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Angiotensin receptive neurones in the subfornical organ structure-activity relations
Brain Research, 149 (1978) 107-116 © Elsevier/North-HollandBiomedical Press
107
A N G I O T E N S I N RECEPTIVE N E U R O N E S IN THE SUBFORNICAL O R G A N .
STRUCTURE-ACTIVITY RELATIONS
DOMINIK FELIX and WERNER SCHLEGEL Institute for Brain Research, University of Ziirich, 8029 Ziirich (Switzerland) and Laboratory of Nutrition and Endocrinology, NIAMDD, NIH, Bethesda, Md. 20014 (U.S.A.)
(Accepted October 27th, 1977)
SUMMARY A microiontophoretic study was performed of the actions of angiotensin II and angiotensin fragments on neurones of the subfornical organ (SFO). Adult cats were anaesthetized and the SFO exposed for penetration by a multibarrelled micropipette. We found that angiotensin II-[2-8]-heptapeptide shows a significantly higher stimulation of firing rate compared to angiotensin II. Angiotensin II-[5-8]-tetrapeptide still produced an excitatory action on single units. Both the action of the heptapeptide and the tetrapeptide were blocked by [Sar 1, AlaS]-angiotensin II (P 113). In contrast, angiotensin II-[6-8]-tripeptide failed to enhance the firing rate of the same neurones. Our data indicate that angiotensin II and some shorter chain peptide fragments can directly affect neurones of the SFO. The study may give new insight in structureactivity relations for angiotensin II. The results support the hypothesis that the subfornical organ is a receptor site which is available to this peptide.
INTRODUCTION The cerebral regulatory function of angiotensin II for blood pressure and hydration is the subject of several recent investigations (see refs. 28, 35) and an endogenous renin-angiotensin system to the brain seems well established4,7,a,ll,12,zg,3L The role of the different parts of the system, their location and their relation to the kidney renin-angiotensin system still is not clear. It seems that angiotensin II expresses cardiovascular effects and plays a role in pituitary function (ADH and ACTH secretion). While a lot of experimental emphasis has been on the study of the elicitation of drinking by angiotensin II (dipsogen response)21,~s, 34, we think that this approach has problems in both the studies on the location of angiotensin receptors as well as for establishing structure-activity relationships for brain angiotensin receptors. In attempting to locate angiotensin receptor carrying structures by insertion
108 ofcannulae to different regions of the brain 2,~the insertion can lead to unphysiological leakage and the position of the tip of the cannulae also may not be the site of angiotensin action. Simpson and Routtenberg 36 have shown that by lesions in the subfornical organ (SFO) the dipsogen response could be blocked and they concluded that the SFO is the essential structure for the dipsogen response. However, this blockage is transient and the response is recovered after 4-14 days .~. Thus it is clear that the SFO is not the only place of the dipsogen response, but that it also might play a role in the transport of angiotensin i[ to sites beyond the interventricular foramen. Structure-activity relationships for angiotensin II action in smooth muscle and on blood pressure have been reviewed recently 23,3°. The results are similar for both actions: the intact carboxyl terminal part (angiotensin lI-[5-8]-tetrapeptide) is necessary for agonistic behaviour and a change for the side chain in position 8 to a non aromatic but lipophilic structure (Phe 8 + Ala, Ileu, Gly, etc) leads to antagonistic properties. The dipsogen response shows markedly different structure-activity relations. The peptides [Ileu8] - and [Ala8]-angiotensin II seem to be agonists, although they antagonise the response to angiotensin IP 1,39 whereas [Sarl] -, [AlaS] - and [Sar 1, lleuS]-angiotensin II are pure antagonists 1°,12,'z2,38. All precursors of angiotensin lI show a dipsogen response when given intracranially is. Dipsogen effects of extracerebrally applied peptides vary a lot with different experimental conditions 2',38. An unexpected finding was that intraventricular application of either an inhibitor of converting enzyme 24,38 or an angiotensin II antagonist 38 does not block thirst evoked by physiological stimuli. Fragments of angiotensin II which are active on blood pressure and myotopic responses do not show the same activity in brain: in the goat angiotensin II-[2-8]-heptapeptide is active, whereas angiotensin lI-[3-8]-hexapeptide seems to be inactivO 4. In rat all the fragments smaller than a heptapeptide are inactive, irrespective of their sequence 18. A more convenient method to look for angiotensin II sensitive neurones seems to be to follow single neurone discharges under the influence of microelectrophoretically applied angiotensin 11. This approach has proven successful mainly in SFO ~'5,1~* but there are other structures in brain which are sensitive to angiotensin I17 as shown by this approach, lateral hypothalamus, zona incerta, ventromedial and dorsomedial hypothalamic nuclei, dental gyrus and thatamus 4°, neurosecretory supraoptic area 26. The response to angiotensin II in SFO has a fast on- and offset, is dose dependent and is completely blocked by [Sar 1, AlaS]-angiotensin II. Other peptides of similar length do not show any responsO 5. With these features the microiontophoretic experimental system is well suited for structure-activity studies with angiotensin II fragments. These studies are of particular interest since from behavioural studies, it is difficult to correlate the structure features of the angiotensin II brain receptors with those of angiotensin II receptors outside the brain. Moreover this study provides additional possibilities of finding other angiotensin II receptors in different parts of the brain. Preliminary results of this study briefly have been reported 17,3a.
109 METHODS
Experimental procedure The experiments were performed on 71 SFO neurones obtained from 13 cats anaesthetized with nitrous oxide (70 %) and fluothane (ICI, 1-2 %). Small i.v. doses of thiopentone sodium (Pentothal, Abbott) were given periodically. The animals were mounted in a stereotaxic frame, with a headwards deflexion of 20°. Body temperature was maintained at 37 °C by a self-regulating heating pad. The electrocardiogram was monitored continuously. In the animals in which the drugs were administered intravenously, the arterial blood pressure was recorded by a pressure transducer (Statham Lab.). The cortical tissue, including the corpus cailosum overlying the ventricle, was removed by suction and the SFO was approached under direct visual control. Extracellular action potentials were recorded through the 4 M NaCl-containing central barrel of a 5-barrel glass micropipette, tip diameter approximately 4 #m. The rate of firing of each neurone was counted by a ratemeter (Nuclear Enterprises NE 4667) and displayed continuously on an UV-oscillograph (Bell and Howell 5-137). The outer channels of the micropipettes contained the compounds to be ejected microiontophoretically: (1) acetylcholine chloride (Fluka), 0.5 M, pH 3.0-3.5; (2) angiotensin II (Calbiochem), prepared as a 10-z M solution in distilled water with a final pH of 4.5, adjusted with NaOH; (3) peptide fragments of angiotensin II: A II[2-8]-heptapeptide, A II-[5-8]-tetrapeptide and A II-[6-8]-tripeptide as mentioned above, prepared as 10-3 M solutions with a final pH of 4.5; (4) [Sar 1, AlaS]-angio tensin II, a specific competitive inhibitor of angiotensin II (P 113, Norwich Pharm.), pH 4.5. All peptides were ejected with cationic currents. A timing circuit was used to administer the drugs for equal periods.
