Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1599-1603, March 1984 Neurobiology

Autoradiographic visualization of angiotensin-converting enzyme in rat with [3H]captopril: Localization to a striatonigral pathway (/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 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 , the median preoptic graphic studies, sections were incubated at 4°C for 5 min in area, and the . 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 (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. + + + ND 0 0 Hypothalamus Median eminence +++ + + + + ++++ Supraoptic n. ++ + ++ Perikarya Paraventricular n., magnocellular + + ++++ Perikarya Suprachiasmatic n. 0 0 +++ + Periventricular n. 0 0 ++++ + Medial habenula + +++ ++ 0 Locus coeruleus + ++ ++ ++ Median + 0 ++ ++ Spinal cord 0 ND 0 ++++ Hippocampus 0(+)$ 0 0 + Cerebellum 0(+)$ + 0 0 Neocortex 0 0 0 + ND, not determined; n, nucleus. *Data from microdensitometry after autoradiography with 3 nM [3H]captopril. + + + + +, >1501 fmol per mg of protein; + + + +, 1500-701 fmol per mg of protein; + + +, 700-301 fmol per mg of protein; + +, 300-151 fmol per mg of protein; +, 150-51 fmol per mg of protein; 0, < 50 fmol per mg of protein. tData from refs. 12 and 13. + + + + +, >200 pmol per ,g of protein FIG. 1. [3H]Captopril binding to rat brain. (A) Toluidine blue per hr; + + + +, 200-101 pmol per ug of protein per hr; + + +, 101- staining; (B) [3H]captopril autoradiography; (C) [3H]captopril auto- 50 pmol per ,g of protein per hr; + +, 50-21 pmol per ug of protein radiography in the presence of 1 ,uM captopril. Note the intense la- per hr; +, 20-11 pmol per ,ug of protein per hr; 0, 10 pmol per ,g of beling of the choroid plexus (Ch) in B. The caudate putamen (Ca) protein per hr. and the substantia nigra (SN) are also visualized by autoradiogra- *A-I1 receptors, data from ref. 4. + + + +, Very high; + + +, high; phy. The streaks of decreased grain density in the caudate putamen + +, moderate to high; +, low to moderate; 0, low or very low. correspond to the location of tracts. The unmarked §Data from refs. 2 and 3. + + + +, Most intense or high; + +, moder- arrows indicate the band that appears to connect the caudate puta- ate or low-moderate; +, widely scattered or scattered; 0, none. men and substantia nigra. 1A low level of labeling was observed in a few sections. Neurobiology: Strittmatter et aL Proc. NatL. Acad. Sci. USA 81 (1984) 1601

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FIG. 2. [3H]Captopril autoradiography of the choroid plexus, subfornical organ, entopeduncular nucleus, median eminence, and of the hypothalamus. (A) The subfornical organ (SFO) and the adjacent choroid plexus (Ch) are intensely labeled in this dark-field view obtained using an emulsion-coated coverslip. The surrounding tissue exhibits few silver grains. (x 80.) (B) [3H]Captopril labeling of the entope- duncular nucleus (EP) and median eminence (ME) are shown in this low-power coronal section. (x2.) Intense staining of the choroid plexus and moderate staining of the tail of the caudate putamen are also visible. (C) Bright field reveals toluidine blue staining. The supraoptic nucleus of the hypothalamus (SO) is apparent as are several blood vessels, indicated with arrows. (x80.) (D) Observation of the same field as in Fig. 3C under dark-field conditions illustrates the presence of silver grains produced -by [3H]captopril in an emulsion-coated coverslip. The supraoptic nucleus of the hypothalamus (SO) and blood vessels seen in Fig. 3C are labeled by [3H]captopril. fornical organ (Fig. 2A). [3H]Captopril binding is present in nority of all blood vessel profiles observed are labeled by high density throughout the anatomical extent of the subfor- [3H]captopril. nical organ, adjacent to the equally dense labeling ofthe cho- Several other brain areas display substantial [3H]capto- roid plexus. The area postrema was not examined. pril-associated grains, although with levels lower than the Outside