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Angiotensin converting enzyme in rat brain visualized by quantitative in vitro autoradiography
NeuroscienceVol. 20, No. 2, pp. 615-627, 1987 Printed in Great Britain
03~~522/87 $3.08+ 0.08 Pergamon Journals Ltd 0
1987 IBRO
ANGIOTENSIN CONVERTING ENZYME IN RAT BRAIN VISUALIZED BY QUANTITATIVE IN VITRO AUTORADIOGRAPHY SIEW YEEN CHAI,
F. A. 0. MENDELSOHNand G. PAXINOG*
University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Victoria, Australia 3084 and *School of Psychology, University of New South Wales, Kensington, New South Wales, Australia, 2033 Abstract-Angiotensin converting enzyme was localized in rat brain by quantitative in u&o autoradiography using an [1251]labelledconverting enzyme inhibitor called “351A”. This radioligand was found to bind with high afhnity and specificity to angiotensin converting enzyme. Very high levels of converting enzyme were observed in the ventricular choroid plexus, ependyma of al1 ventricles and large and medium blood vessels, subfornical organ, and organum vasculosum of the lamina terminals. High levels of converting enzyme were found in the basal ganglia including caudate putamen, nucleus accumbens, globus palhdus, entopenduncular nucleus and substantia nigra pars reticulata. The neurosecretory nuclei, paravent~cular nucleus and supraoptic nucleus, as well as the median eminence and posterior pituitary displayed high levels of the enzyme. In the amygdala, basolaterai, lateral, basomedial, medial and anterior cortical nuclei showed moderate converting enxyme activity. The medial habenula and molectdar layer of the dentate gyrus showed high levels of activity. In the cerebellum, dense labelling was observed in the Purkinje cell layer. Moderate levels of converhrg enzyme occurred in the gelatinosus subnucleus of the caudai part of the nucleus of the spinal tract of the trigeminal. There was a close correspondence between the distribution of angiotensin converting enzyme and angiotensin II in the neurosecretory nuclei ~vent~~~~ and supraoptic nuclei) and median eminence and this suggests a role of angiotensin converting enzyme in the production of angiotensin II in this system. There was also a good correspondence between the distribution of angiotensin converting enzyme and angiotensin II in the subfomical organ, median preoptic nucleus, and organum vasculosum of the lamina terminalis, structures abutting the anterior wall of the third ventricle which are implicated in fluid and electrolyte homeostasis. A striking discrepancy occurs in the basal ganglia which is reported to contain very little angiotensin II or angiotensin II receptors but is very rich in angiotensin converting enzyme. It is concluded that the enzyme may act to (a) convert circulating angiotensin I to angiotensin II in circumventricular organs; (b) generate intraneuronal angiotensin II in pathways such as the hypothalamichypophyseal tract; and (cf process neuro~tid~ other than an~otensin II in regions such as basal ganglia.
Angiotensin converting enzyme (ACE) is a peptidyl dipeptidase which cleaves ~stidyl-leu~ne from the carboxyl terminal of angiotensin I to yield angiotensin II and also cleaves C-terminal peptide from a variety of other peptides including bradykinin,” substance P,6 enkephalins,‘2 and neurotensin.3f ACE has been localized on the huninal surface of endothelial cells in all vascular beds”*q and also in epithelial cells in kidney,** intestine> and testis3’ In the brain, microdissection studies have shown high concentrations of ACE in the choroid plexus, basal ganglia, subfomical organ (SFO), and area postrema.‘JO Moderate amounts of ACE occur in some thalamic and neurosecretory hypothalamic nuclei.’ Immunohistochemical techniques have also detected high con~ntrations of the enzyme in the choroid plexus, the SFO, and in blood vessels.39~ioA study using goat antibody against rabbit ACE did not
detect any enzyme in neural tissues of the rat brain other than in the SFO.* Experiments using rabbit antisera to human ACE detected the enzyme in dendrites of the globus pallidus and substantia nigra in both human and rat brain.‘O Because of these discrepancies, we developed a method to map the distribution of ACE in the rat brain by quantitative in vitro autoradiography using an [12sI]labelled derivative of the potent ACE inhibitor lysinop~l as specific radioligand.24”5 At the same time, St&matter et al. reported similar studies using [3H]captopril as radioligand.” Autoradiography using [‘*‘1]351A (N-[(s)- 1-car~xy-3-phenylpropyl]-L-lysyl-~~osyl-Lproline) offers a number of advantages over [3H]captopril and appears to provide higher resolution which permits detailed anatomi~l l~alization of brain ACE as we report here. EXPERIMENTAL PROCEDURES
~bbr~iufio~: ACE, angiotensin converting enzyme; AII, anaiotensin II: HACBO-GIY, N-(R,S)_3-hydroxyaminocs;bonyl-2-be&yl-1-oxoptopyl-glycine; hydroxamic acid derivative, I-(N-hydroxycarboxamido)-4-methyl-pantanolyl-L-~anyl-Byrne amide; 351A, N-[(s)-l-carboxy-3phenylpropyl]-L-iysyi-t~rosyl-L-proline.
Radio&ml A derivative of the ACE inhibitor lysinopril,37 (kindly donated by Dr C. Sweet, Merck Institute for Therapeutic Research, West Point, PA, U.S.A.), called “35lA” was radio-iodinated by the chloramine T method,” purified on 615
SIEW YEEN CHAI el al
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an SP-C25 sephadex column and eluted with 0.1 M ammonium acetate, pH 3.5. The binding properties of this radio&and to lung membranes, a rich source of ACE, have been described.Z4 Characterization
of binding properties
The sections used for autoradiography were stained with thionin for anatomical localization of the autoradiog~phs. RESULTS
Properties
of [ ‘2s1]35
1A binding
Characteristics of binding of [‘Z*I]351A to brain memThe ability of a number of converting enzyme and branes was evaluated as follows: A lOO&2O,OOOg enkephalinase inhibitors to displace binding of radiomembrane-rich fraction (200 fig protein) prepared from rat caudate putamen was incubated with - 10 nCi of [izSI]351A ligand [12’1]35IA from caudate putamen membranes is shown in Fig. 1. All three of the chemically in OS ml of 10 mM sodium phosphate buffer, pH 7.4, containing ISOmM NaCl and 2 g/l bovine serum albumin 100 (buffer A) and a range of con~ntrations of converting enzyme and enkephalinase inhibitors at 20°C for I h. Free and bound radioligands were separated by filtration through Whatman GF/B glass fibre filters. The [‘251]radioactivity was measured and the binding isotherms analysed by a 80 non-linear, iterative, model-fitting computer programme.*’ The converting enzyme inhibitors were obtained as follows: B/,p lysinopril was donated by Dr C. Sweet, Merck Institute for Therapeutic Research, Westpoint, PA, and captopril and 60 teprotide were donated by the Squibb Insitute for Medical Research, Princetown, NJ. The enkephaiinase inhibitor, 2-AN-hydroxy~rboxamido)-4-methyl ~ntanolyl-L-alanylglycine amide (“hydroxamic acid derivative”)‘” from Calbiochem-Behring, San Diego, CA and phosphor40 amidone” from Sigma Chemical Company, St. Louis, MO. Quantitative in vitro autoradiography
For autoradiography, male Sprague-Dawley rats (* 250 g) were anaesthetized with sodium methohexitone (0.5 mg/kg), perfused via the left ventricle with 200 ml of isotonic saline and then 1% parafo~aldehyde in saline. The brains were then removed, snap frozen in isopentane at -40°C. Coronal sections were cut at 20pm and sagittal sections at 1Opm in a cryostat maintained at -20°C. Sections were thaw-mounted onto gelatin-coated slides and dried in a desiccator for 2 h at 4”Cn The sections were preincubated in buffer A for 15 min at 20°C and then incubated in buffer A containing 0.3 nCi/ml of [‘251]3SlAfor 1 h at 20°C. Non-specific binding was determined in parallel incubations containing either 1mM ethyien~iami~etetraacetate (EDTA) or 1 PM lysinopril. After incubation, the sections were transferred through 4 successive i min washes of 50mM Tris-HCl buffer, pH 7.4 at 0°C. The slides were dried under a stream of cold air, loaded into X-ray cassettes and exposed to Kodak NMB-1 X-ray film for 7 days at room temperature. In each cassette, a set of [rZsI]radioactivity standards was included. These were prepared by applying known amounts of [‘251]radioactivity to 5 mm diameter discs of 20/rm thickness brain sections mounted on coated slides. In addition, the system was calibrated for enzyme activity using a set of enzyme standards. The standards were prepared by serial dilutions of caudate putamen membranes in 1% gelatin solution in buffer A; five ~1 aliquots were applied to subbed slides and dried at 4°C in a dessicator. These were then carried through the same incubation procedure as for the brain sections and exposed to X-ray film for the same period of time. Samples of the membrane suspensions were also assayed for ACE by a fluorimetric method.” The protein content of the membranes was determined by a modified Lowry method.i4 The optical densities obtained from these standards were then converted to ACE activity. The X-ray films were processed in a Kodak RP X-OMAT automatic developer and the optical density quantitated using an EyeCom model 850 image analysis system (Spatial Data Systems, Springfield, VA) coupled to a DEC 1t/23 LSI computer. The optical density of the autoradiographs was calibrated both in terms of dpm per square mm using the radioactivity standards or in terms of the ACE activity (pmol/min/mg protein) by fitting calibration curves with the computer.