Peptides Bovine angiotensin II (Asp-Arg-Val-Tyr-Val-His-Pro-Phe-OH) was supplied by Calbiochem Chem. or Beckman. A II-[2-8]-heptapeptide: Des-Aspl-angiotensin II was prepared by Edmann degradation of angiotensin II and purified on a weakly acid cation exchanger (Merck IV) and desalted on BIOGEL P2 (BIORAD) in 0.2 M acetic acid and lyophilized. The biological activity of the peptide was tested on the blood pressure of the rat and it corresponded well with the results from previous investigations ez. A II-[5-8]-tetrapeptide: Des-Asp1, Des-ArgO, Des_Val a, Des.Tyr4.angiotensin II was prepared by splitting angiotensin II by Chymotrypsin and subsequent purification of the two peptides on CM-Sephadex in 10 mM citrate buffer pH 4.7 and gel filtration on BIOGEL P2. The desalted product was lyophilized. A II-[6-8]-tripeptide: Des-Aspl, Des-Arg2, Des.Val a, Des.Tyr 4, Des.Val 5. angiotensin II was kindly provided by Mario Caviezel, Institut ffir Molekularbiologie und Biophysik, Eidg. Technische Hochschule, 8049 Zfirich, Switzerland, and purified like angiotensin II-[2-8]-heptapeptide. Purity and identity of the fragment peptides were checked by thin layer chromatography in 2 different systems, thin layer electrophoresis and amino acid analysis (Table I).
110 TABLE [
Amino acid analysis' of the fragment peptides used Asp A I I - [2-8]-heptapeptide A I I - [5- 8]-tetrapeptide A II-[6-8]-tripeptide
Arg
Vul
Tyr
His
Pro
Phe
0.95
2.1 0.99
0.91
1.04 0.98 0.86
1.05 0.97 1.13
0.92 1.15 1.01
RESULTS
The majority of the neurones studied were firing spontaneously on a relatively low rate, ranging from 0.2 to 6 spikes/sec. A small number could only be detected by administration of either angiotensin II or acetylcholine. Since the aim of this study was the investigation of the angiotensin II action on SFO neurones we left out neurones which were specifically responsive to acetylcholine. It has to be pointed out that not all SFO neurones from which extracellular action potentials could be registered were sensitive to the microiontophoretic application of angiotensin II or acetylcholine. Fig. 1 gives an overview of the excitatory actions of the different peptides used in this study. The first column in Fig. 1B represents our control experiments with angiotensin II. Of the tested neurones, 85 % (60 out of 71 cells) were activated by angiotensin II,
®
® i
J
,~ n=71
l
~
A~-E2-8] A~-ES-8J n=32 n=22
i
i
f
I
i
A~-[6-81 n=20
Fig. 1. A: amino acid sequences of the peptide fragments. B: excitatory action of the peptides on SFO neurones. The percentages are based on the number of cells (n) on which the fragments were tested.
111
®
30"j
x_ o. t/'l 0 -
®
1
Aa-E2-8]
30-
e ~
During
''''--'e
P-113
20to
~0 "~
30 sec
~/ sj~ ~
~u') 10-
e - - e A II-[2-8] o---o A ~
Control
~
,
.
I
10
~ 20
= 410 J ~ 30 50 60 ANGIOTENSIN (nA)
.
,
i
~
=
7J0
Fig. 2. The action of angiotensin II (All) and angiotensin II-[2-8]-heptapeptide. A: equal amounts of current (70 nA) show differential effect on the firing rate. B • 'dose--response'-curvesof the two peptides. Each point represents the firing rate produced by the peptides ejected with different currents. C: the effect of angiotensin II-[2-8]-heptapeptide (60 nA) and the action on this response by P 113 ejection (150 nA).
using currents between 20 and 100 nA. Thirty-five of the tested SFO-cells responded specifically to angiotensin II. Similar percentages could be obtained by angiotensin II-[2-8]-heptapeptide (78 ~ , 25/32 cells) or angiotensin II-[5-8]-tetrapeptide (73 ~o, 16/22 cells). The results from the two angiotensin II-fragments do not differ significantly from those obtained by angiotensin II. In contrast, a clear cut drop of excitatory action could be seen with angiotensin II-[6-8]-tripeptide. Only 2 out of 20 ceils showed a slight enhancement of firing rate. In both cases, currents of 120 nA were necessary to influence these neurones. In the following, the action of iontophoreticaUy administered angiotensin II peptide fragments was compared to that of similarly applied angiotensin II. Only those neurones were collected on which first an excitatory response was observed by angiotensin II.
Angiotensin H and angiotensin II-[2-8]-heptapeptide (Fig. 2) We found that Des-Asp 1, [ValS]-angiotensin II shows a slightly shorter latency and significantly higher stimulation of firing rate compared to angiotensin II (Fig. 2A). Less than 1 sec after the beginning of the ejection, angiotensin led to the marked acceleration in the discharge rate with a gradual recovery after withdrawal of the ejecting current. Using equal periods of administration of regular intervals, 13 out of 25 neurones showed a more pronounced activation with the heptapeptide; 9/25 neurones showed similar effects and only 3/25 were more strongly affected with angio-
112
@ ~ o
3oj
®
I--1 A1T
~I:l__
An-E.5-8] =
30 sec
I
o A~-[6-81 15
r - ~ sec
Fig~ 3. Effects of shorter chain peptide fragments derived from angiotensin II (A II) on three individual SFO neurones. A : integrated firing frequency of a neurone excited by ejection of angiotensm II (10 nA) and angiotensin II-[5-81-tetrapeptide (40 nA). B: microiontophoresis of angiotensin II (50 nA) leads to an enhanced firing whereas equal amount of angiotensin II-[5-8]tetrapeptide shows only weak response. Application of angiotensin II-[6-8]-tripeptide (100 nA) did not show any effect. At the end of the tracing another ejection of the tetrapeptide (80 nA) produces an excitatory action. C: the effect of the tetrapeptide (100 nA) tested on another SFO neurone. Again the tripeptide ejected with 150 nA, tested in between, failed to enhance the firing frequency. Note different time base in A and B, C as well as different scales of ordinate in A, B and C.
