The Brain Renin-Angiotensin System and Extrarenal Renin Detlev Ganten Title of thesis: STUDIES ON THE EXISTENCE OF AN INDEPENDENT BRAIN RENIN-ANGIOTENSIN SYSTEM: A MODEL FOR EXTRARENAL TISSUE RENIN. Name: Detlev Ganten. Department: Experimental Medicine, McGill University. Degree: Ph.D. SUMHARY An enzyme, capable of catalyzing the reaction with renin substrate to form angiotensin is described in the brain of dogs, rats, sheep and man. This brain tissue iso-renin differs in important characteristics from renal renin. The enzyme persists at control-levels 12 days after nephrectomy. Renin substrate has been found equally in brain tissue of dogs. Angiotensin was extracted from brain of dogs which had been nephrectomized and in which plasma angiotensin was undetectable. This shows that angio­ tensin can be formed locally in the brain. An iso-renin of similar characteristics as brain iso-renin is described and partially characterized in aortic tissue and in adrenal glands. The independence of tissue iso-renin from kidney renin is supported by the fact that the two enzymes react diffe­ rently to the same physio-pathological stimuli. STUDIES ON THE EXISTENCE OF AN INDEPENDENT BRAIN RENIN­ ANGIOTENSIN SYSTEM: A MODEL FOR EXTRARENAL TISSUE RENIN. Det1ev Ganten, M.D. Submitted to the Facu1ty of Graduate Studies and Research of McGi11 University in partial fu1fi1ment of the requirement for the degree of Doctor of Phi1osophy. Department of Experimental Medicine, C1inica1 Research Institute, McGi11 University, Montreal, Canada. Montreal, Canada. August 1972. Det1ev Ganten 1973 FOR URSULA TABLE OF CONTENT ACKNOWLEDGMENTS l LIST OF FIGURES II LIST OF TABLES V LIST OF ABBREVIATIONS VII 1. INTRODUCTION ••.•••.•.•.••...••.•.••.••.•.•.•...•.•••... 1 2~ REVIEW OF THE LITERATURE 2.1. Components of the Renin-l1ngiotensin-System ..• 4 2.1. 1. Renin ........................................ 4 2.1. 2. Tissue Iso-Renin 6 2.1. 2.1. Uterus 7 2.1. 2.2. Salivary Glands 8 2.1. 2.3. Adrenal Glands 9 2.1. 2.4. Arterial lval! 10 2.1. 3. Pseudorenin ••••••••••••••.••••••••••••••••••• Il 2.1. 4. Renin Substrate (Angiotensinogen) 14 2.1. 5. Angiotensin 16 2.1. 6. Angiotensin l Converting Enzymes 18 2.1. 7. Antirenin .................................... 21 2.l. 8. Antiangiotensin .............................. 22 2.1. 9. Renin Inhibitors 23 2.1. 10. Angiotensinases 25 2.2. CZeQPanoe of Renin and Angiotensin from PZasma •...••..••.•..••.••••.•.•....•..•..•..• 26 2.3. Interaotion of the Components and CritioaZ EvaZuation of the CZassioaZ Renin-Angiotensin- System .........••....................•....... 29 2.4. ~ze BZood-Brain-Barrier 36 3. INVESTIGATIVE SECTION . ................................ 40 3.1. Methods for Renin and Angiotensin .••.•...••.. 40 3.1. 1. Plasma ....................................... 43 3.1. 2. Cerebrospina1 F1uid •.••••••.•••••••••.••..•.• 43 3.1. 3. Brain Tissue .•••••••••••••••••••••.•••.•••.•• 43 3.1. 4. Hesenteric Artery Branches .••••••••••••.•.••• 44 3.1. 5. Aortic Tissue •••••••••.••••••••••••••••••.••• 45 3.1. 6. Carotid Artery 45 3.1. 7. Adrena1 Glands 45 3.2. Measuren;ent.of Angiotensin in PZasma, CSF, and Bra"n T"ssue •...•.•.••••.•...•.••....••.. 1.6 3.3. Method for Measurement of Renin Substrate in PZasma and CSF .•..••••••....•.••••...••...•.. 48 3.4. Method for Measurement of Renin Substrate in Brain Tissue ................................. 48 3.5. Preparation of Different Renin Substrates •••. 51 3.5. 1. Dog Plasma "Standard" Substrate •••••••••••••• 51 3.5. 2. Purified Dog Plasma Substrate •••••••••••••••• 52 3.5. 3. lletero1ogous Substrates from Rabbit, Rat and Sheep ........................................ 53 3.5. 4. Synthetic Renin Substrate •••••••••••••••••••• 54 3.6. Pu~ification of Tissue Iso-Renins ..•........ 56 3.7. UZt~acent~ifugation of B~ain and Kidney Tissue ....................................... 57 3.8. '~isc" EZect~opho~esis on PoZyac~yZamide Ge Z. ••••••••••••••••••••••••••••••••••••••••• 60 3.9. Othe~ ftlethods ............................... 62 4. RESULTS (METHODOLOGICAL) •...••••.•...••.•...•.•.••.... 64 4.1. Specificity of the Renin Assay •............. 64 4.2. Identification of Angiotensin as the Froduct of the B~ain-Enzyme Subst~ate Reaction ...... 76 4.3. Compa~ison of B~ain Iso-Renin with Kidney' Renin ....................................... 80 4.4. ~e Pe~/eabiZity of the BZood-B~ain-B~~ie~ fol" Renin ................................... 91 4.5. UZt~acent~ifugation Studies .....•.......•... 94 5. RESULTS (PHYSIO-PATHOLOGICAL) .•••••••..•.••••.•.••.•.• 104 5.1. B~ain Iso-Renin in Cont~oZ and Neph~ectomized Dogs .••.•..•••••.•••••.•••.•..•.•••.••••••.. 104 5.2. nze P~esence of Renin Subst~ate and Angio- tensin in B~ain Tissue .........•............ Ill! 5.3. Iso-Renin, EZect~oZytes and CatechoZamines in B~ains of Dogs at Diffe~ent Ages ......... 120 5.4. Iso-Renin in A~te~iaZ Tissue ..........•..... 133 5.4. 1. Characterization .•.•..•••.•••..••..•.•..•... 133 5.4. 2. Independence of Iso-Renin in Arteria1 Tissue from Plasma and Kidney Renin ....••••••••...• 136 5.4. 2.1. Hemorrhage in Nephrectomized Dogs ••.••• 137 5'.4. 2.2. lIemorrhage in Nephrectomized Dogs with Reduced Sp1anchnic Vascu1ar Bed •••••••• 145 5.4. 2.3. Iso-Renin in Arteria1 Tissue from Normal and Nephrectomized Dogs ••••••••• 147 5.4. 2.4. Stimulation of Iso-Renin in Arteria1 Tissue by C1ipping of the Superior Mesenteric Artery ••••••••••.••••••••••• 151 5.5. Effeat of Aldosterone Administration on Plasma Renin AativitY3 Tissue Iso-Renins and Plasma EZeatrolytes •........................ 159 5.6. Effeat of Progesterone Administration on Plasma Renin AativitY3 Tissue Iso-Renins and Plasma EZeatrolytes .......•.•.•.......•...... 162 5.7. Effeat of Potassium Defiaienay on Plasma Renin Aativity3 Tissue Iso-Renins and Plasma EZectroZytes ................................. 166 5.8. Renin Aativity in other Extrarenal Tissues and Tissue Renin Aativity in Speaies other than Doga .................................................... 169 6. GENERAL DISCUSSION •••••••••••••••••••••••••••••••••••• 176 7. CLAIMS TO ORIGINALITY ••••••••••••••••••••••••••••••••• 194 8. SmmARY ........................................................................................... 197 9. RESl.Jr1.E ................................................................................................ 198 10.. BIBLIOGRAPHY.. .. .. .. .. .. .. .. .. .. .. • .. • .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .... 199 l ACKNOWLEDGMENTS l am deeply indebted to the following: Dr. Jacques Genest and Dr. Roger Boucher for providing the laboratory facilities and for their continued interest and support of my work. Dr. Daniel Trachewsky and Dr. John Grose for many improvements in the English presentation of the manuscript. l am also indebted to my colleagues for their friendly and invaluable collaboration: Dr. H.M. Brecht, Dr. G. Constantopoulos, Dr. P. Granger, Dr. K. Hayduk, Dr. K.P. Karsunky, Dr. M. Kusumoto, Dr. A. Marquez-Julio, and Dr. James Minnich. l wish to extend my gratitude to Miss C. Labarre, Mr. E. Pierre, Mr. D. Charbonneau, Mrs. D. Pazzi and Mrs. S. Olivieri for occasional and skillful technical assistance, Miss I. Morin for preparing the illustrations and Mrs. o. Desroches for typing this thesis. II LIST OF FIGURES FIGURE 1: Sahematia outZine of the renin-angiotensin system ............................................. 31 FIGURE 2: GeneraZ outZine of the proaedure for the measu- rement of pZasma and tissue renin aativity .••....•• 41 FIGURE 3: Effeat of inaubation time of brain iso-renin on angiotensin ,formation ....•....•.•..•...••.•.•.•.••. 67 FIGURE 4: Effeat.of in~reasing ~ounts of brain iso-renin 68 on ang~otens~n format~on ...••.•.•..•....•.•.••.••.. FIGURE 5: Effeat of different substrates on angiotensin formation •.•..•....•.•••.••.•......•••.•••.••.•..•. 75 FIGURE 6: ~ffeat of angiotensin antibodies on pressor materiaZ from 1:naubation of brain iso-renin •.•••..•....•...• 78 FIGURE 7: ~ffeat o.f pH on c;ngiotens~n formation with tissue ~so-ren~ns and k~dney ren~n .•••••..•••.•..•..••..•• 81 FIGURE 8: Comparison of angiotensin formation with purified renaZ renin and iso-renin from brain tissue of dogs •.•........•...............•................... 84 FIGURE 9: Effeat of tetradeaapeptide aonaentration on angio- tens'În fopmation ................................... 86 FIGURE 10: qisa-eZeatrophoresis of purified renin extraats 89 FIGURE Il: UZtraaentrifugation studies: renin aativity in primary ae ZZ fraations ....•..••...•.•.••••..•.•...• 97 FIGURE 12: UZtraaentrifugation studies: renin aativity in subfraations of prima~ mitoahondriaZ fraations 98 FIGURE 13: Effeat of bi ZateraZ nephreatomy on brain iso-renin in doge ............................................ 108 FIGURE 14: Po Zyethy Zene tubing sys tem for repeated peri tonea'l diaZysis ........................................... 109 FIGURE 15: Effeat of bi ZateraZ nephreatomy on iso-renin aon·, tent of aaudate nua Zeus in brain tissue of doga .•.. 113 FIGURE 16: ~(feat of angiotensin antibodies on angiotensin extraat from brain tissue ....•.••..•••.....•.•.••.. 117 III FIGURE 17: f.0~hologiaal aspeat of dog brains at different ages ............................................ 121 FIGURE 18: Comparison of brain iso-renin and plasma renin in dogs of different ages •....•..••.••..••..••.•. 123 FIGURE 19: Water and eleatrolyte aontent of brain tissue (frontal aortex) from dogs of different ages ••••. 126 FIGURE 20: Potassium aontent and iso-renin of brain tissue (aœ<date nualeus)