rats (Gronan and York, 1978; Sterling 22_21,. 27 1980; DiNicolantonio 21_21,, 1982); the mechanisms involved in the hypertensive response to such infusions have yet to be identified. b. Thirst and fluid/electrolyte effects Intracranial administration of angiotensin II is a potent dipsogenic stimulus in many species. Many studies have been undertaken to localize the brain site(s) at which AII acts to induce drinking. Initial studies implicated the preoptic area as an important receptor site for the dipsogenic effect of All (Epstein 22_21,, 1970). However, subsequent studies demonstrated that the extent to which intracranial AII injections produced drinking was related to the accessibility of the injectate to the ventricular system (Johnson and Epstein, 1975). Both the SF0 and structures within the AV3V have been implicated as important receptor areas for the dipsogenic effects of CSF-borne AII. The evi- dence for the SFO as a receptor site for blood-borne AII is described above. Experiments implicating the SFO as a receptor site for the dipsogenic effects of ivt AII are considerably less convincing. Drink- ing can be elicited by injection of All directly into the SF0, and SFO lesion blocks the drinking produced by AII injection into the preoptic area (Simpson and Routtenberg, 1973). However, SFO lesion does not produce consistent thirst deficits to lateral ventricular AII injection (Buggy 21_21,, 1975; Hoffman and Phillips, 1976). Many other studies (as reviewed by Johnson and Buggy, 1977) support the view that access of CSF-borne AII to the SFO is neither necessary nor sufficient for AII- induced drinking. On the other hand, the use of ventricular plugging has demonstrated that access of CSF-borne AII to the AV3V is an absolute requirement for stimulation of drinking (Hoffman and Phillips, 1976; 28 Buggy 21_21,, 1975). In addition, electrolytic ablation of the AV3V region produces drinking deficits to intraventricular AII and hypertonic saline (Buggy and Johnson, 1977; Fink and Bryan, 1980). With regard to the dipsogenic response to chronic ivt AII infusion, one report has shown that this response is not sustained for more than a few days (DiNicolantonio 22_21,, 1982). However, most investigators report that chronic ivt AII infusions produce sustained increases in water intake (Gronan and York, 1978; Fink and Bryan, 1980; Buckley 21_21,, 1981). Intracerebroventricular AII administration, in addition to eliciting pressor and drinking responses, can result in a number of other changes related to fluid and electrolyte status. Effects that have been reported to occur after ivt AII administration include na- triuresis (Severs 22_21,, 1971; Jandhyala 22_21,, 1979; Halperin 22_21,, 1981; Brooks and Malvin, 1982; Fink 22_21,, 1982), stimulation of ACTH release (Maran and Yates, 1977; Eguchi and Bravo, 1984), suppression of plasma renin activity (Malayan 22 21,, 1979; Eguchi and Bravo, 1984), and stimulation of sodium appetite (Avrith and Fitzsimons, 1980). Plasma aldosterone has been reported to increase (Nicholls 22_21,, 1983) or decrease (Brooks and Malvin, 1980) after acute ivt AII infu- sion. C. Central Effects of Angiotensin II in Hypertension As discussed above, AII can exert a centrally-mediated pressor effect from the blood or from the CSF, in the latter case possibly as the result of All generation by the endogenous brain renin-angiotensin system. Two lines of experimental evidence have implicated a central 29 pressor effect of AII as an important pathogenetic factor in many forms of experimental hypertension. First, electrolytic ablation of discrete AII-sensitive brain areas has been demonstrated to prevent the develop- ment of hypertension in response to several experimental interventions, or to reverse hypertension once it has become established. Second, central administration of converting enzyme inhibitors or AII receptor antagonists has been reported to decrease blood pressure in hypertensive rats. A discussion of results obtained with each of these methods, and their limitations, is presented below. 1. Brain lesion studies In rats, electrolytic ablation of the AV3V region will both prevent and reverse one-kidney, one-wrap Grollman hypertension (Buggy 21_ 21,, 1977, 1978) and will reduce the severity of aortic coarctation (Hartle 22 21,, 1979), two-kidney one-clip Goldblatt (Haywood 22_21,, 1983), and deoxycorticosterone-salt hypertension (Fink 22_21,, 1977; Berecek 21_21,, 1982). AY3V lesion also has been shown to retard the development of NaCl hypertension in the Dahl salt-sensitive rat strain (Goto 22_21,, 1982). However, lesion of the AV3V does not effect the development of hypertension in young spontaneously hypertensive rats (SHR) or decrease established blood pressure in adult SHR (Buggy 21_21,, 1978; Gordon 21 21,, 1982). SEC lesion also has been shown to attenuate the severity of 2-kidney one-clip renal hypertension (Buggy 22 21,, 1984) and l-kidney Grollman hypertension (Kneupfer gt_21,, 1984) in the rat. Perhaps one of the most intriguing observations to come from a lesion study is that AV3V lesions in rats largely will prevent the development of hypertension in response to chronic intravenous infusion 30 of acutely subpressor doses of AII (Fink 22_21,, 1982b). This suggests that, as opposed to the pressor response to acute iv AII which is pre- dominantly the result of direct vascular constriction, chronic eleva- tions in blood-borne AII produce hypertension by an action on the brain, mediated through the AV3V region. A species difference between the rat and rabbit with regards to the ability of AV3V lesion to prevent hypertension development has been observed. In contrast to the rat, destruction of the AV3V in the rabbit has no effect on DOCA-salt (Mann 2; 21,, 1984) or 2-kidney one- clip renal hypertension (Fink and Bryan, 1982). However, AV3V lesion does prevent the development of one-kidney one-clip renal hypertension in the rabbit (Fink and Mann, 1983). An important distinction bewteen the rat and rabbit lies in the effect of AV3V destruction on pressor responses to acute or chronic intravenous AII infusion. In the rat, the pressor response to acute iv AII infusion is slightly but significantly reduced after AV3V lesion (Buggy 22_21,, 1977), whereas the same re- sponses in the rabbit are unaltered by AV3V lesion (Fink and Bryan, 1980). Furthermore, as mentioned above, AV3V lesion largely prevents the hypertensive response to chronic elevations of plasma AII in the rat. In contrast, the hypertension observed in the rabbit with prolonged intravenous AII infusion is not attenuated in rabbits with prior AV3V lesions (Fink and Mann, 1984). This species difference in the effects of AV3V lesion on various models of hypertension, especially iv AII- induced hypertension, may reflect the relatively greater importance of the area postrema as a central pressor site for circulating AII in the rabbit. Alternatively, the different effects of AV3V lesion in the two 31 species may relate to the relative size and extent of the lesion. The AV3V lesion is relatively large in the rat, encompassing the OVLT, subcommissural MnPO, and parts of the periventricular and medial preop- tic areas, whereas the area of damage in the rabbit is largely confined to the OVLT and immediately surrounding structures. Since the AV3V region in the rat contains both AII-receptive elements (in OVLT and MnPO) and neural pathways from other AII-sensitive sites (notably the SFO), small discrete lesions within the AV3V region will be necessary to discern which specific areas are responsible for "sensing" chronic changes in blood-borne or CSF-borne All in the rat. One limitation inherent in lesion studies is the possibility that the lesion produces "non-specific" effects unrelated to the primary intent (in this case, interruption of forebrain AII-sensitive mechaa nisms). In the case of AV3V lesion, the acute post-lesion period is characterized by adipsia coupled with an inappropriate antidiuretic response to the reduced water intake. As a result, dehydration ensues (Johnson and Buggy, 1978; Brody and Johnson, 1980). Within three weeks, water intake and fluid balance return to normal, but plasma sodium, osmolarity and plasma renin activity remain chronically elevated (Buggy and Johnson, 1977; Shrager and Johnson, 1980). Although fluid balance returns to normal after AV3V lesion, functional deficits persist. Pressor, antidiuretic and dipsogenic responses to ivt or systemic administration of hypertonic solutions or AII are attenuated (Buggy 21_ 21,, 1977; Bealer 21_21,, 1979; Brody and Johnson, 1980; Fink and Bryan, 1980). In addition, excretion of water and sodium is impaired in re- sponse to a volume load (Brody and Johnson, 1980; Bealer 22_21,, 1983). 32 In short, a rat with an AV3V lesion is not a "normal" rat minus an AII- sensitive brain region, rather it is also a rat with functional deficits in osmoregulation. These "non-specific" lesion effects confound inter- pretation of experiments in which a centrally-mediated AII pressor effect is estimated by the difference between normal and AV3V lesion animals. 2. Central administration of inhibitors of the renin-angiotensin system The second method that frequently has been used to assess the effects of All on the brain is ivt administration of AII receptor anta- gonists or converting enzyme inhibitors. Acute bolus ivt injections of All receptor antagonists (most commonly used are 1sar,8ala-AII and Isar,8ile-AII) will produce slight transient depressor responses in SHR (Ganten 21_21,, 1975; Phillips 22 21,, 1975; Mann 22__1,, 1978; Suzuki 22_21,, 1981), stroke-prone SHR (Phillips 21_21,, 1977), 2-kidney, one- clip renal hypertensive rats (Mann 21_21,, 1978; Suzuki 21_21,, 1981), 2-kidney, 2-c1ip renal hypertensive rats (Schoelkens 21_21,, 1976) and malignant hypertensive rats (Sweet 22_21,, 1976). In SHR, acute ivt injection of the converting enzyme inhibitor captopril also has been reported to produce decreases in blood pressure (Hutchinson 22_21,, 1980), although this is not a universal finding (Crofton 22_21,, 1981). Since acute ivt injections of AII antagonists do not completely reverse hypertension due to chronic elevation of plasma or CSF AII (Mann gt_21,, 1978; Suzuki 22_21,, 1981; Fink 21_21,, 1982a), such injections may not be sufficient to completely reveal a centrally-mediated AII pressor effect in chronic hypertensive states. This is especially important in view of the suggestion that the central effects of AII are slow to 33 develop (Brown 21_21,, 1981). Some investigators have reported that the development of hypertension in young SHR is attenuated by chronic ivt infusion of the renin inhibitor N-acetyl-pepstatin (Tonnaer 22_21,, 1981), or captopril (Okuno 21 21,, 1983). Established hypertension in adult SHR can be partially reversed by chronic ivt infusion of lsar, 8ile-AII (McDonald 22_21,, 1980) and a depressor response to chronic ivt infusion of 1sar,8a1a-AII has been observed in stroke-prone SHR (Ganten 22_21,, 1979). However, these depressor responses are greatest within the first 24 hours; blood pressure tending to return toward control levels over the remaining days of ivt antagonist infusion. Although all of the above-mentioned studies support a role for a central pressor effect of AII in hypertension, the acute nature of many of the experi- ments coupled with the lack of determination to what extent the ivt AII antagonist injections or infusions produce functional blockade of central or peripheral AII responses are limitations of these studies. STATEMENT OF PURPOSE Many recent studies have implicated a central pressor effect of AII (of either peripheral or central origin) as an important contributor to some forms of hypertension. Therefore, the first hypothesis tested in this thesis will be whether chronic stimulation of brain AII receptors will produce hypertension. The cardiovascular and fluid/electrolyte responses to chronic stimulation of central AII receptors by ivt AII infusion will be characterized. The sodium dependency of this form of hypertension will be assessed using both rats and rabbits as animal models. Since the sympathetic nervous system and vasopressin contri- bute to the pressor response to acute ivt AII in the rat, the hypothe- sis that these mechanisms contribute to the hypertension produced by chronic ivt AII infusion will be investigated. The response to pharma- cologic blockade of each of these systems will be used as an index of their contribution to the hypertensive state. The importance of the sympathetic system will be studied further in chemically sympathecto- mized rats. The role of aldosterone and of the direct vasoconstrictor effects of blood-borne AII also will be examined. Finally, the hypo- thesis that interactions of the sympathetic nervous system, AVP, and peripheral AII are involved in the maintenance of hypertension in this model will be studied. 34 35 Methods will be developed to produce chronic, selective pharmaco- logic blockade of brain AII receptors or brain angiotensin converting enzyme, in an effort to determine the extent to which central AII effects contribute to several forms of hypertension. These pharmaco- logic methods should represent an advantage over previous electrolytic lesion studies, since non-specific lesion effects will be dissociated from blockade of central AII receptors. The following models of hyper- tension will be examined: 1) genetic hypertension: chronic ivt infusion of an AII recep- tor antagonist or converting enzyme inhibitor will be per- formed in adult rats with established hypertension 2) DOC-salt hypertension: chronic ivt infusion of AII receptor antagonist prior to and during DOC-salt treatment 3) chronic intravenous AII infusion: chronic ivt infusion of AII receptor antagonist prior to and during chronic iv AII infusion. The hypothesis that chronic elevations of circulating AII produce hypertension by an action on the brain will be further examined by determining the effects of discrete brain lesions on chronic iv AII- induced hypertension. The brain lesions studied in this regard in- clude: l) subfornical organ lesion, 2) knife cut of subfornical organ efferents, 3) median preoptic nucleus lesion. MATERIALS AND METHODS A. General Methods 1. Animals Except where otherwise noted, male Sprague-Dawley rats (250- 350 g) were used in all experiments. Rats were obtained from either Harlan, SASCO, or Charles River breeding farms. Prior to entry into an experimental protocol they were group housed on corn cob bedding in light-cycled, temperature controlled quarters. Access to standard laboratory chow (Wayne Lab Blox) and distilled water was provided 22_ libitum. Male spontaneously hypertensive rats (SHR) derived from the strain developed by Okamoto and Aoki (1963) were obtained from SASCO and were between 250 and 350 g at the time of experimentation. Male albino New Zealand rabbits supplied by Bailey were housed individually in metal metabolism cages and offered 100 g of Purina high fiber rabbit chow (Lab Rabbit Chow HF) per day with 2g_112_access to distilled water prior to entry into a study. 2. General Surgical Procedures All major surgical procedures were performed under pento- barbital anesthesia (50 mg/kg, ip for rats; 30 mg/kg, iv for rabbits). Rats also received 0.2 mg atropine sulfate ip to reduce bronchial congestion. Minor procedures were performed under one of the following: 36 37 1) gaseous anesthesia with ether or halothane, or 2) methohexital, 10 mg/kg, iv. Postoperatively, rats received a single intramuscular in- jection of 20,000 U procaine penicillin G and 25 mg dihydrostreptomycin (100,000 U penicillin and 125 mg dihydrostreptomdcin for rabbits). Rats that were subsequently housed in metabolism cages received twice daily injections of ampicillin (10 mg, iv) throughout the experiment. A recovery period of at least 2-3 days after surgery preceded entry into an experimental protocol. 