STEPHEN M. STRITTMATTER, MATHEW M. S. Lo, JONATHAN A. JAVITCH, AND SOLOMON H. ..... Mendelsohn, F. A. O., Quirion, R., Saavedra, J. M., Agilera,.
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
We have visualized angiotensin-converting ABSTRACT enzyme (ACE; dipeptidyl carboxypeptidase, peptidylpeptide hydrolase, EC 3.4.15.1) in rat brain by in vitro [3H~captopril autoradiography. [3H]Captopril binding to brain slices displays a high affinity (Kd = 1.8 x 10-9 M) and a pharmacological profile similar to that of ACE activity. Very high densities of [ H]captopril binding were found in the choroid plexus and the subfornical organ. High densities were present in the caudate putamen and substantia nigra, zona reticulata. Moderate levels were found in the entopeduncular nucleus, globus pallidus, and median eminence of the hypothalamus. Lower levels were detectable in the supraoptic and paraventricular nuclei of the hypothalamus, the medial habenula, the median preoptic area, and the locus coeruleus. Injection of ibotenic acid or colchicine into the caudate putamen decreased [3Hlcaptopril-associated autoradiographic grains by 85% in the ipsilateral caudate putamen and by >50% in the ipsilateral substantia nigra. Thus, ACE in the substantia nigra is located on presynaptic terminals of axons originating from the caudate putamen, and ACE in the caudate putamen is situated in neuronal perikarya or at the terminals of striatal interneurons. The lack of effect of similar injections into the substantia nigra confirmed that the caudate putamen injections did not cause trans-synaptic changes. The presence of [3lH]captopril binding is consistent with an ACE-mediated production of angiotensin II in some brain regions. Although [31H]captopril autoradiography reveals ACE in a striatonigral pathway, there is no evidence for angiotensin II involvement in such a neuronal pathway.
Angiotensin II (A-IT) is an octapeptide that increases blood pressure peripherally by direct vasoconstriction and stimulates aldosterone release and, hence, salt reabsorption. The central actions of A-IT include stimulation of drinking, increased salt appetite, increase of blood pressure, and release of several pituitary hormones (1). A-IT immunoreactivity (2, 3) and A-TI receptor binding (4) have been identified in the central nervous system. Angiotensin-converting enzyme (ACE; dipeptidyl carboxypeptidase, peptidylpeptide hydrolase, EC 3.4.15.1) is the dipeptidylcarboxypeptidase that produces circulating AII by removing histidylleucine from angiotensin I. Captopril is an extremely potent and selective ACE inhibitor that is highly effective in treating hypertension (5). Recently, we described the binding of [3H]captopril to ACE in membrane fractions of the brain and in various peripheral tissues (6). In the present study, we have visualized ACE in the brain by autoradiographic analysis of [3H]captopril binding and compared its distribution to that of endogenous A-TI and A-II receptors. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1599
MATERIALS AND METHODS
[Prolyl-3,4-3H]-S-acetylcaptopril (New England Nuclear; 50
