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A micromethod for the measurement of renin in brain nuclei: Its application in spontaneously hypertensive rats
~28-3908~82/05~55-09~3.~~0 pergamon Press Ltd
Neuroph~rm~colog~ Vol. 21, pp. 4.55 to 463, 1982
Printed in Great Britain
A MICROMETHOD FOR THE MEASUREMENT OF RENIN IN BRAIN NUCLEI: ITS APPLICATION IN SPONTANEOUSLY HYPERTENSIVE RATS P. SCHELLING*, D. MEYER, H. E. Loos, G. SPECK, M. I. PHILLIPS, A. K. JOHNSON and D. GANTEN Department of Pharmacology and German Institute for High Blood Pressure Research, University of Heidelberg, D-6900 Heidelberg, Germany and *Institut de Recherche Cardio-Ang~iologique, University of Fribourg, CH-1700 Fribourg, Switzerland (Accepted 10 November 1981) Summary-The
aim of this study was to develop a method for the measurement of renin activity in small tissue samples obtained from rat brains by the micropunch technique and to investigate the activity of brain renin in spontaneously hypertensive rats.
The assay satisfied sensitivity and specificity requirements. Angiotensin I was generated at a pH of 6.0; complete recovery of angiotensin I and kinetic studies supported the specificity of the method. Angiotensinase and cathepsin D-like acid protease activity were measured in parallel with renin. Renin was present in all brain regions studied and decreased with the age of the animals. An increased activity of renin was measured in several nuclei of the brain stem and in the neurohypophysis of young hypertensive rats when compareu with age-matcbea normotensive control animals, These differences disappeared in older rats. There was a dissociation between renin and cathepsin D-like acid protease activity. No correlation existed between the distribution of renin and angiotensinase activity. The increased renin activity in brain stem nuclei of spontaneously hypertensive animals is in agreement with previous findings that the brain renin-angiotensin system contributes to the maintenance of high blood pressure in these rats.
There is evidence that angiotensin II (ANG II) in the brain arises from a pathway which resembles described for the plasma renin-angiotensin
the one system
(RAS).
The characterization of components of the RAS within the brain and the ANG II-related central effects of intracerebrally injected renin and angiotensinogen respectively are in support of this hypothesis (for review see Ganten and Speck, 1978; Schelling, Speck, Unger and Ganten, 1980). A renin-like enzyme in brain was first reported by Fischer-Ferraro, Nahmod, Goldstein and Finkielman (1971) and by Ganten, Marquez-Julio, Granger, Hayduk, Karsunky, Boucher and Genest (1971), and was definitely proved to be true renin more recently by its purification and separation from cathepsin D-like acid proteases (Dworschack, Gregory and Printz, 1978; Ganten and Speck, 1978; Inagami, Yokosawa and Hirose, 1978; Osman, Smeby and Sen, 1979), by the demonstration of the in viuo activity of pure brain renin (Speck, Poulsen, Unger, Rettig, Bayer, Schijlkens and Ganten, 1981) and by the immunhistochemical localization of renin in the brain of various species using monospecific renin antibodies (Fuxe, Ganten, Hijkfelt, Locatelli, Poulsen, Stock, Rix and Taugner, 1980; Celio, Clemens and Inagami, 1980; Slater, Defendini and Zimmerman, 1980). Key words: renin, cathepsin D, angiotensinase, spontaneously hypertensive rats.
brain,
In view of the suggested role of the brain reninangiotensin system in blood pressure regulation (Ganten, Hutchinson and Schelling, 1975; Mann, Phillips, Dietz, Haebara and Ganten, 1978; McDonald, Wickre, Aumann, Ban and Moffitt, 1980; Unger, Kaufmann-B~hler, Schiilkens and Ganten, 1981), it was of interest to investigate brain renin in spontaneously hypertensive rats (SHR) (Okamoto, Yamori and Nagaoka, 1974) in comparison to their normotensive Wistar Kyoto (WKY) counterparts. A method was therefore developed to measure renin activity in small tissue samples obtained from rat brains by the technique of micropunches (Palkovits, 1973). The cathepsin D-like acid protease and angiotensinase activity were determined in parallel in the same samples. METHODS
Subjects
Male stroke-prone spontaneously hypertensive rats (SHR-sp) which are derived from the Kyoto strain (Okamoto et al., 1974) and bred in Heidelberg since 1974 were investigated at the age of 9 and 20 weeks and compared with age-matched normotensive WKY control rats. Each experimental group consisted of 20 animals. Commercially available male Wistar rats (Ivanovas, Kisslegg, Germany) of about 300g body weight were used for the methodological studies. 45.5
456
P.
S~HELLING et al.
Dissection of the brain and preparation of the tissue extract All rats were bilaterally nephrectomized. Sixteen hours later they were killed by decapitation between 8 and 10 a.m.. The brains were quickly removed within less than 2 min and immediately frozen in a powder of dry-ice. The pineal gland, the adenohypophysis and the neurohypophysis were also dissected and deep frozen. Tissue from several brain regions was obtained following the punch technique (Palkovits, 1973). For this, the frozen brains were cut on a cryotome into frontal sections of 3OOpm. Defined brain areas of 1 mm in diameter were punched out from the slices using stereotaxic atlases for reference (K&rig and Klippel, 1963; Palkovits and Jacobowitz, 1974). Four punches per brain region, obtained from two rats were pooled in 200~1 of bidistilled ice-cold water, homogenized by sonication in a cold water bath for 10 set (Branson B 12 sonifier, microtip) and then centrifuged in the cold at 5000 g for 10 min. The supernatant was stored at -20°C. The neurosecretory glands were treated like the punches, except that they were not pooled and the supernatants obtained were diluted with bidistilled water (adenohypophysis, 1: 5; neurohypophysis, 1: 5; pineal gland, 1: 20) before storing at -20°C. For methodological studies a pool of brain extract was prepared from brain punches as described above. Purification of angiotensinogen Angiotensinogen was purified from plasma of 24 previously nephrectomized dogs and rats according to the method of Haas, Goldblatt, Gipson and Lewis (1966). A further purification was obtained by gel filtration (Sephadex G 150) and by ion exchange chromatography (Whatman DE 52) as recently reported (Eggena, Chu, Barrett and Sambhi, 1976). The substrate preparations were free of protease and peptidase activity. The lyophilized substrate was stable when stored at -30°C and yielded 45 pmol ANG I per mg (rat angiotensinogen) and 35 pmol ANG I per mg (dog angiotensinogen) when exhausted with excess hog kidney renin (NBC, Cleveland, Ohio, U.S.A.). Measurement of renin activity For the determination of renin activity, 50 ~1 of the brain extract was incubated with an excess of purified rat angiotensinogen (45 pmol) in 0.1 mol/l citratephosphate buffer at pH 6 and 37°C in a final volume of 750 ~1, containing 50 ~1 of an angiotensinase inhibitor mixture (Ganten, Schelling, Hoffman, Phillips and Ganten, 1978) and 100 ~1 90mmol dithiothreitol (DTT). Aliquots were taken from the incubation mixture at 0, 3 and 6 hr of incubation, diluted 1: 3 with cold 0.1 mol/l trishydroxymethylaminomethan (TRIS)-acetate buffer, pH 7.4 (radioimmunoassay (RIA) buffer) and boiled immediately for 5 min to stop
the enzyme activity. After centrifugation, the ANG I generated during the incubation was measured in the enzyme-free supernatant by a specific radioimmunoassay. The crossreactivity of the ANG I antibody with ANG II and ANG II fragments was less than 0.001%. Values are expressed as pmol ANG I generated per mg protein and per hr incubation. Measurement of angiotensinase activity Synthetic ANG II (20pmol) was dissolved in 1000 ~1 0.2 mol/ml TRIS-maleate buffer, pH 7.4, and added to 12.5 ~1 of brain tissue extract. Samples were incubated for 0, 13 and 3 hr at 37°C. Aliquots of 200~1 were pipetted each time into 400~1 of ice-cold RIA buffer and boiled for 5 min. After centrifugation ANG II was measured in the enzyme-free supernatant by radioimmunoassay. The angiotensinase activity was calculated from the disappearance rate of ANG II and is expressed as pmol ANG II degraded per mg protein and per hr incubation. Determination of acid protease activity The cathepsin D-like acid protease activity was measured by a modification of the method of Anson (1937). Briefly, 50~1 of the brain extract was incubated with 200 ~1 acid denatured bovine haemoglobin which was prepared fresh daily. The final concentration of the substrate was 2 x 10m4 mol/l in 0.2 mol/l acetic acid at pH 3.5. Aliquots of 50 ~1 were pipetted after 0, 2 and 4 hr of incubation at 37°C into 100 ~1 of ice-cold 0.3 mol/l trichloroacetic acid. The precipitated protein was centrifuged and the non-precipitable peptides were measured in the neutralized supernatant (Lowry, Rosenbrough, Farr and Randall, 1951). The acid protease activity is expressed as nmol bovine serum albumin (BSA) equivalents generated per mg protein and per hr incubation. Measurement of protein The protein content was measured in appropriate dilutions of 25 ~1 of brain tissue extract using BSA as a standard (Lowry et al., 1951). Methodological studies and kinetics of the brain reninangiotensinogen reaction The standard procedure of renin measurement was varied for some methodological investigations as follows: (1) The renin-angiotensinogen reaction was studied using rat and dog angiotensinogen preparations and different buffers (0.2 mol/l TRIS-maleate and 0.1 mol/‘l citrate-phosphate buffers) at various pH with and without the addition of dithiothreitol. (2) Angiotensin I (0.77 and 1.54 pmol respectively) was added to the incubation to investigate the recovery. (3) Increasing amounts of purified rat angiotensinogen were incubated under standard conditions to determine the Michaelis constant (K,) and the maximal velocity (urnaX)according to Lineweaver and Burk (1934). (4) The influence of several competitive renin inhibitors (Kokubu, Hiwada, Ito, Ueda, Yamamura,
Brain renin in spontaneously protein It --fl -50
-10
-30
-20
-10
\
0 0 somcatmn
30 time
s
-0
60 lscc)
Fig. 1. Effect of sonication time on enzyme activity and on protein extraction; (ho), renin activity (pmol ANG I/punch/hr); (m--m), acid protease activity (nmol BSA equivalents/punch/hr);
protein (X-*), protein/punch).