3. Blood pressure measurement a. Direct catheterization: rats Chronic indwelling catheters were implanted in the ab- dominal aorta and vena cava via the left femoral vessels. Catheters were fashioned from either polyvinyl chloride tubing or polyvinyl- silicone rubber. The arterial catheter was filled with a heparinized (1000 U/ml) solution and both catheters were plugged when not in use. Catheters were tunnelled subcutaneously to the suprascapular region and either left beneath the skin or exteriorized and anchored to the skin with a small amount of cyanoacrylate glue. For measurement of arterial pressure, rats were briefly anesthetized with halothane or methohexital and placed in a tethered rat jacket in their home cage. The tether was attached to a swivel mounted above the cage to allow the rat free move- ment. Arterial and venous catheters were connected to long lengths of polyvinyl chloride tubing that exited the cage via the protective tether. The arterial catheter was connected to a small volume dis- placement pressure transducer (Gould-Statham P2310 or P50) and pulsatile arterial pressure recorded on a Grass polygraph. When necessary, the 38 venous catheter was attached to a Harvard syringe infusion pump for intravenous infusion. The duration of anesthesia was typically 1-2 min, and after this time, direct recording of arterial pressure was obtained in the conscious rats. Rats were allowed to sit undisturbed for at least 10-15 minutes before any experimental manipulation was begun. Mean arterial pressure (MAP) was calculated as 1/3 (pulse pressure) plus diastolic pressure. Heart rate (HR) was counted directly from the arterial pressure tracing. When rats were to be housed in metabolic cages, arterial and venous catheters were tunnelled subcutaneously to the skull, where they were anchored with dental acrylic. Catheters were protected by a flexible metal spring led out the top of the cage and connected to a hydraulic swivel to allow the rat free movement within its cage. A continuous intravenous fluid infusion (either 5% dextrose or 0.9% saline) was maintained throughout the experiment at a rate of 40 m1/24 hr. Mean arterial pressure and heart rate were determined each day between 8:00 and 11:00 a.m., and were taken to be the lowest stable values obtained during a 15-30 min recording session. b. Indirect blood pressure measurement Systolic blood pressure was estimated by an indirect tail cuff plethysmographic method (IITC Pulse Amplifier) with photoelectric detection. Rats were restrained in a plexyglass restrainer and warmed under a Tensor lamp for approximately 10 min to obtain dilation of the tail artery. Tail cuff blood pressure was taken as the average of five determinations. 39 c. Direct measurement: rabbits Mean arterial pressure was measured directly in conscious rabbits loosely restrained in a head stock. Dilation of a central ear artery was obtained by infiltration of the base of the ear with 1 ml of a 2% lidocaine solution and application of 5% lidocaine ointment to the back of the ear. The artery was punctured percutaneously with a 25- gauge butterfly infusion set. Tubing from the infusion set was con- nected to a Gould-Statham 2310 pressure transducer and output recorded on a Grass polygraph. Mean arterial pressure and heart rate were cal- culated in the same manner as for rats. 4. Implantation of intracerebroventricular cannulae Lateral cerebral ventricular cannulae were implanted in rats under pentobarbital anesthesia. The head was immobilized in a Kopf small animal stereotaxic apparatus. The incisor bar was set at a level 5.0 mm above the interaural line. The skull was exposed by a midline incision and 2-3 small jewelers screws were burrowed a short distance into the skull. A small burr hole was made 1.5 mm lateral to bregma, and a stainless steel 23-guage cannula (12 mm length) was lowered so that its tip was located 4.5 mm ventral to the dura. Dental acrylic was used to anchor the cannula to the skull. In some experiments, bilateral ventricular cannulae were implanted by lowering one cannula 1.5 mm lateral to bregma on either side. Cannulae were occluded with 30-gauge stainless steel obturators when not in use. At the conclusion of each experiment, correct cannula placement was verified by injection of Evans Blue dye into the cannula and visualization of the dye in the ventricu- lar system after the brain had been cut in the frontal plane. 40 A single lateral ventricular cannula was implanted in rabbits under pentobarbital anesthesia. The skull was leveled in a Kopf stereo- taxic apparatus and a burr hole made 1.0 mm anterior and 2.0 mm lateral to bregma. A 23-gauge stainless steel cannula was lowered 8.0 mm ven- tral to the dura. The cannula was anchored to the skull with jewelers screws and dental acrylic, and was occluded with an obturator when not in use. 5. Metabolic measurements In some experiments in which rats were housed in metabolic cages, daily measurements of variables related to fluid/electrolyte status were obtained. Fluid intake was quantified as the sum of the volume obtained by intravenous infusion (40 ml) and the volume of water ingested from a calibrated drinking tube. Urine output (U0) was measured by collection in calibrated tubes under the cage. Water balance was calculated as the difference of fluid intake and urine output (assuming a constant insensible loss). Urinary sodium and potas- sium concentrations were measured by flame photometry and daily urinary sodium and potassium excretions calculated by multiplying urine volume by electrolyte concentration. 6. Statistical analysis A variety of statistical tests appropriate for the experimen- tal design were used. Many of the experiments take the format of re- peated measurements within animals. In these experiments, a randomized block analysis of variance (ANOVA) or a mixed design ANOVA (for compari- son or 2 or more groups) was used to detect differences in treatment means. Except where otherwise stated, the "protected" least significant 41 difference test (lsd) was used for nonconfounded individual within- and between-group comparisons. Values given in the text are mean :_standard error of the mean (within groups) or, when a t-test is used, mean :_ standard error of the difference. Specific statistical procedures are mentioned within the context of the particular experiment in the Results section. In all cases a p value of 0.05 was used as the criterion of statistical significance. B. Experimental Protocols 1. Chronic ivt AII infusion: rat a. Sodium dependency In an initial surgical procedure, rats received chronic indwelling arterial and venous catheters and a right lateral cerebral ventricular cannula. The top of the ventricular cannula was fitted with a piece of polyvinyl chloride tubing filled with 0.9% saline and tun- nelled subcutaneously to the scapular region, where it was plugged. Rats were housed in metabolic cages and the catheters protected by a metal spring led out the top of the cage and attached to a hydraulic swivel. Rats had free access to standard laboratory chow (0.1 mEq Na, 0.3 mEq K/g). Sodium intake was controlled by the composition of the intravenous infusion; rats maintained on normal Na intake received 40 ml of 5% dextrose/24 hr and high Na intake was achieved by infusion of 40 ml of 0.9% saline/24 hr (equivalent to 6.2 mEq Na). Daily measurements of mean arterial pressure (MAP), heart rate (HR), urine output (U0), water intake (WI), water balance (WB) and urinary sodium (UNaV) and potassium (UKV) excretions were obtained. After two days of control measurements, rats were briefly anesthetized with methohexital and a 42 prefilled osmotic minipump (Alzet, Model 2001, Alza Corp., Palo Alto, CA) containing a solution of All (angiotensin II amide, HypertensinR, CIBA) in isotonic saline (1 or 6 mg/ml) was implanted subcutaneously in the suprascapular region and connected via polyvinyl chloride tubing to the ventricular cannula. Control rats received ivt infusions of iso- tonic saline. These minipumps deliver solution at a rate of l ul/hr, therefore AII was infused into the ventricular system at a dose of l or 6 ug/hr. The stability of A11 in minipumps over a 7-day period has been previously verified (DiNicolantonio 22_21,, 1982). Intraventricular infusion was maintained for 5 days, after which time the minipumps were removed under methohexital anesthesia, and two recovery days followed. This general protocol was followed in groups of rats receiving the following treatments: 1) All ivt (6 ug/hr, n=5 or 1 pg/hr, n=5) or saline ivt (n=7), high sodium intake, and access to drinking water only during the final 24 hours of ivt infusion; 2) AII ivt (6 ug/hr, n=6 or 1 pg/hr, n=6) or saline ivt (n=6), high sodium intake and 22_112_access to drinking water, and 3) All ivt (6 ug/hr, n=5 or 1 ug/hr, n=6), normal sodium intake and 2g_112_water intake. b. Plasma hormone levels Rats were maintained on a high sodium intake with 22_112 access to water and received ivt infusions of AII (6 ug/hr, n=6) or saline (n=7) for 5 days preceded by two control and followed by two recovery days. MAP and HR were measured daily. On the second control day, the fifth day of ivt infusion, and the second recovery day, after MAP was determined, a 1 ml blood sample was rapidly withdrawn from the arterial catheter into a chilled syringe containing EGTA and glutathione 43 for measurement of plasma norepinephrine and epinephrine concentration (Cat-a-Kit, Upjohn). Immediately following this first sample, a second arterial sample (3 ml) was withdrawn into a chilled syringe over EDTA for aldosterone determination (radioimmunoassay, Damon). The samples were spun in a refrigerated centrifuge at 7,000 x g for 10 min and the plasma was stored at -70°C until assay. A 60 ul plasma sample was reserved for determination of plasma osmolality (Micro OsmetteR) and plasma Na and K concentrations. Hematocrit and body weight also were determined on these days. In a separate group of rats maintained on a high sodium intake (n=12), arterial blood samples (3 ml) were obtained on the second control day and fifth day of ivt AII infusion for measurement of plasma AII concentration by radioimmunoassay. Red blood cells were resuspended in an equal volume of isotonic saline and returned to the rat. Plasma samples were stored at -70°C until they were sent to the laboratory of Dr. Ian Reid, University of California at San Francisco, for analysis. c. Adrenalectomy Bilateral adrenalectomy was performed via a retroperi- toneal approach under pentobarbital anesthesia. Rats received a single postoperative injection of dexamethasone (0.2 mg, im), and were main- tained on 22_112_o.9% saline drinking fluid and standard rat chow. Rats were housed individually in clear plastic cages. Control rats did not undergo sham operation. Three to four weeks after adrenalectomy, a right lateral ventricular cannula was implanted in adrenalectomized and control rats. Two days after cannula implantation, blood pressure measurements were begun. Blood pressure was measured three times weekly 44 by tail cuff plethysmography. Body weight and saline intake were measured daily. After one week of control measurements prefilled osmo- tic minipumps (Alzet Model 2001) containing AII (6 mg/ml) were implanted subcutaneously under ether anesthesia and connected via polyvinyl chloride tubing to the ivt cannula. The ivt AII infusion (6 ug/hr) was maintained for 7 days, during which time blood pressure, body weight, and saline intake were measured. After blood pressure was measured on the seventh day of ivt AII infusion, a plasma sample for aldosterone assay was obtained by cutting the tip of the tail and allowing 1 ml of blood to flow freely into a heparinized tube. Blood samples were cen- trifuged, the plasma removed and stored at -70°C until assay. Osmotic minipumps were removed under ether anesthesia after the blood sample was taken. One week of recovery measurements followed. On the final day of blood pressure measurement, a second plasma sample was obtained for aldosterone determination. d. Acute blockade of vascular AII receptors and ganglionic blockade A series of acute interventions was performed to assess the contributionsof the direct vasoconstrictor actions of peripheral All and of neurogenic tone to maintenance of elevated arterial pressure in response to chronic ivt AII infusion. These interventions were performed in a group of 6 rats maintained on high Na intake in metabolic cages on the second control day, the first, third, and fifth days of ivt AII infusion (6 pg/hr) and the second recovery day. After measurement 8ala- of basal MAP and HR, rats received an intravenous infusion of 1sar, angiotensin II (saralasin, 300 ng/min) for 10 min. The change in MAP and HR to saralasin was calculated as the difference of preinfusion 45 values and the values obtained during the final minute of saralasin infusion. After the iv saralasin infusion was stopped, 10-15 min was allowed for recovery. Hexamethonium (20 mg/kg, iv) was then admini- stered to produce ganglionic blockade, and the change in MAP and HR was determined at 5 min after hexamethonium injection. e. Acute blockade of vascular AVP receptors In a group of 5 rats maintained on high Na intake in metabolic cages, an assessment was made of the contribution of the vasoconstrictor effects of AVP to the maintenance of elevated arterial pressure in response to to ivt AII infusion. On the second control day, the first, third and fifth day of ivt AII infusion (6 pg/hr) and the second recovery day, after measurement of basal MAP and HR, a specific antagonist for the vascular AVP receptor (l-(B-mercapto-B,B cyclopenta- methylene propionic acid), 2-(o-methyl)tyrosine) arginine-B-vasopressin, 10 ug/kg, iv) was administered. The changes in MAP and HR to the AVP antagonist were measured at 5 min after its administration. The ability of this dose of AVP antagonist to block the effects of exogenous infusions of AVP was determined in rats on a high sodium intake and housed in metabolic cages (n=5). After measurement of basal MAP and HR, AVP was infused intravenously at successive doses of 0.3, 1.0, and 3.0 mU/min. Each dose was infused for 10 min or until a steady-state blood pressure had been achieved. One to two hours later, the AVP antagonist was given (10 pg/kg, iv), and the dose-response curve to exogenous AVP was repeated starting 5 min after administration of the antagonist. Changes in MAP and HR in response to AVP were calculated as the difference between basal and steady-state values for each infusion rate. 46 f. Peripheral sympathectomy Chronic peripheral sympathectomy was produced in rats by the method of Johnson 22_21, (1976). Pregnant Sprague-Dawley rats were procured at approximately 1 week before parturition. Starting one week after birth, neonatal rats were treated with guanethidine sulfate (50 mg/kg/day, sc) or with an equal volume of isotonic saline vehicle 5 days per week for 3 weeks (total of 15 injections). Solutions were admini- stered in a volume of 5 ul/g body weight. After weaning, rats were separated according to sex and group housed in standard cages with free access to food and water. When rats (of either sex) had reached a body weight of 275-325 g, bilateral adrenal demedullation was performed by a retroperitoneal approach under pentobarbital anesthesia. A small inci- sion was made in the adrenal cortex and the medulla extruded by lightly squeezing the gland with a pair of smooth-tip forceps. After recovery to pre-surgery body weight (approximately 2 wk), a cannula was implanted in the right lateral cerebral ventricle. Rats were housed individually in clear plastic cages. In order to maintain all rats on a relatively fixed, high sodium intake, they were given 50 m1 of 0.9% saline to drink per day and had free access to regular rat chow. This regimen provided a daily Na intake of roughly 8.0 mEq. After a two-day recovery period, one week of control blood pressure and body weight measurements were obtained. Blood pressure was measured three times weekly by a tail cuff plethysmographic method. The control period was followed by a 7-day ivt infusion of AII (6 ug/hr). Osmotic minipumps were implanted and removed under ether anesthesia. One week of recovery measurements followed the ivt AII infusion period. 