= 37 GBq) was converted to [3H~captopril by treatment with 0.1 M NaOH for 20 min at 23°C as described (6). Male Sprague-Dawley rats (150-200 g) were anesthetized with pentobarbital and perfused via the left ventricle of the heart with 0.9% NaCl/50 mM sodium phosphate, pH 7.5, and then with 50 mM sodium phosphate/0.3 M sucrose. Brains were removed, embedded in brain paste, and rapidly frozen at -70°C on chucks. Sections (8 ,um) were cut at -15°C and thaw-mounted on gelatin-coated slides. The slides were dessicated and stored at -20°C. For autoradiographic studies, sections were incubated at 4°C for 5 min in 50 mM Tris HCl, pH 7.9 (4°C)/100 mM NaCl/2 mg of bovine serum albumin per ml (Sigma, RIA grade) and then incubated for 40 min at 4°C in the same buffer with [3H]captopril (standard concentration, 3 nM) and any inhibitors. Nonspecific binding was determined in the presence of 1 ,M captopril. After two consecutive 1-min washes in the same buffer, the slides were dipped in water and immediately dried under a stream of cold air. Autoradiograms were generated by exposing LKB Ultrofilm to the slides for 12 days at 4°C (7) or by apposition of emulsion-coated coverslips for 14 days at 4°C (8). Tissue was stained after autoradiography with 0.1% toluidine blue. Density of autoradiograms on Ultrofilm was quantified by microdensitometry and converted to fmol of [3H]captopril bound per mg of protein (7). Saturation analysis of binding used 0.22, 0.67, 2, 6, and 18 nM [3H]captopril. The highest level of binding in the serial sections of caudate putamen was quantified as described above. Total and nonspecific binding for each concentration were averaged from two sections from each of two brains that varied by less than 15%. For lesion studies, 4 ,ug of colchicine (Sigma), 15 ,ug of ibotenic acid (Regis, Morton Grove, IL), or 8 ug of 6-hydroxydopamine hydrobromide (Sigma) in 2 ,ul of 0.9% NaCl were injected over 1 min into the center of the left caudate putamen or the left substantia nigra using stereotaxic coordinates measured from the interaural line. The location of the needle tip in an age-matched rat was confirmed by dissection after the injection of a dye. Coronal sections at the level of the caudate putamen and of the substantia nigra were obtained as described above either 7 days or 14 days after the
Ci/mmol; 1 Ci
injections.
For inhibition studies, caudate putamen sections were incubated with 3 nM [3H]captopril and inhibitor concentrations that varied by factors of 10. The highest density of binding in the caudate putamen was determined by autoradiography and microdensitometry. Concentrations of inhibitors that produced 50% inhibition were determined graphiAbbreviations: ACE, angiotensin-converting enzyme; A-TI, angiotensin II.
1600
Proc. NatL Acad Sci. USA 81
Neurobiology: Strittmatter et al.
cally and Ki values were calculated assuming competitive inhibition and a Kd of 1.8 x 10-9 M for [3H]captopril. The results for each inhibition concentration were averages of two sections from each of two brains that varied by 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 per hr; + + + +, 200-101 pmol per ug of protein per hr; + + +, 10150 pmol per ,g of protein per hr; + +, 50-21 pmol per ug of protein per hr; +, 20-11 pmol per ,ug of protein per hr; 0, 10 pmol per ,g of protein per hr. *A-I1 receptors, data from ref. 4. + + + +, Very high; + + +, high; + +, moderate to high; +, low to moderate; 0, low or very low. §Data from refs. 2 and 3. + + + +, Most intense or high; + +, moderate or low-moderate; +, widely scattered or scattered; 0, none. 1A low level of labeling was observed in a few sections.
Neurobiology: Strittmatter et aL
Proc. NatL. Acad. Sci. USA 81 (1984)
1601
.:SO fl.