20
0 Concentration ( MI Fig. 1. Specificity of binding displacement of [“‘1]351A to
amide), A phosphoramidone. Abbreviations wed in jigures
3v 4v 7n ac aca Acb ACo AHi AID AOP APit APT Aq asc7 AV bas BL BM BST BV CA1 CA3 CB Cb cc CeL CeM CG Cg
third ventricle fourth ventricle facial nerve or root of facial nerve anterior commissure anterior commissure, anterior part accumbens nucleus anterior cortical amygdaloid nucleus amygdalohippocampa1 area agranular ittsutar cortex, dorsal part anterior olfactory nucleus, posterior part anterior lobe of the pituitary anterior pntectal area cerebral aqueduct (Sylvius) ascending fibers of the facial nerve anteroventral thalamic nucleus basilar artery basolateral amygdaloid nucleus
baaomaW 8mygdalbid nudeus bed m&us of the stria terminalis blood vessel field CA1 of Amman’s horn field CA3 of Atnmon’s horn cell bridges of the ventral striatum cuebeliurn corpus caiIosum oenttml arny@oid nucleus, lateeal part astral m nucleur, medial part central (pctiqualuctal) gray cingulate cortex
Angiotensin converting enzyme in rat brain CGD CGLD CGLV CGM CL CLi CM CnF CPU DC0
DG DLG DLL DPB DR DTg ECU Ent EP k fr G ?Z GP HDP IAM IC ic icp IF InG Int rnwh IO IF IPit La Lat LD IfP LH LHb LM IO
LOT LP LR4V LS LV LVe MD ME me5 Med MG MiTg ml mlf MM MnPO MnR MoDG MP MPO MS mt MTu MVE OP
central gray, dorsal part central gray, lateral dorsal part central gray, lateral central part central gray, medial part centrolateral thalamic nucieus caudal (central) linear nucleus of the raphe central medial thalamic nucleus cuneiform nucleus caudate putamen (striatum) dorsal cochlear nucleus dentate gyrus dorsal lateral geniculate nucleus dorsal nucleus of the lateral lemniscus dorsal parabrachial nucleus dorsal raphe nucleus dorsal tegmental nucleus external cuneate nucleus entorhinal cortex entopeduncular nucleus fomix fimbria of the hippocampus fasciculus retroflexus (habenulointerpeduncular tract) gelatinosus thalamic nucleus genu of the facial nerve gigantocellular reticular nucleus globus pallidus nucleus of the horizontal limb of the diagonal band (Broca) interanterome~al thalamic nucleus inferior colliculus internal capsule inferior cerebellar peduncle {restiform body) interfa~icular nucleus intermediate gray layer of the superior colliculus interposed (inte~ediate) cerebellar nucleus intermediate white layer of the superior colliculus inferior olive interpeduncular nucleus intermediate lobe of the pituitary lateral amygdaloid nucleus lateral (dentate cerebellar nucleus) Iaterodorsal thalamic nucleus longitudinal fasiculus of the pons lateral hypothalamic area lateral habenular nucleus lateral mammillary nucleus lateral olfactory tract nucleus of the lateral olfactory tract lateral posterior thalamic nucleus lateral recess of the fourth ventricle lateral septal nucleus lateral ventricle lateral vestibular nucleus mediodorsal thalamic nucleus median eminence mesencephalic tract of the trigeminal nerve medial (fastigial) cerebellar nucleus medial geniculate nucleus microcellular tegmental nucleus medial lemniscus media1 longitudinal fasciculus medial mammillary nucleus, medial part median preoptic nucleus median raphe (superior central) nucleus molecular layer of the dentate gyrus medial mammilla~ nucleus, posterior part medial preoptic area medial septal nucleus mammillothalamic tract medial tuberal nucleus medial vestibular nucleus optic nerve layer
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optic tract nucleus of the optic tract otfactory ventricle (olfactory part of lateral ventricle) optic chiasm parabigeminal nucleus k paracentral thalamic nucleus PC posterior commissure parvocellular reticular nucleus ERt PDTg posterior dorsal tegmental nucleus paragigantocellular reticular nucleus PGi PH posterior hypothalamic nucleus Pir piriform cortex (primary olfactory cortex-“PO”) posteromedial cortical amygdaloid nucleus (C3) PMCo pontine nuclei Pn PnC pontine reticular nucleus, caudal part PIIO pontine reticular nucleus, oral part PO posterior thalamic nuclear group PP peripeduncular nucleus PPit posterior lobe of the pituitary pedunculopontine tegmental nucleus PPTg Pr5 principal sensory trigeminal nucleus prepositus hypoglossal nucleus PrH presubiculum PrS paraventricular thalamic nucleus PV paraventricular thalamic nucleus, posterior part PVA pyramidal tract red nucleus PRY reuniens thalamic nucleus Re rhomboid thalamic nucleus Rh raphe magnus nucleus RMg reticular thalamic nucleus Rt superior colliculus SC supr~hiasmatic nucleus SCh superior cerebellar peduncle ~brachium conjunc=P tivum) subfomicai organ SF0 stria medullaris of the thalamus sm substantia nigra, compact part SNC SNR substantia nigra, reticular part so supraoptic nucleus Sol nucleus of the solitary tract superior olive so1 spinal tract of the trigeminal nerve sP5 SPFPC subparafascicular nucleus, parvocellular part sphenoid nucleus Sph spinal vestibular nucleus SpVe st stria terminalis SuG superficial gray layer of the superior colliculus SUM supramammillary nucleus SuVe superior vestibular nucleus T-f taenia tecta (anterior hippocampal rudiment) Tu olfactory tubercle VCoA ventral cochlear nucleus, anterior part VDB nucleus of the vertical limb of the diagonal band (Broca) VL ventrolateral thalamic nucleus VLGMC ventral lateral geniculate nucleus, magnocellular part VLGPC ventral lateral geniculate nucleus, parvocellular part VLL ventral nucleus of lateral lemniscus VM ventrom~ial thalamic nucfeus VMH ven~om~ial h~thalamic nucleus VP ventral pallidurn VPB ventral (medial) parabrachial nucleus VPL ventral posterolateral thalamic nucleus VPM ventroposteromedial thalamic nucleus VTA ventral tegmental area (Tsai) decussation of the superior cerebellar peduncte x=P Y nucleus Y ZI zona incerta zo zona1 layer of the superior colliculus opt OT ov
SEW YEEN CHAI cf d.
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unrelated converting enzyme inhibitors completely displaced the radioligand. Computer analysis of the binding isotherms 27 for the different ACE inhibitors gave the following Ka values: lysinopril, 7.8 t_ 3.4 x 109; enalaprilat, 3.6 f 1.0 x IO*; captopril, 4.4 f 1.4 x 10’ and teprotide, 9.5 f 1.5 x IO6M.. ‘. Specific binding of the radioligand was abolished by 1 mM EDTA. Non-specific binding in the presence of an excess (1 PM) of lysinopril or 1 mM EDTA was 1.O% of total binding. The enkephalinase inhibitors (the hydroxamic acid derivative and phosphoramidone) were very weak in inhibiting binding of the radioligand with IC~‘s of approximately 4 and 50pM, respectively. Autoradiographic localization of angiotensin converting enzyme
The levels of ACE in selected structures are shown in Table 1. For descriptive purposes the density of ACE was scored as follows: very high density, > 1000 pmol/min/mg protein; high density 400-l 000; moderate density, 2W399; low density 50-199; undetectable < 50. Non-specific binding, measured in the presence of 1 PM lysinopril or I mM EDTA, was completely undetectable and produced no visible image on the X-ray films. The autoradiographs shown in Figs 2-4 therefore represent specific binding only. The ventricles and periventricular structures
The choroid plexus of all ventricles contains very high densities of ACE (Figs 2D,H: 3H: 4A,B). The ependymal walls of the olfactory, lateral, third, and fourth ventricles, and central canal has high densities of ACE. The SF0 and vascular organ of the lamina terminalis show a very high density of ACE (Figs 2E and 4A),- while the area postrema only a low density. Large blood vessels show dense labelling on their endothelial walls (Fig. 3F). Medium blood vessels are also densely labelled and can be seen in all figures as longitudinal streaks or spots particularly in the cortex against the low background. The olfactory system
The olfactory bulb, the accessory olfactory bulb and the anterior olfactory nusleus display very low levels of ACE. The lateral olfactory tract (Fig. 23,C) and the anterior part of the anterior commissure (Fig 2B,C) are devoid of ACE activity. The basal ganglia
The caudate intensely labelled the reticular part and 4B,C). The
putamen (striatum) is the most neural structure in the brain after of the substantia nigra (Figs 2A-G distribution of ACE within the
caudate putamen is uneven with patches of high and moderate density. The accumbens nucleus displays dense labelling in its ventrolateral part and only slight binding in its dorsomedial part (Fig. 2 B,C). The olfactory tubercle displays no activity although the finger-like extensions of the striatum into the tubercle (the striatal bridges) are moderately labelled (Fig. 2C). The globus pallidus had very high density of ACE in its lateral part which abuts the striatum and was moderate in its medial parts in which the pallidally associated basal nucleus of Meynert is found (Fig. 2 E-G). The bundles of fibres penetrating the striatum and gathering in the internal capsule were negative. A few such bundles were positioned bctween the striatum and globus pallidus and provide a demarcation of these structures (Fig. 2E). The internal capsule is negative except for its ventral part which includes the striatonigral ACE-containing projection. The ventral pallidurn contains moderate activity which becomes progressively weaker with increased distance from the globus pallidus (Fig. 4C). The reticular part of the substantia nigra displays the most intense activity of any nucleus in the brain (Fig. 3A,B). The compact part of the nigra does not label intensely (Fig. 3A,B). Intense binding is present in the entopeduncular nucleus (Figs 2F,G and 4C). The striatonigral projection containing ACE courses through the entopeduncular nucleus and therefore some of the activity seen in the region of the nucleus may be associated with the fascicles that pass through the nucleus and not with the nucleus itself. However, the binding immediately rostra1 and caudal to the nucleus is less than in the nucleus itself and we can conclude that the nucleus itself is labelled as well. The subthalamic nucleus displays only low activity. The septum and hypothalamus
The medial septum displays a slight labelling which is continuous with the labelling expressed by the nuclei of the vertical limbs of the diagonal band (Fig. 2D). The lateral septum has undetectable activity as does the septohippocampal nucleus (Fig. 2D). The nucleus of the horizontal limb of the diagonal band displays a slightly less activity than the nucleus of the vertical limb of the diagonal band. The median preoptic area is moderately labelled (Fig. 4A) and so is most of the medial preoptic area (Fig. 4A). The lateral preoptic area is not labelled. The neurosecretory nuclei, supraoptic, paraventricular and accessory neurosecretory cells groups show high levels of ACE activity. The suprachiasmatic nucleus has very low ACE activity which
Fig. 2 (opposite). Autoradiographic localization of angiotensin converting enzyme (ACE) in rat forebrain (coronal plane) using [‘29]351A(3 days exposure). Non-specific binding in the presence of 1 mh4 EDNA or I ,uM lysinopril eompktely abolished these patterns of binding.
Fig. 4. Autoradiographic localization of ACE in rat brain (sagittal plane) using [‘z’I]351A(7 da]IS exposure).
Fig. 3 (opposire). Autoradiographic localization of ACE in rat forebrain, midbrain and cerebellum (coronal plane) using [‘2s1]351A (3, 3 and 7 days exposure, respectively). 621
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SEW YEEN CHAI et al.