47 9. Verification of degree of sympathectomy Three procedures were used to assess the degree of peri- pheral sympathectomy produced by neonatal guanethidine treatment plus adrenal demedullation at adulthood. In one group of sympathectomized (n=8) and control rats (n=5) a functional assessment of neurogenic tone was made by measuring the depressor response to sequential ganglionic blockade with hexamethonium and alpha-adrenergic receptor blockade with phentolamine. Rats were briefly anesthetized with methohexital and loosely restrained in a tethered rat jacket in their home cage. After 10-15 min of baseline MAP and HR measurements in the conscious rats, a bolus injection of hexamethonium (20 mg/kg, iv) was administered. The change in MAP and HR to hexamethonium was measured at l and 5 min after the injection. Phentolamine (2 mg/kg, iv) was then administered and MAP and HR responses were measured at l and 5 min. In another group of sympathectomized (n=9) and control rats (n=5), the MAP response to graded stimulation of sympathetic vaso- motor outflow was evaluated in the pithed rat preparation (Gillespie and Muir, 1967). Rats were anesthetized with pentobarbital (50 mg/kg, ip), and the trachea cannulated. Previously implanted arterial and venous catheters were used for blood pressure measurement and iv drug admini- stration. Rats were given atropine (0.2 mg, ip) and were paralyzed with gallamine (10 mg/kg, iv). The animals were then artificially ventilated (Harvard rodent respirator) with room air. Rats were pithed at the level of the seventh cervical vertebra with a steel rod. The change in mean arterial pressure elicited by stimulation of sympathetic vasomotor outflow was determined by stimulating the steel rod with monophasic 48 square wave pulses (cathodal current, 60 V, l msec duration) of varying (l, 2, 3, 5, and 10 Hz) frequency for 20 sec. The stimulations were separated by a period of 5 min. After the stimulus-response curve was obtained, hexamethonium (20 mg/kg, iv) was administered and the 10 Hz stimulation repeated to verify that the pressure rises were mediated by stimulation of sympathetic outflow from the spinal cord. Norepinephrine and dopamine content also was determined in selected tissues of normal (n=5) and sympathectomized (n=8) rats. Rats were killed by decapitation and the following tissues quickly dissected and frozen on dry ice: renal cortex, pineal gland, hypothala- mus, cerebellum, and frontal cortex. Tissues were homogenized in 2.E perchloric acid with 100 mg% EGTA. Norepinephrine and dopamine were analyzed in the supernatant by a radioenzymatic method. Protein was analyzed by the method of Lowry 22_21, (1951). Results were expressed as ng norepinephrine or dopamine per mg protein. h. Pharmacologic assessment of sympathetic tone Rats used in this protocol were instrumented with chronic indwelling arterial and venous catheters and a lateral cerebral ven- tricular cannula. At the time of surgery, an osmotic minipump (Alzet Model 2001) was implanted subcutaneously and connected via polyvinyl chloride tubing to the ivt cannula. Rats received ivt infusions of either AII (6 ug/hr; n=7) or isotonic saline (n=5). Rats were housed individually and had 22_112_access to isotonic saline drinking fluid and standard laboratory chow. Blood pressure was measured in the conscious rats while they were loosely restrained in a rat jacket in their home cage. On day 4 of the ivt infusion, the change in MAP to phentolamine 49 (2 mg/kg, iv) was determined. On day 5, the change in MAP to sequential administration of propranolol (1 mg/kg, iv) and phentolamine (2 mg/kg, iv) was measured. Phentolamine was given 1 min after the propranolol injection. The depressor response to hexamethonium (20 mg/kg, iv) was determined on day 6 of ivt infusion. Blood pressure responses to each intervention were determined at l and 5 min after administration. In 2 of the rats that received ivt AII, the order of the interventions was reversed (i.e., day 4-hexamethonium, day S-propranolol plus phentol- amine, day 6-phentolamine alone). In a separate group of 4 rats, the depressor response to a 5 minute nitroprusside infusion (12 ug/min, iv) was determined. The MAP response to nitroprusside was measured prior to, and on the fifth day of ivt AII infusion. The rats used in this experiment had 22_112. access to isotonic saline drinking fluid and standard rat chow. i. Interactions of AVP, sympathetic nervous system and peripheral renin-angiotensin system This experiment followed a 2x2 factorial design. Four groups of rats were used: normal rats received ivt infusions of either AII (n=5) or saline (n=5) and sympathectomized rats (neonatal guane- thidine treatment plus adrenal demedullation, see section 1f above) similarly received either AII (n=8) or saline ivt (n=5). The sympa- thectomized rats used in this study were a mixture of males and females (AII ivt - 3 male, 5 female; saline ivt - 2 male, 3 female). Female rats were ovariectomized at the same time that adrenal demedullation was performed. In a single surgical procedure, chronic indwelling arterial and venous catheters and a lateral cerebral ventricular cannula were implanted. Osmotic minipumps (Alzet Model 2001) containing AII (6 50 mg/ml) or saline also were implanted at this time and connected to the ivt cannula via polyvinyl chloride tubing. Rats were housed indivi- dually and had 22_112_access to isotonic saline drinking fluid and standard rat chow. On the fifth day of ivt infusion, rats were loosely restrained in a tethered rat jacket in their home cage for measurement of basal MAP and HR. After a steady-state MAP and HR had been recorded (10-15 min), the following series of interventions was performed. First, AVP antagonist (10 ug/kg, iv) was administered, and MAP and HR were determined at 1 and 5 min after the injection. Five min after the AVP antagonist was given, a 15 min infusion of saralasin (10 ug/min, iv) was begun. MAP and HR were determined at 5, 10, and 15 min after starting saralasin infusion. After 15 min of saralasin infusion, phen- tolamine (2 mg/kg, iv) was administered. The saralasin infusion was continued and MAP and HR were measured at l and 5 min after phentol- amine. On day 6 of ivt AII or saline infusion in normal rats, MAP and HR responses to a 15 min infusion of saralasin (10 pg/min, iv followed by AVP antagonist injection (10 ug/kg, iv) were determined. j. Spinal cord stimulation Rats were prepared with chronic indwelling arterial and venous catheters and a lateral cerebral ventricular cannula. Osmotic minipumps containing either All (6 mg/ml; n=9) or isotonic saline (n=8) were implanted subcutaneously and connected by tubing to the ivt can— nula. Rats were housed individually and had 22_112_access to isotonic saline drinking fluid and standard rat chow. On the fifth day of ivt AII or saline infusion, rats were placed in a tethered rat jacket in 51 their home cage and MAP and HR were measured. After basal MAP and HR were measured in the conscious rats, they were anesthetized with pento- barbital (50 mg/kg, ip). Rats were pithed and MAP responses to elec- trical stimulation of the spinal cord were determined as described in section lg. k. Acute ivt sarthran/chronic ivt All In an initial surgical procedure, chronic indwelling arterial and venous catheters and bilateral ivt cannulae were implanted in rats. At the same time, an ivt AII infusion (6 ug/hr, n=5) was started by means of osmotic minipump. Rats were housed individually in clear plastic cages with 22_112_access to standard rat chow and 0.9% saline drinking fluid. 0n the fifth day of ivt infusion basal MAP and HR were measured while rats were loosely restrained in a tethered rat jacket in their home cage. Three doses of sarthran (0.3, 1.0, 3.0 ug in 5 ul of isotonic saline) were administered ivt at 15 min intervals. MAP was measured at 5, 10, and 15 min after injection of each dose. Minipumps were then removed, and the same protocol of ivt sarthran injections was repeated two days later. On the fifth day of ivt AII infusion (6 ug/hr) in a separate group of 4 rats, three 5 u1 injections of isotonic saline were administered with a 15 min interval between injections. MAP was measured every 5 min. 1. Acute ivt sarthran/acute ivt AII A group of 4 rats with bilateral ivt cannulae and ar- terial and venous catheters were used in this experiment. They were housed individually in clear plastic cages with 22_112_access to stand- ard rat chow and distilled water. MAP and HR were determined in the 52 conscious rats while restrained in a tethered rat jacket in their home cage. After a stable MAP had been recorded, an injection of 150 ng AII was administered ivt. One minute after ivt AII injection, sarthran (1 pg in 5 pl volume) was administered ivt in the contralateral cannula. The peak change in MAP to ivt AII was recorded, and MAP also was deter- mined at 2.5, 5, 10, 15, and 20 min after ivt sarthran injection. This protocol was repeated in the same group of rats two days later, with the exception that 5 ul of isotonic saline was administered instead of sarthran. m. Effect of iv saralasin on response to acute ivt AII A group of 5 rats was instrumented with chronic indwell- ing arterial and venous catheters and a lateral cerebral ventricular cannula. They were housed individually and maintained on a normal sodium intake. MAP was measured in the conscious rats while loosely restrained in a tethered rat jacket in their home cage. The peak pressor response to an acute ivt injection of All (50 ng) was recorded. After a 2-3 hour interval, MAP was recorded again and saralasin (300 ng/min) was infused intravenously for 15 min. The pressor response to ivt AII (50 ng) was retested l min after stopping the iv saralasin infusion. 2. Chronic ivt AII infusion in rabbits: Sodium dependency Male albino rabbits weighing 2.5-3.5 kg were used in these experiments. At least one week prior to the beginning of the study, a cannula was implanted in a lateral cerebral ventricle as described in section A.4. Sodium intake was controlled by the amount of sodium in the diet; all rabbits were offered 100 g/day of either sodium-deficient 53 (l mEq Na, 12 mEq K/100 g) or sodium-enriched (163.5 mEq Na, 30.8 mEq K/lOO 9) rabbit chow (Bioserv, Frenchtown, NJ). Tap water was available 22_libitum from calibrated tubes. Rabbits were maintained on either low or high dietary sodium regimens for at least one week prior to study. Daily determinations were made of the following parameters: water in- take, urine output, urinary and fecal Na and K excretion, and food intake. Water balance was calculated as the difference of water intake and urine output, assuming constant insensible loss. Sodium and potas- sium balances were calculated as the difference of dietary intake and the sum of urinary and fecal excretion. The electrolyte content of food and feces was determined by flame photometry after ashing in nitric acid. Rabbits were brought to the laboratory once weekly for deter- mination of the following variables: mean arterial pressure (MAP), heart rate (HR), body weight (B.Wt.), plasma Na (PNa) and K (PK) con- centrations, plasma osmolality (POSM), hematocrit (HCT), plasma volume (PV), extracellular fluid volume (ECFV), blood urea nitrogen (BUN) and creatinine clearance (CCl). MAP and HR were determined in the conscious, lightly restrained rabbits by percutaneous needle puncture of a central ear artery as described in section A.3.c. PNA and PK were determined in triplicate by flame photometry. POSM was determined by the method of freezing point depression (Micro OsmetteR). HCT was determined in triplicate by microcentrifugation. PV and ECFV were estimated by deter- mining the lO-min distribution space of Evans Blue dye and the 30-min distribution space of thiocyanate, respectively (Aikawa, 1950). Ar- terial blood samples (2 ml) were obtained immediately before, and at 10 54 and 30 min after intravenous injection of 1 m1 of a solution containing 5 mg Evans Blue dye and 50 mg sodium thiocyanate. Blood samples were centrifuged, and the concentration of Evans Blue in the lO-min sample and of thiocyanate in the 30-min sample were determined spectrophoto- metrically against the plasma blank. Fluid volumes were calculated by dividing the concentration of indicator in the plasma by the amount injected intravenously, and subsequently expressed per kg body weight. Blood urea nitrogen was determined using a modified Urease-Berthelow reaction (Sigma Kit #660). Creatinine determinations (Jaffe method; Sigma Kit #555) were made on a plasma aliquot taken during the weekly measurements and on a urine sample taken from the previous 24 hr collec- tion. Creatinine clearance was then calculated as UV/P where U=urine creatinine concentration, V = urine volume/24 hr, and P = plasma creati- nine concentration. Baseline measurements of all above-mentioned parameters were made over a two-week period (i.e., 2 weekly determina- tions of MAP, HR, B.Wt., PNA, PK, POSM, HCT, PV, ECFV, BUN, CCl, and daily determinations of other fluid/electrolyte variables). After the second weekly determination of MAP, an osmotic minipump (Alzet Model 2002) containing either AII (6 mg/ml) or isotonic saline vehicle was implanted subcutaneously at the back of the neck under pentobarbital anesthesia. The minipump was connected to the ivt cannula by a short length of polyvinyl chloride tubing tunnelled subcutaneously. This minipump delivers solution at the rate of 0.5 ul/hr, therefore the nominal infusion rate of AII was 3 ug/hr. Four groups of rabbits were studied (n=5 in each group): AII ivt-high Na intake, saline ivt-high Na intake, AII ivt-low Na intake, and saline ivt-low Na intake. Intraventri- cular infusion was maintained for two weeks, during which time daily and 55 weekly measures were obtained. Minipumps were removed and the tubing plugged under local lidocaine anesthesia after the second set of weekly determinations. One week of recovery measures followed the ivt infusion period. 3. Chronic pharmacological blockade of brain AII receptors or brain converting enzyme a. Chronic ivt saralasin 1) Dose determination. Rats were housed individually with 22_112_access to distilled drinking water and standard rat chow. Chronic indwelling arterial and venous catheters were anchored at the neck with a small length of fine copper wire. A lateral cerebral ven- tricular cannula also was implanted. A control set of pressor responses to iv and ivt administration of AII were determined for each rat in the following manner. Basal MAP and HR were measured while the rat was conscious in its home cage. AII was then infused in successive doses of 10, 30, and 100 ng/min, iv. Each dose was infused for 5-10 min or until arterial pressure had stabilized. After the pressor responses to iv AII were determined, blood pressure was allowed to return to control levels. A 30-gauge cannula was connected to a Hamilton microsyringe by a long, flexible piece of tubing and the syringe and tubing were prefilled with a solution of AII (50 ng/pl). The 30-gauge cannula was introduced into the 23-gauge ivt cannula and a single bolus of AII (150 ng) was de- livered into the lateral ventricle and the pressor response recorded. After this control set of pressor responses to iv and ivt AII was determined, rats were anesthetized with ketamine HCl (13 mg/kg, ip) and an osmotic minipump (Alzet Model 2001) filled with a solution of sara- lasin (6 or 12 mg/ml) in isotonic saline was implanted subcutaneously in 56 the scapular region. The minipump was connected to the 30-gauge ven- tricular cannula with a small length of polyvinyl chloride tubing that was tunnelled subcutaneously to the head. Saralasin was infused ivt continuously at doses of 6 ug/hr (n=5) or 12 ug/hr (n=5). Sham rats did not receive an ivt infusion. After 5 days of ivt saralasin infusion (or a 5-day sham period), pressor responses to ivt and iv AII infusion were retested in the same manner as the control measurements were made. Pressor responses to ivt All were retested by removing the tubing leading to the minipump from the ventricular cannula and attaching the prefilled Hamilton microsyringe and tubing to the ivt cannula. 2) Effects in normal- and high-sodium rats. Rats with chronic indwelling arterial and venous catheters and a lateral cerebral ventricular cannula were housed individually in metabolic cages with 22_ 112_access to standard lab chow. Sodium intake was controlled by the composition of a 40 ml/24 hr fluid infusion; rats maintained on normal sodium intake received dextrose and high sodium intake was achieved by iv infusion of isotonic saline. Four groups of rats were used in the experiment. Two groups were maintained on normal sodium intake and two groups were maintained on high sodium intake. Daily measurements of MAP, HR, urine output, and urinary sodium and potassium excretions were obtained. After two days of control measurements rats were briefly anesthetized with methohexital and osmotic minipumps were implanted subcutaneously and connected to the ivt cannula. Rats received ivt infusions of saralasin, 12 ug/hr (normal Na, n=5; high Na, n=7) or saline vehicle (normal Na, n=5; high Na, n=6) for 5 days, followed by two recovery days. Rats had 22_112_access to distilled water from a 57 calibrated drinking tube only during the final 24 hr of the ivt infusion period. 3) Chronic iv saralasin. Rats were housed in metabo- lic cages and maintained on high sodium intake as described in the previous section. After 2 days of cardiovascular and fluid/electrolyte measurements, rats received saralasin (18 ug/hr, iv; n=5) for 7 days, followed by 2 recovery days. The saralasin was added directly to the 40 m1/24 hr iv saline infusion. Rats were not allowed to drink during the course of the experiment and received their fluid requirements from the intravenous infusion. b. Chronic ivt sarthran 1) Dose determination: Sprague-Dawley. In an initial surgical procedure chronic indwelling polyvinyl chloride-silicone rubber arterial and venous catheters and a lateral cerebral ventricular cannula were implanted in male Sprague-Dawley rats (300-325 9). At least two days were allowed for recovery from the surgical procedures. Control pressor responses to intravenous and ivt AII were obtained in the following manner. Rats were briefly anesthetized with methohexital and loosely restrained in a tethered rat jacket. After measurement of basal mean arterial pressure in the conscious rats, AII was infused intra- venously at successive doses of 10, 30, and 100 ng/min. Each dose of AII was infused for 5 min or until a steady-state arterial pressure was reached. After the iv pressor response curve was obtained, 10-15 min was allowed for recovery. A prefilled 30-gauge length of stainless steel tubing was then introduced into the ivt cannula so that its tip was at the end of the cannula. The 30-gauge steel tubing was connected to a Hamilton microsyringe by a long length of polyvinyl chloride 58 tubing. The maximal pressor response to a 150 ng ivt bolus injection of AII (5 pl volume) was then measured. After this set of control re- sponses was obtained, a prefilled osmotic minipump (Alzet Model 2001) was implanted subcutaneously in the suprascapular region and connected to the 30-gauge steel tubing by a length of polyvinyl chloride tubing. This minipump delivers solution at a rate of 1 pl/hr. Minipumps were filled with solutions of sarthran in isotonic saline in varying concen- trations to yield doses of 100 ng/hr (n=5), 300 ng/hr (n=6), 1 pg/hr (n=5) or 6 pg/hr (n=4). The ivt sarthran infusion was maintained for 5 days during which time rats were housed individually with 22_112_access to food and water. The pressor responses to acute iv and ivt All were tested after the 5-day ivt infusion period according to the above pro- tocol. 