Angiotensin-converting enzyme labeled with [3H]captopril. Tissue localizations and changes in different models of hypertension in the rat. S K Wilson, … , D R Lynch, S H Snyder J Clin Invest. 1987;80(3):841-851. https://doi.org/10.1172/JCI113142. Research Article In vitro autoradiography with [3H]captopril was used to localize and quantitate angiotensin-converting enzyme (ACE) in various tissues in two-kidney, one-clip (2K-1C) hypertension, one-kidney, one-clip (1K-1C) hypertension, desoxycorticosterone acetate (DOCA)-salt hypertension, and a normotensive control group. There were no significant differences in mean systolic blood pressure among the hypertensive groups. Plasma renin activity (PRA) was highest in the 2K-1C group (6.20 +/- 2.17 ng/ml per h), intermediate in the 1K-1C group (2.19 +/- 0.62 ng/ml per h) and control group (3.20 +/- 0.53 ng/ml per h), and lowest in the DOCA-salt group (0.07 +/- 0.06 ng/ml per h). In the lungs, aorta, mesenteric arteries, and adrenal medulla, ACE labeling was highest in the 2K-1C group, intermediate in the 1K-1C and control groups, and lowest in the DOCA-salt group. ACE levels in these tissues correlated positively with PRA. In the kidney, anterior pituitary, testis, and choroid plexus of the brain, ACE levels correlated negatively with PRA, with lowest ACE levels in the 2K-1C group and highest levels in the DOCA-salt group. In the epididymis, posterior pituitary, and other regions of the brain, ACE levels did not differ significantly among the groups. Find the latest version: https://jci.me/113142/pdf Angiotensin-converting Enzyme Labeled with [3H]Captopril Tissue Localizations and Changes in Different Models of Hypertension in the Rat Stephen K. Wilson, David R. Lynch,* and Solomon H. Snyder* Departments ofPathology and *Neuroscience, The Johns Hopkins University School ofMedicine, Baltimore, Maryland 21205 Abstract walls, for example, ACE may generate ANG II locally as part of the vascular RAS (3-7). ACE and other components of this In vitro autoradiography with [3Hjcaptopril was used to local- vascular system may be involved in maintaining certain forms ize and quantitate angiotensin-converting enzyme (ACE) in of hypertension, perhaps independently of the plasma RAS various tissues in two-kidney, one-clip (2K-1C) hypertension, (3-5). ACE is also responsible for local ANG II production in one-kidney, one-clip (1K-1C) hypertension, desoxycorticoster- some regions of the brain (8, 9). ACE occurs in other tissues, one acetate (DOCA)-salt hypertension, and a normotensive such as adrenal, kidney, and pituitary gland (10), where it may control group. There were no significant differences in mean also generate ANG II, but its endogenous substrate is not systolic blood pressure among the hypertensive groups. clearly established. Plasma renin activity (PRA) was highest in the 2K-1C group The intent of the present study was to examine the rela- (6.20±2.17 ng/ml per h), intermediate in the 1K-1C group tionship of ACE to changes in the plasma RAS in hyperten- (2.19±0.62 ng/ml per h) and control group (3.20±0.53 ng/ml sion. To do so, we studied three models of hypertension asso- per h), and lowest in the DOCA-salt group (0.07±0.06 ng/ml ciated with high, normal, or low plasma renin levels. ACE was per h). In the lungs, aorta, mesenteric arteries, and adrenal labeled by in vitro autoradiography with [3H]captopril, which medulla, ACE labeling was highest in the 2K-1C group, inter- binds exclusively to ACE both in tissue homogenates and sec- mediate in the 1K-iC and control groups, and lowest in the tions (11-15), permitting the localization of particulate ACE DOCA-salt group. ACE levels in these tissues correlated posi- in tissue as well as quantitation of ACE levels by densitometry tively with PRA. In the kidney, anterior pituitary, testis, and (16, 17). choroid plexus of the brain, ACE levels correlated negatively with PRA, with lowest ACE levels in the 2K-IC group and highest levels in the DOCA-salt group. In the epididymis, pos- Methods terior pituitary, and other regions of the brain, ACE levels did not differ significantly among the groups. Male Wistar rats with initial weights of 200-220 g were obtained from Charles River Laboratories, Inc., Wilmington, MA. For each rat a baseline systolic blood pressure was recorded before surgery. Baseline Introduction blood pressures and all subsequent pressures were taken by the tail cuff method (18), during which rats were lightly anesthetized with ether. Angiotensin-converting enzyme (ACE)' plays a role in blood Pressure tracings were recorded on a physiograph (Narco Biosystems, pressure regulation as a component of the plasma renin-an- Houston, TX). Rats