Table 1. Angiotensin converting enzyme in rat brain quantitated by in vitro autoradiography ACE activity (pmol/min/mg prot.) + SD Ventricles and periventricular structures Choroid plexus Subfomical organ Vascular organ of the lamina terminalis Median blood vessels Basal ganglia Caudate putamen Accumbens nucleus (ventrolateral) Globus pallidus Entopeduncular nucleus Substantia nigra, reticular part Anterior commissure Internal capsule Pituitary Anterior Posterior Intermediate lobe Hypothalamus and septum Nucleus of the vertioal limb of the diagonal band Median preoptic nucleus Supraoptic nucleus Paraventricular nucleus Median eminence Median preoptic area Anterior hypothalamic area Septohypothalamic nucleus Retrochiasmatic area Fornix Thalamus Reuniens thalamic nucleus Paraventricular thalamic nucleus Anteroventral thalamic nudeus Laterodorsal thalamic nucleus Medial habenular nucleus Zona incerta Amygdala nuclei Basolateral Central Anterior cortical Anterior amygdaloid area Bed nucleus of the stria terminalis Hippocampus Dentate gyrus Cerebral cortex Striate Frontoparietal Cingulate Corpus callosum Cerebellum Purkinje cell layer Molecular layer Granular layer Midbrain Central gray Interpeduncular nucleus Pontine nucleus Superior colBcuIus (itcmwdiate gray) Inferior colliculus Brain stem Nucleus of the solitary tract Solitary tract Inferior olive Nucleus ambiguus Spinal tract of the t&em&al nerve
> 1000 > 1000 395 + 54 605 + 54 702+ 18 460+18 424 k 18 425 + 19 825 + 200 < 50 < 50 406+40 >I000 287 + 8 414 + 16 334 It 42 444 Itr3s 436 + 30 757 f 12 250 f 10 384i9 229 f 22 256 + 23 < 50 407&12 256 ?; 28 358 & 21 174+20 413+23 290+30 320 f 17 384 + 12 368 &-14 383+11 352 f 14 635 k 53 268 + 23 291 & 12 326 f 10 < 50 570 f 16 35Of8 326 4 12 384 + 16 350 f 32 325 205* 11 238 f 12 370+6 < 50 294 + 7 375 220 f 8
Angiotensin converting enzyme in rat brain
was lower than the adjacent anterior hypothalamic area. The median eminence is also densely labelled in the region abutting the third ventricle and on its external surface (Fig. 4A). The posterior putuitary contains very high levels of ACE (Fig. 4 A,B) while the anterior pituitary and the intermediate lobe of the pituitary display moderate binding (Fig. 4A,B). In the hypothalamus, binding is expressed in the arcuate, ventromedial, dorsomedial, lateral, perifornical and mammillary nuclei (Fig. 2F-H). The supramammillary nucleus is moderately labelled (Fig. 3A) and the posterior hypothalamic nucleus displays slightly less binding (Fig. 3A). The thalamus
The ventral nuclear complex nuclei are homogeneously negatively labelled. The ventrolateral, ventromedial, ventroposterolateral, ventroposteromedial, ventroposteromedial parvocellular and the gelatinosus nuclei (Fig. 2 F-G) all display low ACE activity. The reticular thalamic nucleus is only lightly labelled (Figs 2G and 4C) but stands out against its nonpositive neighbours (internal capsule and ventral nuclear complex). The anterior nuclear group displays some binding in its anterodorsal and anteroventral nuclei (Fig. 4B). The mediodorsal nucleus is nearly devoid of activity (Figs 2 F, G and 4A). The lateral nuclear groups (laterodorsal and lateroposterior nuclei) are negative (Fig. 2F,G). The intralaminar group displays some binding in its central medial, paracentral, central lateral and parafascicular divisions (Figs 2FG and 4A). The midline nuclear group displays some labelling in its paraventricular, paratenial, rhomboid, and reuniens divisions but is nearly devoid of activity in its interanteromedial division (Figs 2FG and 4A). The subparafascicular nucleus and its parvocellular part display a slight activity which is detectable against the extremely low activity of the ventroposterior group and the absence of activity in the medial lemniscus (Fig. 2H). The medial habenula and medial part of the lateral habenula display a moderate amount of binding (Figs 2G and 4A) and the fasciculus retroflexus is positive in its core (Figs 2H and 3A) suggesting that this fibre bundle carries fibres containing ACE. Part of the lateral subnucleus of the interpeduncular nucleus is moderately labelled (Fig. 3B) but it cannot be deduced that the fasciculus retroflexus fibres containing ACE terminate there without experimental interventions. The amygdala
The basolateral amygdaloid nucleus is the most densely labelled nucleus of the amygdala (Fig. 2F). The lateral nucleus also displays moderate to low activity in some of its subdivisions (Fig. 2H). The MC.20/2--R
623
lateral part of the central nucleus stands out by its lack of activity (Fig. 2F,G). The medial part of the central nucleus displays labelling similar to that of the adjacent medial amygdaloid nucleus (Fig. 2G). The basomedial nucleus shows moderate binding (Fig. 2G) as does the anterior cortical. The nucleus of the lateral olfactory tract is conspicuous by its low activity contrasted by the lightly labelled anterior amygdaloid area and anterior cortical amygdaloid nucleus (Fig. 2F). The posterior cortical amygdaloid nucleus shows very low activity (Fig. 3A). The piriform cortex shows some activity in its plexiform layer but no activity in its pyramidal layer (Fig. 2B,C). The bed nucleus of the stria terminalis shows only slight binding (Figs 2D and 4B) as does the sublenticular substantia innominata (Fig. 2E). The amygdalohippocampal area is devoid of activity (Fig. 2H). The visual system
The ventral lateral geniculate is moderately labelled while the dorsal lateral geniculate is nearly devoid of binding (Fig. 2H). The anterior pretectal (Fig. 2H) and olivary pretectal areas show moderate ACE activity. The superior colliculus showed differential distribution in its layers with very low activity in the optic nerve and deep white layer, low density in the zonal and deep gray layers and slightly higher density in the intermediate gray and white layers (Fig. 3B). The ventral tegmental area
The ventral tegmental area displays low binding (Fig. 3B). The interpeduncular nucleus shows moderate to high activity in its lateral subnucleus (Fig. 3B). The hippocampai region
The most striking feature of the hippocampus was the high density of ACE in the molecular layer of the dentate gyrus (Figs 2H; 3A,B; 4C). The granule cell layer of the dentate gyrus and the pyramidal cell layer of Ammon’s horn were devoid of activity (Fig. 3B). Similarly the stratum oriens, radiatum, and laconosum moleculare of Ammon’s horn are negative (Figs 2H and 3A). The subiculum, presubiculum, and parasubiculum show low levels of labelling (Fig. 3B,C). The entorhinal cortex shows ACE activity in some of its layers (Fig. 3C,D). The cerebral cortex The cerebral cortex displays only low activity. The greatest labelling is found in the granular insular cortex (Fig. 2B,C), and the anterior cingulate cortex (Fig. 2A,B). The rest of the cortex displays very low activity. The reticular formation and the central gray
The reticular nucleus of the medulla displays very low ACE activity in the dorsal and ventral parts.
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SIEW YEEN CHAI et u/.
The gigantocellular and parvocellular reticular nuclei display low binding (Fig. 3G). The pontine caudal and oral nuclei and the paragigantocellular nuclei are similarly negative (Figs 3D,E and 4B). The central gray and the pedunculopontine tegmental nucleus show moderate activity (Fig. 3A-D). The dorsal tegmental nucleus shows low labelhng with some differences in its subnuclei (Fig. 4A). The posterior dorsal tegmental nucleus shows moderate binding (Fig. 4A). The sphenoid nucleus*’ (Fig. 4A) and the locus coeruieus also display moderate binding. The raphe nuclei
The dorsal and medial raphe display low ACE activity (Fig. 3D) but the raphe pallidus, obscurus and magnus are nearly devoid of activity (Fig. 3&I&G). The interfascicular and caudal linear nuclei show some ACE activity (Figs 3B,C and 4A). Brain stem nuclei ~sociated with respiratory, cardio vascular and other auto~mi~ f~~etions
The nucleus of the solitary tract displays moderate to low binding in both its medial and lateral parts (Fig. 3G,H). The ambiguus nucleus is moderately labelled. The dorsal parabrachial, ventral parabrachial and Kolliker-Fuse nuclei display a low but significant level of ACE activity (Fig. 4B).
low activity while the lateral and spinal vestibular nuclei show very low activity (Fig. 3F.G). The precerebellar nuclei and the red nucleus
The inferior olive shows moderate labelling {Fig. 4A) as do the pontine nuciei (Figs 3C and 4B) while the lateral reticular nucleus shows very little binding. The red nucleus is nearly devoid of ACE activity (Fig. 3B). The cerebellum
The lateral, interpasitus and medial nuclei show light labelling (Fig. 3F,G). The cerebellar cortex presents an interesting binding pattern. There is a line of high density labelling at the site of the Furkinje cells (Fig. 3E-H). The rest of the molecular layer as well as the plexiform layer of the folia is moderately labelfed. Fibre tracts
All fibre tracts, for example: corpus callosum, fomix, fimbria, medial lemniscus, longitudinal ftbers of the pons, and superior cerebeltar p&&e are negative except for the fasciculus retroflexus and the solitary tract which expressed some ACE activity and the segment of the internal capsule containing the striatonigral ACE projection which is very densely labelled (Fig. 2G).