content
(mg
Mizoguchi and Shigezane, 1973; Poulsen, Burton and Haber, 1973) on the brain enzyme-angiotensinogen interaction was studied. The peptide inhibitors investigated were [His-Pro-Phe-His-Leu~D)Leu-Val-Tyr], [ Leu-Leu-Leu-Tyr[Leu-mu-mu-Phe-O-Me], O-Me], [His-Leu-Leu-Val-Phe-O-Me], all purchased from Serva, Heidelberg, Germany, and [Leu-Leu-ValPhe-O-Et] kindly supplied to us by Drs Kokubu and Mizoguchi, Tokyo, Japan, The inhibitors were dissolved in 1 moljl acetic acid as a stock solution (10e3 mol/l) and diluted in the incubation buffer to concentrations ranging from 10-O mol/l-lo-’ mol/l. The incubations were performed with either 45 pmol or 22.5 pmol of substrate. The results were calculated as indicated by Dixon (1953) to define the inhibitor constants (&). Statistics
Data are expressed as means + standard error of the mean. They consist of 4-6 values for the methodological studies and of 7-10 values (14-20 brains) for the enzyme distributions. The significances of differences were tested (1) by the two-way analysis of variance on whole brain renin versus strain and versus age respectively and (2) by Student’s unpaired t-tests. RESULTS ~~ethodo~ogicai studies
The influence of sonication time on enzyme extraction and on protein content in the supernatant was investigated (Fig. 1). The largest renin and acid protease activities were obtained within 20 set of sonication. The enzyme activities decreased when the treatment was continued, while the protein content showed a plateau up to 60 set of sonication. The ANG I-forming activity in the brain tissue extract was studied as a function of pH (Fig. 2). The incubation with purified rat angiotensinogen resulted
hypertensive rats
457
in a single peak between pH 5.5 and pH 6.0 compar-
able to the pH curve obtained with plasma renin. A pH optimum at pH 5.5 with a shoulder at pH 4.0 was observed with dog substrate. Quantitatively twice the amount of ANG I was generated at pH 5.5 from dog as compared to rat angiotensinogen; at pH 6.0 this difference was significantly reduced. Less ANG I was formed when TRIS-maleate buffer (0.09 _rt0.02 pmol ANG I/ml/hr) instead of the citrate-phosphate buffer (0.26 + 0.02 pmol ANG I/ml/hr) was used. The addition of dithiothreitol to the citrate-phosphate buffer medium increased the yield of ANG I to 1.01 4: O.lOpmol ANG Ijml/hr without affecting the pH optimum of the enzyme-substrate reaction. Kinetics (Figs 3,4, and 5 and Table 1) were studied with rat angiotensinogen in citrate-phosphate buffer, pH 6, containing dithiothreitol. A linear product formation over time of incubation and an 809; recovery of exogenously added ANG I was found after 8 hr of incubation (Fig. 3). The reaction of brain renin with increasing substrate concentrations followed the Michaelis-Menten kinetics (Fig. 4). The u,,, and K, were determined from the Lineweaver-Burk plot, a,,,,, and K, was 7.15. lo-r3 mol/ml/hr was 4.2’ 10-s mol/l. Several peptide inhibitors of renin interfered in a competitive way with the brain enzyme-angiotensinogen reaction (Table 1 and Fig. 5). The active site directed octapeptide inhibitor [His-ProPhe-His-Leu-(D)Leu-Val-Tyr] against kidney renin had no inhibitory effect. Topographical studies
Systolic blood pressure was 180.4 + 3.9 mmHg in 9-week old SHR-sp and 238.4 k 3.1 mmHg in 20-week old SHR-sp. Blood pressure was the same in both age groups of the normotensive WKY rats (114.0 f 3.5 mmHg). Renin activity was present in all brain regions of spontaneously hypertensive (SHR-sp) and control rats ranging from 3.1 to 15.1 pmol ANG I/mg prot./hr in 9-week old animals and from 2.1 to 10.1 pmol ANG I/mg prot./hr in 20-week old rats (Table 2). The pineal gland was the richest source of enzyme followed by the adenohypophysis and the neurohypophysis. A high level of activity was also measured in the brain stem and in the circumventricular organs; the smallest values were found in the cerebellar cortex, the septal area, the nucleus caudatus putamen and nucleus amygdaloideus. The overall enzyme profile in brains of hypertensive rats was comparable to that in normotensive controls and the analysis of variance on whole brain versus rat strain revealed the same distribution of values in control and spontaneously hypertensive rats (SHR-sp) respectively (F = 0.03; d.f. 1,93; NS). There were, however, specific differences between 9-week old spontaneously hypertensive rats and control rats in details (analyzed by Student’s t-test) which did not persist in the older animals (Table 2). Renin was increased in brain stem regions such as the nucleus reticularis lateralis (Al), nucleus tractus solitarii
458
P.
SC~~ELLING
et al.
10'12 mol
ANG llmllh
I2
10
08
06
6 01
02
0
Fig. 2. Renin activity in rat brain incubated with 0.1 mol/l citrate-phosphate equimolar (A---A)
quantities of purified rat angiotensinogen (+o) or in comparison with plasma renin activity using the homologous a substrate.
(A2), A5 and locus coeruleus (A6). Renin was also elevated in the neurohypophysis, but was less in the frontal cortex. A significant decrease of renin activity was observed with age in both the control and spontaneously hypertensive animals (analysis of variance: F = 4.50; d.f. 1,95; P < 0.05). Angiotensinase activity was estimated in the same tissue samples. There was no relationship between renin and angiotensinase activity. The values varied
.10-l?mo,
ANG
buffer of different pH and purified dog angiotensinogen angiotensinogen (W-D) as
between 210 and 640pmol ANG II degraded/mg prot./hr (Table 3). High levels of angiotensinase activity were measured in the pineal gland. There was a wide range of cathepsin D-like acid 1 ” 10 -3
20
I
l/ml 15
10
I
5
II
P
/I
/
/
I d
-1
Km
Fig. 3. Product formation as a function of incubation time (+a) and when ANG I was added to the incubation; (W---m),
0.77 pmol ANG
I; (S-_),
1.54 pmol ANG
I.
01
02
’
0.3
T
Fig. 4. Kinetics of brain renin with increasing levels of homologous antiotensinogen; Lineweaver-Burk plot, v = 10-i’ mol ANG I/ml/hr; s = low9 mol/l.
Brain renin in spontaneously
I
I I
I
10
-K,
50
100
I
Fig. 5. Dixon plot of the reaction between rat brain renin and homologous angiotensinogen in presence of the competitive [Leu-Leu-Leu-Phe-O-Me] tetrapeptide inhibitor; 45.10-” mol (-0) and 22.5.10-” mol (m-m)
angiotensinogen were added I = 1.33. 10e6 mol/l; v = lo-”
to
the
mol ANG
incubation; I/ml/hr.
protease activity in the brain from 5.4 to 40.4 nmol BSA equivalents/mg prot./hr (Table 4). The pineal gland and the adenohypophysis exhibited high levels of enzyme activity. Some strongly vascularized regions such as the subfornical organ, the median eminence the organum subcommissurale and the area postrema also contained high levels of acid protease activity, while small values were measured in the nucleus caudatus putamen and in the cerebellar cortex. The acid protease content was decreased in the fornix and in the supraoptic nucleus of young hypertensive rats. In some other brain areas of 20-week old hypertensive rats e.g. the adenohypophysis, lower values were observed than in control rats. The acid protease activity did not decrease with the age of animals and no parallelism of the differences existed between renin and cathepsin activity in hypertensive and normotensive rats in both age groups (Tables 2 and 4). DISCUSSION
The separation of brain renin from acid proteases (Dworschack et al., 1978; Ganten and Speck, 1978; Inagami et al., 1978; Osman et al., 1979; Speck et al., 1981) and its immunohistochemical localisation in the Table 1. Interference
of peptide
inhibitors
hypertensive
central nervous system (Celio et al., 1980; Fuxe et a[., 1980; Slater et al., 1980) made it worthwhile to study the renin distribution enzymatically within the brain in detail. Thus, a method was developed which allowed the measurement of brain renin in micropunches. An optimal and reproducible enzyme extraction was obtained when the samples were sonicated under the defined conditions described. Multiple thawing and freezing of micropunches resulted in loss of enzyme activity. Dithiothreitol has been reported to increase plasma renin activity (Poisner and Hong, 1977; Funae, Sasaki and Yamamoto, 1979). The addition of dithiothreitol to the citrate-phosphate buffer induced a threefold rise in brain renin activity under the standard incubation conditions but had a negligible effect when TRIS-maleate buffer was used. The sensitivity of this assay was further enhanced by the use of purified rat plasma angiotensinogen. This may be due to the elimination of renin inhibitors from the crude plasma substrate preparation (Schaechtelin, Baechtold, Haefeli, Regoli, Gaudry-Paredes and Peters, 1968; Kotchen, Welch and Talwalkar, 1978). Less pure rat angiotensinogen had a low affinity for brain renin, requiring heterologous substrates for enzyme measurement in rat brain (Ganten, Ganten, Schelling, Boucher and Genest, 1975). A lower pH optimum of brain renin around pH 4.5 was reported under those conditions (Ganten et al., 1971) arousing doubts about the significance of these measurements, since ubiquitous cathepsin D-like acid proteases were also found to generate ANG I from angiotensinogen at low pH (Day and Reid, 1976; Hackenthal, Hackenthal and Hilgenfeldt, 1978). Cathepsin D-like activity was found to be very low, however, at pH 6 (Day and Reid, 1976). In this study a single pH optimum between pH 5.5 and 6.0 was found when brain renin was incubated with purified rat angiotensinogen. A comparable pH curve was obtained for plasma renin under identical incubation conditions. Contamination of the brain renin measurements with plasma renin can be excluded, however, since the rats were nephrectomized and because of biochemical differences between both enzymes as discussed elsewhere (Speck et al., 1981). Thus, the method reported here provides a specific measurement of rat brain renin. The kinetic data in this study are comparable with
with the rat brain Max. inhibitor concentration (mol/l)
[His-Pro-Phe-His-Leu-(D)Leu-Val-Tyr] [Leu-Leu-Val-Phe-O-Et] [Leu-Leu-Leu-Phe-O-Me] [His-Leu-Leu-Val-Phe-O-Me] [Leu-Leu-Leu-Tyr-O-Me] Incubated N.P.2115-F
with rat angiotensinogen
1.33.10-4 1.17’10-4 1.33’10-4 2.67.10-4 2.67. 1O-4 * 22.5. lo-”
459
rats
mol t 45.10-‘*
renin-rat
angiotensinogen
Inhibition (percentage)
K, (mol/l)
Type of inhibition
1.4. 10m4 5.5.1o-5 1.0. 1o-4 8.6. 1O-5
Competitive Competitive Competitive Competitive
None 51.9* 66.5* 50.0* 63.0* mol.
41.9t 51.9t 33.0t 5O.O.t
reaction
-
460
P. Table 2. Renin activity
S~HELLING et ai.