tensin II. The relationship between the firing frequency of an individual neurone and varying intensities of ejecting currents is represented by a dose-response curve, illustrated in Fig. 2B. In the cases where the heptapeptide had a more p r o n o u n c e d activation, this effect was usually 1.5-2-fold higher. The same differential effect could be observed when the heptapeptide was ejected prior to angiotensin II. Both the action of angiotensin II and angiotensin II-[2-8]-heptapeptide were blocked by [Sar 1, AlaS]-angio tensin II (Fig. 2C, P 113). The antagonist alone produced either no effect or a decrease in unit firing. The most sensitive antagonism on angiotensin II-[2-8]-heptapeptide with respect to dose of P 113 was on neurones responsive to angiotensin and not to both angiotensin and acetylcholine. This finding is in agreement with previously reported antagonism on angiotensin II ~7.
Angiotensin H and angiotensin II-[5-8]-tetrapeptide (Fig. 3) In the second series of experiments consisting of angiotensin II-[5-8]-tetrapeptide ejection, successful results were obtained from 16 S F O neurones. A l t h o u g h the neurones were responsive to the ejection o f the angiotensin II-[5-8]-tetrapeptide it was difficult to make a reliable comparison with the effect of angiotensin II. Whereas
113 relatively small amounts (30-70 nA) of the octapeptide led to a strong excitatory action, very high amounts of currents (often exceeding 100 nA) were necessary to produce a similar effect with the tetrapeptide. On the other hand angiotensin II ejected with doses of 100 nA often produced bursts leading to uncontrollable recordings. Fourteen out of 16 cells showed a stronger response to angiotensin II than to angiotensin II-[5-8]-tetrapeptide. The mean ratio between the two drugs was 1:5. Only two cells responded to both substances with similar enhancement of firing frequency. The weak excitant action by the tetrapeptide was also expressed by the latencies of the occurring responses. The mean delay observed with the tetrapeptide was within the range of 10 sec. Since already very high doses of the fragment peptide were necessary to produce a decent acceleration of firing rate it was difficult to demonstrate whether this change of frequency could be antagonized by P 113. On two occasions, however, such an antagonism could be observed; where the ratio between the peptide and the antagonist was 60:150 and 50:180 respectively. At the end of 2 experiments angiotensin II-[5-8]-tetrapeptide was tested on blood pressure responses by intravenous injection into the radial vein. Angiotensin II (Hypertensin, Ciba) in quantities of 0.5 #g angiotensin/injection served as a control. Whereas i.v. injection of angiotensin II produced a rise in blood pressure (increase of 30 mm Hg) lasting about 3 min, application of the tetrapeptide using doses up to 90 #g/injection failed to produce a marked pressure response. In the second case only a blood pressure rise of 5 mm Hg was caused by injecting 110 #g ofangiotensin 11-[5-8]tetrapeptide.
Angiotensin H and angiotensin II-[6-8]-tripeptide (Fig. 3) Angiotensin II-[6-8]-tripeptide was applied microiontophoretically using ejection currents of 20-250 nA and a direct comparison with angiotensin II was made on 20 SFO neurones. As already mentioned earlier, only two cells could be activated slightly by this drug. On both cells, currents of 120 nA were necessary to produce this effect. In all other cases currents up to 250 nA were tested and an excitant effect was never observed. Angiotensin II-[6-8]-tripeptide was also administered intravenously at the end of two experiments. Although enormous doses (up to 430/~g) were used, in both studies the tripeptide failed to produce a blood pressure change. DISCUSSION The present experiments were undertaken in order to test the sensitivity of SFO neurones to various shorter chain peptide fragments derived from the octapeptide angiotensin II. Simpson and Routtenberg a6 focused attention on this organ as a site of angiotensin dipsogenic action. Our observationslS, 16 of short latency activation of SFO units by microiontophoretic administration of angiotensin II and those on isolated SFO in vitro preparation 6 indicated that this structure contains angiotensin receptors. This was strengthened by the fact that the angiotensin II analogue [Sar 1, AlaS]-angiotensin II (P 113) showed specific blockade of angiotensin II sensitive neurones in the SFO 27. The present data demonstrate that the biologically active
114 Des-Aspl-angiotensin II shows a significantly higher stimulation of firing rate compared to angiotensin II. Angiotensin ll-[5-8]-tetrapeptide still produced, although very weak, an excitatory action on single units. Both the action of the heptapeptide and the tetrapeptide were blocked by P 113. In contrast, the tripeptide failed to enhance the firing rate of the same neurones. The significance of the present results is evident. They demonstrate that angiotensin has a specific action on certain brain cells. In recent years techniques have been developed for the direct study of the interaction of radioactively labelled peptide hormones and their specific target receptors 31. Hubbard and coworkers z7 examined angiotensin II binding activity of rat brain particles using [125I]angiotensin II. Specific angiotensin binding was confirmed to the diencephalon and in particular to the lateral septal region whereas very low levels were found in cortex, hippocampus and striatum. Using the indirect immunohistochemical method it was possible to demonstrate angiotensin like immunoreactivity in the medulla oblongata and the substantia gelatinosa of the spinal cord, suggesting the existence of angiotensin II-containing nerve terminals in the brain and spinal cord 19. With the