The orofacial motor nuclei
The motor trigeminal and facial nuclei are nearly devoid of activity (Fig. 3F,G). The h~glos~~ nucleus shows low ACE activity as does the ntteleus of Roller. The somatosensory system
The gracilis and cuneate nuclei show very low labelling. The external cuneate nucleus shows low ACE activity (Fig. 3H). The principal nucleus of the trigeminakas well as the nucleus of the spinal tract of the trigeminal show very low binding (Figs 3&H and 4C) except for the subnucleus gelatinosus of the caudal part of the nucleus of the @inal tract of the trigeminal which is si~i~c~tly lab&& (not shown). The auditory system
The cochlear nuclei show a low ACE activity (Figs 3E,F and 4C). The lateral superior olive shows very low activity (Fig. 3E) and the nuclei of the trapezoid body are nearly devoid of activity as are tbe nuclei of the lateral lemniscus, the inferior C&C&W and the medial geniculate (Figs 3A,B,D,E, and 4C). The vestibular system
The superior vestibular and infracerebelhtr nucleus as well as nucteus X show moderate binding which is the highest observed in the vestib&ar nuclei (Fig. 3E,F). The medial ~ti~lar n~~~Fi~ 3 F,G} and the p~positus .~~l~l nudeus (Fig. 3F) show
Pro~rt~s
of the [L25~351A radiol~g~d
The ra~oliga~ [“51]351A used in this study, a derivative of the potent spe&Ic ACE i~~or ly*
sinopril,” was found to bind with ~~~~ to saturable sites in brain membranes. This bin&g was abolished by I mM EDTA, which is known to.&&te the essential Zn atom at the catalytic site of ACE a& block its enzymatic activity.’ Binding of the radioligand was also completely inhibited by a range of converting enzyme inhibitors of diverse cben&al structures. The potency of these compounds in displacing the radiohgand closely paraWed their anticataiytic activity ap;ainst converting enxyme.324 The inhibitor, phos~or~~one*6 and ibe hydrox~ic acid derivative of Ala-Gly ~id~which are active against enkephalinase with 1~~‘s of 40 and 3 nM, in di~l~~n~ weak very respectively5-were [r*‘I]351A with tcs,“s at least 1000 times higher. These findings indicate that this radiofiesaMf labels the active cataIytic site of a vetting enzyme in brain membranes and &o S$OW that it does not appreciably label en under these conditions. Autora~r~h~c verting enzyme
localization of arzgiotensin con -
The present auto~a~a~~ extend earlier observations
i-es&s coobtained by
and micro-
Angiotensin converting enzyme in rat brain
dissection and [3H]captopril autoradiography in which high levels of ACE activity were found in the choroid plexus,7.u subfornical organ,30 striatum and the reticular part of the substantia nigra,7Y and the neurosecretory hypothalamic nuclei and the posterior pituitary.3’ In the present autoradio~aphic study, a comprehensive mapping of brain ACE was undertaken which reveals many previously unknot reactive sites and elaborates on the dist~bution in regions known to contain the enzyme. A very high density of ACE evident in the choroid plexus, subfomical organ and blood vessels suggests a likely role for ACE in converting circulating angiotensin I to angiotensin II. Possible roles of bloodborne angiotensin in regulating blood pressure, drinking and vasopressin release have been reviewed.zo~32It should also be noted that very high levels of ACE were found in the ependymal lining in all ventricles and the cerebral aqueduct. A strikingly high ACE activity was observed in all major components of the basal ganglia. The striatum, accumbens nucleus, globus pallidus, entopenduncular nucleus and substantia nigra. The distribution within these nuclei was not homogenous. Compartments that are rich and poor in ACE were observed in the striatum. The accumbens nucleus displayed highest activity in its ventrolateral part. The globus pallidus displayed greater ctincentrations of the enzyme in its lateral portion. Dense labelling in the reticular part of the substantia nigra was not continued in the compact part. It is significant to note that the ventrom~ial segments of the internal capsule displayed intense ACE activity. Descending fibres of the longitudinal fasciculus of the pons (posterior to the substantia nigra) showed no detectable ACE. This supports the suggestion that the enzyme is contained in the striatonigral projection neurons. 34The topographic relation of ACE in the basal ganglia and the proposed pathway is seen at an advantage in the sagittal plane (Fig. 4C). The presence of ACE in the hypothalamic neurosecretory nuclei (supraoptic and paraventricular nuclei), median eminence and posterior pituitary is consistent with the proposed role of angiotensin II in the regulation of vasopressin and oxytocin release.18.20 The distribution of ACE in a number of other brain regions showed excellent correspondence to the known parcellation of these regions. For example, in the hippocampus, the enzyme was localized to the molecular layer of the dentate gyrus. A laminar distribution was also evident in the superior colliculus and the cerebellum. In the cerebellum, a remarkable strip of activity was found in the Purkinje cell layer. The medial habenular nuclei as well as the medial part of the lateral habenular nuclei showed high ACE activity. The lateral subnucleus part of the inter~nduncular nucIeus was positive. The fasciculus retroflexus showed a positive core and this suggests
625
that the known projection of the habenular nuclei to the interpeduncular nucleus may contain ACE.” There is a major problem in reconciling the distribution of ACE as determined here with the distribution of its putative major product, angibtensin II, which has been recently mapped by immunohistochemistry.22 While there are areas of correspondence where both substances are found in high concentrations, notably the subfomi~l organ, paraventricular and supraoptic nucleus and median preoptic nucleus, there are striking incongruities. For example, high densities of ACE are found in the striatum, globus pallidus, entopeduncular nucleus, substantia nigra reticulata and dentate gyrus areas reported to be devoid of angiotensin II cells and fibres.” Conversely, very low ACE activity was observed in the lateral part of the central nucleus, of the amygdala, a region reported to have very dense concentrations of AII cell bodies and fibres.” A paradox exists if the high density of immuno~activity for AI1 cells and fibres reported in the lateral part of the central amygdaloid nucleus is indeed specific AII. This dissociation would suggest the presence of a product (AH) without its generating enzyme (ACE) and would necessitate the postulation of alternative pathways of AI1 formation. While there are gross discrepancies in the overall distribution of ACE and AII, in some specific systems the correspondence was striking. For instance in the paraventricular and supraoptic nuclei of the hypothalamus as well as median eminence and posterior pituitary, both ACE (current study) and angiotensin II receptors. A similar correspondence of AII, AI1 receptors and ACE holds for the subfomi~l organ, vascular organ of the lamina terminalis and the median preoptic area. However, some of the most reactive regions for ACE such as the choroid plexus, caudate putamen, accumbens nucleus, globus pallidus, entopeduncular nucleus, substantia nigra and dentate gyrus and Purkinje cell layer of the cerebellum were primarily found to be conspicuously free of AI1 receptors. 26 Conversely, high AI1 receptor concentrations previously found in the suprachiasmatic nucleus, lateral olfactory tract and locus coeruleus were not paralleled by corresponding en~chment of ACE in the current study. The pattern of ACE dist~bution displays a remarkable similarity to the labelling of enkephalinase with the specific inhibitor [3H]HACBO-Gly3s However there are some differences in that high levels of ACE were detected in paraventricular, supraoptic, median preoptic, median eminence and vascular organ of the lamina terminalis, which are not rich in enkephalinase. 38 Conversely, the olfactory tubercle, which has a high concentration of enkephalinase, contains low levels of ACE. It is also clear that the two radioligands label different specific enzymes since the potent angiotensin converting enzyme inhibitor, captopril, did not displace the enkepha~na~ radioligand in the experiments of Waksman3$ and in our
626
Smw YEENCHAI
studies, the potent enkephahnase inhibitors, phosphoramidone and hydroxamic acid were very weak inhibitors of the binding of (‘251]3S1A. used to label ACE. Abundant substance P-immunoreactive fibres have been Iocalized in the striatonigral pathway” which also contained high levels of ACE in the current study. Since substance P has been shown to be a substrate for brain ACE33 it is possible the ACE in this pathway is involved in the hydrolysis of this peptide. Indeed, support for this concept comes from the finding that an isoenzyme of ACE recently purified from rat brain corpus striatum differed from lung ACE in molecular weight and peptide substrate specificity although the two enzymes displayed very similar immunological properties and susceptibility to inhibitors.36 The striatal enzyme cleaved substance P, substance K and bombesin at a relatively faster rate than ACE and showed a differed pattern of cleavage products when hydrolysing substance P,36A high concentration of substance P-immunoreactive fibres has also been demonstrated in medial and central amygdaloid nucleus,z3 and the substantia gelatinosa of the spinal cord,8 brain regions which display moderate densities of ACE. However, there are also marked differences in the distribution of substance P as compared to ACE. For example, in the cerebellum, ACE is detected in high ievels in the Purkinje cell layer whereas only a very low levels of substance P and immunoreactive fibres are present.
fv d.
Similarly, the olfactory bulb and tuber&, structures which are rich in substance P positive nerve terminals” contain relatively low levels of ACE.
CONCLUSfON
In the brain, the high ~on~ntraiion of ACE detected in some regions such as the subfomical organ and vascular organ of the iamina terminalis suggests that the enzyme may convert circuiating angiotensin I to angiotensin II which could then interact with the high density of AI1 receptors localized in these organs” to elicit actions on drinking, vasopressin release and blood pressure controLzo The existence of ACE with other components of the angiotensin system in the paraventricular and median preoptic, and supraoptic nuclei and median eminence is compatible with a role for the enzyme in generating neuronal angiotensin II in these structures. ACE was also found in high levels in the basal ganglia, dentate gyrus and cerebellum which are not well endowed with AI1 or its receptors; it is therefore likely that ACE in these structures might be involved in the processing of some other neuropeptide. Acknowledgements-Supported
by grants from the National Heart Foundation of Australia and the Austin Hospital Medical Research Foundation to F. A. 0. Mendeisohn and National Health and Medical Research Council to G. Paxinos
REVERENCE
enzyme: presence of higb activity in choroid plexus of mammaiian brain. Eur. J. Piuzrmac.52, 147-150. Browniield M. S., Ried I. A., Ganten D. and Ganong W. F. (1982) Differential dis~ibution of immunore~ti~ angiotcnsin and an~otensin~onve~ing enzyme in rat brain. Neuroscience 1, 1759-1769. Bull H. G., Thornberry N. A.. Co&s M. H. J., Patchett A. E. and Cordes E. H. (1985) Inhibition. of rabbtt lung angiotensin-converting enzyme by N”-[(s)-l-carboxy-3-phenyl-propyl]-t-ala~yl-L-proline and N”-[(S)-l-carboxy-Jpbeny$ropylJ-t-lysyl+proline. J. biol. Chem. 260, 2952-2962. Bunning P. and Riordan J. F. (1985) The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism. J. Inorgun. B&hem. 24, 183-198. Caldwcll P. R. B., Seegal B. C., Hsu K. C., Das M. and Soffer R. L. (1976) Angiotensin-converting enzyme: vascular endothelial localization. Science 191, 1050-1051. Caseieri M. A., Bull H. G., Mumford R. A., Patchett A. E., Thornberry N. A. and Liang T. (1984) Carboxy-terminal tripeptidyl hydrolysis of substance P by purified rabbit lung angiotensin-converting enzyme and the potentiation of substance P activity in viw by captopri1 and MK-422. Molec. Pharmoc. 23, 287-293. Chevillard C. and Saavedra J. M. (1982) Distribution of angiotensin-converting enzyme a;:ivity in specific areas of the rat brain stem. J. Neurochem. 38, 281-284.
1. Arregui A. and Iversen L. L. (1978) An~o~nsin~onve~ing
2. 3.
4. 5. 6.
I.
8. Cuello A. C. and Kanazawa I. (1978) The distribution of substance P immunoreactive tibers in the rat central nervous system. J. camp. Neural. 178, 129-156. 9. Danielsen E. M., Vyas J. P. and Kenny A. J. (1980) A neutral endopeptidase in the microvillar membrane of pig intestine. Biochem. J. 191, W-648. 10. Defadini R., Zimmerman E. A.. Wean J. A., AIhenc-Gelas F. and Erdos E. G. (1983) Angiotensin-converting enzyme in epitbelial and nettroepitbelial cells. Neuradacrindogy 37, 32-40. 11. Doter F. E., Kahn J. R., Lentz K. E., Levine M. and Skeggs L. T. (1974) Hydrolysis of bradytinin by angiotensin converting enzyme. Circ. Res. X 824-827. 12. Erdos E. G., Johnson A. R. and Boyden N. T. (1978) Hydrolysis of enlrephalin by cultured human endotheiiai cells se. &i&em. Pkmac. 27,843-848. and by purifkl pcptidyt d enzyme. Am. J. 13. Friuibtnd J. and Silver&n E. (1976) A sensitive fluorimetric assay for serum an~o~nsin~on~~ing din. F&id. 66,416624. of protein: a m~ifi~tion of the Lowry method that gives a linear p~to~t~ 14. Hzatree E. F. (1972) ~t~~tion msponse, Anldyt. Bkdem. (r& 422-427. of brain opiate receptors: a general auto15. Herlcenham M. and Pert C. B. (1982) Light microscopic localiition radiographic method which preserves tissue quality. J. Neurosci. 2, 1129-l 149.
