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Autoradiographic Visualization of Angiotensin-Converting Enzyme In Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1599-1603, March 1984 Neurobiology Autoradiographic visualization of angiotensin-converting enzyme in rat brain with [3H]captopril: Localization to a striatonigral pathway (hypothalamus/circumventricular organs/dipeptidylcarboxypeptidase/ibotenic acid/colchicine) STEPHEN M. STRITTMATTER, MATHEW M. S. Lo, JONATHAN A. JAVITCH, AND SOLOMON H. SNYDER Departments of Neuroscience, Pharmacology and Experimental Therapeutics, Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205 Contributed by Solomon H. Snyder, November 29, 1983 ABSTRACT We have visualized angiotensin-converting MATERIALS AND METHODS enzyme (ACE; dipeptidyl carboxypeptidase, peptidylpeptide [Prolyl-3,4-3H]-S-acetylcaptopril (New England Nuclear; 50 hydrolase, EC 3.4.15.1) in rat brain by in vitro [3H~captopril Ci/mmol; 1 Ci = 37 GBq) was converted to [3H~captopril by autoradiography. [3H]Captopril binding to brain slices dis- treatment with 0.1 M NaOH for 20 min at 23°C as described plays a high affinity (Kd = 1.8 x 10-9 M) and a pharmacologi- (6). Male Sprague-Dawley rats (150-200 g) were anesthe- cal profile similar to that of ACE activity. Very high densities tized with pentobarbital and perfused via the left ventricle of of [ H]captopril binding were found in the choroid plexus and the heart with 0.9% NaCl/50 mM sodium phosphate, pH 7.5, the subfornical organ. High densities were present in the cau- and then with 50 mM sodium phosphate/0.3 M sucrose. date putamen and substantia nigra, zona reticulata. Moderate Brains were removed, embedded in brain paste, and rapidly levels were found in the entopeduncular nucleus, globus palli- frozen at -70°C on chucks. Sections (8 ,um) were cut at dus, and median eminence of the hypothalamus. Lower levels -15°C and thaw-mounted on gelatin-coated slides. The were detectable in the supraoptic and paraventricular nuclei of slides were dessicated and stored at -20°C. For autoradio- the hypothalamus, the medial habenula, the median preoptic graphic studies, sections were incubated at 4°C for 5 min in area, and the locus coeruleus. Injection of ibotenic acid or col- 50 mM Tris HCl, pH 7.9 (4°C)/100 mM NaCl/2 mg of bovine chicine into the caudate putamen decreased [3Hlcaptopril-as- serum albumin per ml (Sigma, RIA grade) and then incubat- sociated autoradiographic grains by 85% in the ipsilateral cau- ed for 40 min at 4°C in the same buffer with [3H]captopril date putamen and by >50% in the ipsilateral substantia nigra. (standard concentration, 3 nM) and any inhibitors. Nonspe- Thus, ACE in the substantia nigra is located on presynaptic cific binding was determined in the presence of 1 ,M capto- terminals of axons originating from the caudate putamen, and pril. After two consecutive 1-min washes in the same buffer, ACE in the caudate putamen is situated in neuronal perikarya the slides were dipped in water and immediately dried under or at the terminals of striatal interneurons. The lack of effect a stream of cold air. Autoradiograms were generated by ex- of similar injections into the substantia nigra confirmed that posing LKB Ultrofilm to the slides for 12 days at 4°C (7) or the caudate putamen injections did not cause trans-synaptic by apposition of emulsion-coated coverslips for 14 days at changes. The presence of [3lH]captopril binding is consistent 4°C (8). Tissue was stained after autoradiography with 0.1% with an ACE-mediated production of angiotensin II in some toluidine blue. Density of autoradiograms on Ultrofilm was brain regions. Although [31H]captopril autoradiography re- quantified by microdensitometry and converted to fmol of veals ACE in a striatonigral pathway, there is no evidence for [3H]captopril bound per mg of protein (7). angiotensin II involvement in such a neuronal pathway. Saturation analysis of binding used 0.22, 0.67, 2, 6, and 18 nM [3H]captopril. The highest level of binding in the serial Angiotensin II (A-IT) is an octapeptide that increases blood sections of caudate putamen was quantified as described pressure peripherally by direct vasoconstriction and stimu- above. Total and nonspecific binding for each concentration lates aldosterone release and, hence, salt reabsorption. The were averaged from two sections from each of two brains central actions of A-IT include stimulation of drinking, in- that varied by less than 15%. creased salt appetite, increase of blood pressure, and release For lesion studies, 4 ,ug of colchicine (Sigma), 15 ,ug of of several pituitary hormones (1). A-IT immunoreactivity (2, ibotenic acid (Regis, Morton Grove, IL), or 8 ug of 6-hy- 3) and A-TI receptor binding (4) have been identified in the droxydopamine hydrobromide (Sigma) in 2 ,ul of 0.9% NaCl central nervous system. were injected over 1 min into the center of the left caudate