2) Effects in high-sodium rats. A group of 8 rats was instrumented with indwelling arterial and venous catheters and a lateral cerebral ventricular cannula. Rats were housed individually in metabo- 1ic cages. A continuous iv infusion of isotonic saline (40 ml/24 hr) provided a sodium intake of 6.2 mEq/day, and was the sole source of fluid intake during the experiment. Low sodium rat chow (10-15 g/day; 0.002 mEq Na, 0.3 mEq K/g) was provided so that sodium intake could be fixed at the level provided by the iv saline infusion. This regimen maintained sodium intake at a level approximately 4-5 times normal, and fluid intake (40 m1/day) at a level comparable to that observed in freely-drinking rats (Halperin 21_21,, 1981). A three-day recovery period followed the surgical procedures. The experimental period con- sisted of two control days, 5 days of ivt sarthran infusion, and two 59 post-infusion recovery days. During the experiment, the following cardiovascular and fluid/electrolyte measures were obtained daily: MAP, HR, U V, U V, and U0. After MAP and HR were determined on the second Na K control day, rats were briefly anesthetized with methohexital and pre- filled osmotic minipumps (Alzet Model 2001) were implanted subcutaneous- ly in the suprascapular region and connected to the tubing leading to the ivt cannula. Minipumps were removed in a similar manner after measurements had been obtained on the fifth day of ivt infusion. Mini- pumps were filled with a solution of sarthran (1 mg/ml in isotonic saline), thus the dose of sarthran infused was 1 pg/hr. Correct place- ment of the ivt cannula was verified at the end of the experiment by injecting Evans Blue dye into the cannula and visualization of dye in the ventricular system after the brain had been cut in the frontal plane. Rats were included in the study only if dye could be visualized in the right and left lateral and third cerebral ventricles. 3) Dose determination: Spontaneously hypertensive rats. Male spontaneously hypertensive rats (SHR) were surgically prepared with chronic indwelling arterial and venous catheters and bilateral ivt cannulae. The day prior to the start of the experiment, correct location of ivt cannulae was verified by measuring the drinking latency to an ivt injection of 150 ng AII. The criterion for acceptable cannula placement was a drinking latency of less than 3 min. The following day, MAP was recorded in the conscious rats while they were loosely restrained in a tethered rat jacket. After a stable blood pressure was recorded, AII was infused iv at successive doses of 10, 30, and 100 ng/min. Each dose was infused for 5-10 min or until a steady- state blood pressure was achieved. Blood pressure was allowed to return 60 to control levels after termination of iv AII infusion (approximately 10 min). For the purpose of ivt injection of AII, a Hamilton microsyringe was connected to the left lateral ventricular cannula by a long length of polyvinyl chloride tubing. The tubing and syringe were prefilled with a solution of A11 and a bolus ivt injection of AII (150 ng) was administered in a 5 pl volume over a 2-3 sec period. Changes in MAP in response to iv infusion and ivt injection of All were calculated as the difference of the peak steady-state MAP and the resting or pre-injection MAP. After this control set of pressor responses to peripheral and central administration of All was obtained, a prefilled osmotic minipump (Alzet Model 2001) was implanted subcutaneously under methohexital anesthesia and connected to the right lateral ventricular cannula by polyvinyl chloride tubing. The tubing and right ivt cannula were covered with dental acrylic, leaving the left cannula accessible. Two groups of rats were used in this experiment: one group received sar- thran, l pg/hr ivt (n=5), and a second group received sarthran, 6 pg/hr ivt (n=6). The vehicle for all infusions was isotonic saline. Intra- ventricular infusions were maintained for 5 days, and on the fifth day, pressor responses to iv and ivt AII administration were retested in the were then removed, and after a 2-day recovery period, pressor responses to iv and ivt AII were tested a third time. c. Chronic ivt teprotide 1) Dose determination: Sprague-Dawley. Male Sprague- Dawley rats were instrumented with chronic indwelling arterial and venous catheters and a lateral cerebral ventricular cannula. Changes in MAP in response to successive intravenous infusions of AI (3, 10, 30, 61 100 ng/min) were recorded in conscious rats. Each dose was infused for 5-10 min or until a steady-state arterial pressure was reached. After the iv pressor response curve was obtained, 10—15 min was allowed for recovery. A prefilled 30-gauge length of stainless steel tubing was then introduced into the ivt cannula so that its tip was at the end of the cannula. The 30-gauge steel tubing was connected to a Hamilton microsyringe by a long length of polyvinyl chloride tubing. The maximal pressor response to a 100 ng ivt bolus injection of AI (5 pl volume) was then measured. After this set of control repsonses was obtained, a prefilled osmotic minipump (Alzet Model 2001) was implanted subcuta- neously in the suprascapular region and connected to the 30-gauge steel tubing by a length of polyvinyl chloride tubing. This minipump delivers solution at a rate of l p1/hr. Minipumps were filled with solutions of teprotide in isotonic saline in varying concentrations to yield doses of 1 (n=4), 3 (n=3) and 10 pg/hr (n=5). The ivt teprotide infusion was maintained for 5 days during which time rats were housed individually with 22_112 access to food and water. The pressor responses to acute iv and ivt AI were retested after 5-day ivt infusion period according to the above protocol. 2) Dose determination: Spontaneously hypertensive rats. The protocol for this experiment was similar to that described for sarthran dose determination in SHR (Section 3.b.3.), except that pressor responses to iv AI (10, 30, 100 ng/min) and ivt AI (100 ng) were measured. These responses to peripheral and central AI administration were measured prior to, on the fifth day of, and 2 days after a 5-day ivt teprotide infusion (10 pg/hr). The pressor response to ivt AII (150 ng) also was determined 30 min after the ivt AI injection was given. 62 d. Chronic ivt sarthran/chronic iv angiotensin II Rats used in this experiment were surgically prepared with indwelling arterial and venous catheters and a lateral cerebral ventricular cannula and housed individually in metabolic cages. A minipump (Alzet Model 2002) containing sarthran (2 mg/ml) or isotonic saline was implanted subcutaneously and connected to the ivt cannula at the time of the initial surgery. These minipumps deliver solution at the rate of 0.5 pl/hr for a l4-day period, therefore sarthran was in- fused at a dose of l pg/hr ivt. Rats were maintained on a high sodium intake by continuous iv saline infusion (40 ml/day) and were given 12-15 g of low sodium rat chow per day. After three recovery days, daily measurements of MAP, HR, U0, and urinary sodium and potassium excretions were begun. The experimental period consisted of two control days, 5 days of continuous iv AII infusion, and finally two recovery days after the iv AII infusion was stopped. The ivt infusion of sarthran or saline was maintained throughout the entire experimental protocol (i.e., during the 3-day post-surgery recovery period, and the control, iv AII-infu- sion, and recovery days). Four groups of rats (n=8 in each group) were used in this experiment: two groups received iv AII infusions at a dose of 10 ng/min for 5 days and two groups received AII at 20 ng/min, iv. At each dose level of iv AII, one group of rats received sarthran 1 pg/hr ivt and the other group received isotonic saline ivt as a control. Correct ivt cannula placement was verified at the end of the experiment by injection of Evans Blue dye in the cannula and visualization of the dye in the ventricular system. 63 e. Chronic ivt sarthran/DOC-salt hypertension Right nephrectomy was performed in male Sprague-Dawley rats by a retroperitoneal approach under pentobarbital anesthesia 2 weeks prior to study. Rats were provided with 0.9% saline drinking fluid and standard rat chow 22_112_for the duration of the experiment. Two control blood pressure measurements were obtained (tail cuff ple- thysmography), after which a lateral cerebral ventricular cannula was implanted and an ivt infusion of saline (n=10) or sarthran (l pg/hr, n=6) was begun. Two days after the start of ivt infusion, DOC (deoxy- corticosterone, Percorten pivalate, CIBA) was administered at a dose of 50 mg/kg, sc. This dose of DOC was administered once weekly for the remainder of the study. Blood pressure and body weight were measured twice weekly for 4 weeks, starting on the fourth day after the first DOC injection. Spent minipumps (Alzet Model 2002) were replaced with freshly-filled pumps 14 days after they were implanted. After the final tail cuff blood pressure measurement, femoral arterial and venous cathe- ters were implanted under pentobarbital anesthesia. Two days were allowed for recovery from surgery, at which time MAP and HR were measured in the conscious rats while they were loosely restrained in a rat jacket. Changes in MAP and HR were determined at l and 5 min after the following interventions: 1) AVP antagonist, 10 pg/kg, iv; and 2) hexamethonium, 20 mg/kg, iv. f. Chronic ivt sarthran/spontaneously hypertensive rats SHR used in this experiment underwent an initial surgi- cal procedure for placement of a cannula in the right lateral cerebral ventricle. Rats were housed individually and had 22_112_access to 64 standard rat chow and distilled water. Blood pressure was measured indirectly by a tail cuff plethysmographic method in conscious, re- strained rats. Control blood pressure measurements were made on days 1 and 3 of the experiment. On day 6, an osmotic minipump (Alzet Model 2002) containing sarthran (2 mg/ml; n=7) or isotonic saline vehicle (n=9) was implanted subcutaneously and connected to the ivt cannula. Since this minipump delivers solution at the rate of 0.5 p1/hr, the dose of sarthran infused ivt was 1 pg/hr. This infusion was maintained for 14 days, and blood pressure was measured on the second, fourth, ninth, and eleventh days of ivt sarthran infusion. On the fourteenth day of ivt infusion, the spent minipumps were removed under ether anesthesia and replaced with a minipump (Alzet Model 2001) filled with sarthran (6 mg/ml) or isotonic saline. The ivt sarthran infusion was continued for another five days at a dose of 6 pg/hr. After minipump removal, 2 recovery blood pressure measurements were obtained. 9. Chronic ivt teprotide/spontaneously hypertensive rats SHR were prepared as described in the previous section for chronic ivt sarthran infusion. Blood pressure was measured by tail cuff plethysmography. Two control blood pressure measurements were obtained on days 1 and 4 of the experiment. On day 6, a prefilled osomotic minipump (Alzet Model 2001) containing teprotide (10 mg/ml, n=10) or isotonic saline vehicle (n=15) was implanted subcutaneously and connected to the ivt cannula. Blood pressure measurements were obtained on days 7, 9, 11, and 13 during ivt teprotide or saline infusion. Minipumps were removed on day 14 under ether anesthesia, and one re- covery blood pressure determination was obtained on day 18. 65 4. Brain lesion studies: chronic intravenous AII infusion a. Chronic intravenous AII infusion protocol Male Sprague-Dawley rats (300-400 g) were housed indivi- dually in metabolic cages after surgery for implantation of arterial and venous catheters. A sodium deficient rat chow was provided (12-15 g/day; 0.002 mEq Na, 0.3 mEq K per 9) so that sodium intake could be strictly controlled at 6.2 mEq/day by iv infusion of 40 m1 isotonic saline/24 hr. Distilled water was available 22_112_from calibrated drinking tubes. After three recovery days, the experimental protocol was begun. This consisted of 2 control days, five days during which All was infused continuously iv at a rate of 10 ng/min, then two post- infusion recovery days. Daily measurements of MAP, HR, WI, U0, UNaV’ and UKV were obtained. b. Subfornical organ lesion Three groups of rats were studied in the preceding chronic iv AII infusion protocol. The first group (n=7) received only saline during the 5-day "hormone infusion" period. The second group of rats (n=8) received All (10 ng/min) during the same 5-day infusion period. A third group (n=9) was subjected to electrolytic destruction of the SFO 2-4 weeks prior to a 5-day infusion of All (10 ng/min). Electrolytic ablation of the SFO was accomplished in the following manner. Rats were anesthetized with a pentobarbital-chloral hydrate mixture and immobilized in a Kopf stereotaxic instrument. A trephine hole was drilled in the skull dorsal to the lesion site and a 30-gauge Teflon-insulated monopolar tungsten electrode was lowered to the lesion site. Because the anterior stalk of the SFO is quite ventral to the posterior stalk, three electrode penetrations were required. A 66 total of 21 millicoulombs (1 mA for 21 sec) of anodal current was passed, with 7 m6 passed per penetration. All penetrations were made in the midline after the superior sagittal sinus had been retracted. The first penetration was to 5.0 mm ventral to the dura, and 0.3 mm pos- terior to bregma. Each successive penetration was made to a point 0.3 mm posterior and 0.2 mm dorsal to the preceding one. Following surgery, each rat received 100,000 IU of procaine penicillin (im) and was re- turned to its home cage. Lesions were produced in 22 rats by Dr. Michael Mangia- pane, University of Rochester, following which the animals were sent to our laboratory for AII infusion. At the end of the infusion protocol, the brain of each rat was perfused with buffered formalin, coded, and returned to Dr. Mangiapane for a blind histological analysis of lesion size and location. Frozen sections (35 microns) were cut through the lesion site and stained with cresyl violet. The sections were then examined carefully in a light microsc0pe to determine the location of the lesion, and the examiner had no knowledge of the data for any parti- cular animal. Only after confirmation that the lesion destroyed greater than 80% of the SFO was a given rat included in the "SFO lesion" group. Sixteen rats completed the AII infusion protocol, and nine of these had greater than 80% destruction of the SFO. These nine were included in the analysis. None of these had damage to the median preoptic nucleus. As is typical of SFO lesions (Mangiapane 22 21,, 1984), all sustained a minor degree of damage to the fornical commissure, triangular septal nucleus, and paraventricular nucleus of the thalamus. 