Angiotensin converting enzyme in rat brain
621
16. Hudgin R. L., Charleson S. E., Zimmerman M., Mumford R. and Wood P. L. (1981) Enkephalinase: Selective peptide inhibitors. Life Sci. 29, 2593-2601. 17. Hunter W. M. and Greenwood F. C. (1962) Preparation of iodine-13 1 labelled human growth hormone of high specific activity. Nature 194, 495-596. 18. Iovino M. and Steardo L. (1985) Thirst and vasopressin secretion following central administration of angiotensin II in rats with lesions of the septal area and subfomical organ. Neuroscience 15, 6167. 19. Kanazawa I., Emson P. C. and Ceullo A. C. (1977) Evidence for the existence of substance P-containing fibers in striato-nigral and pallido-nigral pathways in rat brain. Brain Rex 119, 447453. 20. Lang R. E., Unger T., Rascher W. and Ganten D. (1983) Brain angiotensin. In Handbook of Psychopharmacology, Vol. 16, Neuropeptides (eds Iversen L. L., Iversen S. D. and S. H. Snyder), pp. 307-361. Plenum Press, New York. 21. Lind R. W., Swanson L. W. and Ganten D. (1984) Angiotensin II immunoreactivity in the neural afferents and efferents of the subfomical organ of the rat. Brain Res. 321, 209215. 22. Lind R. W., Swanson L. W. and Ganten D. (1985) Organization of angiotensin II immunoreactive cells and fibers in the rat central nervous system. Neuroendocrinology 49, 2-24. 23. Ljungdahl A., Hokfelt T. and Nilsson G. (1978) Distribution of substance P-like immunoreactivity in the central nervous system of the rat-I. Cell bodies and nerve terminals. Neuroscience 3, 861-943. 24. Mendelsohn F. A. 0. (1984) Localization of angiotensin converting enzyme in rat forebrain and other tissues by in vitro autoradiography using ‘251-labelled MK351A. Clin. exp. Pharmac. Physiol. 11, 431436. 25. Mendelsohn F. A. O., Chai S. Y. and Dunbar M. (1984) In vitro autoradiographic localization of angiotensin-converting enzyme in rat brain using ‘*‘I-labelled MK351A. J Hypertension 2, 4144. 26. Mendelsohn F. A. O., Quirion R., Saavedra J. M., Aguilera G. and Catt K. J. (1984) Autoradiographic localization of angiotensin II receptors in rat brain. Proc. natn. Acad. Sci. U.S.A. 81, 1575-1579. 27. Munson P. J. and Rodbard D. (1980) Ligand: A versatile computerized approach for characterization of ligand-binding systems. Analyt. Biochem. 107, 22&239. 28. Oshima G., Geose A. and Erdos E. G. (1974) Angiotensin I converting enzyme of the kidney cortex. Biochim. biophys. Acta 359, 26-37. 29. Paxinos G. and Butcher L. L. (1985) Organizational
principles of the brain as revealed by choline acetyltransferase and acetylcholinesterase distribution and projections. In The Rat Nervous Sysfem, Vol. 1: Forebrain and Midbrain (ed. Paxinos G.), pp. 487-508. Academic Press, Sydney. 30. Saavedra J. M. and Chevillard C. (1982) Angiotensin-converting enzyme is present in the subfomical organ and other circumventricular organs of the rat. Neurosci. Lett. 29, 123-127. 3 1. Saavedra J. M., Femandez-Pardal J. and Chevillard C. (1982) Angiotensin-converting enzyme in discrete areas of the rat forebrain and pituitary gland. Brain Res. 245, 317-325. 32. Severs W. B. and Daniels-Severs A. E. (1973) Effects of angiotensin on the central nervous-system. Pharmac. Rev. 23, 415-449. 33. Skidgel R. A., Engelbrecht S., Johnson A. R. and Erdos E. G. (1984) Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase. Peptides 5, 769-776. 34. Strittmatter S. M., Lo M. M. S., Javitch J. A. and Snyder S. H. (1984) Autoradiographic visualization of angiotensin-converting enzyme in rat brain with [3H]captopril: localization to a striato-nigral pathway. Proc. natn. Acad. Sci. U.S.A. 81, 1599-1603.
35. Strittmatter S. M. and Snyder S. H. (1984) Angiotensin-converting enzyme in the male rat reproductive system: autoradiographic visualization with [‘Hlcaptopril. Endocrinology 115, 2332-2341. 36. Strittmatter S. M., Thiele E. A., Kapiloff M. S. and Snyder S. H. (1985) A rat brain isozyme of angiotensin-converting enzyme. Unique specificity for am&ted peptide substrates. J. biol. Chem. 260, 9825-9832. 37. Sweet C. S., Gross D. M., Glaine E. H., Ulm E. M. and Stone C. A. (1981) A new class ofconverting enzyme inhibitors. In New Trends in Arterial Hypertension (eds Worcel M. et al.), ISERM Symposium No. 17. Elsevier, Amsterdam. 38. Waksman G., Hamel E., Foumie-Zaluski M.-C. and Roques B. P. (1986) Comparative distribution of the neutral endopeptidase “enkephalinase” and of mu and delta opioid receptors in rat brain by autoradiography. Proc. natn. Acad. Sci. U.S.A. in press. 39. Wigger M. J. and Stalcup S. A. (1978) Distribution and development of angiotensin converting enzyme in the fetal and newborn rabbit. Lab. Invest. 38, 581-585. 40. Yang H. Y. T. and Neff N. H. (1972) Distribution and properties of angiotensin converting enzyme of rat brain. J. Neurochem. 19. 2443-2450. (Accepted 5 June 1986)
03~~522/87 $3.08+ 0.08 Pergamon Journals Ltd 0
1987 IBRO
ANGIOTENSIN CONVERTING ENZYME IN RAT BRAIN VISUALIZED BY QUANTITATIVE IN VITRO AUTORADIOGRAPHY SIEW YEEN CHAI,
F. A. 0. MENDELSOHNand G. PAXINOG*
University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Victoria, Australia 3084 and *School of Psychology, University of New South Wales, Kensington, New South Wales, Australia, 2033 Abstract-Angiotensin converting enzyme was localized in rat brain by quantitative in u&o autoradiography using an [1251]labelledconverting enzyme inhibitor called “351A”. This radioligand was found to bind with high afhnity and specificity to angiotensin converting enzyme. Very high levels of converting enzyme were observed in the ventricular choroid plexus, ependyma of al1 ventricles and large and medium blood vessels, subfornical organ, and organum vasculosum of the lamina terminals. High levels of converting enzyme were found in the basal ganglia including caudate putamen, nucleus accumbens, globus palhdus, entopenduncular nucleus and substantia nigra pars reticulata. The neurosecretory nuclei, paravent~cular nucleus and supraoptic nucleus, as well as the median eminence and posterior pituitary displayed high levels of the enzyme. In the amygdala, basolaterai, lateral, basomedial, medial and anterior cortical nuclei showed moderate converting enxyme activity. The medial habenula and molectdar layer of the dentate gyrus showed high levels of activity. In the cerebellum, dense labelling was observed in the Purkinje cell layer. Moderate levels of converhrg enzyme occurred in the gelatinosus subnucleus of the caudai part of the nucleus of the spinal tract of the trigeminal. There was a close correspondence between the distribution of angiotensin converting enzyme and angiotensin II in the neurosecretory nuclei ~vent~~~~ and supraoptic nuclei) and median eminence and this suggests a role of angiotensin converting enzyme in the production of angiotensin II in this system. There was also a good correspondence between the distribution of angiotensin converting enzyme and angiotensin II in the subfomical organ, median preoptic nucleus, and organum vasculosum of the lamina terminalis, structures abutting the anterior wall of the third ventricle which are implicated in fluid and electrolyte homeostasis. A striking discrepancy occurs in the basal ganglia which is reported to contain very little angiotensin II or angiotensin II receptors but is very rich in angiotensin converting enzyme. It is concluded that the enzyme may act to (a) convert circulating angiotensin I to angiotensin II in circumventricular organs; (b) generate intraneuronal angiotensin II in pathways such as the hypothalamichypophyseal tract; and (cf process neuro~tid~ other than an~otensin II in regions such as basal ganglia.
Angiotensin converting enzyme (ACE) is a peptidyl dipeptidase which cleaves ~stidyl-leu~ne from the carboxyl terminal of angiotensin I to yield angiotensin II and also cleaves C-terminal peptide from a variety of other peptides including bradykinin,” substance P,6 enkephalins,‘2 and neurotensin.3f ACE has been localized on the huninal surface of endothelial cells in all vascular beds”*q and also in epithelial cells in kidney,** intestine> and testis3’ In the brain, microdissection studies have shown high concentrations of ACE in the choroid plexus, basal ganglia, subfomical organ (SFO), and area postrema.‘JO Moderate amounts of ACE occur in some thalamic and neurosecretory hypothalamic nuclei.’ Immunohistochemical techniques have also detected high con~ntrations of the enzyme in the choroid plexus, the SFO, and in blood vessels.39~ioA study using goat antibody against rabbit ACE did not
detect any enzyme in neural tissues of the rat brain other than in the SFO.* Experiments using rabbit antisera to human ACE detected the enzyme in dendrites of the globus pallidus and substantia nigra in both human and rat brain.‘O Because of these discrepancies, we developed a method to map the distribution of ACE in the rat brain by quantitative in vitro autoradiography using an [12sI]labelled derivative of the potent ACE inhibitor lysinop~l as specific radioligand.24”5 At the same time, St&matter et al. reported similar studies using [3H]captopril as radioligand.” Autoradiography using [‘*‘1]351A (N-[(s)- 1-car~xy-3-phenylpropyl]-L-lysyl-~~osyl-Lproline) offers a number of advantages over [3H]captopril and appears to provide higher resolution which permits detailed anatomi~l l~alization of brain ACE as we report here. EXPERIMENTAL PROCEDURES
~bbr~iufio~: ACE, angiotensin converting enzyme; AII, anaiotensin II: HACBO-GIY, N-(R,S)_3-hydroxyaminocs;bonyl-2-be&yl-1-oxoptopyl-glycine; hydroxamic acid derivative, I-(N-hydroxycarboxamido)-4-methyl-pantanolyl-L-~anyl-Byrne amide; 351A, N-[(s)-l-carboxy-3phenylpropyl]-L-iysyi-t~rosyl-L-proline.