(pmol ANG I/mg prot./hr) in various brain regions of nephrectom~zed (SHR-sp) and normotensive (WKY) rats at different ages 9-week old rats WKY SHR-sp
Frontal cortex Septum Fornix Nucleus caudatus putamen Nucleus amygdaloideus Organum vasculosum lamina terminalis Organum subfornicale Median eminence Organum subcommissurale Area postrema Nucleus supraopticus Nucleus paraventricularis Nucleus anterior hypothaiami Nucleus posterior hypothalami Substantia nigra reticularis Formatio reticularis Cerebellar cortex Locus coeruleus A5 area Nucleus tractus solitarii (A2) Nucleus reticularis lateralis (Al) Pineal gland Neurohypophysis Adenohypophysis Values are means
i: SEM as measured
4.69 3.98 3.98 3.23 4.03 4.92 5.24 5.67 5.55 5.00 4.91 4.16 4.14 3.98 4.00 3.77 3.14 4.05 4.31 4.37 4.20 12.98 4.76 7.30
_I + + + + + & & f + ? + + + f + + + + * f + i f
0.27 0.20 0.32 0.15 0.42 0.19 0.58 0.57 0.53 0.43 0.18 0.32 0.29 0.35 0.34 0.32 0.28 0.33 0.39 0.40 0.39 1.33 0.54 0.85
3.78 3.58 3.85 3.27 3.43 4.54 4.86 5.72 5.54 5.61 4.58 4.29 4.34 3.71 4.04 3.71 3.59 5.46 5.56 5.52 5.33 15.07 6.18 9.26
20-week WKY
) 0.30* & 0.32 rt: 0.25 + 0.15 1: 0.27 & 0.40 i 0.39 +_ 0.54 i: 0.48 5 0.24 + 0.40 Ifr 0.33 i: 0.28 j, 0.29 + 0.34 ) 0.34 & 0.18 + 0.37** ) 0.35* + 0.33* & 0.27* + 1.03 zt 0.30* + 0.83
t rt f + ; f 2 + + 2 2 f + + + It * It * * + + + i
0.10 0.12 0.12 0.10 0.08 0.04 0.30 0.23 0.20 0.23 0.14 0.20 0.15 0.16 0.10 0.13 0.10 0.19 0.21 0.15 0.23 0.50 0.30 0.42
in various brain regions of nephrec(WKY) rats at different ages
9-week old rats WKY SHR-sp
+ SEM as measured
3.01 2.68 2.79 2.30 2.94 3.45 3.54 4.05 4.76 3.66 3.63 2.93 3.16 2.79 2.83 2.61 2.11 3.02 3.31 3.37 3.29 10.12 2.94 4.90
while the tetrapeptide and pentapeptide renin inhibitors inhibited the rat brain renin-angiotensinogen reaction in a competitive way as has been demonstrated for kidney renin (Kokubu et al., 1973). Renin showed a differential distribution in various
Table 3. Angiotensinase activity (pm01 ANG II degradedlmg prot./hr) tomized spontaneously hypertensive (SHR-sp) and normotensive
Values are means
+_ 0.24 f 0.16 k 0.11 f 0.10 : 0.15 f 0.23 f 0.25 f 0.10 f 0.26 + 0.16 f 0.18 f 0.10 + 0.17 k 0.08 + 0.05 k 0.09 k 0.20 + 0.14 + 0.15 * 0.15 k 0.15 f 0.66 f 0.27 + 0.51
old rats SHR-sp
from 7 to 10 tissue pools. *P < 0.05, **P < 0.02.
those reported for kidney and plasma renin (Page and McCubbin, 1968; Lee, 1969; Poulsen, 1978). The active site-directed octapeptide inhibitor of renin (Poulsen et a[., 1973) had no effect on rat brain renin and rat kidney renin alike at the concentrations used,
Frontal cortex Septum Fornix Nucleus caudatus putamen Nucleus amygdaloideus Organum vasculosum lamina terminalis Organum subfornicale Median eminence Organum subcommissurale Area postrema Nucleus supraopticus Nucleus paraventricularis Nucleus anterior hypothalami Nucleus posterior hypothalami Substantia nigra reticularis Formatio reticularis Cerebeliar cortex Locus coeruleus A5 area Nucleus tractus solitarii (A21 Nucleus reticularis laterahs (Al) Pineal gland Neurohypophysis Adenohypophysis
3.34 3.09 2.92 2.49 2.93 3.70 3.54 4.60 3.87 3.68 4.10 2.91 3.06 2.95 2.77 2.82 2.46 3.49 3.45 3.47 3.80 10.36 3.33 6.15
hypertensive
286.4 284.7 296.5 280.5 263.0 231.8 311.9 270.5 264.4 253.8 275.2 250.3 227.5 235.6 283.8 237.7 237.7 269.9 204.8 218.4 261.1 479.4 319.7 264.9
* 27.6 I 24.5 k 27.3 +_ 20.7 f 18.8 k 15.8 + 31.0 f 24.8 + 17.6 5 24.1 + 25.2 k 25.9 f 20.8 i 12.6 + 15.9 f 18.1 f 16.6 f 24.9 i 20.6 + 21.2 + 22.0 Ifr 25.1 + 28.1 + 29.7
254.2 229.1 287.1 253.6 248.0 222.5 284.0 269.6 294.9 227.4 206.1 245.4 218.5 221.9 287.1 240.4 216.2 251.2 217.4 207.8 245.7 379.0 245.6 233.4
+ 22.4 i- 19.5* ; 11,7 + 22.7 + 22.1 + 19.7 & 8.8 + 32.9 & 33.1 Ifr 25.7 + 16.5* + 25.5 ? 13.5 + 13.7 i: 309 + 23.8 f 23.6 + 27.8 + 26.0 + 21.5 ; 39.1 * 19.7** + 16.1* + 9.7
20-week WKY 359.0 377.2 387.9 451.9 410.9 356.9 404.2 411.7 400.2 374.8 391.1 406.3 330.7 384.4 410.2 370.2 368.7 394.8 346.4 371.9 338.6 527.8 335.9 306.4
+ + ; f * + + + + f + + : f f i f + + $ ; f + +
17.5 23.1 26.0 28.9 35.5 22.8 37.5 29.4 27.8 27.8 31.3 22.3 23.5 29.2 27.3 22.9 19.4 26.4 20.3 30.0 22.7 54.7 29.4 32.6
from 7 to 10 tissue pools. *P < 0.05, **P < 0.01.
old rats SHR-sp 367.5 389.1 399.0 415.6 399.3 332.0 384.3 386.9 392.7 349.7 331.0 375.8 361.1 367.7 379.7 344.7 336.6 365.7 333.7 350.6 334.8 639.4 384.9 315.4
i 28.8 + 24.4 z 23.3 + 25.3 rl: 24.0 & 20.4 + 21.3 f 21.5 + 31.5 + 19.0 f 22.6 f 30.6 ? 21.8 + 25.8 * 25.7 rt 21.9 + 19.3 t 16.6 + 24.2 + 22.6 ; 22.9 + 62.5 + 32.1 IcL67.5
Brain renin in spontaneously
461
hypertensive rats
Table 4. Cathepsin D-like acid protease activities (nmoi BSA equivaients/mg prot./h) in various brain regions of nephrectomized spontaneously hypertensive (SHR-sp) and normotensive (WKY) rats at different ages g-week
WKY
old rats SHR-sp
20-week old rats SHR-sp WKY
Frontal cortex Septum Fornix Nucleus caudatus putamen Nucleus amygdaloideus Organum vasculosum lamina terminalis Organum subfornicale Median eminence Organum su~ommissurale Area postrema Nucleus supraopticus
7.09 f 7.13 f 7.32 + 5.57 * 7.36 + 9.15 + 9.82 + 12.73 + 11.61 f 10.35 f 9.88 +
0.55 0.28 0.28 0.21 0.39 0.28 0.69 0.68 0.56 0.43 0.28
7.39 & 0.25 6.79 + 0.28 6.18 + 0.42* 5.59 rt 0.32 6.80 + 0.30 9.12 + 0.42 10.05 i: 0.47 12.59 & 0.60 12.70 k 0.56 11.48 rf: 0.46 8.41 & 0.30**
7.97 * 0.15 7.18 k 0.15 6.94 + 0.23 6.22 + 0.32 7.36 + 0.21 10.39 f 0.61 10.36 + 0.28 13.93 & 0.25 11.39 i 0.25 12.13 i 0.34 12.12 + 1.02
7.51 + o.io* 7.63 i 0.16 6.75 + 0.22 5.46 + 0.05* 7.34 + 0.13 9.45 + 0.28 11.25 rt 0.22* 14.01 + 0.37 15.12 + 0.66*** 12.64 -i: 0.18 9.92 f 0.60
Nucleus paraventricularis Nucleus anterior hypothalami Nucleus posterior hypothalami Substantia nigra reticularis Formatio reticularis Cerebellar cortex Locus coeruleus A5 area Nucleus tractus solitarii (A2) Nucleus reticularis lateralis (Al) Pineal gland Neurohypophysis Adenohypophysis
8.21 8.12 7.51 7.51 7.06 5.88 8.70 8.59 9.21 8.70 33.72 8.92 17.11
0.39 0.34 0.27 0.30 0.31 0.27 0.38 0.41 0.59 0.46 1.33 0.50 0.72
8.13 7.86 7.19 7.67 7.11 5.37 9.09 8.93 9.08 8.13 32.76 9.26 16.40
8.08 8.62 8.09 7.68 7.49 5.47 9.54 8.96 9.12 8.17 39.84 9.55 25.33
8.58 8.61 7.96 7.44 7.47 5.57 9.31 9.90 9.43 7.73 40.39 9.36 21.18
Values are means
+ SEM as measured
+ f k * + + + + + + * + f
rt 0.33 rt 0.37 t 0.21 rt 0.26 + 0.28 + 0.33 It 0.40 + 0.30 + 0.40 + 0.28 Lt: 1.52 i 0.79 + 0.92
f & + f k _t _t f f k + f +
0.12 0.29 0.14 0.20 0.23 0.15 0.17 0.22 0.16 0.39 1.34 0.12 1.51
from 7 to 10 tissue pools. *p < 0.05, **P < 0.02, ***P
brain regions and levels decreased with age. The pineal organ followed by the adenohypophysis exhibited by far the highest level of enzyme activity within the central nervous system. This confirms previous reports in rats, dogs and pigs obtained by different methods including direct radioimmunoassay for renin (Haulica, Branisteanu, Rosca, Stratone, Berbeleu, Balan and Ionescu, 1975; De Agostini, Reinharz and Vallotton, 1980; Hirose, Yokosowa, fnagami and Workman, 1980). The distribution of renin in the brain of rats is quite similar to that recently published for hog brain, differing essentially in the values for nucleus amygdaloideus and neurohypophysis (Hirose et al., 1980). The immunohistochemical mapping studies demonstrated localization of renin in neuronal cells and confirmed a wide distribution of the enzyme within the brain of rats, mice and humans (Celio et al., 1980; Fuxe et al., 1980; Slater et al., 1980). They differ, however, in details between each other as well as in comparison with the biochemical measurements of renin reported here. Thus, renin-like staining was found to be intensive in the cerebellum and to be low or absent in the neuroh~ophysis, whereas just the reverse is true for renin measured enzymatically. On the other hand, immunohistochemical and biochemical results were in agreement in the pineal gland, adenohypophysis, brain stem and hypothalamic regions. Species related differences as well as methodological reasons, e.g. specificity of renin antibodies or activation of prorenin (Ganten and Speck, 1978; Hirose et al., 1980) during tissue preparation and incubation may contribute to these discrepancies.