the subfornical organ, the neuronal areas with the areas mentioned above. Within the hypothalamus, grains are greatest levels of [3H]captopril binding are the corpus stria- highly localized to the median eminence (Fig. 2B), the su- tum and substantia nigra (Table 1; Fig. 1). Extremely dense praoptic nucleus (Fig. 2 C and D), and the paraventricular [3H]captopril-associated silver grains are apparent through- nucleus (not shown). Negligible levels are present elsewhere out the caudate putamen. Streaks of grain-free zones reflect in the hypothalamus. white matter tracks passing through the caudate putamen. In the thalamic area, the highest grain densities occur in There is some variation in grain density in the caudate puta- the medial habenula with undetectable levels in the lateral men with highest levels being anterior and lateral. Gray mat- habenula. No portion of the itself possesses detect- ter regions of the corpus striatum examined under high mag- able concentrations of grains. nification show a-homogeneous distribution of silver grains, The median preoptic area and the locus coeruleus have as opposed to the localized binding to blood vessels ob- grain densities just above the level of detection. Ependymal served in Fig. 2 C and D. A lesser degree of [3H]captopril cells lining the ventricles exhibit low to moderate grain den- binding occurs in the globus pallidus, about one-half that of sity. the highest levels in the caudate putamen. The selectivity of [3H]captopril binding localization is ap- Autoradiographic grains can be observed in a band that parent in the many parts of the central nervous systems that appears to connect the caudate putamen and the zona reticu- display few if any grains. Negligible levels of binding occur lata of the substantia nigra (Fig. 1). An enlargement of this throughout the cerebral cortex, cerebellum, most of the band is detectable in the area of the entopeduncular nucleus brain stem, spinal cord, and hipjpocampus (Table 1). (Fig. 2B). Effects of Brain Lesions on ['HICaptopril Binding in the Within the substantia nigra, grains occur throughout the Caudate Putamen and Substantia Nigra. To explore the pos- zona reticulata with negligible grain density in the zona com- sibility of a descending striatonigral localization of [3H]cap- pacta. At some levels of the substantia nigra, variations in topril sites, we injected colchicine or ibotenic acid unilateral- grain density are apparent within the zona reticulata (Fig. 3). ly into the caudate putamen (Fig. 4). Colchicine destroys A small zone of increased grain density occurs in the most cells by interfering with microtubular function. Ibotenic acid dorsal portion of the zona reticulata. Examination at high selectively destroys neuronal cells intrinsic to the site of in- magnification reveals a homogenous distribution of silver jection (14). By day 7 after these injections, a marked deple- grains in the substantia nigra, zona reticulata, as in the gray tion of [3H]captopril-associated grains is readily apparent in matter of the corpus striatum. the caudate putamen at the site of injection (Fig. 4; Table 2). In some brain sections [3H]captopril labels blood vessels Grain density in the ipsilateral substantia nigra is markedly (Fig. 2 C and D). It is not possible to determine whether the depleted 14 days after the injections into the corpus striatum, grains are overlying endothelium or muscle layer. Only a mi- while only partial depletion is apparent after 7 days. The de- 1602 Neurobiology: Strittmatter et aLPProc. Nad Acad Sci. USA 81 (1984) Table 2. Effect of lesions on [3H]captopril binding [3H]Captopril binding, fmol per mg of protein Caudate putamen Substantia nigra Control Lesion Control Lesion Drug Day side side side side Caudate putamen lesions Ibotenic acid 7 706 85 875 680 14 695 128 646 375 Colchicine 7 638 85 841 536 14 833 85 782 306 Substantia nigra lesions Ibotenic acid 7 926 859 604 758 14 808 800 629 646 Colchicine 7 850 850 790 799 14 800 808 620 629 I 