Radio&ml A derivative of the ACE inhibitor lysinopril,37 (kindly donated by Dr C. Sweet, Merck Institute for Therapeutic Research, West Point, PA, U.S.A.), called “35lA” was radio-iodinated by the chloramine T method,” purified on 615
SIEW YEEN CHAI el al
616
an SP-C25 sephadex column and eluted with 0.1 M ammonium acetate, pH 3.5. The binding properties of this radio&and to lung membranes, a rich source of ACE, have been described.Z4 Characterization
of binding properties
The sections used for autoradiography were stained with thionin for anatomical localization of the autoradiog~phs. RESULTS
Properties
of [ ‘2s1]35
1A binding
Characteristics of binding of [‘Z*I]351A to brain memThe ability of a number of converting enzyme and branes was evaluated as follows: A lOO&2O,OOOg enkephalinase inhibitors to displace binding of radiomembrane-rich fraction (200 fig protein) prepared from rat caudate putamen was incubated with - 10 nCi of [izSI]351A ligand [12’1]35IA from caudate putamen membranes is shown in Fig. 1. All three of the chemically in OS ml of 10 mM sodium phosphate buffer, pH 7.4, containing ISOmM NaCl and 2 g/l bovine serum albumin 100 (buffer A) and a range of con~ntrations of converting enzyme and enkephalinase inhibitors at 20°C for I h. Free and bound radioligands were separated by filtration through Whatman GF/B glass fibre filters. The [‘251]radioactivity was measured and the binding isotherms analysed by a 80 non-linear, iterative, model-fitting computer programme.*’ The converting enzyme inhibitors were obtained as follows: B/,p lysinopril was donated by Dr C. Sweet, Merck Institute for Therapeutic Research, Westpoint, PA, and captopril and 60 teprotide were donated by the Squibb Insitute for Medical Research, Princetown, NJ. The enkephaiinase inhibitor, 2-AN-hydroxy~rboxamido)-4-methyl ~ntanolyl-L-alanylglycine amide (“hydroxamic acid derivative”)‘” from Calbiochem-Behring, San Diego, CA and phosphor40 amidone” from Sigma Chemical Company, St. Louis, MO. Quantitative in vitro autoradiography
For autoradiography, male Sprague-Dawley rats (* 250 g) were anaesthetized with sodium methohexitone (0.5 mg/kg), perfused via the left ventricle with 200 ml of isotonic saline and then 1% parafo~aldehyde in saline. The brains were then removed, snap frozen in isopentane at -40°C. Coronal sections were cut at 20pm and sagittal sections at 1Opm in a cryostat maintained at -20°C. Sections were thaw-mounted onto gelatin-coated slides and dried in a desiccator for 2 h at 4”Cn The sections were preincubated in buffer A for 15 min at 20°C and then incubated in buffer A containing 0.3 nCi/ml of [‘251]3SlAfor 1 h at 20°C. Non-specific binding was determined in parallel incubations containing either 1mM ethyien~iami~etetraacetate (EDTA) or 1 PM lysinopril. After incubation, the sections were transferred through 4 successive i min washes of 50mM Tris-HCl buffer, pH 7.4 at 0°C. The slides were dried under a stream of cold air, loaded into X-ray cassettes and exposed to Kodak NMB-1 X-ray film for 7 days at room temperature. In each cassette, a set of [rZsI]radioactivity standards was included. These were prepared by applying known amounts of [‘251]radioactivity to 5 mm diameter discs of 20/rm thickness brain sections mounted on coated slides. In addition, the system was calibrated for enzyme activity using a set of enzyme standards. The standards were prepared by serial dilutions of caudate putamen membranes in 1% gelatin solution in buffer A; five ~1 aliquots were applied to subbed slides and dried at 4°C in a dessicator. These were then carried through the same incubation procedure as for the brain sections and exposed to X-ray film for the same period of time. Samples of the membrane suspensions were also assayed for ACE by a fluorimetric method.” The protein content of the membranes was determined by a modified Lowry method.i4 The optical densities obtained from these standards were then converted to ACE activity. The X-ray films were processed in a Kodak RP X-OMAT automatic developer and the optical density quantitated using an EyeCom model 850 image analysis system (Spatial Data Systems, Springfield, VA) coupled to a DEC 1t/23 LSI computer. The optical density of the autoradiographs was calibrated both in terms of dpm per square mm using the radioactivity standards or in terms of the ACE activity (pmol/min/mg protein) by fitting calibration curves with the computer.
20
0 Concentration ( MI Fig. 1. Specificity of binding displacement of [“‘1]351A to
amide), A phosphoramidone. Abbreviations wed in jigures
3v 4v 7n ac aca Acb ACo AHi AID AOP APit APT Aq asc7 AV bas BL BM BST BV CA1 CA3 CB Cb cc CeL CeM CG Cg
third ventricle fourth ventricle facial nerve or root of facial nerve anterior commissure anterior commissure, anterior part accumbens nucleus anterior cortical amygdaloid nucleus amygdalohippocampa1 area agranular ittsutar cortex, dorsal part anterior olfactory nucleus, posterior part anterior lobe of the pituitary anterior pntectal area cerebral aqueduct (Sylvius) ascending fibers of the facial nerve anteroventral thalamic nucleus basilar artery basolateral amygdaloid nucleus
baaomaW 8mygdalbid nudeus bed m&us of the stria terminalis blood vessel field CA1 of Amman’s horn field CA3 of Atnmon’s horn cell bridges of the ventral striatum cuebeliurn corpus caiIosum oenttml arny@oid nucleus, lateeal part astral m nucleur, medial part central (pctiqualuctal) gray cingulate cortex
Angiotensin converting enzyme in rat brain CGD CGLD CGLV CGM CL CLi CM CnF CPU DC0
DG DLG DLL DPB DR DTg ECU Ent EP k fr G ?Z GP HDP IAM IC ic icp IF InG Int rnwh IO IF IPit La Lat LD IfP LH LHb LM IO
LOT LP LR4V LS LV LVe MD ME me5 Med MG MiTg ml mlf MM MnPO MnR MoDG MP MPO MS mt MTu MVE OP
central gray, dorsal part central gray, lateral dorsal part central gray, lateral central part central gray, medial part centrolateral thalamic nucieus caudal (central) linear nucleus of the raphe central medial thalamic nucleus cuneiform nucleus caudate putamen (striatum) dorsal cochlear nucleus dentate gyrus dorsal lateral geniculate nucleus dorsal nucleus of the lateral lemniscus dorsal parabrachial nucleus dorsal raphe nucleus dorsal tegmental nucleus external cuneate nucleus entorhinal cortex entopeduncular nucleus fomix fimbria of the hippocampus fasciculus retroflexus (habenulointerpeduncular tract) gelatinosus thalamic nucleus genu of the facial nerve gigantocellular reticular nucleus globus pallidus nucleus of the horizontal limb of the diagonal band (Broca) interanterome~al thalamic nucleus inferior colliculus internal capsule inferior cerebellar peduncle {restiform body) interfa~icular nucleus intermediate gray layer of the superior colliculus interposed (inte~ediate) cerebellar nucleus intermediate white layer of the superior colliculus inferior olive interpeduncular nucleus intermediate lobe of the pituitary lateral amygdaloid nucleus lateral (dentate cerebellar nucleus) Iaterodorsal thalamic nucleus longitudinal fasiculus of the pons lateral hypothalamic area lateral habenular nucleus lateral mammillary nucleus lateral olfactory tract nucleus of the lateral olfactory tract lateral posterior thalamic nucleus lateral recess of the fourth ventricle lateral septal nucleus lateral ventricle lateral vestibular nucleus mediodorsal thalamic nucleus median eminence mesencephalic tract of the trigeminal nerve medial (fastigial) cerebellar nucleus medial geniculate nucleus microcellular tegmental nucleus medial lemniscus media1 longitudinal fasciculus medial mammillary nucleus, medial part median preoptic nucleus median raphe (superior central) nucleus molecular layer of the dentate gyrus medial mammilla~ nucleus, posterior part medial preoptic area medial septal nucleus mammillothalamic tract medial tuberal nucleus medial vestibular nucleus optic nerve layer
617
optic tract nucleus of the optic tract otfactory ventricle (olfactory part of lateral ventricle) optic chiasm parabigeminal nucleus k paracentral thalamic nucleus PC posterior commissure parvocellular reticular nucleus ERt PDTg posterior dorsal tegmental nucleus paragigantocellular reticular nucleus PGi PH posterior hypothalamic nucleus Pir piriform cortex (primary olfactory cortex-“PO”) posteromedial cortical amygdaloid nucleus (C3) PMCo pontine nuclei Pn PnC pontine reticular nucleus, caudal part PIIO pontine reticular nucleus, oral part PO posterior thalamic nuclear group PP peripeduncular nucleus PPit posterior lobe of the pituitary pedunculopontine tegmental nucleus PPTg Pr5 principal sensory trigeminal nucleus prepositus hypoglossal nucleus PrH presubiculum PrS paraventricular thalamic nucleus PV paraventricular thalamic nucleus, posterior part PVA pyramidal tract red nucleus PRY reuniens thalamic nucleus Re rhomboid thalamic nucleus Rh raphe magnus nucleus RMg reticular thalamic nucleus Rt superior colliculus SC supr~hiasmatic nucleus SCh superior cerebellar peduncle ~brachium conjunc=P tivum) subfomicai organ SF0 stria medullaris of the thalamus sm substantia nigra, compact part SNC SNR substantia nigra, reticular part so supraoptic nucleus Sol nucleus of the solitary tract superior olive so1 spinal tract of the trigeminal nerve sP5 SPFPC subparafascicular nucleus, parvocellular part sphenoid nucleus Sph spinal vestibular nucleus SpVe st stria terminalis SuG superficial gray layer of the superior colliculus SUM supramammillary nucleus SuVe superior vestibular nucleus T-f taenia tecta (anterior hippocampal rudiment) Tu olfactory tubercle VCoA ventral cochlear nucleus, anterior part VDB nucleus of the vertical limb of the diagonal band (Broca) VL ventrolateral thalamic nucleus VLGMC ventral lateral geniculate nucleus, magnocellular part VLGPC ventral lateral geniculate nucleus, parvocellular part VLL ventral nucleus of lateral lemniscus VM ventrom~ial thalamic nucfeus VMH ven~om~ial h~thalamic nucleus VP ventral pallidurn VPB ventral (medial) parabrachial nucleus VPL ventral posterolateral thalamic nucleus VPM ventroposteromedial thalamic nucleus VTA ventral tegmental area (Tsai) decussation of the superior cerebellar peduncte x=P Y nucleus Y ZI zona incerta zo zona1 layer of the superior colliculus opt OT ov
SEW YEEN CHAI cf d.