FIG. 2. [3H]Captopril autoradiography of the choroid plexus, subfornical organ, entopeduncular nucleus, median eminence, and supraoptic nucleus 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 entopeduncular nucleus (EP) and median eminence (ME) are shown in this low-power coronal section. (x 2.) 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 high density throughout the anatomical extent of the subfornical organ, adjacent to the equally dense labeling of the choroid plexus. The area postrema was not examined. Outside the subfornical organ, the neuronal areas with the greatest levels of [3H]captopril binding are the corpus striatum and substantia nigra (Table 1; Fig. 1). Extremely dense [3H]captopril-associated silver grains are apparent throughout the caudate putamen. Streaks of grain-free zones reflect white matter tracks passing through the caudate putamen. There is some variation in grain density in the caudate putamen with highest levels being anterior and lateral. Gray matter regions of the corpus striatum examined under high magnification show a -homogeneous distribution of silver grains, as opposed to the localized binding to blood vessels observed in Fig. 2 C and D. A lesser degree of [3H]captopril binding occurs in the globus pallidus, about one-half that of the highest levels in the caudate putamen. Autoradiographic grains can be observed in a band that appears to connect the caudate putamen and the zona reticulata of the substantia nigra (Fig. 1). An enlargement of this band is detectable in the area of the entopeduncular nucleus (Fig. 2B). Within the substantia nigra, grains occur throughout the zona reticulata with negligible grain density in the zona compacta. At some levels of the substantia nigra, variations in grain density are apparent within the zona reticulata (Fig. 3). A small zone of increased grain density occurs in the most dorsal portion of the zona reticulata. Examination at high magnification reveals a homogenous distribution of silver grains in the substantia nigra, zona reticulata, as in the gray matter of the corpus striatum. In some brain sections [3H]captopril labels blood vessels (Fig. 2 C and D). It is not possible to determine whether the grains are overlying endothelium or muscle layer. Only a mi-
nority of all blood vessel profiles observed are labeled by
[3H]captopril.
Several other brain areas display substantial [3H]captopril-associated grains, although with levels lower than the areas mentioned above. Within the hypothalamus, grains are highly localized to the median eminence (Fig. 2B), the supraoptic nucleus (Fig. 2 C and D), and the paraventricular nucleus (not shown). Negligible levels are present elsewhere in the hypothalamus. In the thalamic area, the highest grain densities occur in the medial habenula with undetectable levels in the lateral habenula. No portion of the thalamus itself possesses detectable concentrations of grains. The median preoptic area and the locus coeruleus have grain densities just above the level of detection. Ependymal cells lining the ventricles exhibit low to moderate grain density. The selectivity of [3H]captopril binding localization is apparent in the many parts of the central nervous systems that display few if any grains. Negligible levels of binding occur throughout the cerebral cortex, cerebellum, most of the brain stem, spinal cord, and hipjpocampus (Table 1). Effects of Brain Lesions on ['HICaptopril Binding in the Caudate Putamen and Substantia Nigra. To explore the possibility of a descending striatonigral localization of [3H]captopril sites, we injected colchicine or ibotenic acid unilaterally into the caudate putamen (Fig. 4). Colchicine destroys cells by interfering with microtubular function. Ibotenic acid selectively destroys neuronal cells intrinsic to the site of injection (14). By day 7 after these injections, a marked depletion of [3H]captopril-associated grains is readily apparent in the caudate putamen at the site of injection (Fig. 4; Table 2). Grain density in the ipsilateral substantia nigra is markedly depleted 14 days after the injections into the corpus striatum, 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 Caudate putamen lesions Ibotenic acid
Colchicine
Day
side
side
side
side
7 14 7 14
706 695 638 833
85 128 85 85
875 646 841 782
680 375 536 306
Substantia nigra lesions Ibotenic acid
I
FIG. 3. The effect of substantia nigra injections on [3Hlcaptopril autoradiography. Ibotenic acid (A and B), colchicine (C and D), or 6hydroxydopamine (E and F) was injected into the left substantia nigra. After 14 days, autoradiography was done. Note the equality of 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 within one substantia nigra, which can be observed in B, D, and F is present whether or not an injection has been made into the left substantia nigra. The intensely labeled structure in A, C, and E is the choroid plexus.
pletion of binding throughout the substantia nigra is at least as great as that shown in Table 2, which reports the maximum density found at any one point in the substantia nigra as opposed to the average density. [3H]Captopril-associated grains are not affected in the choroid plexus closely adjacent
I
758 604 859 7 926 646 800 629 808 14 799 790 850 7 850 Colchicine 629 620 808 14 800 706 791 1054 960 6-OH-Dopamine 14 Autoradiography was carried out with 3 nM [3H]captopril and followed by microdensitometry. The values are the average from two sections of each of two rats that varied by