were then divided into four experimental groups. giotensin system (RAS) by converting angiotensin I (ANG I) to Group 1: two-kidney, one-clip (2K-IC) hypertension. After rats were the potent vasoconstrictor angiotensin II (ANG II) and de- anesthetized with sodium pentobarbital (40 mg/kg body wt, i.p.), an grading the vasodilator bradykinin (1). In the plasma RAS, abdominal incision was made and a 0.2-mm silver clip placed on the ACE conversion of ANG I occurs primarily in the lungs, pro- left renal artery. The right kidney and renal artery remained un- ducing changes in circulating ANG II levels that vary in ac- touched. After surgery, blood pressures were taken twice weekly (tail cordance with plasma renin levels (2). ACE also influences the cuff method) for 4 wk. Animals were given free access to tap water and local production of ANG II in various tissues. In blood vessel rat chow. Group 2: one-kidney, one-clip (1K-IC) hypertension. In this group a 0.2-mm silver clip was placed on the left renal artery and the right twice and Address reprint requests to Stephen K. Wilson, M.D., Department of kidney was removed. Blood pressures were recorded weekly, Pathology, The Johns Hopkins Hospital, Baltimore, MD 21205. the rats received tap water and normal rat chow. Receivedfor publication 10 February 1987 and in revisedform 27 Group 3: desoxycorticosterone acetate (DOCA)-salt hypertension. April 1987. Animals in this group underwent excision of the right kidney; then silicone rubber molds containing DOCA (Sigma Chemical Co., St. Louis, MO; 200 mg/kg body wt) were implanted subcutaneously (19). 1. Abbreviations used in this paper: ACE, angiotensin-converting en- This group was given 1% NaCl (instead of tap water) and normal rat zyme; ANG I and ANG II, angiotensin I and II; DOCA, desoxycorti- chow. Blood pressures were recorded twice weekly. costerone acetate; lK-lC, one-kidney, one-clip hypertension; 2K-1C, Group 4: nonhypertensive (control) group. Sham operations were two-kidney, one-clip hypertension; PRA, plasma renin activity; RAS, performed in which the left renal artery was manipulated (but not renin-angiotensin system. clipped) and the right kidney was left intact. Animals were given tap water and normal rat chow, and blood pressures were recorded twice J. Clin. Invest. weekly. © The American Society for Clinical Investigation, Inc. 4 wk after surgery all animals were sacrificed by decapitation. - 2 0021-9738/87/09/0841/11 $2.00 ml arterial blood from each rat was collected in chilled tubes contain- Volume 80, September 1987, 841-851 ing 0.2 ml EDTA for determination of plasma renin activity (PRA); all Angiotensin-converting Enzyme in Hypertension 841 samples were collected in one morning to avoid diurnal variation in if 00 ( renin levels (20). Serum was frozen at -20'C, and at a later time PRA o - - was determined by a modification of the microradioimmunoassay +1 gN e 6e0 (70% IIII, e -,NH to ,-.H en,m 11 00 .1 I4 a: 4 ;sotf .- _4 - _" _ method of Husain and Jones (21). Sections of lung, aorta, mesentery, _w _. .. -, kidney, adrenal gland, testis, brain, and pituitary gland were rapidly removed, embedded in Tissue-Tek (Miles Laboratories Inc., Naper- p p S;;. 4 Z;;;;. 0 ville, IL) or brain paste (except lung), and quickly frozen in liquid +1 -H +i -H 11 11 00 II 11 W it 2: it nitrogen. Tissue sections (8 Am) were cut on a cryostat, mounted on Irl --, .c -, t- -, § z gelatin-coated slides, and stored at -20'C. For in vitro autoradiographic studies, (prolyl-3, 4-[3H])-S-acetyl- r t. C , F4, -Sfn captopril (New England Nuclear, Boston, MA; 50 Ci/mmol) was con- v verted to [3Hlcaptopril with 0.1 M NaOH for 20 min at 230C as previously described (15). Slide-mounted tissue was first incubated in a 4i-v -HIo - buffer of 50 mM Tris-HCl, pH 7.9 (40C), and 100 mM NaCl at 4VC 0.- for 5 min, then transferred to a solution of [3H]captopril (8 nM) in the XtX-II e same buffer for 40 min at 4VC. Slides were given two 1-min washes in A R .- .J buffer, briefly dipped in distilled water, and dried in a stream of cool t -j .0= air. To assess nonspecific binding, slide-mounted tissue was treated as ToA,- skr described above except that the incubation solution contained 1 UM 0.- ,e 11 11 +n mu 1 1 unlabeled captopril or enalaprilat. ii Autoradiograms were produced by placing either Ultrofilm or emulsion-coated coverslips over the slide-mounted tissue for 12 d at - A. - - (N ^, 't e 4VC. Density of [3H]captopril labeling on Ultrofilm was quantitated by M- 0.-. microdensitometry and converted to femtomoles of [3H]captopril 0Q 0It 00 t Ito 1s 00 bound per milligram protein using [3H] standards obtained from Amersham (Arlington Heights, IL) (16).