+ 0.25 * 0.30 + 0.21 + 0.15 rf: 0.25 + 0.10 * 0.33 + 0.25** + 0.23 * 0.35 k 1.22 + 0.21 i: 0.99* < 0.001.
The angiotensinase activity showed uniform distribution within the brain and increased with age. There was no apparent relationship between ANG II degradation and renin, if anything angiotensinase was lower in spontaneously hypertensive rats. Cathepsin D-like acid protease activity paralleled renin to some extent, but differed from the latter in several important aspects: the acid protease activity did not decrease with the age of the animals and no differences as described for renin existed between the hypertensive and normotensive rats in both age groups. Cathepsin D was recently reported to split neuropeptides such as opiates from their high molecular weight precursors in vitro (Benuck, Grynbaum, Cooper and Marks, 1978). This group of enzymes could therefore also be of interest for the regulation of synthesis and turnover of neuropeptides. The renin content in spontaneously hypertensive (SHR-sp) as compared to normotensive WKY rats was elevated in catecholaminergic nuclei of the brain stem and in the neurohypophysis during development of hypertension; this difference disappeared when hypertension reached a plateau and became stable. These data could be meaningful in view of the changes described for the catecholamine content and turnover in cardiovascular control centres of the brain stem (Versteeg, Palkovits, Van Der Gugten, Wijnen, Smeets and De Jong, 1976; Nakamura and Nakamura, 1978; Fuxe, Ganten, Jonsson, Agnati, Andersson, Hokfelt, Bolme, Goldstein, Hallman, Unger and Rascher, 1979; Saavedra, 1979). Angiotensin II was reported to stimulate the synthesis of catecholamines and to facilitate neurotransmission
462
P.
SCHELLING et al.
(see !&helling et al., 1980). The blood pressure increase after central applicaiion of ANG II appears, in fact, to be mediated by central catecholaminergi~ mechanisms, since it was inhibited by central pretreatment with 6-hgdroxydopamine (Hoffman, Phillips and Schmid. 1977bj and central beta-adrenorecentor blockade (Simon, ‘Schaz, Mann, Ganten, John’son, Unger, Rascher and Ganten, 1981). The present results of increased renin in the brain of spontaneously hypertensive rats are in support of, and extend. earlier findings demonstrating (1) the blood pressure lowering effect of central ANG II receptor blockade and converting enzyme inhibition in hypertensive rats (Ganten et al., 1975; Mann et al., 1978; McDonald et al., 1980; Unger et ol., 1981), (2) increased levels of ANG-like material in the cerebrospinal fluid of hypertensive rats (Ganten et ul., 1975) and hypertensive patients (Finkielman, FischerFerraro, Diaz, Goldstein and Nahmod, 1972; Kaneko, Ohnishi, Fujishima, Tanaka and Umemura, 1979) and (3) enhanced blood pressure responses to centrally applied ANG II in spontaneously hypertensive rats (Hoffman, Phillips and Schmid, 1977a). It has also to be mentioned in this resnect that the olasma renin-angiotensin system is not stimulated in adult hypertensive rats (Matsunaga, Yamamoto, Hara, Yamori, Ogino and Okamoto, 197.5; Mann et af., 1978) that the blood cerebrospinal fluid barrier is impermeable for plasma ANG II (Schelling, Ganten, Sponer, Unger and Ganten, 1980) and that the blood pressure fall in spontaneously hypertensive rats following central administration of the ANG II receptor antagonist is still present when the rats were nephrectomized (Mann et al., 1978). In conclusion, a specific and sensitive method was developed which allowed the investigation of brain renin in spontaneously hypertensive rats. Renin activity showed a typical pattern of distribution in various brain regions. The enzyme activity decreased with the age of animals. An elevated renin content was measured in catecholaminergic nuclei of the brain stem and in the neurohypophysis of young spontaneously hypertensive rats. The possible link between brain renin and central blood pressure control is evident and the blood pressure lowering effects of centrallv , administered inhibitors of the reninangiotensin system may be explained by interference with the stimulated brain r~nin-angiotensin system in hypertensive rats. 1
1
Ackno,c,ledyrmenrs---These
studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 “Cardiovaskullres System”, by grant No. 3.033 -0.76 of the Swiss National Science Foundation, by the Emil Bare11 Foundation and the Swiss Foundation for Cardiology. M.I.P. and A.K.J. were recipients of a Humboldt fellowship. We wish to thank C. Bayer, S. Sowarka and S. Miiller for excellent technical assistance and F. Liard for typing the manuscript.
Gen. Fhysiol.
20: 565-574.
_
Benuck M., Grynbaum A., Cooper T. B. and Marks N. (1978) Conversion of Iipotropic peptides by purified cathepsin D of human pituitary: release of y-endorphin by cleavage of the leu77-uhe78 bond. Neurosci. Lett. 10: 3-9. Celio M. R., Clemens D. L. and Inagami T. (1980) Renin in anterior pituitary, pineal, and neuronal cells of mouse brain, immunohistochemical localization. Biomed. Res. 1: 427-431.
Day R. and Reid I. A. (1976) Renin activity in dog brain: etymological similarity to cathepsin D. ~n~o~~~~~~~~y 99: 93--100, De Agostini A., Reinharz A. and Vallotton M. B. (1980) Cathepsin D and renin-like activity in bovine pineal gland. Exprrirntia 36: 28. Dixon M. (1953) The determination of enzyme inhibitor constants. Biochem. 1. 55: 17&171. Dworschack R. T., Gregory T. J. and Printz M. P. (1978) Identification of neutral pH renin-like activity from brain homogenates by chromatography of phenylethylamine-Sepharose, Fed. Proc. 38: 1385. Eggena P., Chu C. L., Barrett J. D. and Sambhi P. (1976) Purification and partial characterization of human angiotensinogen. Biochim. biophys. Acra 427: 208-217. Finkielman S., Fischer-Ferraro C., Diaz A., Goldstein D. J. and Nahmod V. E. (1972) A pressor substance in the cerebrospinal fluid of normotensive and hypertensive patients. Proc. nuts Acad. Sci., U.S.A. 69: 3341-3344. Fischer-Ferraro C., Nahmod V. E., Goldstein D. J. and Finkielman S. (1971) Angiotensin and renin in rat and docl brain. 1. ~X.YD. Med. 133: 353-361. Funa: Y.. Sasaki ‘D. and Yamamoto K. (1979) EfTect of Ditb~othreitol on the reaction of renin and an8iotensinogen. Clinicff chim. Acta 91: 183-189. Fuxe K., Ganten D., Hiikfelt T., Locatelli V., Paulsen K., Stock G., Rix E. and Taugner R. (1980) Renin-like immunocytochemical activity in the rat and mouse brain. Neurosci. Lett. 18: 245-250. Fuxe K., Ganten D., Jonsson G., Agnati L. F., Andersson K., Hiikfclt T., Bolme P., Goldstein M., Hallman H., Unger T. and Rascher W. (1979) Catecholamine turnover changes in hypothalam~is and dorsal midline area of the caudal medulla oblong&a of spontaneously hypertensive rats. Neurosci. Left. 15: 283-288. Ganten D., Ganten U., Schelling P., Boucher R. and Genest J. (1975) The renin and iso-renin-an~iot~nsin systern in rats with experimental pi~~~itary tumors. Proc. SM. up.
Biol. Med. 148: 568-572.
Ganten D., Hutchinson J. S. and Schelling P. (1975) The intrinsic brain iso-renin-angiotensin system in the rat: its possible role in central mechanisms of blood pressure regulation. Clin. Sci. m&c. Med. 48: 26%268s. Ganten D., Marquez-Julio A., Granger P., Hayduk K., Karsunkv K. P.. Boucher R. and Genest J. (1971) Renin in dog biain. Am. f. Pltysioi. 221: 1733-1737. Ganten D., Schelling P., Hoffman W. E., Phillips M. I. and Ganten U. (1978) The measurement of extrarenal isorenins. in: RfrdioimmunoassaJ;-Rrfiin-Angiolensin (Krause I>. K. CIC ul., Eds), pp. 144155. Georg Thieme, Stuttgart. Ganten D. and Speck G. (1978) The brain renin-angiotensin system: a model for the synthesis of peptides in the brain. B~ocil~rn‘~~lar~nuc. 27: 2379-2389. Haas E., GoIdb~tt H., Gipson A. C. and Lewis L. (1966) Extraction, purification and assay of human renin free of angiotensinase. Circulation Rrs. 19: 739-749. Hackenthal E., Hackenthal R. and Hilgenfeldt U. (1978) Purification and partial characterization of rat brain acid proteinase (isorenin). Biochim. hiophys. Acta 522: 561-573.
REFERENCES Anson M. L. (1937) The estimation
hemoglobin and the partial purification of cathepsin. J.
of cathepsin
with
Haulica I., Branisteanu D. D.. Rosca V.. Stratone A.. Berbeleu V., Balan G. and Ionescu L. (1975) A renin-like
Brain renin in spontaneously activity in pineal gland and hypophysis. Endocrinology %: 508-510. Hirose S., Yokosawa H., Inagami T. and Workman R. J. (1980) Renin and prorenin in hog brain: ubiquitous distribution and high concentration in the pituitary and pineal. Bruin Res. 191: 489-499. Hoffman W. E., Phillips M. I. and Schmid P. G. (lfl7a) Central angiotensin II-induced responses in spontaneously hypertensive rats. Am. .I. Physiol. 232: H426H433. Hoffman W. E., Phillips M. I. and Schmid P. (1977b) The role of catecholamines in central antidiuretic and pressor mechanisms. Ne~~~o~~ar~a~o~o~~16: 5633569. Inagami T., Yokosawa H. and Hirose S. (1978) Definitive evidence for renin in rat brain by affinity chromatographic separation from protease. Clin. Sci. m&c. Med. 55: 121s.-123s. Kaneko Y., Ohnishi T., Fujishima S., Tanaka T. and Umemura S. (1979) Relation of angiotensin II concentrations in cerebrospinal fluid to systemic blood pressure levels in essential hypertension. Proc. 6th Scientific Meeting I of I the Int. Ser. Of‘Hypertension, Giiteborg. K.okubu T., Hiwada K., Ito T., Ueda E., Yamamura Y., Mizoguchi T. and Shigezane K. (1973) Peptide inhibitors of renin angiotensinogen reaction system. Biochem. Phurmac. 22: 3217-3223.