67 b. Knife cut of SFO efferents A knife cut of SFO efferents was performed in rats by Dr. R.W. Lind according to a previously published method (Lind and Johnson, 1982). Briefly, under ether anesthesia and using stereotaxic guidance, a knife cut of SFO efferents was made with a rotating Knigge wire knife. In sham knife-cut rats, the knife was lowered into the brain, but the blade of the knife was not extruded. Rats were then shipped to our laboratory for performance of the chronic iv AII infusion protocol (10 ng/min, iv for 5 days, see Section 4.a.). On the first control day, MAP and HR changes were determined to acute iv AII infusion (10, 30, and 100 ng/min). After the chronic iv AII infusion protocol was completed, the brain of each rat was perfused with buffered formalin, coded, and returned to Dr. Lind for blind histological analysis. Brains were embedded in an albumin/gelatin matrix, frozen, and.40 micron sagittal sections were cut. Cresyl violet-stained sections were examined light microscopically to determine position of the knife cut. Five rats completed the iv AII infusion protocol and were found to have knife cuts immediately ventral to the SFO; these rats comprised the "knife cut" group. As described above, sham rats (n=5) underwent surgery but the knife blade was not rotated. c. Median preoptic nucleus lesion Chronic iv AII infusion was performed according to the above protocol (Section 4.a.) in two groups of rats: 1) a sham group (n=12), and 2) a gropu which had undergone electrolytic destruction of 68 the median preoptic nucleus (MnPO) approximately three weeks prior to study (n=5). Electrolytic destruction of the MnPO was performed by Dr. Michael Mangiapane, University of Rochester, in the following manner. Rats were anesthetized with a pentobarbital-chloral hydrate mixture and immobilized in a Kopf stereotaxic apparatus. The skull was leveled and a hole was drilled in the skull 0.1 mm posterior to bregma. After retraction of the superior sagittal sinus, a monopolar Teflon-coated tungsten electrode (225 micron diameter) was lowered 6.7 mm ventral to the dura on the midline. The MnPO was lesioned by passing 1 mA of anodal current for 15 sec. Following surgery, each rat received 100,000 IU of procaine penicillin (im) and was returned to its home cage. Rats were then shipped to our laboratory for iv AII infusion. After the iv AII infusion protocol was completed, the brain of each rat was perfused with 10% formalin, coded, and returned to Dr. Mangiapane for a blind histological analysis of lesion size and location. Lesion extent was determined by light microscopic examination of cresyl violet-stained sections through the MnPO. The criterion for inclusion of a rat in the “MnPO lesion" group was greater than 90% destruction of the MnPO (ven- tral to the anterior commissure) with no OVLT damage. Five rats met this criterion and thus were included in the MnPO lesion group. The sham group (n=12) consisted of rats which underwent lesion surgery but no damage to the MnPO or surrounding structures was observed upon histo- logical examination, and rats that had no sham surgery. RESULTS A. Chronic ivt AII Infusion: Rat 1. Sodium dependency_ In rats maintained on a high sodium intake with free access to drinking water, ivt infusion of A11 for 5 days resulted in dose-depen- dent increases in MAP (Figure l). Elevations in MAP were not asso- ciated with any consistent changes in HR. Rats receiving the lower dose of AII (l pg/hr) exhibited 3 days of natriuresis, whereas this effect was not seen in rats receiving the higher dose of AII (6 pg/hr). Al- though WI increased significantly in rats receiving the low dose of AII, and tended to increase in rats receiving the high dose of AII, water balance did not change significantly over the course of the experiment. Similar changes in MAP in response to ivt AII infusion were observed in rats whose fluid intake was restricted to the 40 ml/day provided by the iv infusion (Figure 2). Again, there were no consistent changes in HR or UNaV' Urine output remained constant except on the fifth day of ivt infusion, when rats were allowed to drink. The volume of water ingested over 24 hr was significantly greater in rats receiving AII 6 pg/hr ivt than in saline controls (p<0.05, rank sum test). The effect of sodium intake on cardiovascular and fluid and electrolyte responses to chronic ivt AII are depicted in Figure 3 (l pg/hr) and Figure 4 (6 pg/hr). The magnitude of the ivt AII-induced 69 70 Figure 1. Effect of chronic ivt AII infusion on cardiovascular and fluid/electrolyte parameters in the rat. MAP = mean arterial pressure, HR = heart rate, UNA = urinary sodium excretion, WI = water intake, WB = water balance. Points are mean responses in groups of 6 rats main- tained on 7.5 mEq Na/day. Cl and C2 are 2 control days, Al-A5 are 5 days of ivt infusion (shaded area), R1 and R2 are recovery days. Vertical bars on C2 value represent standard errors for within groups comparisons. Asterisk (*) represents significant (p<0.05) difference from the average of Cl and C2 values (randomized block ANOVA and lsd). 71 <>8ALHUE OATH UG/HR 170 150 MAP (MM we) 13° 11o HR (BEATS/MIN) 300 UNA (MEG/DAY) W8 (ML/DAY) . .., TIME (DAYS) Figure l 72 Figure 2. Chronic ivt AII infusion in rats on restricted fluid intake. Rats were maintained on 7.5 mEq Na/day and allowed access to drinking water only during the final 24 hr of ivt infusion. Vertical bars represent average SEM for within groups comparisons. Units of the abcissa are days. The volume of water ingested during the final 24 hr of ivt infusion was as follows: AII 6 ug/hr ivt: 92 :_11 m1, AII l ug/hr ivt: 79 1.30 ml, saline ivt: 44 :_13 ml. Asterisk (*) represents significant (p 0.05) difference from the average of Cl and C2 values (randomized block ANOVA and lsd). Other symbols as in Figure l. 73 MAP (MM HG) HR , (ants/um) IJlmA (NEG/DAY) UO (ml/do!) unaware) I ”Langmuir-5) I: Salmon-1) 0 All. 1 MIG-5) Figure 2 3.4 o 74 Figure 3. Cardiovascular and fluid/electrolyte responses to chronic ivt AII infusion (l pg/hr) in rats maintained on normal and high sodium intake. Values represent mean responses in groups of 6 rats. Vertical bars on C2 point represent SEM for individual within groups comparisons. Asterisk (*) represents significant (p<0.05) difference from average of C1 and C2 values (mixed design ANOVA and lsd). Other symbols as in Figure 1. 75 170 150 MAP (MM HG) 13° 110 w~ ‘9 "LIE" 3591?" I; I 1 I A1 A2 A3 A4 A5 R1 R2 Ci c2) 3 HR (BEATS/MIN) LflflA (MEG/DAY) W1 (ML/DAY) we (ML/DAY) Figure 3 76 Figure 4. Cardiovascular and fluid/electrolyte responses to chronic ivt AII infusion (6 pg/hr) in rats maintained on normal and high sodium intake. Points represent mean responses in groups of 5 (normal sodium) and 6 (high sodium) rats. Units of the abscissa are days. Vertical bars on C2 value represent SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from average of Cl and C2 values. Cross (+) indicates significant difference between high sodium and normal sodium groups (mixed design ANOVA and lsd). Other symbols as in Figure l. 77 oNormal NI » o _I-Ilgh Na MAP (MMHm i u MEG NNDAY s s a a.-...... WI (ML/DAY) Figure 4 78 increase in arterial pressure was significantly greater in high sodium rats than in normal sodium rats, regardless fo the dose of AII in- fused. This difference was especially pronounced in rats receiving the 6 pg/hr dose of AII (Figure 4). Water balance remained unchanged and a tendency for sodium loss was observed during ivt AII infusion. The dipsogenic response to ivt infusion of AII at 6 pg/hr was identical to ivt AII infusion at the lower dose (1 pg/hr), WI was increased in high sodium rats and slightly but significantly decreased in normal sodium rats (Figure 3). 2. Plasma hormone leve1§_ The MAP and HR response to 5-day ivt infusion of AII (6 pg/hr, n=6) or isotonic saline (n=7) in rats maintained on a high sodium in- take with free access to drinking water are shown in Figure 5. Ar- terial blood samples were obtained on the second control day, the fifth day of ivt infusion and the second recovery day for determination of plasma aldosterone and catecholamine levels, Na and K concentrations, osmolality, and hematocrit. Plasma Na and K concentrations and osmo- lality were not affected by chronic ivt AII infusion (Figure 6). Hema- tocrit and body weight decreased in both AII and saline-treated rats, probably due in part to the repeated blood sampling. Plasma concentra- tions of norepinephrine and epinephrine on the second control day, fifth day of ivt infusion and second recovery day are reported in Table 1. There were no significant changes in plasma norepinephrine or epine- phrine in rats receiving either ivt saline or AII when compared to con- trol levels. Plasma aldosterone was significantly elevated on the fifth day of ivt AII infusion when compared to control levels (Table 2). 79 Figure 5. Cardiovascular response to chronic ivt AII infusion in rats used for plasma hormone determination. Rats received either AII, 6 pg/hr ivt (n=6) or saline ivt (n=7) and were maintained on high sodium intake. Vertical bars on C1 value indicate SEM for individual within groups comparisons. Asterisk (*) represents significant (p<0.05) difference from average of Cl and C2 values and cross (+) represents significant difference between AII- and saline-treated rats (mixed design ANOVA and lsd). Other symbols as in Figure 1. MAP (MM HG) HR (BPM) 180‘ 160 140 120 100 80- 500 300 200. 80 O SALINE IVT D AIIVT TIME (DAYS) Figure 5 81 Figure 6. Effect of chronic ivt AII infusion on plasma electrolytes, osmolality, hematocrit and body weight. PNa = plasma sodium concen- tration, PK = potassium concentration, P0 M = plasma osmolality, HCT = hematocrit, B.WT. = body weight. This da a was obtained from rats whose MAP and HR responses to ivt AII (n=6) and saline (n=7) are depicted in Figure 5. C2 is the second control day, A5 is the fifth day of ivt infusion and R2 is the second recovery day. Vertical bars on C2 value represent SEM for within groups comparisons. Asterisk (*) denotes significant (p<0.05) difference from C2 value and cross (+) indicates significant difference between saline and AII rats (mixed design ANOVA and lsd). 82 O SALINE NT (:1 A11 IVT 140 PNA . 130 (MEG/L) 120 . 5 PK (MEG/L) POSM (MOSMHJ HCT (%) TIME (DAYS) Figure 6 83 TABLE 1 Plasma Catecholamine Levels Before, During and After ivt Angiotensin II Infusion C2 A5 R2 Plasma Norepinephrine (pg/m1) AII ivt 77:94 101 86 saline ivt 86:60 164 117 Plasma Epinephrine (pg/m1) AII ivt 42:91 92 40 saline ivt 45:84 84 192 Values are mean + SEM (for within group comparisons). Rats received either AII (6 pg/hr ivt; n=6) or saline ivt (n=7) for 5 days. Plasma was sampled for catecholamine determinations on the second control day (C2), fifth day of ivt infusion (A5) and second recovery day (R2). no significant difference in plasma norepinephrine or epi- nephrine levels on the fifth day of ivt AII or saline infu- sion when compared to control levels. There was 84 TABLE 2 Plasma Aldosterone Levels Before, During, and After ivt Angiotensin II Infusion C2 A5 R2 Plasma Aldosterone (ng /dl) AII ivt 17.7129.o 137.8* 49.1 saline ivt 24.6:_9.1 21.8 34.2 Values are mean :_SEM (within groups). Rats received either A11 6 pg/hr ivt (n=6) or saline ivt (n=7) for 5 days. Plasma was sampled for aldosterone determinations on the second control day (C2), fifth day of ivt infusion (A5) and second recovery day (R2). Asterisk (*) indicates a signifi- cant difference from C2 value (p<0.05). 85 Within 2 days after termination of ivt AII infusion, plasma aldosterone returned to pre-infusion levels. Plasma aldosterone did not change in response to ivt infusion of isotonic saline. Plasma AII levels were measured on the second control day and fifth day of ivt AII infusion in a separate group of 12 rats (Figure 7). The rats were divided into two groups depending on their blood pressure response to the ivt AII infu- sion. Rats in the hypertensive group (average MAP = 151 mmHg; n=7) were found to have ventricular cannulae located in the lateral ventricu- lar space upon post-mortem exam, and thus were receiving the AII infu- sion directly into cerebrospinal fluid. Normotensive rats (average MAP = 123 mmHg; n=5) had ventricular cannulae tips located in periventri- cular brain tissue. Therefore, this group of rats did not become hyper- tensive in response to AII infusion. Regardless of whether AII was in- fused directly into cerebrospinal fluid or into periventricular brain tissue, plasma AII levels were not significantly elevated on the fifth day of All infusion when compared to control levels. 3. Adrenalectomy_ The effect of adrenalectomy on the response to chronic ivt AII infusion is depicted in Figure 8. The increase in blood pressure seen in response to a 7-day ivt AII infusion was the same in adrenalecto- mized and intact rats. Blood pressure was still elevated two days after termination of ivt AII infusion in both groups, but returned to control levels by the fourth day after ivt AII was stopped. Saline intake in- creased in both groups during the period of ivt AII infusion. Body weight was stable in intact rats over the course of the experiment whereas body weight fell in adrenalectomized rats. Plasma aldosterone was undetectable in adrenalectomized rats, and there was no elevation 86 Figure 7. Plasma AII concentration in rats receiving chronic ivt AII infusion. C2 = second control day; A5 = fifth day of ivt A11 (6 pg/hr) infusion. The hypertensive group (n=7) had ivt cannulae loca- ted in the ventricular space and the normotensive group (n=5) had cannulae located outside the ventricle. Numbers below data points are means :_SEM. Mixed design ANOVA followed by lsd was performed on the log transform of the data. There were no significant (p<0.05) differ- ences between plasma AII concentrations measured on C2 and A5 in either group. PLASMA AII (Pohnn §§§§ 338§ a as a §:§ 8888 10 87 t ) 195.9 1 33.4 88.5 112.6 __ $54.7 $63.2 $25.3 :41.1 I J l 1 HYPERTENSIVE NORMOTENSIVE Figure 7 88 Figure 8. Effect of adrenalectomy on the hypertensive response to chronic ivt AII infusion. Adrenalectomized (ADX, n=7) and control (n=4) rats received ivt infusions of AII (6 pg/hr) for the 7-day period denoted by shading. Asterisk (*) represents significant (p<0.05) difference from average of all pre-infusion values (mixed design ANOVA and lsd). 89 220 200 ThHIJunr 180’ P2316. 1 50 (mm Hg) 1 4O 1 20 1 00 1 50 35.55555? ' '":95=3=53"2‘“"‘7‘7'7“'i"-‘="':3* " Sella. 100 “ ‘i' .. ,.. 3 Intake o 375 Body 350 “Mom 325 (gm) 300 275 13 5 7 91113151719 TIMEIDAYS) Figure 8 90 in plasma aldosterone in intact rats on the final day of ivt AII infu- sion when compared to levels measured one week after the end of ivt infusion. 4. Acute blockade of vascular AII receptors and2gangjionic blockade Cardiovascular and fluid/electrolyte responses to a 5-day ivt infusion of A11 are depicted in Figure 9. A sustained increase in MAP was observed throughout the ivt infusion period. Blood pressure re- turned to control levels within 24 hr after the infusion was stopped. Heart rate was not altered by ivt AII infusion, although a significant tachycardia occurred on the 2 post—infusion recovery days. Urinary sodium and potassium excretions were not consistently changed by ivt AII infusion. Since sodium intake was held at a fixed level (6.2 mEq/day), sodium balance (assuming constant fecal losses) did not change in response to ivt infusion of AII. Water intake slowly increased during the period of ivt AII infusion, reaching a statistically signi- ficant elevation on day 3 of the infusion. Water intake dropped to control levels on the first day after stopping the ivt infusion. Water balance remained unchanged throughout the experiment. The response of the group of rats shown in Figure 9 to block- ade of the direct vasoconstrictor effects of AII is depicted in Figure 10. During the control period, acute iv infusion of saralasin (300 ng/min for 10 min) produced a small increase in MAP and a decrease in HR. When saralasin was administered on the first, third, and fifth days of ivt AII—induced hypertension, a moderate decrease in MAP occurred, associated with tachycardia. The decreases in MAP and increases in HR produced by iv saralasin during the ivt AII infusion period were 91 Figure 9. Cardiovascular and fluid/electrolyte response to chronic ivt AII in rats that were subject to acute saralasin and hexamethonium treatment. Rats were maintained on a high sodium intake and received AII, 6pg/hr ivt for 5 days (n= 6). MAP = mean arterial pressure, HR = heart rate. Units of the abscissa are days. Cl and C2 are control days, Al—A5 are 5 days of ivt infusion, R1 and R2 are recovery days. Vertical bars on C2 value represent SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from average of C1 and C2 values (randomized block ANOVA and lsd). 170 150 MAP (MM HG) ‘30 110 o" 90, 500 HR (BEATS/MIN) 300 o c..o.---nu-...un on .----.- 556 NA EXERETION --rv:a. ..,., WATER-MAKE ‘IOOb Figure 9 93 Figure 10. Change in mean arterial pressure and heart rate in re- sponse to acute iv saralasin infusion before, during, and after chronic ivt AII infusion. C2 is the second control day; Al, A3, and A5 are the first, third and fifth days of ivt AII infusion; R2 is the second recovery day. Saralasin was infused at the rate of 300 ng/min for 10 min. Data was obtained from rats (n=6) whose cardiovascular and fluid/electrolyte response to chronic ivt AII is depicted in Figure 9. Vertical lines on C2 value represent SEM for individual within groups comparisons. Asterisk (*) indicates significant differ- ence from C2 value (randomized block ANOVA and lsd). 94 CHANGE IN MAP (MM HG) ‘TIIIIIIT I'UUIIIIT CHANGE IN HR (BEATS/MIN) p I I- I- n- p I- D - b l- I- p p- 1- b P Figure l0 95 significantly different from the response to saralasin observed in the control period. By the second recovery day, the MAP response to iv saralasin was not significantly different from the control response. Acute ganglionic blockade with hexamethonium produced a decrease in MAP and HR during the control period (Figure ll). Hexamethonium admini- stration on the first, third, and fifth days of ivt AII infusion re- sulted in a greater depressor response than that observed during the control period, but this difference was not statistically significant. 5. Acute blockade of vascular AVP receptors The change in MAP and HR in response to acute iv administra- tion of the vascular AVP receptor antagonist (l-(B-mercapto-B,B cyclo- pentamethylene propionic acid), 2-(o-methyl)tyrosine) arginine-8-vaso- pressin, l0 ug/kg) is depicted in Figure 12. Blockade of the vasocon- strictor actions of blood-borne AVP on the second control day did not effect MAP or HR. Similarly, administration of AVP antagonist on the first, third or fifth day of ivt AII infusion or the second post- infusion recovery day resulted in no change in MAP or HR. Shown in Figure 13 is the effect of AVP antagonist (l0 ug/kg, iv) on the MAP and HR responses to exogenous AVP infusion. At 2 hours after administration of the AVP antagonist, exogenous AVP infusion produced negligible changes in MAP and HR when compared to control responses (blocked fac- torial ANOVA followed by lsd). 