Angiotensin-converting enzyme (ACE; dipeptidyl car- putamen or the left substantia nigra using stereotaxic coordi- boxypeptidase, peptidylpeptide hydrolase, EC 3.4.15.1) is nates measured from the interaural line. The location of the the dipeptidylcarboxypeptidase that produces circulating A- needle tip in an age-matched rat was confirmed by dissection II by removing histidylleucine from angiotensin I. Captopril after the injection of a dye. Coronal sections at the level of is an extremely potent and selective ACE inhibitor that is the caudate putamen and of the substantia nigra were ob- highly effective in treating hypertension (5). Recently, we tained as described above either 7 days or 14 days after the described the binding of [3H]captopril to ACE in membrane injections. fractions of the brain and in various peripheral tissues (6). In For inhibition studies, caudate putamen sections were in- the present study, we have visualized ACE in the brain by cubated with 3 nM [3H]captopril and inhibitor concentra- autoradiographic analysis of [3H]captopril binding and com- tions that varied by factors of 10. The highest density of pared its distribution to that of endogenous A-TI and A-II binding in the caudate putamen was determined by autoradi- receptors. ography and microdensitometry. Concentrations of inhibi- tors that produced 50% inhibition were determined graphi- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: ACE, angiotensin-converting enzyme; A-TI, angio- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tensin II. 1599 1600 Neurobiology: Strittmatter et al. Proc. NatL Acad Sci. USA 81 (1984) cally and Ki values were calculated assuming competitive in- evaluating the potency of various ACE inhibitors to decrease hibition and a Kd of 1.8 x 10-9 M for [3H]captopril. The binding levels in striatal brain slices. MK-422, the results for each inhibition concentration were averages of active diacid of enalapril, and N-(l(S)-carboxy-3-phenyl- two sections from each of two brains that varied by <20%. propyl)-L-lysyl-L-proline, the lysyl analogue of MK-422, are potent ACE inhibitors (9) and potent in decreasing RESULTS [3H]captopril binding with Ki values of 7.5 and 15 x 10-9 M, respectively. Teprotide, a somewhat weaker ACE inhibitor Biochemical Properties of [3H]Captopril Binding to Slices of displays a Ki value of 235 x 10-9 M. By contrast, thiorphan, the Corpus Striatum. To ensure that [3H]captopril labels an extremely potent inhibitor of enkephalinase A (10), an en- ACE in brain slices, we evaluated the pharmacological prop- zyme also called endopeptidase 24.11 (EC 3.4.24.11) (11), is erties of [3H]captopril binding to slices of rat corpus striatum quite weak at [3H]captopril binding sites, failing to give 50% by microdensitometric analysis of autoradiograms. As we inhibition in concentrations as high as 100 ,uM. The chelating observed previously in homogenates of brain tissue (6), agent EDTA, which inhibits ACE activity, also decreases [3H]captopril binds saturably and with high affinity to stria- [3H]captopril binding to negligible levels at 1 mM. The selec- tal slices. Total binding with 3 nM [3H]captopril is about 100 tivity of these agents and the similarity of their potencies at times greater than nonspecific binding measured in the pres- [3H]captopril binding sites to their influences on ACE cata- ence of 1 AM captopril (Fig. 1C). Specific binding, the differ- lytic activity indicates that the sites visualized in the corpus ence between total and nonspecific binding, begins to pla- striatum by [3H]captopril autoradiography represent ACE. teau at about 6 nM and reaches half-maximal values at about Autoradiographic Localization of [ HICaptopril Sites in Rat 2 nM. Scatchard analysis reveals a single component of bind- Brain. Table 1 summarizes the distribution of specific ing (correlation coefficient of 0.99) with a dissociation con- [3H]captopril binding obtained by autoradiography and mi- stant (Kd) of 1.8 x 10-9 M and a maximal number of binding crodensitometry. The densest localization of [ H]captopril sites (Bmax) of 1500 fmol per mg of protein. These values binding sites occurs in the choroid plexus throughout the agree closely with those obtained in homogenate studies (6) brain (Figs. 1 and 2A). The choroid plexus is labeled in the and with the known potency of captopril as an inhibitor of lateral ventricles, the third ventricle, and the fourth ventri- ACE catalytic activity (5). cle. The specificity of [3H]captopril binding was examined by Almost as densely labeled as the choroid plexus is the sub- A Table 1. Distribution of ACE, A-II receptors, and A-II [3H]Captopril Region binding* ACEt A-IIRt A-h1 Choroid plexus +++++ +++++ 0 0 Subfornical organ +++++ +++++ ++++ 0 Caudate putamen ++++ ++++ 0 + Substantia nigra, zona reticulata ++++ +++ 0 + Globus pallidus +++ +++ 0 0 Entopeduncular n.
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