K&rig J. F. R. and Klippel R. A. (1963) The Rat Brain a Stereotaxic Arias oftbe Forebrain azrd Lower Parts of the Brain Stem. R. E. Krieger Publishing, New York. ”
Kotchen T. A.. Welch W. J. and Talwalkar R. T. (1978) In citro and in &I inhibition of renin bv fatty_ acids. Ai. J.
Physiol. 234: E593-E599. Lee M. R. (1969) Renin and Hvuertension: thesis. Williams & Wilkins, Baltimore. r.
A Modern
Svn-
Lineweaver H. and Burk D. (1934) The determination of enzyme dissociation constants. J. Am. k.hem. Sac. 56: &8-666.
Lowry D. M., Rosenbrough N. J., Farr A. I. and Randall R. J. (1951) Protein measurement with the phenol reagent. J. bid/. Gem. 193: 265-275. Mann J. F. E., Phillips M. I., Dietz R., Haebara H. and Ganten D. (1978) Effects of central and peripheral angiotensin blockade in hypertensive rats. Am. f. P~z.vsjo~. 234: H629-H637. Matsunaga M., Yamamoto J., Hara A., Yamori Y., Ogino K. and Okamoto K. (1975) Plasma renin and hypertensive vascular complications: an observation in the stroke-prone spontaneously hypertensive rat. Japan. Circ. 3. 39: 1305-1311. McDonald W., Wickre C., Aumann S.. Ban D. and Moffitt B. (1980) The sustained antihypertensive effect of chronic cerebroventricuiar infusion of angiotensin antagonist in spontaneously hypertensive rats. Endocrinology 107: 1305-1308.
Nakamura K. and Nakamura K. (1978) Role of brainstem and spinal noradrenergic and adrcnergic neurons in the development and maintenance of hypertension in spontaneously hypertensive rats. Naanyn-Se~m~ede~ergs Arch. Pharmac. 305: 127-133.
Okamoto K., Yamori Y. and Nagaoka E. (1974) Establishment of the stroke-prone spontaneously hypertensive rats. Circ. Res. 34/35: Suppl. I, 143-153.
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hypertensive rats
Osman M. Y., Smeby R. R. and Sen S. (1979) Separation of dog brain renin-like activity from acid protease activity. Hypertension 1: 53-60. Page I. H. and McCubbin J. W. (1968) Renal Hypertension. Year Book Medical Publishers, Chicago. Palkovits M. (1973) Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res. 59: 449450. Palkovits M. and Jacobowitz D. M. (1974) Topographic acetylcholinesteraseatlas of catecholamine and containing neurons in the rat brain. J. comp. Neuroi. 157: 29-42. Poisner A. M. and Hong J. S. (1977) Dithiothreitol augments renin activity by an action on renin substrate. Proc. Sot. exp. Biol. Med. 154: 180-183. Paulsen K. (1978) Kinetics of the renin substrate reaction in plasma as basis for measuring. In: RadioimmunoussayRenin-Angiotensin (Krause D. K. et ui.. Eds), pp. 76-81. Georg Thieme, Stuttgart. Poulsen K., Burton J. and Haber E. (1973) Competitive inhibitors of renin. ~jac~~e}~isfr~ 12: 3877-3882. Saavedra J. M. (1979) Adrenaline levels in brain stem nuclei and effects of a PNMT inhibitor on spontaneously hypertensive rats. Brain Res. 166: 2833292. Schaechtelin G., Baechtold N., Haefeli L., Regoli D., Gaudry-Paredes A. and Peters G. (1968) A renin-inactivating system in rat plasma. Am. J. Physiol. 215: 632-636. Schelling P., Ganten U., Sponer G., Unger 1. and Ganten D. (1980) Components of the renin-angiotensin system in the cerebrospinal fluid of rats and dogs with special consideration of the origin and the fate of angiotensin II. Neuroendocrinoiogy
31: 297-308.
Schelling P., Speck G., Unger T. and Ganten D. (1980) The brain renin-angiotensin system: biochemistry, localisation and functional aspects. In: Adzances in Experimental Medicine (Parve2 H. and Parvez_S., Eds), Vol. I, pp. 243-288. Elsevier/North-Holland Biomedical Press, Amsterdam. Simon W., Schaz K., Mann J. F. E., Ganten U., Johnson A. K., Unger Th., Rascher W. and Ganten D. (1981) The effects of beta-adrenergic receptor blockers on blood pressure responses to central angiotensin II. Neuropharmacology 20: 7 19-726.
Slater E. E., Defendini R. and Zimmerman E. A. (1980) Wide distribution of immunoreactive renin in nerve cells of human brain. Proc. natn. Acad. Sci., U.S.A. 77: 5458-5460.
Speck G., Poulsen K., Unger Th., Rettig R., Bayer C., Scholkens B. and Ganten D. (1981) in Go activity of purified mouse brain renin. Brain Res. 21% 371384.
Unger T., Kaufmann-Biihler I., Scholkens B. and Ganten D. (1981) Brain-converting enzyme inhibition: a possible mechanism for the antihypertensive action of captopril in spontaneously hypertensive rats. Eur. J. Pharmac. 70: 467478.
Versteeg D. H. G., Palkovits M., Van Der Gugten J., Wijnen H. L. J. M., Smeets G. W. M. and De Jong W. (1976) Catecholamine content of individual brain regions of spontaneously hypertensive rats (SH-rats). Bruin Res. 112: 429-434.
Neuroph~rm~colog~ Vol. 21, pp. 4.55 to 463, 1982
Printed in Great Britain
A MICROMETHOD FOR THE MEASUREMENT OF RENIN IN BRAIN NUCLEI: ITS APPLICATION IN SPONTANEOUSLY HYPERTENSIVE RATS P. SCHELLING*, D. MEYER, H. E. Loos, G. SPECK, M. I. PHILLIPS, A. K. JOHNSON and D. GANTEN Department of Pharmacology and German Institute for High Blood Pressure Research, University of Heidelberg, D-6900 Heidelberg, Germany and *Institut de Recherche Cardio-Ang~iologique, University of Fribourg, CH-1700 Fribourg, Switzerland (Accepted 10 November 1981) Summary-The
aim of this study was to develop a method for the measurement of renin activity in small tissue samples obtained from rat brains by the micropunch technique and to investigate the activity of brain renin in spontaneously hypertensive rats.
The assay satisfied sensitivity and specificity requirements. Angiotensin I was generated at a pH of 6.0; complete recovery of angiotensin I and kinetic studies supported the specificity of the method. Angiotensinase and cathepsin D-like acid protease activity were measured in parallel with renin. Renin was present in all brain regions studied and decreased with the age of the animals. An increased activity of renin was measured in several nuclei of the brain stem and in the neurohypophysis of young hypertensive rats when compareu with age-matcbea normotensive control animals, These differences disappeared in older rats. There was a dissociation between renin and cathepsin D-like acid protease activity. No correlation existed between the distribution of renin and angiotensinase activity. The increased renin activity in brain stem nuclei of spontaneously hypertensive animals is in agreement with previous findings that the brain renin-angiotensin system contributes to the maintenance of high blood pressure in these rats.
There is evidence that angiotensin II (ANG II) in the brain arises from a pathway which resembles described for the plasma renin-angiotensin
the one system
(RAS).
The characterization of components of the RAS within the brain and the ANG II-related central effects of intracerebrally injected renin and angiotensinogen respectively are in support of this hypothesis (for review see Ganten and Speck, 1978; Schelling, Speck, Unger and Ganten, 1980). A renin-like enzyme in brain was first reported by Fischer-Ferraro, Nahmod, Goldstein and Finkielman (1971) and by Ganten, Marquez-Julio, Granger, Hayduk, Karsunky, Boucher and Genest (1971), and was definitely proved to be true renin more recently by its purification and separation from cathepsin D-like acid proteases (Dworschack, Gregory and Printz, 1978; Ganten and Speck, 1978; Inagami, Yokosawa and Hirose, 1978; Osman, Smeby and Sen, 1979), by the demonstration of the in viuo activity of pure brain renin (Speck, Poulsen, Unger, Rettig, Bayer, Schijlkens and Ganten, 1981) and by the immunhistochemical localization of renin in the brain of various species using monospecific renin antibodies (Fuxe, Ganten, Hijkfelt, Locatelli, Poulsen, Stock, Rix and Taugner, 1980; Celio, Clemens and Inagami, 1980; Slater, Defendini and Zimmerman, 1980). Key words: renin, cathepsin D, angiotensinase, spontaneously hypertensive rats.
brain,
In view of the suggested role of the brain reninangiotensin system in blood pressure regulation (Ganten, Hutchinson and Schelling, 1975; Mann, Phillips, Dietz, Haebara and Ganten, 1978; McDonald, Wickre, Aumann, Ban and Moffitt, 1980; Unger, Kaufmann-B~hler, Schiilkens and Ganten, 1981), it was of interest to investigate brain renin in spontaneously hypertensive rats (SHR) (Okamoto, Yamori and Nagaoka, 1974) in comparison to their normotensive Wistar Kyoto (WKY) counterparts. A method was therefore developed to measure renin activity in small tissue samples obtained from rat brains by the technique of micropunches (Palkovits, 1973). The cathepsin D-like acid protease and angiotensinase activity were determined in parallel in the same samples. METHODS
Subjects
Male stroke-prone spontaneously hypertensive rats (SHR-sp) which are derived from the Kyoto strain (Okamoto et al., 1974) and bred in Heidelberg since 1974 were investigated at the age of 9 and 20 weeks and compared with age-matched normotensive WKY control rats. Each experimental group consisted of 20 animals. Commercially available male Wistar rats (Ivanovas, Kisslegg, Germany) of about 300g body weight were used for the methodological studies. 45.5
456
P.
S~HELLING et al.