6. Peripheral sympathectomy_ The role of the peripheral sympathetic nervous system in the hypertension produced by a 7-day ivt AII infusion was assessed in rats which had undergone neonatal guanethidine treatment plus adrenal 96 Figure ll. Change in mean arterial pressure and heart rate in response to hexamethonium before, during, and after chronic ivt AII infusion. Hexamethonium was administered as a bolus (20 mg/kg, iv). Data was obtained from rats (n=6) whose cardiovascular and fluid/ electrolyte response to chronic ivt AII is depicted in Figure 9. Vertical lines on C2 value represent SEM for individual within groups comparisons (mixed design ANOVA and lsd). Other symbols as in Figure 10. 97 2C) o CHANGE IN MAP '20 (MM HG) -40 ~60 -8C) 8C) 40 CHANGE IN HR (BEATS/MIN) ° .5 0 IT "'l'llllllllllllll I'll (1 no 1> A J> (a J> (A II he Figure ll 98 Figure l2. Change in mean arterial pressure and heart rate in re- sponse to AVP antagonist before, during, and after chronic ivt AII infusion. The top panel depicts mean arterial pressure (MAP) in response to a 5-day ivt AII infusion (6 ug/hr) in high sodium rats (n=5). The middle and bottom panels depict the change in MAP and heart rate (HR) to acute administration of the AVP antagonist (l0 ug/kg, iv). Vertical lines on C2 value represent SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from average of Cl and C2 values (MAP) or from C2 value (AMAP and AHR). Data was analyzed by randomized block ANOVA followed by lsd. 180 1 60 MAP 140 (mmHg) 1 20 100 +5 A MAP (mmHg) -5 +20 A HR (barn) -20 V I xvufi ..411 ...--n ... . c~o o. TTT I'UI 'UUVV TIM E (DAYS) Figure 12 100 Figure 13. Efficacy of AVP antagonist in blocking responses to exogenous AVP infusion. Change in mean arterial pressure (AMAP) and heart rate (AHR) in response to lO-min iv AVP infusions (0.3, 1.0, 3.0 mU/min) was determined before, and 5 min after AVP antagonist admini- stration (n=5). Crosses (+) represent significant (p<0.05) difference between pre- and post-antagonist values (mixed design ANOVA and lsd). 101 +50 " HPRE-ANTAGONIST +40 __ (::-50)POST-ANTAGONIST A MAP +30 mmH ' ( 9) +20 +10 A HR -150 - ~200 AVP(mU/min) Figure 13 102 demedullation at adulthood. Chronic ivt AII infusion produced similar elevations in blood pressure in sympathectomized and normal rats (Figure 14). The onset of hypertension was significantly delayed in the sympathectomized group. On the second day of ivt AII infusion, the sympathectomized group was normotensive while normal rats exhibited a significant elevation in blood pressure at that time. By the fourth day of ivt infusion, both groups were hypertensive. The offset of the hypertensive response to chronic ivt AII also was significantly delayed in sympathectomized rats. 7. Verification of degree of sympathectomy The effect of sequential ganglionic blockade and alpha—adrener- gic blockade on MAP and HR of sympathectomized and normal rats is shown in Figure 15. The drop in MAP at l and 5 min after hexamethonium ad- ministration was significantly less in sympathectomized rats than in normal rats. Subsequent administration of phentolamine resulted in no further depressor response in either group of rats. The overall de- pressor response to combined ganglionic and alpha-adrenoceptor blockade was less in sympathectomized rats than control rats. Changes in heart rate in response to these interventions did not differ in the two groups. Increases in MAP elicited by electrical stimulation of sympa— thetic vasomotor outflow in the pithed rat preparation are shown in Figure 16. Normal rats exhibited graded increases in MAP in response to spinal cord stimulation over the range of stimulation frequencies (l-10 Hz). Sympathectomized rats exhibited pressor responses that were significantly less than those seen in normal rats at all stimulation 103 Figure 14. Effect of peripheral sympathectomy on hypertensive re- sponse to chronic ivt AII infusion. Sympathectomized (SYMX, n=5) and control (n=6) rats were given ivt infusions of AII (6 ug/hr) for 7 days (shaded area). Vertical bars on first data point represent SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from average of the three pre-infusion values. Cross (+) indicates significant difference between sympa- thectomized and control rats (mixed design ANOVA and lsd). Tall Cuff Blood Pressure (mini-lg) BOdY Weight (gm) 220 200 1 80 1 60 140 120 1 00 325 275 250 225 104 o-- smx (n = 5 ; ‘- CONTROL ------ ------ ..... ...... . . ~ ..... 1 11 13 15 17 19 TI M E (DAYS) Figure 14 105 Figure 15. Blood pressure and heart rate response to hexamethonium and phentolamine in normal and sympathectomized rats. Changes in mean arterial pressure (AMAP) and heart rate (AHR) were measured at 1 and 5 min after sequential administration of hexamethonium (20 mg/kg, iv) and phentolamine (2 mg/kg, iv) in normal (n=5) and sympathectomized (SYMX, n=8) rats. Points represent means :_SEM. Differences between normal and sympathectomized rats were tested at each time point using Student's t-test. Cross (+) indicates significant (p<0.05) difference between normal and sympathectomized rats. 106 HEXAMETHONIUM PHENTOLAMINE 1 mm 5 min 1 min 5 min f r T O INTACT (n=5) OSYMX (n=8) A MAP ‘\*~~~ (mmHg) A HR 1139'") Figure 15 107 Figure 16. Change in mean arterial pressure in response to elec- trical stimulation of sympathetic vasomotor outflow in normal and sympathectomized pithed rats. Points represent average changes in mean arterial pressure (AMAP) in intact (n=5) and sympathectomized (SYMX, n=8) rats. Vertical bars represent SEM for individual between groups pairwise comparisons. Cross (+) indicates si nificant (p<0.05) difference between normal and sympathectomized rats 1mixed design ANOVA and lsd). AMAP (MMHO) 20 10 107b " C INTACT (n=5) 1— O SYMX (n=9) L 1 l l l I l l L 2 3 4 5 6 7 8 9 1O Stimulation frequency (Hz) Figure 16 108 frequencies. The pressor response to the 10 Hz stimulation was com- pletely abolished by hexamethonium (20 mg/kg, iv) in all rats, indicat- ing that pressor responses were neurogenically mediated. Tissue norepinephrine and dopamine content from normal and sympathectomized rats is reported in Table 3. Norepinephrine content was significantly lower in the pineal and higher in the cerebellum of sympathectomized rats than control rats. Dopamine content of hypothala- mus was lower in sympathectomized rats than in normal rats. 8. Pharmacologic assessment of sympathetic tone Resting MAP of rats used in these experiments is presented in Table 4. The depressor responses to phentolamine, propranolol plus phentolamine, hexamethonium, and nitroprusside in rats receiving chronic ivt AII infusions are depicted in Figure 17. In control rats (saline ivt, n=5), phentolamine (2 mg/kg, iv) produced an average fall in MAP of 47:6 mmHg. The combination of propranolol (1 mg/kg, iv) followed by phentolamine produced a depressor response of 19 mmHg, which was signi— ficantly different than the response to phentolamine alone. Hexametho- nium (20 mg/kg, iv) decreased MAP by 30 mmHg, a value not different from the response to phentolamine alone. The depressor responses to all of these interventions were significantly greater in rats receiving AII ivt (n=7), averaging 64:] mmHg for phentolamine alone, 43 mmHg for pro- pranolol plus phentolamine, and 66 mmHg for hexamethonium. In the rats that received AII ivt, the depressor response to propranolol plus phen- tolamine was significantly less than the response to phentolamine alone or hexamethonium. 109 .Apmmpnp m.pcovzpmv mum; vawEouomzomaExm ncm Passe: :mogpmn wucmgmwwwv ucmovwwcmwm mmumowvcw Akv xmwgmpm< .Amn: "mum; vaPEopuwcamaexm .mu: "mum; _msgocv 2mm + some ucmmwcamg mm:_m> em Qm_m._ mm.QM¢o.o .mm.gm_u.~ mo.gmmm.o eo.ngN.o emNPEopuagpmasxm Ak.m+mm.© eo.o+Nm.o mm.o+en.m P_.o+mfi.o Np.o+_e.o _aseoz Acwmpoga ms\mcv chEmaoo me.anm.m .mm.nmNN.A _m.mmwo.m .¢¢.Qmw_._ ¢A.mmoo.m emuweoouagpagsxm mm._+oe.m AA.o+¢m.m mm.m+m¢._m Am.o+eo.m an._+mo.e Pastoz A:_ouoga me\mcv mcwccawcwawgoz xmpgou Fences; E:_anmLou m35mpmcpoax: Fmocwm xwpgou chmm mpmm uonEouomcpmaezm ucm —mELoz cw pcmpcoo mewEmaoo ucm wcwgcamcwamgoz mammwh m m4m<~ 110 TABLE 4 Resting Mean Arterial Pressure Before Various Vasodepressor Interventions Resting MAP (mmHg) Intervention Saline ivt (n=5) AII ivt (n=7) Phentolamine 111:6 115:5* Propranolol plus Phentolamine 111 148* Hexamethonium 116 144* Values represent mean :_SEM for within groups compari— sons. Asterisk (*) indicates significant difference from saline ivt value (mixed design ANOVA and lsd). 111 Figure 17. Pharmacologic assessment of sympathetic tone in rats receiving chronic ivt infusions of AII. Change in mean arterial pressure (AMAP) was measured at 5 min after each intervention (see text for details of experimental protocol). PHENT = phentolamine, PROP = propranolol, HEX = hexamethonium, NITRO = nitroprusside. Vertical lines on each pair of bars represent SEM for individual between groups comparisons. Asterisk (*) indicates significant (p<0.05) difference between A11- and saline-infused rats (mixed design ANOVA and Tukey's test). Nitroprusside data was analyzed using a paired t-test. 112 PROP/ PHENT HEX NITROT -1o - 1’ - .20- I o. ‘61 '30 b l < I '40 - ' .. -60 - -'- -7O - - DControl _ : g*.1 hi4 l_*.J : I Day 5 '1‘” A" IAllivt(n==7) (n=4) Ci Saline ivt (n = 5) Figure 17 113 In a separate group of rats (n=4), the decrease in MAP pro- duced by a 5 min iv nitroprusside infusion (12 ug/min) was not signifi- cantly different before, and on the fifth day of ivt AII infusion (39:4 :5, 47 mmHg, paired t-test). Resting MAP in these rats before nitro- prusside infusion was 118:6 mmHg on the control day and 155 mmHg on the fifth day of ivt AII infusion (p<0.05, paired t-test). 9. Interactions of AVP, sympathetic nervous system and peripheral renin-angiotensin system The effect of sequential administration of AVP antagonist (10 ug/kg, iv), saralasin (10 ug/min, iv for 15 min) and phentolamine (2 mg/kg, iv) on MAP and HR in normal rats receiving chronic ivt saline (n=5) or AII infusions (n=5) is shown in Figure 18. On the fifth day of ivt infusion, MAP was significantly greater in rats receiving AII (6 ug/hr) than in saline controls (165 ys, 114 mmHg, respectively). Blood pressure was not significantly different at l and 5 min after admini- stration of the AVP antagonist in either group of rats. Heart rate was significantly increased by this intervention in the A11 group but not in the saline group. Subsequent intravenous infusion of saralasin (lO ug/min) for 15 min caused no change in MAP in the ivt-saline control rats, but caused a significant fall in MAP in rats receiving AII ivt. After the combined treatment with AVP antagonist and saralasin, MAP was still significantly higher in All rats when compared to saline rats. Phentolamine administration (2 mg/kg, iv) produced a significant fall in blood pressure in both groups of rats. After the administration of phentolamine, MAP was not significantly different between the ivt saline and ivt AII groups of rats. 114 Figure 18. Effect of sequential administration of AVP antagonist, saralasin, and phentolamine on blood pressure and heart rate in normal rats receivin chronic ivt infusions of AII. Rats received either All (6 ug/hr, n=5? or saline (n=5) ivt and were tested on the fifth day of infusion (see text for complete description of protocol). CON = control; numbers along abscissa indicate time in minutes after each intervention (denoted by arrows). Vertical bars represent SEM for individual within groups comparisons. Asterisk (*) denotes signifi- cant (p<0.05) difference from control value. Cross (+) denotes signi- ficant difference between AII and saline rats (mixed design ANOVA and sd . 115 180 E:— e»llulvt(n==5) 150- o Saline ivt(n=5) i- 4. . 140 '- t + MAP 120 - + + + 100 - 80" * 60 \0 l L 1 1 L 1 i-- .L 550 o In.‘ ‘4 500 r- 113 * mp") 450 - 400 l 1 +1 J L cont 1’ 5' 1 5' 1d 15' 1 1' 5' AVP saralasin phentolamine antag. Figure 18 116 Figure 19 shows the MAP and HR responses to 15 min iv sarala- sin infusion followed by AVP antagonist injection on the sixth day of ivt saline or AII infusion. Saralasin infusion produced a significant depressor response in rats receiving ivt AII, but not in saline control rats. Subsequent injection of AVP antagonist produced no further change in pressure in either group of rats. Mean blood pressure and heart rate responses to sequential administration of AVP antagonist and saralasin in sympathectomized rats on the fifth day of ivt saline or AII infusion is depicted in Figure 20. Resting MAP was significantly higher in sympathectomized rats given ivt AII infusions than in saline-treated sympathectomized rats (154 gs, 121 mmHg, respectively). Injection of the AVP antagonist did not alter blood pressure in either group of rats. Intravenous saralasin infusion, after AVP antagonist administration, produced a significant fall in pressure in the sympathectomized-ivt AII group, but not in the sympa- thectomized-ivt saline group. MAP was not significantly different in AII- and saline-treated sympathectomized rats after combined treatment with AVP antagonist and saralasin. The MAP responses of individual sympathectomized rats given chronic ivt AII infusion (n=8) to sequential administration of AVP antagonist and saralasin are given in Table 5. As can be seen from this table, there was considerable heterogeneity in the response to AVP antagonist plus saralasin in this group of rats. Notably, 3 rats (#2, 4, and 6) experienced large depressor responses to the combination of AVP antagonist and saralasin, while MAP in one rat (#3) remained unchanged in response to these interventions. The other rats exhibited intermediate responses. 117 Figure 19. Effect of sequential administration of saralasin and AVP antagonist on blood pressure and heart rate in normal rats receiving chronic ivt infusions of AII. Rats received either AII (6 ug/hr, n=4) or saline (n=5) ivt and were tested on the sixth day of ivt infusion (see text for complete description of protocol). CON = control; numbers along abscissa denote time in minutes after each intervention (indicated by arrows). Vertical bars represent SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from control value. Cross (+) indicates significant differ- ence between AII and saline rats (mixed design ANOVA and lsd). 180 180 140 MAP (mm H9) 120 100 550 HR 500 bm (P1450 118 _ oAlllvt(n=4) n=5 L. \— J; *oSalitLelvH“ ) P" W L- , b + 1' 1’ + + + r °‘-—°---°---°---°---° 1- 1 L 1 _L l L + " 5- 1 ‘~ -"’9;"-"Q l CON? 5' 10’ is'f 1’ saralasin AVP antag. Figure 19 119 Figure 20. Effect of sequential administration of AVP antagonist and saralasin on blood pressure and heart rate in sympathectomized rats receiving chronic ivt infusions of AII. Sympathectomized rats re- ceived either AII (6 ug/hr, n=8) or saline (n=5) ivt and were tested on the fifth day of ivt infusion (see text for complete description of protocol). CON = control; numbers along abscissa denote time in minutes after each intervention (indicated by arrows). Vertical bars represent SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from control value. Cross (+) indicates significant difference between AII and saline rats (mixed design ANOVA and lsd). 120 SYM PATH ECTOMIZED RATS 180 _ ' All ivt (n = 8) 160 _ o Saline ivt (n = 5) MAP I ” __ + * (mm H9) 120 - T- —o-----o---—--o----’ 100 '- 80 '- 60 " 1 1 I 1 1 l 550 ’3‘“ * H R. 500 ~ * x’ “.0 I (BPM) 450 .. ’;’/’°’ * * 400' I T ' ‘ CONT ‘1' 5' T 5' 10' 15' AVP saralasin Figure 20 121 TABLE 5 Effect of Sequential Administration of AVP Antagonist and Saralasin on Blood Pressure in Sympathectomized Rats Receiving Chronic ivt Infusions of AII Mean Arterial Pressure (mmHg) Rat Resting AVP Antagonist Saralasin 1 min 5 min 5 min 10 min 15 min 1 122 132 131 128 128 126 2 219 177 207 124 121 124 3 178 178 170 179 171 171 4 111 125 119 76 76 8O 5 167 146 145 150 148 146 6 156 143 144 109 105 104 7 132 125 131 115 111 110 8 146 143 138 125 120 125 Values represent mean arterial pressure (MAP). "Resting" denotes basal MAP before any intervention. MAP was measured at 1 and 5 min after administration of AVP antagonist (10 ug/ kg, iv) and at 5, 10, and 15 min after subsequent iv saralasin infusion (10 ug/min). 122 10. Spinal cord stimulation Figure 21 depicts the pressor responses to electrical stimula- tion of the spinal cord in pithed rats on the fifth day of ivt saline (n=8) or AII (6 ug/hr, n=9) infusion. The resting MAP in the conscious rats was measured immediately before the pithing procedure and averaged 152:6 mmHg in AII-infused rats and 120:4 mmHg in saline controls (p<0.05, Student's t-test). Blood pressure after anesthesia and pithing was not significantly different in the two groups (62:5 mmHg for saline controls, 60:6 mmHg for AII-treated rats, Student's t-test). Electrical stimulation of the spinal cord produced frequency-dependent increases in MAP in both groups of rats. There were no significant differences between AII- and saline-treated rats in the pressor response to spinal stimulation. 