6-OH- 14 960 1054 791 706 Autoradiography was carried out with 3 nM [3H]captopril and fol- lowed by microdensitometry. The values are the average from two sections of each of two rats that varied by <20%. From most sec- tions, the highest density observed in the indicated brain region is FIG. 3. The effect of substantia nigra injections on [3Hlcaptopril reported. However, in the caudate putamen sections from brains autoradiography. Ibotenic acid (A and B), colchicine (C and D), or 6- injected in the caudate putamen, binding was measured in the center hydroxydopamine (E and F) was injected into the left substantia ni- of the obvious lesions (Fig. 4) and in a corresponding position on the gra. After 14 days, autoradiography was done. Note the equality of control side. labeling between the left, lesioned side and the right, control side in the caudate putamen sections (A, C, and E) and in the substantia nigra sections (B, D, and F). The variation in the density of labeling to the injected caudate putamen, thus indicating the selectiv- within one substantia nigra, which can be observed in B, D, and F is ity of the colchicine and ibotenic acid injections. Injections present whether or not an injection has been made into the left sub- of saline alone have no effect on ACE levels after 7 days stantia nigra. The intensely labeled structure in A, C, and E is the (data not shown). choroid plexus. We also carried out unilateral injections into the substantia nigra of colchicine, ibotenic acid, or 6-hydroxydopamine. 6- pletion of binding throughout the substantia nigra is at least Hydroxydopamine injections in the substantia nigra selec- as great as that shown in Table 2, which reports the maxi- tively destroy dopamine-containing cells (15). None of these mum density found at any one point in the substantia nigra as three treatments alters [3H]captopril binding levels in either opposed to the average density. [3H]Captopril-associated the substantia nigra or the caudate putamen (Fig. 3; Table 2). grains are not affected in the choroid plexus closely adjacent The effectiveness of these nigral lesions to destroy the ni- grostriatal dopamine system was tested in adjacent sections from the same brains. All three lesions caused a unilateral depletion of [3H]mazindol binding in the ipsilateral caudate putamen (unpublished observations). [3H]Mazindol labels neuronal dopamine uptake sites (16). There appears to be some somatotopic relationship of the [3H]captopril binding sites in the caudate putamen and sub- I stantia nigra (Fig. 4). Thus, injections that cause a loss of [3H]captopril binding in the more medial portions of the cau- date putamen are associated with a greater loss of [3H]capto- pril binding in more medial portions of the substantia nigra. DISCUSSION [3H]Captopril autoradiography reveals ACE in specific brain regions (Table 1). The results agree with localizations ob- tained in microdissection studies measuring ACE catalytic activity (Table 1) (12, 13). However, autoradiographic analy- sis has permitted a more complete localization of the en- zyme. Thus, it has been possible to visualize ACE in the FIG. 4. The effect of caudate putamen lesions on [3H]captopril entopeduncular nucleus, in a band apparently connecting the autoradiography. Ibotenic acid (A and B) or colchicine (C and D) corpus striatum and substantia nigra, and in the gray matter were injected into the caudate putamen. After 14 days, autoradiog- of the caudate putamen. Immunohistochemical techniques raphy was carried out. The arrows indicate the location of the le- have also been used to localize ACE in the brain. Antibody sion. Note the decrease in [3Hlcaptopril-associated grains on the in left, lesioned side in the caudate putamen in A and C and in the directed against rabbit lung ACE detected ACE the cho- substantia nigra in B and D. The right, control side is unaffected. roid plexus, the subfornical organ, and scattered blood ves- The choroid plexus is the intensely labeled structure in A and C; it