618
unrelated converting enzyme inhibitors completely displaced the radioligand. Computer analysis of the binding isotherms 27 for the different ACE inhibitors gave the following Ka values: lysinopril, 7.8 t_ 3.4 x 109; enalaprilat, 3.6 f 1.0 x IO*; captopril, 4.4 f 1.4 x 10’ and teprotide, 9.5 f 1.5 x IO6M.. ‘. Specific binding of the radioligand was abolished by 1 mM EDTA. Non-specific binding in the presence of an excess (1 PM) of lysinopril or 1 mM EDTA was 1.O% of total binding. The enkephalinase inhibitors (the hydroxamic acid derivative and phosphoramidone) were very weak in inhibiting binding of the radioligand with IC~‘s of approximately 4 and 50pM, respectively. Autoradiographic localization of angiotensin converting enzyme
The levels of ACE in selected structures are shown in Table 1. For descriptive purposes the density of ACE was scored as follows: very high density, > 1000 pmol/min/mg protein; high density 400-l 000; moderate density, 2W399; low density 50-199; undetectable < 50. Non-specific binding, measured in the presence of 1 PM lysinopril or I mM EDTA, was completely undetectable and produced no visible image on the X-ray films. The autoradiographs shown in Figs 2-4 therefore represent specific binding only. The ventricles and periventricular structures
The choroid plexus of all ventricles contains very high densities of ACE (Figs 2D,H: 3H: 4A,B). The ependymal walls of the olfactory, lateral, third, and fourth ventricles, and central canal has high densities of ACE. The SF0 and vascular organ of the lamina terminalis show a very high density of ACE (Figs 2E and 4A),- while the area postrema only a low density. Large blood vessels show dense labelling on their endothelial walls (Fig. 3F). Medium blood vessels are also densely labelled and can be seen in all figures as longitudinal streaks or spots particularly in the cortex against the low background. The olfactory system
The olfactory bulb, the accessory olfactory bulb and the anterior olfactory nusleus display very low levels of ACE. The lateral olfactory tract (Fig. 23,C) and the anterior part of the anterior commissure (Fig 2B,C) are devoid of ACE activity. The basal ganglia
The caudate intensely labelled the reticular part and 4B,C). The
putamen (striatum) is the most neural structure in the brain after of the substantia nigra (Figs 2A-G distribution of ACE within the
caudate putamen is uneven with patches of high and moderate density. The accumbens nucleus displays dense labelling in its ventrolateral part and only slight binding in its dorsomedial part (Fig. 2 B,C). The olfactory tubercle displays no activity although the finger-like extensions of the striatum into the tubercle (the striatal bridges) are moderately labelled (Fig. 2C). The globus pallidus had very high density of ACE in its lateral part which abuts the striatum and was moderate in its medial parts in which the pallidally associated basal nucleus of Meynert is found (Fig. 2 E-G). The bundles of fibres penetrating the striatum and gathering in the internal capsule were negative. A few such bundles were positioned bctween the striatum and globus pallidus and provide a demarcation of these structures (Fig. 2E). The internal capsule is negative except for its ventral part which includes the striatonigral ACE-containing projection. The ventral pallidurn contains moderate activity which becomes progressively weaker with increased distance from the globus pallidus (Fig. 4C). The reticular part of the substantia nigra displays the most intense activity of any nucleus in the brain (Fig. 3A,B). The compact part of the nigra does not label intensely (Fig. 3A,B). Intense binding is present in the entopeduncular nucleus (Figs 2F,G and 4C). The striatonigral projection containing ACE courses through the entopeduncular nucleus and therefore some of the activity seen in the region of the nucleus may be associated with the fascicles that pass through the nucleus and not with the nucleus itself. However, the binding immediately rostra1 and caudal to the nucleus is less than in the nucleus itself and we can conclude that the nucleus itself is labelled as well. The subthalamic nucleus displays only low activity. The septum and hypothalamus
The medial septum displays a slight labelling which is continuous with the labelling expressed by the nuclei of the vertical limbs of the diagonal band (Fig. 2D). The lateral septum has undetectable activity as does the septohippocampal nucleus (Fig. 2D). The nucleus of the horizontal limb of the diagonal band displays a slightly less activity than the nucleus of the vertical limb of the diagonal band. The median preoptic area is moderately labelled (Fig. 4A) and so is most of the medial preoptic area (Fig. 4A). The lateral preoptic area is not labelled. The neurosecretory nuclei, supraoptic, paraventricular and accessory neurosecretory cells groups show high levels of ACE activity. The suprachiasmatic nucleus has very low ACE activity which
Fig. 2 (opposite). Autoradiographic localization of angiotensin converting enzyme (ACE) in rat forebrain (coronal plane) using [‘29]351A(3 days exposure). Non-specific binding in the presence of 1 mh4 EDNA or I ,uM lysinopril eompktely abolished these patterns of binding.
Fig. 4. Autoradiographic localization of ACE in rat brain (sagittal plane) using [‘z’I]351A(7 da]IS exposure).
Fig. 3 (opposire). Autoradiographic localization of ACE in rat forebrain, midbrain and cerebellum (coronal plane) using [‘2s1]351A (3, 3 and 7 days exposure, respectively). 621
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SEW YEEN CHAI et al.
Table 1. Angiotensin converting enzyme in rat brain quantitated by in vitro autoradiography ACE activity (pmol/min/mg prot.) + SD Ventricles and periventricular structures Choroid plexus Subfomical organ Vascular organ of the lamina terminalis Median blood vessels Basal ganglia Caudate putamen Accumbens nucleus (ventrolateral) Globus pallidus Entopeduncular nucleus Substantia nigra, reticular part Anterior commissure Internal capsule Pituitary Anterior Posterior Intermediate lobe Hypothalamus and septum Nucleus of the vertioal limb of the diagonal band Median preoptic nucleus Supraoptic nucleus Paraventricular nucleus Median eminence Median preoptic area Anterior hypothalamic area Septohypothalamic nucleus Retrochiasmatic area Fornix Thalamus Reuniens thalamic nucleus Paraventricular thalamic nucleus Anteroventral thalamic nudeus Laterodorsal thalamic nucleus Medial habenular nucleus Zona incerta Amygdala nuclei Basolateral Central Anterior cortical Anterior amygdaloid area Bed nucleus of the stria terminalis Hippocampus Dentate gyrus Cerebral cortex Striate Frontoparietal Cingulate Corpus callosum Cerebellum Purkinje cell layer Molecular layer Granular layer Midbrain Central gray Interpeduncular nucleus Pontine nucleus Superior colBcuIus (itcmwdiate gray) Inferior colliculus Brain stem Nucleus of the solitary tract Solitary tract Inferior olive Nucleus ambiguus Spinal tract of the t&em&al nerve
> 1000 > 1000 395 + 54 605 + 54 702+ 18 460+18 424 k 18 425 + 19 825 + 200 < 50 < 50 406+40 >I000 287 + 8 414 + 16 334 It 42 444 Itr3s 436 + 30 757 f 12 250 f 10 384i9 229 f 22 256 + 23 < 50 407&12 256 ?; 28 358 & 21 174+20 413+23 290+30 320 f 17 384 + 12 368 &-14 383+11 352 f 14 635 k 53 268 + 23 291 & 12 326 f 10 < 50 570 f 16 35Of8 326 4 12 384 + 16 350 f 32 325 205* 11 238 f 12 370+6 < 50 294 + 7 375 220 f 8
Angiotensin converting enzyme in rat brain
was lower than the adjacent anterior hypothalamic area. The median eminence is also densely labelled in the region abutting the third ventricle and on its external surface (Fig. 4A). The posterior putuitary contains very high levels of ACE (Fig. 4 A,B) while the anterior pituitary and the intermediate lobe of the pituitary display moderate binding (Fig. 4A,B). In the hypothalamus, binding is expressed in the arcuate, ventromedial, dorsomedial, lateral, perifornical and mammillary nuclei (Fig. 2F-H). The supramammillary nucleus is moderately labelled (Fig. 3A) and the posterior hypothalamic nucleus displays slightly less binding (Fig. 3A). The thalamus
The ventral nuclear complex nuclei are homogeneously negatively labelled. The ventrolateral, ventromedial, ventroposterolateral, ventroposteromedial, ventroposteromedial parvocellular and the gelatinosus nuclei (Fig. 2 F-G) all display low ACE activity. The reticular thalamic nucleus is only lightly labelled (Figs 2G and 4C) but stands out against its nonpositive neighbours (internal capsule and ventral nuclear complex). The anterior nuclear group displays some binding in its anterodorsal and anteroventral nuclei (Fig. 4B). The mediodorsal nucleus is nearly devoid of activity (Figs 2 F, G and 4A). The lateral nuclear groups (laterodorsal and lateroposterior nuclei) are negative (Fig. 2F,G). The intralaminar group displays some binding in its central medial, paracentral, central lateral and parafascicular divisions (Figs 2FG and 4A). The midline nuclear group displays some labelling in its paraventricular, paratenial, rhomboid, and reuniens divisions but is nearly devoid of activity in its interanteromedial division (Figs 2FG and 4A). The subparafascicular nucleus and its parvocellular part display a slight activity which is detectable against the extremely low activity of the ventroposterior group and the absence of activity in the medial lemniscus (Fig. 2H). The medial habenula and medial part of the lateral habenula display a moderate amount of binding (Figs 2G and 4A) and the fasciculus retroflexus is positive in its core (Figs 2H and 3A) suggesting that this fibre bundle carries fibres containing ACE. Part of the lateral subnucleus of the interpeduncular nucleus is moderately labelled (Fig. 3B) but it cannot be deduced that the fasciculus retroflexus fibres containing ACE terminate there without experimental interventions. The amygdala
The basolateral amygdaloid nucleus is the most densely labelled nucleus of the amygdala (Fig. 2F). The lateral nucleus also displays moderate to low activity in some of its subdivisions (Fig. 2H). The MC.20/2--R
623
lateral part of the central nucleus stands out by its lack of activity (Fig. 2F,G). The medial part of the central nucleus displays labelling similar to that of the adjacent medial amygdaloid nucleus (Fig. 2G). The basomedial nucleus shows moderate binding (Fig. 2G) as does the anterior cortical. The nucleus of the lateral olfactory tract is conspicuous by its low activity contrasted by the lightly labelled anterior amygdaloid area and anterior cortical amygdaloid nucleus (Fig. 2F). The posterior cortical amygdaloid nucleus shows very low activity (Fig. 3A). The piriform cortex shows some activity in its plexiform layer but no activity in its pyramidal layer (Fig. 2B,C). The bed nucleus of the stria terminalis shows only slight binding (Figs 2D and 4B) as does the sublenticular substantia innominata (Fig. 2E). The amygdalohippocampal area is devoid of activity (Fig. 2H). The visual system
The ventral lateral geniculate is moderately labelled while the dorsal lateral geniculate is nearly devoid of binding (Fig. 2H). The anterior pretectal (Fig. 2H) and olivary pretectal areas show moderate ACE activity. The superior colliculus showed differential distribution in its layers with very low activity in the optic nerve and deep white layer, low density in the zonal and deep gray layers and slightly higher density in the intermediate gray and white layers (Fig. 3B). The ventral tegmental area
The ventral tegmental area displays low binding (Fig. 3B). The interpeduncular nucleus shows moderate to high activity in its lateral subnucleus (Fig. 3B). The hippocampai region
The most striking feature of the hippocampus was the high density of ACE in the molecular layer of the dentate gyrus (Figs 2H; 3A,B; 4C). The granule cell layer of the dentate gyrus and the pyramidal cell layer of Ammon’s horn were devoid of activity (Fig. 3B). Similarly the stratum oriens, radiatum, and laconosum moleculare of Ammon’s horn are negative (Figs 2H and 3A). The subiculum, presubiculum, and parasubiculum show low levels of labelling (Fig. 3B,C). The entorhinal cortex shows ACE activity in some of its layers (Fig. 3C,D). The cerebral cortex The cerebral cortex displays only low activity. The greatest labelling is found in the granular insular cortex (Fig. 2B,C), and the anterior cingulate cortex (Fig. 2A,B). The rest of the cortex displays very low activity. The reticular formation and the central gray
The reticular nucleus of the medulla displays very low ACE activity in the dorsal and ventral parts.
624
SIEW YEEN CHAI et u/.