11. Acute ivt sarthranjchronic ivt AII The ability of acute ivt sarthran injection to reverse hyper- tension induced by chronic ivt AII infusion is depicted in Figure 22. On the fifth day of hypertension induced by chronic ivt AII infusion, acute ivt sarthran injection (0.3 ug) had no effect on MAP (n=5). However, successive sarthran injections (1 and 3 pg, ivt), each separated by 15 min, produced a significant decrease in MAP (ivt AII/ sarthran group). This series of ivt sarthran injections had no effect on blood pressure in the same group of rats on the second day after stopping the ivt AII infusion (recovery/sarthran group). Three ivt injections of 5 ul isotonic saline in a separate group of rats (n=4) on the fifth day of ivt AII infusion also had no effect on MAP (denoted ivt AII/saline). 123 Figure 21. Effect of chronic ivt AII infusion on pressor responses to electrical stimulation of sympathetic vasomotor outflow in pithed rats. Points represent average changes in mean arterial pressure (AMAP) of rats treated with saline (n=8) or AII (6 ug/hr, n=9) ivt for 5 days. Vertical bars represent SEM for individual within groups comparisons. There were no significant differences between the AII and saline group (mixed design ANOVA and lsd). 124 19° C SALINE ivt (n= 8) o All ivt (n --- 9) A MAP (mmHm 'ITI'I'I‘I'IFI'I‘l' 011144L1111 12345678910 stimulation frequency (Hz) Figure 21 125 Figure 22. Ability of acute ivt sarthran injection to reverse hyper- tension induced by chronic ivt AII infusion. MAP = mean arterial pressure; BASAL = resting MAP. For explanation of experimental groups, see description in text. Vertical bars represent SEM for within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from basal value (mixed design ANOVA and lsd). MAP - (mmHg) 126 OivtAll/sarthran(n=5) Clivt All/saline (n=4) 1 7O - O recovery/sarthran 160 - ' ‘"=51 150- _ a1: * a: 140-- 130- 120- 110— L’A\ A- - ‘°~~ "On-o" ~°'-'°“~o 100 1 1 i Y i 1 l l l l BASALTS 10 151,120 25 3OT35 4O 45 03:19 1:19 3P9 time (min) after ivt sarthran injection Figure 22 127 12. Acute ivt sarthran/acute ivt AII Figure 23 illustrates the ability of acute ivt sarthran injec— tion to reverse the pressor effect of acute ivt AII injection. There was no significant difference in the time course of the decay of the pressor response to an acute ivt AII injection (150 ng) whether the injection was followed one minute later by an ivt saline injection or an ivt injection of 1 ug sarthran. 13. Effect of iv saralasin on response to acute ivt AII ‘ The pressor response to acute ivt AII (50 ng) was evaluated in a group of 5 rats before and after a 15 min iv saralasin infusion (300 ng/min). Resting MAP was unaltered over the course of the experiment and averaged 120:] mmHg before the first ivt AII injection, 117 mmHg before starting the iv saralasin infusion (after a 2-hour interval) and 124 mmHg at the end of the 15-min saralasin infusion (immediately pre— ceding the second ivt injection of AII). The pressor response to the initial AII injection was 13:2 mmHg, and averaged 9 mmHg after the iv saralasin infusion. This difference was not statistically significant (p>0.05, paired t-test). B. Chronic ivt AII Infusion in Rabbits: Sodium Dependency_ The effect of chronic ivt AII infusion (3 ug/hr) on MAP, HR, and B.Wt. in rabbits on low and high sodium intake is shown in Figure 24. In rabbits on low sodium intake, MAP, HR, and B.Wt. were unaltered during ivt AII or saline infusion. In contrast, ivt AII infusion pro- duced a significant increase in MAP in high sodium rabbits which was evident on both the first and second weeks of measurement. Blood 128 Figure 23. Ability of acute ivt sarthran injection to reverse the pressor effect of acute ivt AII injection. The peak change in mean arterial pressure (AMAP) to acute ivt A11 (150 ng) and the subsequent decay in this response was measured in rats (n=5) receiving saline or sarthran (1 ug) ivt at l min after ivt AII injection. Vertical bar represents SEM for individual between groups comparisons (blocked factorial ANOVA and lsd). 129 [I] saline ivt sarthran ivt 20 1" "=5 15 - AMAP 5; (mmHg) 10 __ g. 5 L ‘ O 2.5 5 1O 15 20 time after ivt All injection (min) Figure 23 130 Figure 24. Mean arterial pressure, heart rate, and body weight responses to chronic ivt AII infusion in rabbits maintained on low and high sodium intake. MAP = mean arterial pressure, HR = heart rate, B.WT. = body weight. Cl and C2 are two control weeks, P1 and P2 are 2 weeks of ivt AII infusion (3 ug/hr, n=5) or saline infusion (n=5), P3 is a recovery week. Vertical bars on C1 value represent SEM for individual within group comparisons. Asterisk (*) indicates signifi- cant (p<0.05) difference from average of C1 and C2 values. Cross (+) indicates significant difference between AII- and saline-infused rabbits (mixed design ANOVA and lsd). MAP (MM HG) HR (BEATS/MIN) 120 110 131 D AIIIVT 0 SALINE lVT . 1.9qu A man NA : 7 - / . / ,,,,, / - / : é - / / . 7 b % - #41 Figure 24 O .0 C2 P1 P2 P3 132 pressure returned to control levels within 1 week after stopping ivt AII infusion. MAP remained unchanged in high sodium rabbits receiving saline ivt. The hypertensive response to chronic ivt AII infusion in the high sodium rabbits was associated with a significant tachycardia and decrease in body weight, neither of which were observed in ivt saline-infused control rabbits. Plasma Na and K concentrations fell significantly during the period of ivt AII infusion in both low and high sodium rabbits (Figure 25). Plasma Na concentration was still sig- nificantly lower than control levels one week after stopping ivt AII infusion, however, plasma K concentration had recovered to a value not significantly different from control by that time. Plasma osmolality was decreased during ivt AII infusion only in the high sodium group. As shown in Figure 26, daily water intake did not change signifi- cantly in high-sodium rabbits over the first week of ivt AII infusion. Water intake slowly increased over the second week of ivt infusion, reaching a statistically significant elevation only on the final day of the ivt infusion period. Urine output paralleled water intake, there- fore water balance did not change over the course of the experiment in this group of rabbits. Low-sodium rabbits exhibited significant in- creases in water intake and urine output during ivt AII infusion, how- ever, water balance remained unchanged (Figure 27). Since food intake tends to decrease in response to ivt AII infusion and drinking is asso- ciated with ingestion of food in rabbits, the ratio of water intake to food intake was calculated and is presented in Figure 28. The ratio of water/food in low-sodium rabbits was dramatically increased over the first week of AII infusion, and decreased to a level not significantly 133 Figure 25. Effect of chronic ivt AII infusion on plasma sodium and potassium concentrations and plasma osmolality in rabbits maintained on low and high sodium intake. PNa = Pplasma sodium concentration, PK= plasma potassium concentration, plasma osmolality. Data was analyzed using a mixed design ANOVRS gollowed by lsd. Other symbols as in Figure 24. NA (MEG/L) . PK (MEG/L) F'osM (MOSH/L) 160 155 150 145 140 135 130 134 n An NT 0 SALINE WT I V U 7 T \\\ n HIGH NA 7 rrtlivmyi1I1 \\\\\Y\ 1 \\\\\\ Figure 25 135 Figure 26. Water intake, urine output, and water balance in response to chronic ivt AII infusion in high-sodium rabbits. Top panel depicts responses in rabbits (n=5) that received AII ivt (3 ug/hr), and lower panel depicts responses in saline control rabbits (n=5). Vertical lines on day 1 value represent SEM for individual within groups com- parisons. Asterisk (*) indicates significant (p<0.05) difference from average)of values obtained during the control week (mixed design ANOVA and lsd . 1200 MUDAY §§§§§ 1200 MUDAY 136 —- WATER lNTAKE “a- URINE OUTPUT LM [+100 WATER O BALANCE 40° (ML/DAY) 0-—-———-—AanT-—-————-c MW ‘100 WATER [O BALANCE -1OO (ML/0“" h--— SALlNE IVT———o 7 14 21 28 TIME (DAYS) Figure 26 137 Figure 27. Water intake, urine output, and water balance in response to chronic ivt AII infusion in low-sodium rabbits. Top panel depicts responses in rabbits (n=5) that received AII ivt (3 ug/hr) and lower panel depicts responses in saline control rabbits (n=5). Vertical lines on day 1 value represent SEM for individual within groups com- parisons. Asterisk (*) indicates significant (p<0.05) difference from average of values obtained during the control week. Cross (+) indi- cates significant difference between AII- and saline-infused rabbits (mixed design ANOVA and lsd). 138 “- WATER INTAKE '--- URINE OUTPUT ', I 100 WATER o 4001' 1: *1 :- 0 Wu AY) ML 3°°‘ h 5717 200 1 00 WATER BALANCE o (ML/DAY) 11, 5‘8“ NE NT“ 1 7 14 21 "ME (DAYS) F1'Siur'e 27 139 Figure 28. Ratio of water intake to food intake in rabbits main- tained on low and high sodium intake in response to chronic ivt AII infusion. Open circles represent ivt AII-infused rabbits and closed circles represent saline controls (n=5 in all groups). Vertical bars on day 1 values represent SEM for individual within groups compari- sons. Asterisk (*) indicates significant (p<0.05) difference from average of control week values. Cross (+) indicates significant difference between AII- and saline-infused rabbits (mixed design ANOVA and lsd . 140 WATE R/ FOOD (3%) WATER] FOOD (1%) TIME (DAYS) Figure 28 141 different from saline-infused rabbits during the second week of ivt AII infusion. Hater/food intake was significantly elevated on 6 out of the 14 days of ivt AII infusion in high-sodium rabbits, but the increase was not as marked as that seen in low-sodium rabbits. Figure 29 illustrates the effect of chronic ivt AII infusion on body fluid compartment volumes and hematocrit. In high-sodium rabbits, plasma volume (PV), extracellular fluid volume (ECFV) and hematocrit (HCT) were unchanged in response to ivt AII infusion. However, in low- sodium rabbits, ECFV increased significantly during the ivt AII infusion and remained elevated one week after the infusion was stopped. PV also was significantly elevated on the recovery week. Hematocrit did not change in the low-sodium rabbits over the course of the experiment. Daily sodium and potassium balance are shown in Figure 30. Sodium balance did not change significantly during ivt AII infusion in high- sodium rabbits, but low-sodium rabbits went into negative sodium balance over the first 8 days of ivt AII infusion. The sodium lost during this time period was not regained once the ivt AII infusion was stopped. Potassium balance decreased significantly in both low- and high-sodium rabbits in response to ivt AII infusion. In high-sodium rabbits, K balance was significantly more negative in the group receiving AII ivt than in saline control rabbits over the first week of ivt AII infusion. Low-sodium rabbits exhibited only one day of decreased potassium balance. Intraventricular AII infusion had no effect on creatinine clearance or blood urea nitrogen in either low- or high-sodium rabbits (Figure 31). 142 Figure 29. Effect of chronic ivt AII infusion on body fluid compart- ment volumes and hematocrit in rabbits maintained on low and high sodium intake. PV = plasma volume, ECFV = extracellular fluid volume, HCT = hematocrit. Vertical bars on C1 value represent SEM for indi- vidual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from average of Cl and C2 values (mixed design ANOVA and lsd). Other symbols as in Figure 24. 143 o All ivt o Saline ivt HIGH NA 70 b -::s;;::5;:; 33:33:53 PV’ 50 P (ml/k9) 50 F. L 40 350 ECFV 30° : (ml/k9) 25° .. 200 50 HCT 4°: C1 C2 P1 P2 P3 Figure 29 144 Figure 30. Effect of chronic ivt AII infusion on sodium and potas- sium balance in rabbits maintained on low and high sodium intake. Upper two panels depict daily sodium balance in high and low sodium rabbits receiving either AII (3 ug/hr) or saline ivt (n=5 in each group). Lower two panels depict daily potassium balance. Vertical bars on day 1 value represent SEM for individual within groups com- parisons. Asterisk (*) indicates significant (p<0.05) difference from average of control week values. Cross (+) indicates significant difference between A11- and saline-infused rabbits (mixed design ANOVA and lsd . 145 0 All WT OSALINE IVT NA BALANCE MEO NA/DAY HIGH NA MEG NA/DAY LOW NA MEG no" ‘° HIGH NA ~10 MEG K/DAY LOW NA "1 w.-m 1; 21 28 T1ME(DAYS) Figure 30 146 Figure 31. Effect of chronic ivt AII infusion on creatinine clear- ance and blood urea nitrogen in rabbits maintained on low and high sodium intake. C1CR = creatinine clearance; BUN = blood urea nitro- gen. Vertical bars on day 1 value indicate SEM for individual within groups comparisons. Other symbols as in Figure 24. 3 C 5 3 a o I BUN (MG 96) LOW NA D AIIIVT O SALINE IVT IuAMIIII-u-ov . u - ...s \\\\\\\\ 20 16 1O 148 C. Chronic Pharmacological Blockade of Brain AII Receptors or Brain Converting Enzyme 1. Chronic ivt saralasin a. Dose determination As shown in Table 6, 5-day ivt infusion of saralasin at a dose of either 6 or 12 ug/hr does not alter resting mean arterial blood pressure in rats maintained on normal sodium intake. To assure that pressor responses to ivt and iv AII are reproducible after 5 days, control pressor responses were measured in a group of 5 rats and were measured again after a 5-day sham period, during which no ivt infusion was performed (Figure 32). The control pressor response to 150 ng AII ivt did not differ from that measured in the same rats after 5 days (19 ys, 21 mmHg, respectively, paired t-test). The iv dose-response curve for AII also did not differ from control after the 5-day sham period (mixed design ANOVA and lsd). Intraventricular saralasin infusion (6 ug/hr) for 5 days did not alter pressor responses to iv AII infusions as illustrated in Figure 33 (mixed design ANOVA and lsd). The pressor response to ivt AII also was not significantly different before and after the 5-day sara- lasin infusion (paired t-test), although the pressor response after the ivt saralasin infusion tended to be less than the control response (15 gs, 24 mmHg, respectively). As shown in Figure 34, ivt infusion of saralasin (12 pg/hr) for 5 days completely abolished pressor responsive- ness to centrally administered AII. Pressor responses to iv AII were modestly reduced after ivt saralasin infusion, significantly so at the 30 ng/min infusion rate of AII. 149 TABLE 6 Resting Mean Arterial Pressure Before and After ivt Saralasin Infusion Mean Arterial Pressure (mmHg) Control Treatment 5-Day Sham Period 120:4 121:6 5-Day Saralasin 6 ug/hr ivt 118:2 127:3 5-Day Saralasin 12 ug/hr ivt 118:3 122:6 Values are mean :_SEM; n=5 in each group. There were no significant differences in resting mean arterial pressure be- fore and after any treatment (p<0.05). 150 Figure 32. Pressor responses to acute intravenous and intraventri- cular AII administration in rats before and after a 5-day sham period. Open symbols represent control pressor responses and filled symbols represent responses in the same group of rats (n=5) after a 5-day sham period. AMAP = change in mean arterial pressure. Values represent mean : SEM. A MAP(mmH9) 30 151 O--- CONTROL .--- SHAM i I L I I 150 119 A ll 10 3° 10° ivt All iv (ng/min) Figure 32 152 Figure 33. Effect of continuous ivt infusion of saralasin (6 ug/hr) on pressor responses to acute intravenous and intraventricular AII administration. Open symbols represent control pressor responses and filled symbols represent responses in the same group of rats (n=5) after a 5-day sham period. AMAP = change in mean arterial pressure. Values represent mean : SEM. 30 A MAP (mmHg) 153 o.— - CONTROL .—— “RAMS". 6 69/1", 1V1 I, l l J 150 119 A II 19 3° 10° 1V1 All iv (ng/min) Figure 33 154 Figure 34. Effect of continuous ivt infusion of saralasin (12 ug/hr) on pressor responses to acute intravenous and intraventricular AII administration. Open circles represent control pressor responses and filled circles represent pressor responses in the same group of rats (n=5) after a 5-day ivt infusion of saralasin (12 ug/hr). Values represent mean :_SEM. Asterisk (*) indicates significant difference from corresponding control value (paired t-test for ivt response, mixed design ANOVA and lsd for iv dose-response). A MAP (mmHg) v—————4L£ 155 0- -- CONTROL .