microiontophoretic technique the effectors are applied directly to the cell under study. Thus binding, transport and breakdown show only a very minor influence on the apparent effectiveness so that the structure-activity relationships found with this technique reflect mainly the hormone-receptor interaction. The side effects mentioned above are of particular importance in the study of short peptides since binding is usually decreased and degradation leads to inactive products. This may explain the better effectiveness of the shorter peptides in our study as opposed to studies of the dipsogen response. Fitzsimons is has shown that the angiotensin II[5-8]-heptapeptide retained about 50 ~ of the dipsogenic activity of the octapeptide, whereas the angiotensin II-[5-8]-tetrapeptide had no effect on drinking when injected into the angiotensin sensitive region of the diencephalon. However, when microiontophoretically applied this tetrapeptide shows activity and the angiotensin 11-[2-8]heptapeptide has been found to be more active than the octapeptide. This is similar to structure-activity relationships in the adrenal where the heptapeptide is as effective as angiotensin II in stimulating aldosterone synthesis. The removal of asparagine or aspartic acid could lead to a higher accessibility of the peptide to the membrane receptors, or there may be specific angiotensin III receptors in the brain, as shown by Meyer and coworkers 9 for rat adrenal. A very interesting feature of angiotensin 1I activity is its potentiation by Na + both for the peripheral z°,32 and the central action 1,2,13. It is not known whether angiotensin II is transported into and through cells; this would require a special mechanism. One can envisage a process which, like other transport processes, would be driven by the unbalanced Na + concentrations inside and outside the plasma membrane and that transport of peptides would lead to a concomitant rise in intracellular Na ÷ and as a consequence to a reduced potential and thus to a higher discharge rate. For the microiontophoretic experiments it is not yet possible to decide whether angiotensin II shows transmitter like or just modulating activity. Using voltage-clamp techniques, Barker and Smith 3 have reported that a peptide, 8-Lysine-vasopressin, alters the cell membrane's steadystate current voltage indicating that such a peptide has a long-
115 t e r m r e g u l a t i o n o f the v o l t a g e - d e p e n d e n t conductances. W h e t h e r the action o f angiotensin II w o u l d be b a s e d o n a similar m e c h a n i s m c a n n o t be decided f r o m o u r experiments. T o conclude, the present studies on the S F O offer new insight in structure-activity relations for angiotensin I I which are o b s c u r e d by side effects in other b i o a s s a y systems a n d suggest the presence o f specific angiotensin receptors in the brain. ACKNOWLEDGEMENTS The a u t h o r s wish to express their sincere t h a n k s to Prof. K. A k e r t . F u r t h e r m o r e the skillful technical assistance o f U. F r a n g i , A. F~ih a n d A. Fid61er as well as the help o f R. Emch, H. Hauser, D. Savini, E. Schneider a n d U. W a l t y is greatly appreciated. W e t h a n k Dr. D a v i d W r i g h t for critical r e a d i n g o f the manuscript. This w o r k was s u p p o r t e d b y grants N o . 3.534.75 a n d 3.636.75 o f the Swiss N a t i o n a l Science F o u n d a t i o n a n d the Dr. Eric S l a c k - G y r F o u n d a t i o n in Z/irich.
REFERENCES 1 Andersson, B., Eriksson, L. and Fernandez, O., Reinforcement by Na + of centrally mediated hypertensive response to angiotensin II, Life Sci., 10 (1971) 633-638. 2 Andersson, B., Eriksson, L., Fernandez, O., Kolomodin, C.-G. and Oltner, R., Centrally mediated effects of sodium and angiotensin II on arterial blood pressure and fluid balance, Acta physiol. scand., 85 (1972) 398-407. 3 Barker, J.L. and Smith, T.G., Peptide regulation of neuronal membrane properties, Brain Research, 103 (1976) 167-170. 4 Bryant, R. W. and Falk, J. L., Angiotensin I as a dipsogen: efficacy in brain independent of conversion to angiotensin II, PharmacoL Biochem. Behav., 1 (1973) 469--475. 5 Buggy, J., Fisher, A. E., Hoffman, W. E., Johnson, A. K. and Phillips, M. I., Ventricular obstruction: effect on drinking induced by intracranial injection of angiotensin, Science, 190 (1975) 72-74. 6 Buranarugsa, P. and Hubbard, J. I., Angiotensin receptors in the rat subfornical organ in vitro, Proc. Univ. Otago Medical School, 54 (1976) 3--4. 7 Cooling, M. J. and Day, M. D., Inhibition of renin-angiotensin induced drinking in the cat by enzyme inhibitors and by analogue antagonists of angiotensin, II, Clin. exp. Pharmacol. Physiol., 1 (1974) 389-396. 8 Daul, C.B., Heath, R.G. and Garey, R.E., Angiotensin-forming enzyme in human brain, Neuropharmacology, 14 (1975) 75-80. 9 Devynck, M.A., Pernollet, M. G., Matthews, P. G. and Meyer, P., Demonstration of specific angiotensin III receptors in rat adrenal, Proc. Int. Union Physiol. Sciences, Paris, 1977, Abstr. 529. 10 Epstein, A. N. and Simpson, J. B., The dipsogenic action of angiotensin, Acta physioL lat. amer., 24 (1974) 405--408. 11 Epstein, A. N., Fitzsimons, J. T. and Rolls, B. J., Drinking induced by injection of angiotensin II into the brain of the rat, J. Physiol. (Lond.), 210 (1970) 457-474. 12 Epstein, A. N., Fitzsimons, J. T. and Johnston, A. K., Peptide antagonists of the renin-angiotensin system and the elucidation of the receptors for angiotensin-induced drinking, J. Physiol. (Lond.), 238 (1974) 34-35P. 13 Eriksson, L., Sodium and angiotensin II in the central control of fluid balance, Acta physioL scand. Suppl., 444 (1976). 14 Eriksson, L. and Fyhrquist, F., A comparison between the central effects of angiotensin II and its fragments, des 1-angiotensin II and des 1,2-angiotensin II, Acta physioL scand., 96 (1976) 134-136. 15 Felix, D., Peptide and acetylcholine action on neurons of the cat subfornical organ, Naunyn-