Dissection of the brain and preparation of the tissue extract All rats were bilaterally nephrectomized. Sixteen hours later they were killed by decapitation between 8 and 10 a.m.. The brains were quickly removed within less than 2 min and immediately frozen in a powder of dry-ice. The pineal gland, the adenohypophysis and the neurohypophysis were also dissected and deep frozen. Tissue from several brain regions was obtained following the punch technique (Palkovits, 1973). For this, the frozen brains were cut on a cryotome into frontal sections of 3OOpm. Defined brain areas of 1 mm in diameter were punched out from the slices using stereotaxic atlases for reference (K&rig and Klippel, 1963; Palkovits and Jacobowitz, 1974). Four punches per brain region, obtained from two rats were pooled in 200~1 of bidistilled ice-cold water, homogenized by sonication in a cold water bath for 10 set (Branson B 12 sonifier, microtip) and then centrifuged in the cold at 5000 g for 10 min. The supernatant was stored at -20°C. The neurosecretory glands were treated like the punches, except that they were not pooled and the supernatants obtained were diluted with bidistilled water (adenohypophysis, 1: 5; neurohypophysis, 1: 5; pineal gland, 1: 20) before storing at -20°C. For methodological studies a pool of brain extract was prepared from brain punches as described above. Purification of angiotensinogen Angiotensinogen was purified from plasma of 24 previously nephrectomized dogs and rats according to the method of Haas, Goldblatt, Gipson and Lewis (1966). A further purification was obtained by gel filtration (Sephadex G 150) and by ion exchange chromatography (Whatman DE 52) as recently reported (Eggena, Chu, Barrett and Sambhi, 1976). The substrate preparations were free of protease and peptidase activity. The lyophilized substrate was stable when stored at -30°C and yielded 45 pmol ANG I per mg (rat angiotensinogen) and 35 pmol ANG I per mg (dog angiotensinogen) when exhausted with excess hog kidney renin (NBC, Cleveland, Ohio, U.S.A.). Measurement of renin activity For the determination of renin activity, 50 ~1 of the brain extract was incubated with an excess of purified rat angiotensinogen (45 pmol) in 0.1 mol/l citratephosphate buffer at pH 6 and 37°C in a final volume of 750 ~1, containing 50 ~1 of an angiotensinase inhibitor mixture (Ganten, Schelling, Hoffman, Phillips and Ganten, 1978) and 100 ~1 90mmol dithiothreitol (DTT). Aliquots were taken from the incubation mixture at 0, 3 and 6 hr of incubation, diluted 1: 3 with cold 0.1 mol/l trishydroxymethylaminomethan (TRIS)-acetate buffer, pH 7.4 (radioimmunoassay (RIA) buffer) and boiled immediately for 5 min to stop
the enzyme activity. After centrifugation, the ANG I generated during the incubation was measured in the enzyme-free supernatant by a specific radioimmunoassay. The crossreactivity of the ANG I antibody with ANG II and ANG II fragments was less than 0.001%. Values are expressed as pmol ANG I generated per mg protein and per hr incubation. Measurement of angiotensinase activity Synthetic ANG II (20pmol) was dissolved in 1000 ~1 0.2 mol/ml TRIS-maleate buffer, pH 7.4, and added to 12.5 ~1 of brain tissue extract. Samples were incubated for 0, 13 and 3 hr at 37°C. Aliquots of 200~1 were pipetted each time into 400~1 of ice-cold RIA buffer and boiled for 5 min. After centrifugation ANG II was measured in the enzyme-free supernatant by radioimmunoassay. The angiotensinase activity was calculated from the disappearance rate of ANG II and is expressed as pmol ANG II degraded per mg protein and per hr incubation. Determination of acid protease activity The cathepsin D-like acid protease activity was measured by a modification of the method of Anson (1937). Briefly, 50~1 of the brain extract was incubated with 200 ~1 acid denatured bovine haemoglobin which was prepared fresh daily. The final concentration of the substrate was 2 x 10m4 mol/l in 0.2 mol/l acetic acid at pH 3.5. Aliquots of 50 ~1 were pipetted after 0, 2 and 4 hr of incubation at 37°C into 100 ~1 of ice-cold 0.3 mol/l trichloroacetic acid. The precipitated protein was centrifuged and the non-precipitable peptides were measured in the neutralized supernatant (Lowry, Rosenbrough, Farr and Randall, 1951). The acid protease activity is expressed as nmol bovine serum albumin (BSA) equivalents generated per mg protein and per hr incubation. Measurement of protein The protein content was measured in appropriate dilutions of 25 ~1 of brain tissue extract using BSA as a standard (Lowry et al., 1951). Methodological studies and kinetics of the brain reninangiotensinogen reaction The standard procedure of renin measurement was varied for some methodological investigations as follows: (1) The renin-angiotensinogen reaction was studied using rat and dog angiotensinogen preparations and different buffers (0.2 mol/l TRIS-maleate and 0.1 mol/‘l citrate-phosphate buffers) at various pH with and without the addition of dithiothreitol. (2) Angiotensin I (0.77 and 1.54 pmol respectively) was added to the incubation to investigate the recovery. (3) Increasing amounts of purified rat angiotensinogen were incubated under standard conditions to determine the Michaelis constant (K,) and the maximal velocity (urnaX)according to Lineweaver and Burk (1934). (4) The influence of several competitive renin inhibitors (Kokubu, Hiwada, Ito, Ueda, Yamamura,
Brain renin in spontaneously protein It --fl -50
-10
-30
-20
-10
\
0 0 somcatmn
30 time
s
-0
60 lscc)
Fig. 1. Effect of sonication time on enzyme activity and on protein extraction; (ho), renin activity (pmol ANG I/punch/hr); (m--m), acid protease activity (nmol BSA equivalents/punch/hr);
protein (X-*), protein/punch).
content
(mg
Mizoguchi and Shigezane, 1973; Poulsen, Burton and Haber, 1973) on the brain enzyme-angiotensinogen interaction was studied. The peptide inhibitors investigated were [His-Pro-Phe-His-Leu~D)Leu-Val-Tyr], [ Leu-Leu-Leu-Tyr[Leu-mu-mu-Phe-O-Me], O-Me], [His-Leu-Leu-Val-Phe-O-Me], all purchased from Serva, Heidelberg, Germany, and [Leu-Leu-ValPhe-O-Et] kindly supplied to us by Drs Kokubu and Mizoguchi, Tokyo, Japan, The inhibitors were dissolved in 1 moljl acetic acid as a stock solution (10e3 mol/l) and diluted in the incubation buffer to concentrations ranging from 10-O mol/l-lo-’ mol/l. The incubations were performed with either 45 pmol or 22.5 pmol of substrate. The results were calculated as indicated by Dixon (1953) to define the inhibitor constants (&). Statistics
Data are expressed as means + standard error of the mean. They consist of 4-6 values for the methodological studies and of 7-10 values (14-20 brains) for the enzyme distributions. The significances of differences were tested (1) by the two-way analysis of variance on whole brain renin versus strain and versus age respectively and (2) by Student’s unpaired t-tests. RESULTS ~~ethodo~ogicai studies
The influence of sonication time on enzyme extraction and on protein content in the supernatant was investigated (Fig. 1). The largest renin and acid protease activities were obtained within 20 set of sonication. The enzyme activities decreased when the treatment was continued, while the protein content showed a plateau up to 60 set of sonication. The ANG I-forming activity in the brain tissue extract was studied as a function of pH (Fig. 2). The incubation with purified rat angiotensinogen resulted
hypertensive rats
457
in a single peak between pH 5.5 and pH 6.0 compar-
able to the pH curve obtained with plasma renin. A pH optimum at pH 5.5 with a shoulder at pH 4.0 was observed with dog substrate. Quantitatively twice the amount of ANG I was generated at pH 5.5 from dog as compared to rat angiotensinogen; at pH 6.0 this difference was significantly reduced. Less ANG I was formed when TRIS-maleate buffer (0.09 _rt0.02 pmol ANG I/ml/hr) instead of the citrate-phosphate buffer (0.26 + 0.02 pmol ANG I/ml/hr) was used. The addition of dithiothreitol to the citrate-phosphate buffer medium increased the yield of ANG I to 1.01 4: O.lOpmol ANG Ijml/hr without affecting the pH optimum of the enzyme-substrate reaction. Kinetics (Figs 3,4, and 5 and Table 1) were studied with rat angiotensinogen in citrate-phosphate buffer, pH 6, containing dithiothreitol. A linear product formation over time of incubation and an 809; recovery of exogenously added ANG I was found after 8 hr of incubation (Fig. 3). The reaction of brain renin with increasing substrate concentrations followed the Michaelis-Menten kinetics (Fig. 4). The u,,, and K, were determined from the Lineweaver-Burk plot, a,,,,, and K, was 7.15. lo-r3 mol/ml/hr was 4.2’ 10-s mol/l. Several peptide inhibitors of renin interfered in a competitive way with the brain enzyme-angiotensinogen reaction (Table 1 and Fig. 5). The active site directed octapeptide inhibitor [His-ProPhe-His-Leu-(D)Leu-Val-Tyr] against kidney renin had no inhibitory effect. Topographical studies
Systolic blood pressure was 180.4 + 3.9 mmHg in 9-week old SHR-sp and 238.4 k 3.1 mmHg in 20-week old SHR-sp. Blood pressure was the same in both age groups of the normotensive WKY rats (114.0 f 3.5 mmHg). Renin activity was present in all brain regions of spontaneously hypertensive (SHR-sp) and control rats ranging from 3.1 to 15.1 pmol ANG I/mg prot./hr in 9-week old animals and from 2.1 to 10.1 pmol ANG I/mg prot./hr in 20-week old rats (Table 2). The pineal gland was the richest source of enzyme followed by the adenohypophysis and the neurohypophysis. A high level of activity was also measured in the brain stem and in the circumventricular organs; the smallest values were found in the cerebellar cortex, the septal area, the nucleus caudatus putamen and nucleus amygdaloideus. The overall enzyme profile in brains of hypertensive rats was comparable to that in normotensive controls and the analysis of variance on whole brain versus rat strain revealed the same distribution of values in control and spontaneously hypertensive rats (SHR-sp) respectively (F = 0.03; d.f. 1,93; NS). There were, however, specific differences between 9-week old spontaneously hypertensive rats and control rats in details (analyzed by Student’s t-test) which did not persist in the older animals (Table 2). Renin was increased in brain stem regions such as the nucleus reticularis lateralis (Al), nucleus tractus solitarii
458
P.
SC~~ELLING
et al.
10'12 mol
ANG llmllh
I2
10
08
06
6 01
02
0
Fig. 2. Renin activity in rat brain incubated with 0.1 mol/l citrate-phosphate equimolar (A---A)
quantities of purified rat angiotensinogen (+o) or in comparison with plasma renin activity using the homologous a substrate.
(A2), A5 and locus coeruleus (A6). Renin was also elevated in the neurohypophysis, but was less in the frontal cortex. A significant decrease of renin activity was observed with age in both the control and spontaneously hypertensive animals (analysis of variance: F = 4.50; d.f. 1,95; P < 0.05). Angiotensinase activity was estimated in the same tissue samples. There was no relationship between renin and angiotensinase activity. The values varied
.10-l?mo,
ANG
buffer of different pH and purified dog angiotensinogen angiotensinogen (W-D) as
between 210 and 640pmol ANG II degraded/mg prot./hr (Table 3). High levels of angiotensinase activity were measured in the pineal gland. There was a wide range of cathepsin D-like acid 1 ” 10 -3
20
I
l/ml 15
10
I
5
II
P
/I
/
/
I d
-1
Km
Fig. 3. Product formation as a function of incubation time (+a) and when ANG I was added to the incubation; (W---m),
0.77 pmol ANG
I; (S-_),
1.54 pmol ANG
I.
