BrainResearch, 375(1986)259-266 259 Elsevier BRE 11763 Quantitative Distribution of Angiotensin-Converting Enzyme (Kininase II) in Discrete Areas of the Rat Brain by Autoradiography with Computerized Microdensitometry FERNANDO M.A. CORREA*, LAURA M. PLUNKETT** and JUAN M. SAAVEDRA Section on Clinical Pharmacology, Laboratory of Clinical Science, NationalInstitute of Mental Health, Bethesda, MD 20892 (U.S.A.) (Accepted October 22nd, 1985) Key words: quantitative enzyme autoradiography-- angiotensin-converting enzyme inhibitor -- kininase II -- brain nucleus -- microdensitometry of brain enzyme -- brain peptide We report the localization of angiotensin-converting enzyme (kininase II, EC 3.4.15.1) in discrete nuclei and areas of the rat brain by a quantitative autoradiographic technique using image processing coupled to computerized microdensitometry, after incubation of brain sections with the specific converting enzyme inhibitor [125I]351A. High angiotensin-converting enzyme levels are present in cir- cumventricular organs (organon subfornicalis and area postrema), the choroid plexus, and extrapyramidal areas (nucleus caudatus, globus pallidus and substantia nigra) with intermediate levels in selected hypothalamic, septal, habenular and brainstem nuclei. Our results support the idea that angiotensin II could be formed in specific brain areas, both outside and inside the blood-brain barrier. In other brain structures, such as the extrapyramidal areas, kininase II could be involved in the processing or metabolism of other brain peptides. INTRODUCTION Blood-borne angiotensin II (ANG), produced in the periphery by the renin-angiotensin system, acts on the brain to stimulate drinking, increase blood pressure, and release vasopressin 19. These effects are mediated, at least partially, through stimulation of ANG receptors located in circumventricular or- gans11,12,18,25. Like a number of other peptides that are synthe- sized and active in the periphery, local synthesis of ANG could also occur in brain. All components of the angiotensin system, including renin, the renin substrate angiotensinogen, the angiotensin-convert- ing enzyme (ACE, kininase II, dipeptidyl carboxy- peptidase, EC 3.4.15.1), ANG, and ANG receptors, have been shown to occur in the central nervous sys- tem3,9,11,12,16,18,20,24. Newly modified autoradiographic methods with image analysis, computerized microdensitometry, and comparison to 125I-standards have been used to localize and quantitate ANG receptors in rat brain n,t2. We extended these techniques to the study of ACE in individual nuclei of the rat brain, after in- cubation of brain sections with the specific ACE in- hibitor [125I]351 msa,8,29,3°. MATERIALS AND METHODS Animals and tissue preparation Adult male Sprague-Dawley rats, (250 g) were kept under standard laboratory conditions for 1 week with lights on from 06.00 to 18.00 h. Rats were sacri- ficed by decapitation between 09.00 and 11.00 h and their brains were immediately removed and frozen by immersion in isopentane (-30 °C). Within 24 h of sacrifice tissue sections (16 ktm) were cut in a cryostat * Department of Pharmacology, School of Medicine of Ribeirao Preto, USP 14100 Ribeirao Preto, Sao Paulo, Brazil. ** Pharmacology Research Associate, National Institute of General Medical Sciences. Correspondence: J.M. Saavedra, Section of Clinical Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, 9000 Rockville Pike, Bldg. 10, Rm. 2D46, Washington, DC 20205-1000, U.S.A.