116 Schmiedeberg's Arch. Pharmacol., 292 (1976) 15--20. 16 Felix, D. and Akert, K., The effect of angiotensin 11 on neurones of the cat subfornical organ, Brain Research, 76 (1974) 350-353. 17 Felix, D. and Schlegel, W., Evidence for brain angiotensin receptors, Experientia (Basel), 33 (1977) 779. 18 Fitzsimons, J. T., The effect on drinking of peptide precursors and of shorter chain peptide fragments of angiotensin I1 injected into the rat'.~ diencephalon, J. Physiol. (Lond.), 214 (1971) 295-303. 19 Fuxe, K., Ganten, D., H6kfelt, T. and Bolme, P., Immunohistochemical evidence for the existence of angiotensin II containing nerve terminals in the brain and spinal cord in the rat, Neurosci. Lett., 2 (1976) 229-234. 20 Glossman, H., Baukal, A. and Catt, K.J., Cation dependence of high-affinity angiotensin 1l binding to adrenal cortex receptors, Science, 185 (1974) 281-283. 21 Johnsson, A. K. and Epstein, A. N., The cerebral ventricles as the avenue for the dipsogenic action of intracranial angiotensin, Brain Research, 86 (1975) 399418. 22 Johnson, A. K. and Schwob, J. E., Cephalic angiotensin receptois mediating drinking to systemic angiotensin 1I, Pharmacol. Biochem. Behav., 3 (1975) 1077-1084. 23 Khosla, M. C., Smeby, R. R. and Bumpus, F. M., Structure-activity relationship in angiotensin 11 analogs. In O. Eichter, H. Herken, A. Farah and A. D. Welch (Eds.) Handbook of Experimental Pharmacology, Vol. 37, Springer, Berlin, 1974, pp. 126-169. 24 Lehr, D., Goldman, H. W. and Casner, P., Renin-angiotensin role in thirst: paradoxical enhancement of drinking by angiotensin converting enzyme inhibitor, Science, 182 (1973) 1031-1034. 25 Myers, R. D., Hall, G. H. and Rudy, T. A., Drinking in the monkey evoked by nicotine or angiotensin 1I microinjected in hypothalamic and mesencephalic sites, Pharmacol. Biochem. Behav., 1 (1973) 15 22. 26 Nicoll, R. A. and Barker, J. L., Excitation of supraoptic neurosecretory cells by angiotensin I1, Nature New Biology, 223 (1971) 172-174. 27 Phillips, M. 1. and Felix, D., Specific angiotensin II receptive neurons in the cat subfornical organ, Brain Research, 109 (1976) 531-540. 28 Phillips, M. 1. and Hoffman, W. E., Sensitive sites in the brain for the blood pressure and drinking responses to angiotensin II. In J. P. Buckley and C. M, Ferrario (Eds.) Central Actions of Angiotensin and Related Hormones, Pergamon Press, New York, 1976, pp. 311-342. 29 Poth, M. M., Heath, R. G. and Ward, M., Angiotensin-converting enzyme in human brain, J. Neurochem., 25 (1975) 83-85. 30 Regoli, D., Park, W. K. and Rioux, F., Pharmacology of angiotensin, Pharmacol. Rev., 26 (1974) 69-123. 31 Roth, J., Peptide hormone binding to receptors: a review of direct studies in vitro, Metabolism, 22 (1973) 1059-1073. 32 Schaechtlin, G., Walter, R., Salomon, H., Jelinek, J., Karen, P. and Cort, J. H., Enhancement of the activity of angiotensin 11 by certain cations, Molec. Pharmacol., 10 (1974) 57-67. 33 Schlegel, W. and Felix, D., Structure-activity relations for angiotensin 1I action in subfornical organ, Experientia (Basel), 32 (1976) 761. 34 Severs, W.B., Angiotensin interaction with thirst mechanisms. In W. Kaufmann and D . K . Krause (Eds.) Central Nervous Control of Na + Balance - Relations to the Renin-Angiotensin System, lnternat. Workshop at Cologne, 1975, Georg Thieme, Stuttgart, 1976. 35 Severs, W. B. and Daniels-Severs, A. E., Effects of angiotensin on the central nervous system, Pharmacol. Rev., 25 (1973) 415~,49. 36 Simpson, J. B. and Routtenberg, A., Subfornical organ: site of drinking elicitation by angiotensin II, Science, 181 (1973) 1172-1175. 37 Sirett, N. E., McLean, A. S., Bray, J. J. and Hubbard, J. I., Distribution of angiotensin I1 receptors in rat brain, Brain Research, 122 (1977) 299-312. 38 Summy-Long, J. and Severs, W. B., Angiotensin and thirst: studies with a converting enzyme inhibitor and a receptor antagonist, Life Sei., 15 (1974) 569-582. 39 Swanson, L. W., Marshall, G. R., Needleman, P. and Sharpe, L. G., Characterization of central angiotensin lI receptors involved in the elicitation of drinking in the rat, Brain Research, 49 (1973) 441~146. 4.0 Wayner, M. J., Ono, T. and Nolley, D., Effects of angiotensin II on central neurons, Pharmacol. Biochem. Behav., 1 (1973) 679-691.
107
A N G I O T E N S I N RECEPTIVE N E U R O N E S IN THE SUBFORNICAL O R G A N .
STRUCTURE-ACTIVITY RELATIONS
DOMINIK FELIX and WERNER SCHLEGEL Institute for Brain Research, University of Ziirich, 8029 Ziirich (Switzerland) and Laboratory of Nutrition and Endocrinology, NIAMDD, NIH, Bethesda, Md. 20014 (U.S.A.)
(Accepted October 27th, 1977)
SUMMARY A microiontophoretic study was performed of the actions of angiotensin II and angiotensin fragments on neurones of the subfornical organ (SFO). Adult cats were anaesthetized and the SFO exposed for penetration by a multibarrelled micropipette. We found that angiotensin II-[2-8]-heptapeptide shows a significantly higher stimulation of firing rate compared to angiotensin II. Angiotensin II-[5-8]-tetrapeptide still produced an excitatory action on single units. Both the action of the heptapeptide and the tetrapeptide were blocked by [Sar 1, AlaS]-angiotensin II (P 113). In contrast, angiotensin II-[6-8]-tripeptide failed to enhance the firing rate of the same neurones. Our data indicate that angiotensin II and some shorter chain peptide fragments can directly affect neurones of the SFO. The study may give new insight in structureactivity relations for angiotensin II. The results support the hypothesis that the subfornical organ is a receptor site which is available to this peptide.