01
02
’
0.3
T
Fig. 4. Kinetics of brain renin with increasing levels of homologous antiotensinogen; Lineweaver-Burk plot, v = 10-i’ mol ANG I/ml/hr; s = low9 mol/l.
Brain renin in spontaneously
I
I I
I
10
-K,
50
100
I
Fig. 5. Dixon plot of the reaction between rat brain renin and homologous angiotensinogen in presence of the competitive [Leu-Leu-Leu-Phe-O-Me] tetrapeptide inhibitor; 45.10-” mol (-0) and 22.5.10-” mol (m-m)
angiotensinogen were added I = 1.33. 10e6 mol/l; v = lo-”
to
the
mol ANG
incubation; I/ml/hr.
protease activity in the brain from 5.4 to 40.4 nmol BSA equivalents/mg prot./hr (Table 4). The pineal gland and the adenohypophysis exhibited high levels of enzyme activity. Some strongly vascularized regions such as the subfornical organ, the median eminence the organum subcommissurale and the area postrema also contained high levels of acid protease activity, while small values were measured in the nucleus caudatus putamen and in the cerebellar cortex. The acid protease content was decreased in the fornix and in the supraoptic nucleus of young hypertensive rats. In some other brain areas of 20-week old hypertensive rats e.g. the adenohypophysis, lower values were observed than in control rats. The acid protease activity did not decrease with the age of animals and no parallelism of the differences existed between renin and cathepsin activity in hypertensive and normotensive rats in both age groups (Tables 2 and 4). DISCUSSION
The separation of brain renin from acid proteases (Dworschack et al., 1978; Ganten and Speck, 1978; Inagami et al., 1978; Osman et al., 1979; Speck et al., 1981) and its immunohistochemical localisation in the Table 1. Interference
of peptide
inhibitors
hypertensive
central nervous system (Celio et al., 1980; Fuxe et a[., 1980; Slater et al., 1980) made it worthwhile to study the renin distribution enzymatically within the brain in detail. Thus, a method was developed which allowed the measurement of brain renin in micropunches. An optimal and reproducible enzyme extraction was obtained when the samples were sonicated under the defined conditions described. Multiple thawing and freezing of micropunches resulted in loss of enzyme activity. Dithiothreitol has been reported to increase plasma renin activity (Poisner and Hong, 1977; Funae, Sasaki and Yamamoto, 1979). The addition of dithiothreitol to the citrate-phosphate buffer induced a threefold rise in brain renin activity under the standard incubation conditions but had a negligible effect when TRIS-maleate buffer was used. The sensitivity of this assay was further enhanced by the use of purified rat plasma angiotensinogen. This may be due to the elimination of renin inhibitors from the crude plasma substrate preparation (Schaechtelin, Baechtold, Haefeli, Regoli, Gaudry-Paredes and Peters, 1968; Kotchen, Welch and Talwalkar, 1978). Less pure rat angiotensinogen had a low affinity for brain renin, requiring heterologous substrates for enzyme measurement in rat brain (Ganten, Ganten, Schelling, Boucher and Genest, 1975). A lower pH optimum of brain renin around pH 4.5 was reported under those conditions (Ganten et al., 1971) arousing doubts about the significance of these measurements, since ubiquitous cathepsin D-like acid proteases were also found to generate ANG I from angiotensinogen at low pH (Day and Reid, 1976; Hackenthal, Hackenthal and Hilgenfeldt, 1978). Cathepsin D-like activity was found to be very low, however, at pH 6 (Day and Reid, 1976). In this study a single pH optimum between pH 5.5 and 6.0 was found when brain renin was incubated with purified rat angiotensinogen. A comparable pH curve was obtained for plasma renin under identical incubation conditions. Contamination of the brain renin measurements with plasma renin can be excluded, however, since the rats were nephrectomized and because of biochemical differences between both enzymes as discussed elsewhere (Speck et al., 1981). Thus, the method reported here provides a specific measurement of rat brain renin. The kinetic data in this study are comparable with
with the rat brain Max. inhibitor concentration (mol/l)
[His-Pro-Phe-His-Leu-(D)Leu-Val-Tyr] [Leu-Leu-Val-Phe-O-Et] [Leu-Leu-Leu-Phe-O-Me] [His-Leu-Leu-Val-Phe-O-Me] [Leu-Leu-Leu-Tyr-O-Me] Incubated N.P.2115-F
with rat angiotensinogen
1.33.10-4 1.17’10-4 1.33’10-4 2.67.10-4 2.67. 1O-4 * 22.5. lo-”
459
rats
mol t 45.10-‘*
renin-rat
angiotensinogen
Inhibition (percentage)
K, (mol/l)
Type of inhibition
1.4. 10m4 5.5.1o-5 1.0. 1o-4 8.6. 1O-5
Competitive Competitive Competitive Competitive
None 51.9* 66.5* 50.0* 63.0* mol.
41.9t 51.9t 33.0t 5O.O.t
reaction
-
460
P. Table 2. Renin activity
S~HELLING et ai.
(pmol ANG I/mg prot./hr) in various brain regions of nephrectom~zed (SHR-sp) and normotensive (WKY) rats at different ages 9-week old rats WKY SHR-sp
Frontal cortex Septum Fornix Nucleus caudatus putamen Nucleus amygdaloideus Organum vasculosum lamina terminalis Organum subfornicale Median eminence Organum subcommissurale Area postrema Nucleus supraopticus Nucleus paraventricularis Nucleus anterior hypothaiami Nucleus posterior hypothalami Substantia nigra reticularis Formatio reticularis Cerebellar cortex Locus coeruleus A5 area Nucleus tractus solitarii (A2) Nucleus reticularis lateralis (Al) Pineal gland Neurohypophysis Adenohypophysis Values are means
i: SEM as measured
4.69 3.98 3.98 3.23 4.03 4.92 5.24 5.67 5.55 5.00 4.91 4.16 4.14 3.98 4.00 3.77 3.14 4.05 4.31 4.37 4.20 12.98 4.76 7.30
_I + + + + + & & f + ? + + + f + + + + * f + i f
0.27 0.20 0.32 0.15 0.42 0.19 0.58 0.57 0.53 0.43 0.18 0.32 0.29 0.35 0.34 0.32 0.28 0.33 0.39 0.40 0.39 1.33 0.54 0.85
3.78 3.58 3.85 3.27 3.43 4.54 4.86 5.72 5.54 5.61 4.58 4.29 4.34 3.71 4.04 3.71 3.59 5.46 5.56 5.52 5.33 15.07 6.18 9.26
20-week WKY
) 0.30* & 0.32 rt: 0.25 + 0.15 1: 0.27 & 0.40 i 0.39 +_ 0.54 i: 0.48 5 0.24 + 0.40 Ifr 0.33 i: 0.28 j, 0.29 + 0.34 ) 0.34 & 0.18 + 0.37** ) 0.35* + 0.33* & 0.27* + 1.03 zt 0.30* + 0.83
t rt f + ; f 2 + + 2 2 f + + + It * It * * + + + i
0.10 0.12 0.12 0.10 0.08 0.04 0.30 0.23 0.20 0.23 0.14 0.20 0.15 0.16 0.10 0.13 0.10 0.19 0.21 0.15 0.23 0.50 0.30 0.42
in various brain regions of nephrec(WKY) rats at different ages
9-week old rats WKY SHR-sp
+ SEM as measured
3.01 2.68 2.79 2.30 2.94 3.45 3.54 4.05 4.76 3.66 3.63 2.93 3.16 2.79 2.83 2.61 2.11 3.02 3.31 3.37 3.29 10.12 2.94 4.90
while the tetrapeptide and pentapeptide renin inhibitors inhibited the rat brain renin-angiotensinogen reaction in a competitive way as has been demonstrated for kidney renin (Kokubu et al., 1973). Renin showed a differential distribution in various
Table 3. Angiotensinase activity (pm01 ANG II degradedlmg prot./hr) tomized spontaneously hypertensive (SHR-sp) and normotensive
Values are means
+_ 0.24 f 0.16 k 0.11 f 0.10 : 0.15 f 0.23 f 0.25 f 0.10 f 0.26 + 0.16 f 0.18 f 0.10 + 0.17 k 0.08 + 0.05 k 0.09 k 0.20 + 0.14 + 0.15 * 0.15 k 0.15 f 0.66 f 0.27 + 0.51
old rats SHR-sp
from 7 to 10 tissue pools. *P < 0.05, **P < 0.02.
those reported for kidney and plasma renin (Page and McCubbin, 1968; Lee, 1969; Poulsen, 1978). The active site-directed octapeptide inhibitor of renin (Poulsen et a[., 1973) had no effect on rat brain renin and rat kidney renin alike at the concentrations used,
Frontal cortex Septum Fornix Nucleus caudatus putamen Nucleus amygdaloideus Organum vasculosum lamina terminalis Organum subfornicale Median eminence Organum subcommissurale Area postrema Nucleus supraopticus Nucleus paraventricularis Nucleus anterior hypothalami Nucleus posterior hypothalami Substantia nigra reticularis Formatio reticularis Cerebeliar cortex Locus coeruleus A5 area Nucleus tractus solitarii (A21 Nucleus reticularis laterahs (Al) Pineal gland Neurohypophysis Adenohypophysis
3.34 3.09 2.92 2.49 2.93 3.70 3.54 4.60 3.87 3.68 4.10 2.91 3.06 2.95 2.77 2.82 2.46 3.49 3.45 3.47 3.80 10.36 3.33 6.15
hypertensive
286.4 284.7 296.5 280.5 263.0 231.8 311.9 270.5 264.4 253.8 275.2 250.3 227.5 235.6 283.8 237.7 237.7 269.9 204.8 218.4 261.1 479.4 319.7 264.9
* 27.6 I 24.5 k 27.3 +_ 20.7 f 18.8 k 15.8 + 31.0 f 24.8 + 17.6 5 24.1 + 25.2 k 25.9 f 20.8 i 12.6 + 15.9 f 18.1 f 16.6 f 24.9 i 20.6 + 21.2 + 22.0 Ifr 25.1 + 28.1 + 29.7
254.2 229.1 287.1 253.6 248.0 222.5 284.0 269.6 294.9 227.4 206.1 245.4 218.5 221.9 287.1 240.4 216.2 251.2 217.4 207.8 245.7 379.0 245.6 233.4
+ 22.4 i- 19.5* ; 11,7 + 22.7 + 22.1 + 19.7 & 8.8 + 32.9 & 33.1 Ifr 25.7 + 16.5* + 25.5 ? 13.5 + 13.7 i: 309 + 23.8 f 23.6 + 27.8 + 26.0 + 21.5 ; 39.1 * 19.7** + 16.1* + 9.7
20-week WKY 359.0 377.2 387.9 451.9 410.9 356.9 404.2 411.7 400.2 374.8 391.1 406.3 330.7 384.4 410.2 370.2 368.7 394.8 346.4 371.9 338.6 527.8 335.9 306.4
+ + ; f * + + + + f + + : f f i f + + $ ; f + +
17.5 23.1 26.0 28.9 35.5 22.8 37.5 29.4 27.8 27.8 31.3 22.3 23.5 29.2 27.3 22.9 19.4 26.4 20.3 30.0 22.7 54.7 29.4 32.6
from 7 to 10 tissue pools. *P < 0.05, **P < 0.01.