is sels of the rat brain (3, 17, 18). This antibody does not detect unaffected by the lesions. There is a degeneration in the size of the the prominent striatonigral ACE system. Antibody to human caudate putamen and an enlargement of the lateral ventricle in A and kidney ACE did stain the substantia nigra and globus palli- C on the lesioned side. dus but revealed little ACE in the caudate putamen of rat or Neurobiology: Strittmatter et aL Proc. Natl. Acad. Sci. USA 81 (1984) 1603 (19). Other regions, such as the neocortex, hip- in the body are the lungs and testes. Recently, we have visu- pocampus, and hypothalamus, which show much lower lev- alized [3H]captopril binding sites associated with ACE in the els of ACE by [3H]captopril autoradiography or by measure- male reproductive system of the rat (unpublished observa- ment of catalytic activity, were stained by this antibody. tions). ACE is highly concentrated in seminiferous tubules of In some regions in which [3H]captopril reveals the pres- the testes and in the epithelial surface and lumen of the epi- ence of ACE, the role of the enzyme appears to be the pro- didymis. duction of A-II. However, the caudate putamen, the sub- stantia nigra, zona reticulata, the entopeduncular nucleus, This work was supported by U.S. Public Health Service Grants and the globus pallidus exhibit high concentrations of MH-18501, DA-00266, NS-16375, and RSA Award DA-00074 to [3H]captopril binding with no evidence for A-TI receptors or S.H.S., Training Grant GM-07309 to S.M.S. and J.A.J., and a grant endogenous A-IT. Striatal lesions carried out using ibotenic of the McKnight Foundation. acid or colchicine show that ACE in the substantia nigra is present on presynaptic terminals of axons originating in the 1. Lang, R. E., Unger, T., Rascher, W. & Ganten, D. (1983) in ipsilateral caudate putamen and that the ACE of the caudate Handbook ofPsychopharmacology, eds. Iversen, L. L., Iver- putamen is present in cell bodies projecting to the substantia sen, S. D. & Snyder, S. H. (Plenum, New York), Vol. 16, pp. nigra or is associated with striatal interneurons. Our autora- 307-361. diographic studies agree with earlier studies showing that 2. Ganten, D., Fuxe, K., Phillips, M. I., Mann, J. F. E. & Gan- striatal ibotenate lesions deplete ACE catalytic activity in ten, U. (1978) in Frontiers in , eds. Gan- ong, W. F. & Martini, L. (Raven, New York), Vol. 5, pp. 61- the caudate putamen (20, 21) and in the substantia nigra (22). 101. Moreover, in Huntington's disease, the massive destruction 3. Brownfield, M. S., Reid, I. A., Ganten, D. & Ganong, W. F. of the corpus striatum is associated with a 75% depletion of (1982) Neuroscience 7, 1759-1769. ACE activity in the substantia nigra (22). The absence of ef- 4. Mendelsohn, F. A. O., Quirion, R., Saavedra, J. M., Agilera, fects on ACE after substantia nigra lesions shows that the G. & Catt, K. J. (1984) Proc. Natl. Acad. Sci. USA 81, 1575- nigral changes observed with striatal lesions are not due to 1579. trans-synaptic effects. 5. Cushman, D. & Ondetti, M. (1980) Biochem. Pharmacol. 29, Because ACE is a dipeptidylcarboxypeptidase with wide 1871-1877. substrate specificity (23), the enzyme may act on a substrate 6. Strittmatter, S. M., Kapiloff, M. S. & Snyder, S. H. (1983) other than Biochem. Biophys. Res. Commun. 112, 1027-1033. angiotensin I to either convert an inactive precur- 7. Unnerstall, J. R., Niehoff, D. L., Kuhar, M. J. & Palacios, sor into a biologically active neuropeptide or to destroy J. M. (1982) J. Neurosci. Methods 6, 59-73. some neuropeptide. The finding that ACE is contained in a 8. Young, W. S., III, & Kuhar, M. J. (1979) Brain Res. 179, 255- descending striatonigral pathway indicates that such a hypo- 270. thetical neuropeptide may also occur in this pathway. Sub- 9. Patchett, A. A., Harris, E., Tristram, E. W., Wyuratt, M. J., stance P immunoreactive projections exist from the striatum Wu, M. T., Taub, D., Petersen, E. R., Ikeler, T. J., ten to the zona reticulata