INTRODUCTION The cerebral regulatory function of angiotensin II for blood pressure and hydration is the subject of several recent investigations (see refs. 28, 35) and an endogenous renin-angiotensin system to the brain seems well established4,7,a,ll,12,zg,3L The role of the different parts of the system, their location and their relation to the kidney renin-angiotensin system still is not clear. It seems that angiotensin II expresses cardiovascular effects and plays a role in pituitary function (ADH and ACTH secretion). While a lot of experimental emphasis has been on the study of the elicitation of drinking by angiotensin II (dipsogen response)21,~s, 34, we think that this approach has problems in both the studies on the location of angiotensin receptors as well as for establishing structure-activity relationships for brain angiotensin receptors. In attempting to locate angiotensin receptor carrying structures by insertion
108 ofcannulae to different regions of the brain 2,~the insertion can lead to unphysiological leakage and the position of the tip of the cannulae also may not be the site of angiotensin action. Simpson and Routtenberg 36 have shown that by lesions in the subfornical organ (SFO) the dipsogen response could be blocked and they concluded that the SFO is the essential structure for the dipsogen response. However, this blockage is transient and the response is recovered after 4-14 days .~. Thus it is clear that the SFO is not the only place of the dipsogen response, but that it also might play a role in the transport of angiotensin i[ to sites beyond the interventricular foramen. Structure-activity relationships for angiotensin II action in smooth muscle and on blood pressure have been reviewed recently 23,3°. The results are similar for both actions: the intact carboxyl terminal part (angiotensin lI-[5-8]-tetrapeptide) is necessary for agonistic behaviour and a change for the side chain in position 8 to a non aromatic but lipophilic structure (Phe 8 + Ala, Ileu, Gly, etc) leads to antagonistic properties. The dipsogen response shows markedly different structure-activity relations. The peptides [Ileu8] - and [Ala8]-angiotensin II seem to be agonists, although they antagonise the response to angiotensin IP 1,39 whereas [Sarl] -, [AlaS] - and [Sar 1, lleuS]-angiotensin II are pure antagonists 1°,12,'z2,38. All precursors of angiotensin lI show a dipsogen response when given intracranially is. Dipsogen effects of extracerebrally applied peptides vary a lot with different experimental conditions 2',38. An unexpected finding was that intraventricular application of either an inhibitor of converting enzyme 24,38 or an angiotensin II antagonist 38 does not block thirst evoked by physiological stimuli. Fragments of angiotensin II which are active on blood pressure and myotopic responses do not show the same activity in brain: in the goat angiotensin II-[2-8]-heptapeptide is active, whereas angiotensin lI-[3-8]-hexapeptide seems to be inactivO 4. In rat all the fragments smaller than a heptapeptide are inactive, irrespective of their sequence 18. A more convenient method to look for angiotensin II sensitive neurones seems to be to follow single neurone discharges under the influence of microelectrophoretically applied angiotensin 11. This approach has proven successful mainly in SFO ~'5,1~* but there are other structures in brain which are sensitive to angiotensin I17 as shown by this approach, lateral hypothalamus, zona incerta, ventromedial and dorsomedial hypothalamic nuclei, dental gyrus and thatamus 4°, neurosecretory supraoptic area 26. The response to angiotensin II in SFO has a fast on- and offset, is dose dependent and is completely blocked by [Sar 1, AlaS]-angiotensin II. Other peptides of similar length do not show any responsO 5. With these features the microiontophoretic experimental system is well suited for structure-activity studies with angiotensin II fragments. These studies are of particular interest since from behavioural studies, it is difficult to correlate the structure features of the angiotensin II brain receptors with those of angiotensin II receptors outside the brain. Moreover this study provides additional possibilities of finding other angiotensin II receptors in different parts of the brain. Preliminary results of this study briefly have been reported 17,3a.
109 METHODS
Experimental procedure The experiments were performed on 71 SFO neurones obtained from 13 cats anaesthetized with nitrous oxide (70 %) and fluothane (ICI, 1-2 %). Small i.v. doses of thiopentone sodium (Pentothal, Abbott) were given periodically. The animals were mounted in a stereotaxic frame, with a headwards deflexion of 20°. Body temperature was maintained at 37 °C by a self-regulating heating pad. The electrocardiogram was monitored continuously. In the animals in which the drugs were administered intravenously, the arterial blood pressure was recorded by a pressure transducer (Statham Lab.). The cortical tissue, including the corpus cailosum overlying the ventricle, was removed by suction and the SFO was approached under direct visual control. Extracellular action potentials were recorded through the 4 M NaCl-containing central barrel of a 5-barrel glass micropipette, tip diameter approximately 4 #m. The rate of firing of each neurone was counted by a ratemeter (Nuclear Enterprises NE 4667) and displayed continuously on an UV-oscillograph (Bell and Howell 5-137). The outer channels of the micropipettes contained the compounds to be ejected microiontophoretically: (1) acetylcholine chloride (Fluka), 0.5 M, pH 3.0-3.5; (2) angiotensin II (Calbiochem), prepared as a 10-z M solution in distilled water with a final pH of 4.5, adjusted with NaOH; (3) peptide fragments of angiotensin II: A II[2-8]-heptapeptide, A II-[5-8]-tetrapeptide and A II-[6-8]-tripeptide as mentioned above, prepared as 10-3 M solutions with a final pH of 4.5; (4) [Sar 1, AlaS]-angio tensin II, a specific competitive inhibitor of angiotensin II (P 113, Norwich Pharm.), pH 4.5. All peptides were ejected with cationic currents. A timing circuit was used to administer the drugs for equal periods.
Peptides Bovine angiotensin II (Asp-Arg-Val-Tyr-Val-His-Pro-Phe-OH) was supplied by Calbiochem Chem. or Beckman. A II-[2-8]-heptapeptide: Des-Aspl-angiotensin II was prepared by Edmann degradation of angiotensin II and purified on a weakly acid cation exchanger (Merck IV) and desalted on BIOGEL P2 (BIORAD) in 0.2 M acetic acid and lyophilized. The biological activity of the peptide was tested on the blood pressure of the rat and it corresponded well with the results from previous investigations ez. A II-[5-8]-tetrapeptide: Des-Asp1, Des-ArgO, Des_Val a, Des.Tyr4.angiotensin II was prepared by splitting angiotensin II by Chymotrypsin and subsequent purification of the two peptides on CM-Sephadex in 10 mM citrate buffer pH 4.7 and gel filtration on BIOGEL P2. The desalted product was lyophilized. A II-[6-8]-tripeptide: Des-Aspl, Des-Arg2, Des.Val a, Des.Tyr 4, Des.Val 5. angiotensin II was kindly provided by Mario Caviezel, Institut ffir Molekularbiologie und Biophysik, Eidg. Technische Hochschule, 8049 Zfirich, Switzerland, and purified like angiotensin II-[2-8]-heptapeptide. Purity and identity of the fragment peptides were checked by thin layer chromatography in 2 different systems, thin layer electrophoresis and amino acid analysis (Table I).