The gigantocellular and parvocellular reticular nuclei display low binding (Fig. 3G). The pontine caudal and oral nuclei and the paragigantocellular nuclei are similarly negative (Figs 3D,E and 4B). The central gray and the pedunculopontine tegmental nucleus show moderate activity (Fig. 3A-D). The dorsal tegmental nucleus shows low labelhng with some differences in its subnuclei (Fig. 4A). The posterior dorsal tegmental nucleus shows moderate binding (Fig. 4A). The sphenoid nucleus*’ (Fig. 4A) and the locus coeruieus also display moderate binding. The raphe nuclei
The dorsal and medial raphe display low ACE activity (Fig. 3D) but the raphe pallidus, obscurus and magnus are nearly devoid of activity (Fig. 3&I&G). The interfascicular and caudal linear nuclei show some ACE activity (Figs 3B,C and 4A). Brain stem nuclei ~sociated with respiratory, cardio vascular and other auto~mi~ f~~etions
The nucleus of the solitary tract displays moderate to low binding in both its medial and lateral parts (Fig. 3G,H). The ambiguus nucleus is moderately labelled. The dorsal parabrachial, ventral parabrachial and Kolliker-Fuse nuclei display a low but significant level of ACE activity (Fig. 4B).
low activity while the lateral and spinal vestibular nuclei show very low activity (Fig. 3F.G). The precerebellar nuclei and the red nucleus
The inferior olive shows moderate labelling {Fig. 4A) as do the pontine nuciei (Figs 3C and 4B) while the lateral reticular nucleus shows very little binding. The red nucleus is nearly devoid of ACE activity (Fig. 3B). The cerebellum
The lateral, interpasitus and medial nuclei show light labelling (Fig. 3F,G). The cerebellar cortex presents an interesting binding pattern. There is a line of high density labelling at the site of the Furkinje cells (Fig. 3E-H). The rest of the molecular layer as well as the plexiform layer of the folia is moderately labelfed. Fibre tracts
All fibre tracts, for example: corpus callosum, fomix, fimbria, medial lemniscus, longitudinal ftbers of the pons, and superior cerebeltar p&&e are negative except for the fasciculus retroflexus and the solitary tract which expressed some ACE activity and the segment of the internal capsule containing the striatonigral ACE projection which is very densely labelled (Fig. 2G).
The orofacial motor nuclei
The motor trigeminal and facial nuclei are nearly devoid of activity (Fig. 3F,G). The h~glos~~ nucleus shows low ACE activity as does the ntteleus of Roller. The somatosensory system
The gracilis and cuneate nuclei show very low labelling. The external cuneate nucleus shows low ACE activity (Fig. 3H). The principal nucleus of the trigeminakas well as the nucleus of the spinal tract of the trigeminal show very low binding (Figs 3&H and 4C) except for the subnucleus gelatinosus of the caudal part of the nucleus of the @inal tract of the trigeminal which is si~i~c~tly lab&& (not shown). The auditory system
The cochlear nuclei show a low ACE activity (Figs 3E,F and 4C). The lateral superior olive shows very low activity (Fig. 3E) and the nuclei of the trapezoid body are nearly devoid of activity as are tbe nuclei of the lateral lemniscus, the inferior C&C&W and the medial geniculate (Figs 3A,B,D,E, and 4C). The vestibular system
The superior vestibular and infracerebelhtr nucleus as well as nucteus X show moderate binding which is the highest observed in the vestib&ar nuclei (Fig. 3E,F). The medial ~ti~lar n~~~Fi~ 3 F,G} and the p~positus .~~l~l nudeus (Fig. 3F) show
Pro~rt~s
of the [L25~351A radiol~g~d
The ra~oliga~ [“51]351A used in this study, a derivative of the potent spe&Ic ACE i~~or ly*
sinopril,” was found to bind with ~~~~ to saturable sites in brain membranes. This bin&g was abolished by I mM EDTA, which is known to.&&te the essential Zn atom at the catalytic site of ACE a& block its enzymatic activity.’ Binding of the radioligand was also completely inhibited by a range of converting enzyme inhibitors of diverse cben&al structures. The potency of these compounds in displacing the radiohgand closely paraWed their anticataiytic activity ap;ainst converting enxyme.324 The inhibitor, phos~or~~one*6 and ibe hydrox~ic acid derivative of Ala-Gly ~id~which are active against enkephalinase with 1~~‘s of 40 and 3 nM, in di~l~~n~ weak very respectively5-were [r*‘I]351A with tcs,“s at least 1000 times higher. These findings indicate that this radiofiesaMf labels the active cataIytic site of a vetting enzyme in brain membranes and &o S$OW that it does not appreciably label en under these conditions. Autora~r~h~c verting enzyme
localization of arzgiotensin con -
The present auto~a~a~~ extend earlier observations
i-es&s coobtained by
and micro-
Angiotensin converting enzyme in rat brain
dissection and [3H]captopril autoradiography in which high levels of ACE activity were found in the choroid plexus,7.u subfornical organ,30 striatum and the reticular part of the substantia nigra,7Y and the neurosecretory hypothalamic nuclei and the posterior pituitary.3’ In the present autoradio~aphic study, a comprehensive mapping of brain ACE was undertaken which reveals many previously unknot reactive sites and elaborates on the dist~bution in regions known to contain the enzyme. A very high density of ACE evident in the choroid plexus, subfomical organ and blood vessels suggests a likely role for ACE in converting circulating angiotensin I to angiotensin II. Possible roles of bloodborne angiotensin in regulating blood pressure, drinking and vasopressin release have been reviewed.zo~32It should also be noted that very high levels of ACE were found in the ependymal lining in all ventricles and the cerebral aqueduct. A strikingly high ACE activity was observed in all major components of the basal ganglia. The striatum, accumbens nucleus, globus pallidus, entopenduncular nucleus and substantia nigra. The distribution within these nuclei was not homogenous. Compartments that are rich and poor in ACE were observed in the striatum. The accumbens nucleus displayed highest activity in its ventrolateral part. The globus pallidus displayed greater ctincentrations of the enzyme in its lateral portion. Dense labelling in the reticular part of the substantia nigra was not continued in the compact part. It is significant to note that the ventrom~ial segments of the internal capsule displayed intense ACE activity. Descending fibres of the longitudinal fasciculus of the pons (posterior to the substantia nigra) showed no detectable ACE. This supports the suggestion that the enzyme is contained in the striatonigral projection neurons. 34The topographic relation of ACE in the basal ganglia and the proposed pathway is seen at an advantage in the sagittal plane (Fig. 4C). The presence of ACE in the hypothalamic neurosecretory nuclei (supraoptic and paraventricular nuclei), median eminence and posterior pituitary is consistent with the proposed role of angiotensin II in the regulation of vasopressin and oxytocin release.18.20 The distribution of ACE in a number of other brain regions showed excellent correspondence to the known parcellation of these regions. For example, in the hippocampus, the enzyme was localized to the molecular layer of the dentate gyrus. A laminar distribution was also evident in the superior colliculus and the cerebellum. In the cerebellum, a remarkable strip of activity was found in the Purkinje cell layer. The medial habenular nuclei as well as the medial part of the lateral habenular nuclei showed high ACE activity. The lateral subnucleus part of the inter~nduncular nucIeus was positive. The fasciculus retroflexus showed a positive core and this suggests
625
that the known projection of the habenular nuclei to the interpeduncular nucleus may contain ACE.” There is a major problem in reconciling the distribution of ACE as determined here with the distribution of its putative major product, angibtensin II, which has been recently mapped by immunohistochemistry.22 While there are areas of correspondence where both substances are found in high concentrations, notably the subfomi~l organ, paraventricular and supraoptic nucleus and median preoptic nucleus, there are striking incongruities. For example, high densities of ACE are found in the striatum, globus pallidus, entopeduncular nucleus, substantia nigra reticulata and dentate gyrus areas reported to be devoid of angiotensin II cells and fibres.” Conversely, very low ACE activity was observed in the lateral part of the central nucleus, of the amygdala, a region reported to have very dense concentrations of AII cell bodies and fibres.” A paradox exists if the high density of immuno~activity for AI1 cells and fibres reported in the lateral part of the central amygdaloid nucleus is indeed specific AII. This dissociation would suggest the presence of a product (AH) without its generating enzyme (ACE) and would necessitate the postulation of alternative pathways of AI1 formation. While there are gross discrepancies in the overall distribution of ACE and AII, in some specific systems the correspondence was striking. For instance in the paraventricular and supraoptic nuclei of the hypothalamus as well as median eminence and posterior pituitary, both ACE (current study) and angiotensin II receptors. A similar correspondence of AII, AI1 receptors and ACE holds for the subfomi~l organ, vascular organ of the lamina terminalis and the median preoptic area. However, some of the most reactive regions for ACE such as the choroid plexus, caudate putamen, accumbens nucleus, globus pallidus, entopeduncular nucleus, substantia nigra and dentate gyrus and Purkinje cell layer of the cerebellum were primarily found to be conspicuously free of AI1 receptors. 26 Conversely, high AI1 receptor concentrations previously found in the suprachiasmatic nucleus, lateral olfactory tract and locus coeruleus were not paralleled by corresponding en~chment of ACE in the current study. The pattern of ACE dist~bution displays a remarkable similarity to the labelling of enkephalinase with the specific inhibitor [3H]HACBO-Gly3s However there are some differences in that high levels of ACE were detected in paraventricular, supraoptic, median preoptic, median eminence and vascular organ of the lamina terminalis, which are not rich in enkephalinase. 38 Conversely, the olfactory tubercle, which has a high concentration of enkephalinase, contains low levels of ACE. It is also clear that the two radioligands label different specific enzymes since the potent angiotensin converting enzyme inhibitor, captopril, did not displace the enkepha~na~ radioligand in the experiments of Waksman3$ and in our
626
Smw YEENCHAI
studies, the potent enkephahnase inhibitors, phosphoramidone and hydroxamic acid were very weak inhibitors of the binding of (‘251]3S1A. used to label ACE. Abundant substance P-immunoreactive fibres have been Iocalized in the striatonigral pathway” which also contained high levels of ACE in the current study. Since substance P has been shown to be a substrate for brain ACE33 it is possible the ACE in this pathway is involved in the hydrolysis of this peptide. Indeed, support for this concept comes from the finding that an isoenzyme of ACE recently purified from rat brain corpus striatum differed from lung ACE in molecular weight and peptide substrate specificity although the two enzymes displayed very similar immunological properties and susceptibility to inhibitors.36 The striatal enzyme cleaved substance P, substance K and bombesin at a relatively faster rate than ACE and showed a differed pattern of cleavage products when hydrolysing substance P,36A high concentration of substance P-immunoreactive fibres has also been demonstrated in medial and central amygdaloid nucleus,z3 and the substantia gelatinosa of the spinal cord,8 brain regions which display moderate densities of ACE. However, there are also marked differences in the distribution of substance P as compared to ACE. For example, in the cerebellum, ACE is detected in high ievels in the Purkinje cell layer whereas only a very low levels of substance P and immunoreactive fibres are present.
fv d.
Similarly, the olfactory bulb and tuber&, structures which are rich in substance P positive nerve terminals” contain relatively low levels of ACE.
CONCLUSfON
In the brain, the high ~on~ntraiion of ACE detected in some regions such as the subfomical organ and vascular organ of the iamina terminalis suggests that the enzyme may convert circuiating angiotensin I to angiotensin II which could then interact with the high density of AI1 receptors localized in these organs” to elicit actions on drinking, vasopressin release and blood pressure controLzo The existence of ACE with other components of the angiotensin system in the paraventricular and median preoptic, and supraoptic nuclei and median eminence is compatible with a role for the enzyme in generating neuronal angiotensin II in these structures. ACE was also found in high levels in the basal ganglia, dentate gyrus and cerebellum which are not well endowed with AI1 or its receptors; it is therefore likely that ACE in these structures might be involved in the processing of some other neuropeptide. Acknowledgements-Supported
by grants from the National Heart Foundation of Australia and the Austin Hospital Medical Research Foundation to F. A. 0. Mendeisohn and National Health and Medical Research Council to G. Paxinos
REVERENCE
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