of the substantia nigra, the globus palli- Broeke, J., Payne, L. G., Ondeyka, D. L., Thorselt, E. D., dus, and the entopeduncular nucleus (24). The fact that sub- Greenlee, W. J., Lohr, N. S., Hoffsomer, R. O., Joshua, H., stance P contains a carboxyl-terminal amide had suggested Ruyle, W. V., Rothrock, J. W., Aster, S. D., Maycock, A. L., that it not be a Robinson, F. M., Hirschmann, R., Sweet, C. S., Ulm, E. H., might substrate for ACE. However, we have Gross, D. M., Vassil, T. C. & Stone, C. A. (1980) Nature recently shown that substance P is readily cleaved by pure (London) 288, 280-283. ACE (unpublished results). Of course, the striatonigral en- 10. Roques, B. P., Fournie-Zalaski, M. C., Soroca, E., Lecomte, dogenous substrate of ACE may be a heretofore unknown I. M., Maltroy, B., Llorens, C. & Schwartz, J.-C. (1980) Na- neuropeptide. ture (London) 288, 286-288. Whereas ACE in the striatonigral system appears unrelat- 11. Fulcher, I. S., Matsas, R., Turner, A. J. & Kenny, A. J. (1982) ed to endogenous angiotensin, the ACE activity of other Biochem. J. 203, 519-522. brain regions may be involved with A-TI production. ACE in 12. Saavedra, J. M., Fernandez-Pardal, J. & Chevillard, C. (1982) the choroid plexus may regulate the A-TI content of the ven- Brain Res. 245, 317-325. 13. Chevillard, C. & Saavedra, J. M. (1982) J. Neurochem. 38, tricles (25) from which the octapeptide can exert its dipso- 281-284. genic and hypertensive effects on the circumventricular or- 14. Schwarcz, R., Hokfelt, T., Fuxe, K., Johnsson, G., Goldstein, gans. The subfornical organ and the median preoptic area, M. & Terenius, L. (1979) Exp. Brain Res. 37, 199-216. where A-IT regulates drinking behavior (1), contain A-TI re- 15. Jonsson, G., Malmfors, T. & Sachs, C., eds. (1975) 6-Hydroxy- ceptors and [3H]captopril binding, suggesting the formation dopamine as a Denervation Tool in Catacholamine Research of A-TI at these sites. A-TI may regulate antidiuretic (North-Holland, Amsterdam). disposition in the median eminence, the supraoptic nucleus, 16. Javitch, J. A., Blaustein, R. 0. & Snyder, S. H. (1983) Eur. J. and the paraventricular nucleus of the hypothalamus where Pharmacol. 90, 461-462. the peptides have been colocalized to the same neurons (26). 17. Rix, E., Ganten, D., Schull, B., Unger, T. & Taugner, R. The medial habenula and locus coeruleus contain both (1981) Neurosci. Lett. 22, 125-130. 18. Wigger, H. J. & Stalcup, S. A. (1978) Lab. Invest. 38, 581-585. [3H]captopril binding and A-TI receptors. Several areas con- 19. Defendini, R., Zimmerman, E. A., Weare, J. A., Alhenc-Ge- tain little ACE but significant levels of A-II receptors-i.e., las, F. & Erdos, E. G. (1983) Neuroendocrinology 37, 32-40. the lateral olfactory tract and its nuclei, the organum vascu- 20. Singh, E. A. & McGeer, E. G. (1978) Ann. Neurol. 4, 85-86. losum of the lamina terminalis, the , and 21. Fuxe, K., Ganten, D., Kohler, C., Schull, B. & Speck, G. the suprachiasmatic and periventricular nuclei of the hypo- (1980) Acta Physiol. Scand. 110, 321-323. thalamus (4). Brain regions with high A-TI levels but no ACE 22. Arregui, A., Emson, P. C. & Spokes, E. G. (1978) Eur. J. include the central nucleus of the amygdala, the bed nucleus Pharmacol. 52, 121-124. of the , and parts of the spinal cord (2, 3). 23. Krutzsch, H. C. (1981) Biochemistry 19, 5290-5296. The use of to in 24. Jessell, T. M. (1983) in Handbook of Psychopharmacology, [3H]captopril binding characterize ACE eds. Iversen, L. L., Iversen, S. D. & Snyder, S. H. (Plenum, homogenates (6) and in brain slices of autoradiographic stud- New York), Vol. 16, pp. 1-105. ies provides a powerful extension of receptor-binding tech- 25. Arregui, A. & Iversen, L. L. (1978) Eur. J. Pharmacol. 52, niques to membrane-associated enzymes. [3H]Captopril 147-150. autoradiography can also be used to localize ACE in other 26. Kilcoyne, M. M., Hoffman, D. L. & Zimmerman, E. A. parts of the body. The two tissues with highest ACE activity (1980) Clin. Sci. 59, 57s-60s.