110 TABLE [
Amino acid analysis' of the fragment peptides used Asp A I I - [2-8]-heptapeptide A I I - [5- 8]-tetrapeptide A II-[6-8]-tripeptide
Arg
Vul
Tyr
His
Pro
Phe
0.95
2.1 0.99
0.91
1.04 0.98 0.86
1.05 0.97 1.13
0.92 1.15 1.01
RESULTS
The majority of the neurones studied were firing spontaneously on a relatively low rate, ranging from 0.2 to 6 spikes/sec. A small number could only be detected by administration of either angiotensin II or acetylcholine. Since the aim of this study was the investigation of the angiotensin II action on SFO neurones we left out neurones which were specifically responsive to acetylcholine. It has to be pointed out that not all SFO neurones from which extracellular action potentials could be registered were sensitive to the microiontophoretic application of angiotensin II or acetylcholine. Fig. 1 gives an overview of the excitatory actions of the different peptides used in this study. The first column in Fig. 1B represents our control experiments with angiotensin II. Of the tested neurones, 85 % (60 out of 71 cells) were activated by angiotensin II,
®
® i
J
,~ n=71
l
~
A~-E2-8] A~-ES-8J n=32 n=22
i
i
f
I
i
A~-[6-81 n=20
Fig. 1. A: amino acid sequences of the peptide fragments. B: excitatory action of the peptides on SFO neurones. The percentages are based on the number of cells (n) on which the fragments were tested.
111
®
30"j
x_ o. t/'l 0 -
®
1
Aa-E2-8]
30-
e ~
During
''''--'e
P-113
20to
~0 "~
30 sec
~/ sj~ ~
~u') 10-
e - - e A II-[2-8] o---o A ~
Control
~
,
.
I
10
~ 20
= 410 J ~ 30 50 60 ANGIOTENSIN (nA)
.
,
i
~
=
7J0
Fig. 2. The action of angiotensin II (All) and angiotensin II-[2-8]-heptapeptide. A: equal amounts of current (70 nA) show differential effect on the firing rate. B • 'dose--response'-curvesof the two peptides. Each point represents the firing rate produced by the peptides ejected with different currents. C: the effect of angiotensin II-[2-8]-heptapeptide (60 nA) and the action on this response by P 113 ejection (150 nA).
using currents between 20 and 100 nA. Thirty-five of the tested SFO-cells responded specifically to angiotensin II. Similar percentages could be obtained by angiotensin II-[2-8]-heptapeptide (78 ~ , 25/32 cells) or angiotensin II-[5-8]-tetrapeptide (73 ~o, 16/22 cells). The results from the two angiotensin II-fragments do not differ significantly from those obtained by angiotensin II. In contrast, a clear cut drop of excitatory action could be seen with angiotensin II-[6-8]-tripeptide. Only 2 out of 20 ceils showed a slight enhancement of firing rate. In both cases, currents of 120 nA were necessary to influence these neurones. In the following, the action of iontophoreticaUy administered angiotensin II peptide fragments was compared to that of similarly applied angiotensin II. Only those neurones were collected on which first an excitatory response was observed by angiotensin II.
Angiotensin H and angiotensin II-[2-8]-heptapeptide (Fig. 2) We found that Des-Asp 1, [ValS]-angiotensin II shows a slightly shorter latency and significantly higher stimulation of firing rate compared to angiotensin II (Fig. 2A). Less than 1 sec after the beginning of the ejection, angiotensin led to the marked acceleration in the discharge rate with a gradual recovery after withdrawal of the ejecting current. Using equal periods of administration of regular intervals, 13 out of 25 neurones showed a more pronounced activation with the heptapeptide; 9/25 neurones showed similar effects and only 3/25 were more strongly affected with angio-
112
@ ~ o
3oj
®
I--1 A1T
~I:l__
An-E.5-8] =
30 sec
I
o A~-[6-81 15
r - ~ sec
Fig~ 3. Effects of shorter chain peptide fragments derived from angiotensin II (A II) on three individual SFO neurones. A : integrated firing frequency of a neurone excited by ejection of angiotensm II (10 nA) and angiotensin II-[5-81-tetrapeptide (40 nA). B: microiontophoresis of angiotensin II (50 nA) leads to an enhanced firing whereas equal amount of angiotensin II-[5-8]tetrapeptide shows only weak response. Application of angiotensin II-[6-8]-tripeptide (100 nA) did not show any effect. At the end of the tracing another ejection of the tetrapeptide (80 nA) produces an excitatory action. C: the effect of the tetrapeptide (100 nA) tested on another SFO neurone. Again the tripeptide ejected with 150 nA, tested in between, failed to enhance the firing frequency. Note different time base in A and B, C as well as different scales of ordinate in A, B and C.
tensin II. The relationship between the firing frequency of an individual neurone and varying intensities of ejecting currents is represented by a dose-response curve, illustrated in Fig. 2B. In the cases where the heptapeptide had a more p r o n o u n c e d activation, this effect was usually 1.5-2-fold higher. The same differential effect could be observed when the heptapeptide was ejected prior to angiotensin II. Both the action of angiotensin II and angiotensin II-[2-8]-heptapeptide were blocked by [Sar 1, AlaS]-angio tensin II (Fig. 2C, P 113). The antagonist alone produced either no effect or a decrease in unit firing. The most sensitive antagonism on angiotensin II-[2-8]-heptapeptide with respect to dose of P 113 was on neurones responsive to angiotensin and not to both angiotensin and acetylcholine. This finding is in agreement with previously reported antagonism on angiotensin II ~7.
Angiotensin H and angiotensin II-[5-8]-tetrapeptide (Fig. 3) In the second series of experiments consisting of angiotensin II-[5-8]-tetrapeptide ejection, successful results were obtained from 16 S F O neurones. A l t h o u g h the neurones were responsive to the ejection o f the angiotensin II-[5-8]-tetrapeptide it was difficult to make a reliable comparison with the effect of angiotensin II. Whereas
113 relatively small amounts (30-70 nA) of the octapeptide led to a strong excitatory action, very high amounts of currents (often exceeding 100 nA) were necessary to produce a similar effect with the tetrapeptide. On the other hand angiotensin II ejected with doses of 100 nA often produced bursts leading to uncontrollable recordings. Fourteen out of 16 cells showed a stronger response to angiotensin II than to angiotensin II-[5-8]-tetrapeptide. The mean ratio between the two drugs was 1:5. Only two cells responded to both substances with similar enhancement of firing frequency. The weak excitant action by the tetrapeptide was also expressed by the latencies of the occurring responses. The mean delay observed with the tetrapeptide was within the range of 10 sec. Since already very high doses of the fragment peptide were necessary to produce a decent acceleration of firing rate it was difficult to demonstrate whether this change of frequency could be antagonized by P 113. On two occasions, however, such an antagonism could be observed; where the ratio between the peptide and the antagonist was 60:150 and 50:180 respectively. At the end of 2 experiments angiotensin II-[5-8]-tetrapeptide was tested on blood pressure responses by intravenous injection into the radial vein. Angiotensin II (Hypertensin, Ciba) in quantities of 0.5 #g angiotensin/injection served as a control. Whereas i.v. injection of angiotensin II produced a rise in blood pressure (increase of 30 mm Hg) lasting about 3 min, application of the tetrapeptide using doses up to 90 #g/injection failed to produce a marked pressure response. In the second case only a blood pressure rise of 5 mm Hg was caused by injecting 110 #g ofangiotensin 11-[5-8]tetrapeptide.