— SARALASIN 12 Mglhr, M 150 ngAll ivt A 11 iv (ng/min) Figure 34 156 b. Effects in normal- and high-sodium rats In rats maintained on a normal sodium intake, 5-day ivt saralasin infusion (12 ug/hr) had no effect on MAP, HR, UNaV and U0 (Figure 35). The amount of water ingested over the final 24 hr of ivt saralasin infusion was not different from saline controls (rank sum test). However, in rats maintained on a high sodium intake, ivt sarala- sin infusion produced a significant increase in MAP by day 3 of the infusion, which was reversed within 24 hr after the infusion was stopped (Figure 36). This increase in MAP was associated with a bradycardia and transient sodium retention. Rats receiving saralasin tended to drink more than saline controls during the final 24 hr of ivt infusion, although this difference was not statistically significant. Intravenous saralasin infusion (18 ug/hr) produced a significant increase in MAP in rats maintained on high sodium intake, although the pressor response was of a lesser magnitude than that seen with ivt saralasin (Figure 37). In addition, the pressor response to iv saralasin was evident within 24 hr after the infusion was started, whereas a 3-day latent period was observed with ivt saralasin infusion. Intravenous infusion of saralasin caused no significant changes in HR, U aV or UO. N A comparison of results obtained in rats receiving chronic ivt saralasin to rats receiving chronic ivt AII infusions is depicted in Figure 38. In rats maintained on a high sodium intake, both ivt saralasin (12 ug/hr) and AII (l ug/hr) infusion increased MAP with approximately a 3-day latency. No remarkable differences in the re- sponse of HR, UNaV’ or UO to infusion of saralasin or AII were seen between the two groups of rats. 157 Figure 35. Cardiovascular and fluid/electrolyte responses to chronic ivt saralasin infusion in normal sodium rats. Rats were maintained on a sodium intake of 1.5 mEq/day. Units on the abscissa are days. Cl and C2 are two control days, Al-A5 are five days of ivt infusion, R1 and R2 are recovery days. Rats received either saralasin (12 ug/hr, n=5) or saline ivt (n=5) during the ivt infusion period. Vertical bars on C2 value represent SEM for individual within groups compari- sons. Numbers in parentheses indicate the volume of water ingested during the final 24 hr of ivt infusion (mean + SEM). Asterisk (*) indicates significant (p<0.05) difference from average of C1 and C2 values (randomized block ANOVA and lsd). 158 H saralasin 12ug/hf ivt. (n=5) Ne+ intake in 1 .5 mEq/day o- - 4 saline ivt (n=5) um MAP (NMHm um um Head - rate (but!!!) an Na+ 2 excrefion ‘ (mEQIday) o I so Uflne output (ml/day) o . Figure 35 159 Figure 36. Cardiovascular and fluid/electrolyte responses to chronic ivt saralasin infusion in high sodium rats. Rats were maintained on a sodium intake of 7.5 mEq/day. Rats received either saralasin (12 ug/hr, n=6) or saline (n=7) during the ivt infusion period. Vertical bars on C2 value indicate SEM for individual within groups compari- sons. Values in parentheses represent the volume of water ingested during the final 24 hr of ivt infusion (mean :_SEM). Asterisk (*) indicates significant (p<0.05) difference from average of Cl and C2 values (randomized block ANOVA and lsd). Other symbols as in Figure 34. 160 MAP (MMH91 Heart rate lem) 160 H saralasin inglhr ivt, (n=8) Ne on .oflsallne ivt (n= Ne" excretion (mEq/day) + intake 2- 7.5 mEq/day Uflne output (ml/day) Figure 36 161 Figure 37. Effect of chronic intravenous saralasin infusion (18 ug/hr) on cardiovascular and fluid/electrolyte parameters. Rats received intravenous infusions of saralasin (18 ug/hr) or saline during the 7day infusion period (denoted Sl-S7). Vertical bars on C2 value indicate SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from average of Cl and C2 values (randomized block ANOVA and lsd). Other symbols as in Figure 34. 162 1 60 °""°Saralasin, 1 8 ug/hr, iv (n=5) e—-—e Saline iv (n=4) N * eke MAP immHm 1 00 400 Heart rate leMI 300 Na 6 excretion (n1 Eq/day) Uflne output (ml/day) o, Figure 37 163 Figure 38. Comparison of cardiovascular and fluid/electrolyte re- sponses to chronic ivt saralasin and AII. Rats were maintained on high sodium intake and received either AII (l ug/hr, n=5) or saralasin (12 ug/hr, n=6) during the ivt infusion period. Vertical bars on C2 value indicate SEM for individual within groups comparisons. Values in parentheses represent the volume of water ingested during the final 24 hr of ivt infusion (mean :_SEM). Asterisk (*) indicates signifi— cant (p<0.05) difference from average of C1 and C2 values (randomized block ANOVA and lsd). Other symbols as in Figure 34. o---o All, ing/hr ivt (n=-=5) H Saralasin, 1 2 119/hr ivt (n= q/day . . a er ..e aeae -. .. a e . . . u a. a . - . . . .. . .... .. a u . v . . A a ' 1 , 0 ea e e Na+ excretion (mEq/day) 4 Urine output (ml/day) .....- A-AA‘IALI Figure 38 165 2. Chronic ivt sarthran a. Dose determination: Sprague-Dawley The effect of chronic ivt sarthran infusion at various doses on the pressor responses to acute iv and ivt AII is depicted in Figure 39. Infusion of sarthran at a dose of 100 ng/hr ivt for 5 days did not effect the pressor response to acute iv AII infusion. However, this dose of sarthran did produce a significant attenuation of the pressor response to an acute ivt bolus of AII. Chronic ivt sarthran infusion at 300 ng/hr for 5 days did not alter the pressor response to iv or ivt AII. Marked suppression of the pressor response to acute ivt AII was obtained after 5 days of ivt sarthran infusion at doses of 1 ug/hr and 6 ug/hr. However, the 6 ug/hr dose produced significant inhi- bition of iv AII pressor responses at all 3 doses of AII infused. In contrast, the 1 ug/hr dose produced significant inhibition of the iv AII dose-response curve only at the highest infusion rate of AII. In view of the above findings, the ivt sarthran dose of l ug/hr for 5 days was chosen as the optimum dose that would produce relatively complete func- tional blockade of brain AII receptors with minimal antagonism of peri- pheral vascular receptors. This dose of sarthran was used in all sub- sequent experiments. b. Effects in high-sodium rats Figure 40 shows the cardiovascular and fluid/electrolyte responses to a 5-day ivt sarthran infusion (l ug/hr) in rats maintained on a high sodium intake (n=8). MAP and HR were not significantly changed by the ivt sarthran infusion. Urinary sodium excretion also was basically unchanged although on the third day of ivt sarthran infusion, 166 Figure 39. Effect of chronic ivt sarthran infusion on pressor re- sponses to acute ivt and iv AII administration. Change in mean arterial pressure (AMAP) was measured in response to 150 ng AII ivt (left panel) and 10, 30, and 100 ng/min AII iv (right panel). Filled symbols are control responses and open symbols are responses in the same group of rats after the 5-day ivt infusion period. Doses of sarthran infused were: 6 ug/hr (n=4, top panel), 1 ug/hr (n=5, second panel), 300 ng/hr (n=6, third panel), and 100 ng/hr (n=5, bottom panel . Vertical bars represent SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from corresponding control value (paired t-test for ivt response; mixed design ANOVA and lsd for iv dose-response curves). AMAP (mmHel ‘0 20 AMAP (mmHg) ‘0 AMAP (mmHg) ‘0 20 167 lVT ‘SAR 8THR All 0--— control o--- after ‘sar athr All 6 Ito/hr (1124) E 0* r1'1'1 1 pg/hr (n=5) 11111] AAA...) 300 ng/hr(n=6) IIIIII iml _ 100 tag/11min) *- 0 T'Tll' Lml 9 l 10 All (no/min), i.v. 150 ng All ivt Figure 39 168 Figure 40. Cardiovascular and fluid/electrolyte responses to chronic ivt sarthran infusion (l ug/hr) in rats on high sodium intake. Cl and C2 are control days, 51-55 are 5 days of ivt sarthran infusion (shaded area, R1 and R2 are recovery days. MAP = mean arterial pressure, HR = heart rate, UO = urine output. Vertical bars on C2 value indicate SEM for within groups comparisons (n=8). Asterisk (*) represents signifi- cant (p<0.05) difference from average of Cl and C2 values (randomized block ANOVA and lsd). 120 MAP (mmHg) ‘00 80 500 HR (beats/min.) ‘00 300 6 meq/day 4 2 O 40 U0 (ml/day) 20 O 169 'V'W'Vr'f 'I'I'Url' Na excretion K excretion TIME (DAYS) Figure 40 170 sodium excretion fell significantly when compared to control values. Urinary potassium excretion and urine output also remained stable throughout the ivt sarthran infusion period. c. Dose determination: spontaneously hypertensive rats The pressor responses to acute ivt and iv AII administra- tion before, during, and after 5-day ivt sarthran infusion (l and 6 ug/ hr) are depicted in Figure 41. The pressor response to 150 ng AII ivt was significantly reduced from a control value of 29 mmHg to 15 mmHg on the fifth day of ivt sarthran infusion (1 ug/hr, n=5; paired t-test). Recovery measurements were obtained 2 days after termination of ivt sarthran in 2 rats and averaged 30 mmHg in response to acute ivt AII. The pressor responses to graded iv infusions of AII were not signifi- cantly different on the fifth day of ivt sarthran infusion when compared to control and recovery days (mixed design ANOVA and lsd). Chronic ivt infusion of sarthran at a dose of 6 ug/hr (n=6) caused a reduction in the acute ivt AII pressor response from 35 mmHg to 9 mm Hg. The re- sponse to ivt AII on the second day after pump removal (29 mmHg) was restored to a value not significantly different from the control value. The dose-response curves for acute iv AII were not significantly differ- ent at any time. As shown in Table 7, resting MAP was elevated by 20- 25 mmHg on the fifth day of ivt sarthran infusion at either dose when compared to control values. 3. Chronic ivt teprotide a. Dose determination: Sprague-Dawley Figure 42 depicts the pressor responses to acute ivt and iv AI administration before and on the fifth day of ivt teprotide infusion 171 Figure 41. Effect of chronic ivt sarthran infusion on pressor re- sponses to acute ivt and iv A11 in SHR. Sarthran was infused con- tinuously ivt at l ug/hr (n=5, upper panel) or 6 ug/hr (n=6, lower panel). C = control response, S = response on fifth day of ivt sar- thran infusion, R = recovery response. Change in mean arterial pressure (AMAP) was measured in response to 150 ng AII ivt (histo- grams) and to 10, 30, and 100 ng/min AII iv (line plots). Vertical bars represent SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from control response (randomized block ANOVA for ivt response, mixed design ANOVA and lsd for iv dose-response). 172 IVT SARTHRAN - SHR 1 Ito/nun = 5) A MAP 30 (mm Hg) Vijlllllil A MAP (mm Hg) 30 '1l|l[I]1]1 cs R10 so 100 150Ar1'g ivt All, iv (ng/min) Figure 41 173 TABLE 7 Resting Mean Arterial Pressure Before, During, and After ivt Sarthran or Teprotide Infusion in SHR Treatment Control Day 5-ivt Recovery Sarthran l ug/hr ivt (n=5) 162:8 183* --- Sarthran 6 ug/hr ivt (n=6) 161:6 186* 155 Teprotide 10 ug/hr ivt (n=6) 152:6 174* 156 Values represent mean :_SEM for individual within groups com- parisons. Asterisk (*) represents significant (p<0.05) difference from control value (randomized block ANOVA and lsd). 174 Figure 42. Effect of chronic ivt teprotide infusion on pressor responses to acute ivt and iv AI administration. Teprotide was in- fused continuously ivt at l ug/hr (n=4, upper panel), 3 u/hr (n=3, middle panel), and 10 ug/hr (n=5, lower panel). Change in mean arterial pressure (AMAP) was measured in response to 100 ng AI ivt (left side) and to 3, 10, 30, and 100 ng/min AI iv (right side). Filled symbols represent control responses and open symbols represent responses in the same group of rats on the fifth day of ivt teprotide infusion. Vertical bars represent SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from control value (paired t-test for ivt response, mixed design ANOVA and lsd for iv dose-response). 175 WT TE PROTIDE O—— control 9 - after teprotide 50 1 pig/m (n=4) ‘0 )- AMAP 3° ' mmH 1 912° ‘ 10. 0 l 50 Spa/hunt” ‘0 b AMAP 3° 5 ("NM") 20 __ O P 10 . 1 5° 10#9/hr(n=5) ‘0 b AMAP 3° " * (mmHg) 20 __ l 10 I- 0 100.119 AI 3 10 so 100 wt Al (119/min). 1.1:. Figure 42 176 at l, 3, or 10 ug/hr. Infusion of teprotide at any dose did not produce a significant reduction in the pressor response to intravenous AI infu- sion. The pressor response to ivt AI injection was not significantly different before or on the fifth day of ivt teprotide infusion at the l and 3 ug/hr doses. However, the pressor response to ivt AI was signi- ficantly reduced on the fifth day of ivt teprotide infusion at 10 ug/hr when compared to the control response. b. Dose determination: spontaneously hypertensive rats The effect of S-day ivt infusion of teprotide (10 ug/hr) on the pressor responses to ivt and iv AI administration is shown in Figure 43. On the fifth day of ivt teprotide infusion, the pressor re- sponse to ivt AI was slightly, but significantly decreased when compared to the control response (22 gs, 30 mmHg, respectively). Two days after stopping the ivt teprotide infusion, the pressor response to ivt Al was significantly elevated when compared to control (38 gs, 30 mmHg, re- spectively). The dose-response relationship for intravenous infusions of AI was unaltered by ivt teprotide infusion. The pressor response to ivt injection of All (150 ng) averaged 26:3 mmHg on the fifth day of ivt teprotide infusion and 27 mmHg on the second recovery day (no signifi- cant difference, paired t-test). The fact that ivt teprotide infusion caused significant changes in the pressor response to ivt AI but not to ivt AII verifies that the effect of teprotide was specific for the conversion of AI to AII, and did not represent a general decrease in responsiveness to AII. 4. Chronic ivt sarthran/chronic iv angiotensin II Cardiovascular and fluid/electrolyte responses to chronic in- travenous AII infusion (10 ng/min) in the presence or absence of chronic 177 Figure 43. Effect of chronic ivt teprotide infusion on pressor responses to acute ivt and iv AI administration in SHR. Teprotide was infused continuously ivt at 10 ug/hr (n=6). C = control response, T = response on fifth day of ivt teprotide infusion, R = recovery re- sponse. Change in mean arterial pressure (AMAP) was determined in response to 100 ng AI ivt (histograms) and to 10, 30, and 100 ng/min AI iv (line plots). Vertical lines represent SEM for individual within groups comparisons. Asterisk (*) indicates significant (p<0.05) difference from control response (randomized block ANOVA for ivt response, mixed design ANOVA and lsd for iv dose-response). A MAP (mm H9) 178 IVT TE PROTIDE - SHR 8 30 20 1o. 1 o pig/hr (n = 6) 1 l1] Figure 43 Al, iv(ng/min) 179 ivt sarthran infusion are shown in Figure 44. Chronic iv AII infusion at this dose produced a sustained increase in MAP in rats receiving saline ivt. The hypertensive response to iv AII infusion in rats re- ceiving sarthran ivt was similar, however, this group of rats did not exhibit a statistically significant increase in MAP until the fourth day of iv AII infusion. In addition, there was a significant difference in MAP between saline- and sarthran-treated rats on the first day of iv AII infusion. Rats receiving sarthran exhibited a significant decrease in UNaV on the first day of A11 infusion, but UNaV was unchanged there- after. There were no significant changes in any of the other variables measured (HR, UKV or UO) in either saline- or sarthran-treated rats in response to chronic iv AII infusion. Figure 45 depicts responses of saline- and sarthran-treated rats to chronic iv AII infusion at a dose Of 20 ng/min. In this experiment, there were virtually no differences between the two groups of rats in their response to AII. MAP was signi- ficantly elevated to the same extent in both groups of rats, while HR, UNaV’ U KV, and U0 were unchanged in both groups over the course of the experiment. 5. Chronic ivt sarthran/DOC-salt hypertension The hypertensive response to DOC-salt treatment in rats re- ceiving chronic ivt sarthran infusion (l ug/hr) is reported in Table 8. A significant increase in tail cuff blood pressure was observed in both saline- and sarthran-treated rats in response to DOC administration. The increase in blood pressure was evident 4 days after DOC injection (the first point at which it was measured) and persisted in both groups of rats for the duration of the study (3.5 wk). At no time was there a significant difference in blood pressure between rats receiving 180 Figure 44. Effect of chronic ivt sarthran infusion (l ug/hr) on cardiovascular and fluid/electrolyte responses to chronic iv AII infusion (10 ng/min). Rats received either sarthran (l ug/hr, n=8) or saline ivt (n=8) during the entire protocol. MAP = mean arterial pressure, HR = heart rate, UNAV = urinary sodium excretion, UKV = urinary potassium excretion, UO = urine output. Units of the abscissa are days. Al-A5 (shaded area) are the 5 days of iv AII infusion. Vertical lines on C2 value represent SEM for within groups compari- sons. Asterisk (*) indicates significant (p<0.05) difference from average of Cl and C2 values. Cross (+) indicates significant differ- ence between sarthran- and saline-infused rats (mixed design ANOVA and lsd . 181 IV AII/WT SARTHRAN MAP (MM HG) HR IB/MI UNAV (MEO) UKV (MEG) UO (ML) Cl C2 A1 A2 A3 A4 A5 R1 R2 TIME I SALINE lVT (n = a) e SARTHRANIVTUT= a) Figure 44 182 Figure 45. Effect of chronic ivt sarthran infusion (l ug/hr) on cardiovascular and fluid/electrolyte responses to chronic iv AII infusion (20 ng/min). Rats received either sarthran (l ug/hr, n=8) or saline ivt (n=8) during the entire protocol. MAP = mean arterial pressure, HR = heart rate, UNAV = urinary sodium excretion, UKV = urinary potassium excretion, U0 = urine output. Units of the absicssa are days. Al-A5 (shaded area) are the 5 days of iv AII infusion. 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