old rats SHR-sp 367.5 389.1 399.0 415.6 399.3 332.0 384.3 386.9 392.7 349.7 331.0 375.8 361.1 367.7 379.7 344.7 336.6 365.7 333.7 350.6 334.8 639.4 384.9 315.4
i 28.8 + 24.4 z 23.3 + 25.3 rl: 24.0 & 20.4 + 21.3 f 21.5 + 31.5 + 19.0 f 22.6 f 30.6 ? 21.8 + 25.8 * 25.7 rt 21.9 + 19.3 t 16.6 + 24.2 + 22.6 ; 22.9 + 62.5 + 32.1 IcL67.5
Brain renin in spontaneously
461
hypertensive rats
Table 4. Cathepsin D-like acid protease activities (nmoi BSA equivaients/mg prot./h) in various brain regions of nephrectomized spontaneously hypertensive (SHR-sp) and normotensive (WKY) rats at different ages g-week
WKY
old rats SHR-sp
20-week old rats SHR-sp WKY
Frontal cortex Septum Fornix Nucleus caudatus putamen Nucleus amygdaloideus Organum vasculosum lamina terminalis Organum subfornicale Median eminence Organum su~ommissurale Area postrema Nucleus supraopticus
7.09 f 7.13 f 7.32 + 5.57 * 7.36 + 9.15 + 9.82 + 12.73 + 11.61 f 10.35 f 9.88 +
0.55 0.28 0.28 0.21 0.39 0.28 0.69 0.68 0.56 0.43 0.28
7.39 & 0.25 6.79 + 0.28 6.18 + 0.42* 5.59 rt 0.32 6.80 + 0.30 9.12 + 0.42 10.05 i: 0.47 12.59 & 0.60 12.70 k 0.56 11.48 rf: 0.46 8.41 & 0.30**
7.97 * 0.15 7.18 k 0.15 6.94 + 0.23 6.22 + 0.32 7.36 + 0.21 10.39 f 0.61 10.36 + 0.28 13.93 & 0.25 11.39 i 0.25 12.13 i 0.34 12.12 + 1.02
7.51 + o.io* 7.63 i 0.16 6.75 + 0.22 5.46 + 0.05* 7.34 + 0.13 9.45 + 0.28 11.25 rt 0.22* 14.01 + 0.37 15.12 + 0.66*** 12.64 -i: 0.18 9.92 f 0.60
Nucleus paraventricularis Nucleus anterior hypothalami Nucleus posterior hypothalami Substantia nigra reticularis Formatio reticularis Cerebellar cortex Locus coeruleus A5 area Nucleus tractus solitarii (A2) Nucleus reticularis lateralis (Al) Pineal gland Neurohypophysis Adenohypophysis
8.21 8.12 7.51 7.51 7.06 5.88 8.70 8.59 9.21 8.70 33.72 8.92 17.11
0.39 0.34 0.27 0.30 0.31 0.27 0.38 0.41 0.59 0.46 1.33 0.50 0.72
8.13 7.86 7.19 7.67 7.11 5.37 9.09 8.93 9.08 8.13 32.76 9.26 16.40
8.08 8.62 8.09 7.68 7.49 5.47 9.54 8.96 9.12 8.17 39.84 9.55 25.33
8.58 8.61 7.96 7.44 7.47 5.57 9.31 9.90 9.43 7.73 40.39 9.36 21.18
Values are means
+ SEM as measured
+ f k * + + + + + + * + f
rt 0.33 rt 0.37 t 0.21 rt 0.26 + 0.28 + 0.33 It 0.40 + 0.30 + 0.40 + 0.28 Lt: 1.52 i 0.79 + 0.92
f & + f k _t _t f f k + f +
0.12 0.29 0.14 0.20 0.23 0.15 0.17 0.22 0.16 0.39 1.34 0.12 1.51
from 7 to 10 tissue pools. *p < 0.05, **P < 0.02, ***P
brain regions and levels decreased with age. The pineal organ followed by the adenohypophysis exhibited by far the highest level of enzyme activity within the central nervous system. This confirms previous reports in rats, dogs and pigs obtained by different methods including direct radioimmunoassay for renin (Haulica, Branisteanu, Rosca, Stratone, Berbeleu, Balan and Ionescu, 1975; De Agostini, Reinharz and Vallotton, 1980; Hirose, Yokosowa, fnagami and Workman, 1980). The distribution of renin in the brain of rats is quite similar to that recently published for hog brain, differing essentially in the values for nucleus amygdaloideus and neurohypophysis (Hirose et al., 1980). The immunohistochemical mapping studies demonstrated localization of renin in neuronal cells and confirmed a wide distribution of the enzyme within the brain of rats, mice and humans (Celio et al., 1980; Fuxe et al., 1980; Slater et al., 1980). They differ, however, in details between each other as well as in comparison with the biochemical measurements of renin reported here. Thus, renin-like staining was found to be intensive in the cerebellum and to be low or absent in the neuroh~ophysis, whereas just the reverse is true for renin measured enzymatically. On the other hand, immunohistochemical and biochemical results were in agreement in the pineal gland, adenohypophysis, brain stem and hypothalamic regions. Species related differences as well as methodological reasons, e.g. specificity of renin antibodies or activation of prorenin (Ganten and Speck, 1978; Hirose et al., 1980) during tissue preparation and incubation may contribute to these discrepancies.
+ 0.25 * 0.30 + 0.21 + 0.15 rf: 0.25 + 0.10 * 0.33 + 0.25** + 0.23 * 0.35 k 1.22 + 0.21 i: 0.99* < 0.001.
The angiotensinase activity showed uniform distribution within the brain and increased with age. There was no apparent relationship between ANG II degradation and renin, if anything angiotensinase was lower in spontaneously hypertensive rats. Cathepsin D-like acid protease activity paralleled renin to some extent, but differed from the latter in several important aspects: the acid protease activity did not decrease with the age of the animals and no differences as described for renin existed between the hypertensive and normotensive rats in both age groups. Cathepsin D was recently reported to split neuropeptides such as opiates from their high molecular weight precursors in vitro (Benuck, Grynbaum, Cooper and Marks, 1978). This group of enzymes could therefore also be of interest for the regulation of synthesis and turnover of neuropeptides. The renin content in spontaneously hypertensive (SHR-sp) as compared to normotensive WKY rats was elevated in catecholaminergic nuclei of the brain stem and in the neurohypophysis during development of hypertension; this difference disappeared when hypertension reached a plateau and became stable. These data could be meaningful in view of the changes described for the catecholamine content and turnover in cardiovascular control centres of the brain stem (Versteeg, Palkovits, Van Der Gugten, Wijnen, Smeets and De Jong, 1976; Nakamura and Nakamura, 1978; Fuxe, Ganten, Jonsson, Agnati, Andersson, Hokfelt, Bolme, Goldstein, Hallman, Unger and Rascher, 1979; Saavedra, 1979). Angiotensin II was reported to stimulate the synthesis of catecholamines and to facilitate neurotransmission
462
P.
SCHELLING et al.
(see !&helling et al., 1980). The blood pressure increase after central applicaiion of ANG II appears, in fact, to be mediated by central catecholaminergi~ mechanisms, since it was inhibited by central pretreatment with 6-hgdroxydopamine (Hoffman, Phillips and Schmid. 1977bj and central beta-adrenorecentor blockade (Simon, ‘Schaz, Mann, Ganten, John’son, Unger, Rascher and Ganten, 1981). The present results of increased renin in the brain of spontaneously hypertensive rats are in support of, and extend. earlier findings demonstrating (1) the blood pressure lowering effect of central ANG II receptor blockade and converting enzyme inhibition in hypertensive rats (Ganten et al., 1975; Mann et al., 1978; McDonald et al., 1980; Unger et ol., 1981), (2) increased levels of ANG-like material in the cerebrospinal fluid of hypertensive rats (Ganten et ul., 1975) and hypertensive patients (Finkielman, FischerFerraro, Diaz, Goldstein and Nahmod, 1972; Kaneko, Ohnishi, Fujishima, Tanaka and Umemura, 1979) and (3) enhanced blood pressure responses to centrally applied ANG II in spontaneously hypertensive rats (Hoffman, Phillips and Schmid, 1977a). It has also to be mentioned in this resnect that the olasma renin-angiotensin system is not stimulated in adult hypertensive rats (Matsunaga, Yamamoto, Hara, Yamori, Ogino and Okamoto, 197.5; Mann et af., 1978) that the blood cerebrospinal fluid barrier is impermeable for plasma ANG II (Schelling, Ganten, Sponer, Unger and Ganten, 1980) and that the blood pressure fall in spontaneously hypertensive rats following central administration of the ANG II receptor antagonist is still present when the rats were nephrectomized (Mann et al., 1978). In conclusion, a specific and sensitive method was developed which allowed the investigation of brain renin in spontaneously hypertensive rats. Renin activity showed a typical pattern of distribution in various brain regions. The enzyme activity decreased with the age of animals. An elevated renin content was measured in catecholaminergic nuclei of the brain stem and in the neurohypophysis of young spontaneously hypertensive rats. The possible link between brain renin and central blood pressure control is evident and the blood pressure lowering effects of centrallv , administered inhibitors of the reninangiotensin system may be explained by interference with the stimulated brain r~nin-angiotensin system in hypertensive rats. 1
1
Ackno,c,ledyrmenrs---These
studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 “Cardiovaskullres System”, by grant No. 3.033 -0.76 of the Swiss National Science Foundation, by the Emil Bare11 Foundation and the Swiss Foundation for Cardiology. M.I.P. and A.K.J. were recipients of a Humboldt fellowship. We wish to thank C. Bayer, S. Sowarka and S. Miiller for excellent technical assistance and F. Liard for typing the manuscript.
Gen. Fhysiol.
20: 565-574.
_
Benuck M., Grynbaum A., Cooper T. B. and Marks N. (1978) Conversion of Iipotropic peptides by purified cathepsin D of human pituitary: release of y-endorphin by cleavage of the leu77-uhe78 bond. Neurosci. Lett. 10: 3-9. Celio M. R., Clemens D. L. and Inagami T. (1980) Renin in anterior pituitary, pineal, and neuronal cells of mouse brain, immunohistochemical localization. Biomed. Res. 1: 427-431.
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