Angiotensin H and angiotensin II-[6-8]-tripeptide (Fig. 3) Angiotensin II-[6-8]-tripeptide was applied microiontophoretically using ejection currents of 20-250 nA and a direct comparison with angiotensin II was made on 20 SFO neurones. As already mentioned earlier, only two cells could be activated slightly by this drug. On both cells, currents of 120 nA were necessary to produce this effect. In all other cases currents up to 250 nA were tested and an excitant effect was never observed. Angiotensin II-[6-8]-tripeptide was also administered intravenously at the end of two experiments. Although enormous doses (up to 430/~g) were used, in both studies the tripeptide failed to produce a blood pressure change. DISCUSSION The present experiments were undertaken in order to test the sensitivity of SFO neurones to various shorter chain peptide fragments derived from the octapeptide angiotensin II. Simpson and Routtenberg a6 focused attention on this organ as a site of angiotensin dipsogenic action. Our observationslS, 16 of short latency activation of SFO units by microiontophoretic administration of angiotensin II and those on isolated SFO in vitro preparation 6 indicated that this structure contains angiotensin receptors. This was strengthened by the fact that the angiotensin II analogue [Sar 1, AlaS]-angiotensin II (P 113) showed specific blockade of angiotensin II sensitive neurones in the SFO 27. The present data demonstrate that the biologically active
114 Des-Aspl-angiotensin II shows a significantly higher stimulation of firing rate compared to angiotensin II. Angiotensin ll-[5-8]-tetrapeptide still produced, although very weak, an excitatory action on single units. Both the action of the heptapeptide and the tetrapeptide were blocked by P 113. In contrast, the tripeptide failed to enhance the firing rate of the same neurones. The significance of the present results is evident. They demonstrate that angiotensin has a specific action on certain brain cells. In recent years techniques have been developed for the direct study of the interaction of radioactively labelled peptide hormones and their specific target receptors 31. Hubbard and coworkers z7 examined angiotensin II binding activity of rat brain particles using [125I]angiotensin II. Specific angiotensin binding was confirmed to the diencephalon and in particular to the lateral septal region whereas very low levels were found in cortex, hippocampus and striatum. Using the indirect immunohistochemical method it was possible to demonstrate angiotensin like immunoreactivity in the medulla oblongata and the substantia gelatinosa of the spinal cord, suggesting the existence of angiotensin II-containing nerve terminals in the brain and spinal cord 19. With the microiontophoretic technique the effectors are applied directly to the cell under study. Thus binding, transport and breakdown show only a very minor influence on the apparent effectiveness so that the structure-activity relationships found with this technique reflect mainly the hormone-receptor interaction. The side effects mentioned above are of particular importance in the study of short peptides since binding is usually decreased and degradation leads to inactive products. This may explain the better effectiveness of the shorter peptides in our study as opposed to studies of the dipsogen response. Fitzsimons is has shown that the angiotensin II[5-8]-heptapeptide retained about 50 ~ of the dipsogenic activity of the octapeptide, whereas the angiotensin II-[5-8]-tetrapeptide had no effect on drinking when injected into the angiotensin sensitive region of the diencephalon. However, when microiontophoretically applied this tetrapeptide shows activity and the angiotensin 11-[2-8]heptapeptide has been found to be more active than the octapeptide. This is similar to structure-activity relationships in the adrenal where the heptapeptide is as effective as angiotensin II in stimulating aldosterone synthesis. The removal of asparagine or aspartic acid could lead to a higher accessibility of the peptide to the membrane receptors, or there may be specific angiotensin III receptors in the brain, as shown by Meyer and coworkers 9 for rat adrenal. A very interesting feature of angiotensin 1I activity is its potentiation by Na + both for the peripheral z°,32 and the central action 1,2,13. It is not known whether angiotensin II is transported into and through cells; this would require a special mechanism. One can envisage a process which, like other transport processes, would be driven by the unbalanced Na + concentrations inside and outside the plasma membrane and that transport of peptides would lead to a concomitant rise in intracellular Na ÷ and as a consequence to a reduced potential and thus to a higher discharge rate. For the microiontophoretic experiments it is not yet possible to decide whether angiotensin II shows transmitter like or just modulating activity. Using voltage-clamp techniques, Barker and Smith 3 have reported that a peptide, 8-Lysine-vasopressin, alters the cell membrane's steadystate current voltage indicating that such a peptide has a long-
115 t e r m r e g u l a t i o n o f the v o l t a g e - d e p e n d e n t conductances. W h e t h e r the action o f angiotensin II w o u l d be b a s e d o n a similar m e c h a n i s m c a n n o t be decided f r o m o u r experiments. T o conclude, the present studies on the S F O offer new insight in structure-activity relations for angiotensin I I which are o b s c u r e d by side effects in other b i o a s s a y systems a n d suggest the presence o f specific angiotensin receptors in the brain. ACKNOWLEDGEMENTS The a u t h o r s wish to express their sincere t h a n k s to Prof. K. A k e r t . F u r t h e r m o r e the skillful technical assistance o f U. F r a n g i , A. F~ih a n d A. Fid61er as well as the help o f R. Emch, H. Hauser, D. Savini, E. Schneider a n d U. W a l t y is greatly appreciated. W e t h a n k Dr. D a v i d W r i g h t for critical r e a d i n g o f the manuscript. This w o r k was s u p p o r t e d b y grants N o . 3.534.75 a n d 3.636.75 o f the Swiss N a t i o n a l Science F o u n d a t i o n a n d the Dr. Eric S l a c k - G y r F o u n d a t i o n in Z/irich.
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