132
Brain Research. s 12 (b190) 132- 137
BRES 15301
Binding of atrial and brain natriuretic peptides in brains of hypertensive rats John Brown and Andrzej Czarnecki Physiological Laboratory, Cambridge (U. K.) (Accepted 15 August 1989)
Key words: Brain natriuretic peptide; Atrial natriuretic peptide; Receptor; Subfornical organ; Cboroid plexus; Arachnoid mater; Hypertension
Displacement of bound [125I]a-atrialnatriuretic peptide (a-ANP) by brain natriuretic peptide (BNP) was used to map receptors common to both peptides in rat brain by in vitro autoradiography. Both spontaneously hypertensive rats (SHR) and the normotensive Wistar-Kyoto control strain (WKY) were studied. In both strains, [125I]a-ANP bound densely to subfornical organ, choroid plexus and arachnoid mater. Binding at these sites in either strain was displaced similarly by 1 #M unlabelled a-ANP or BNP. However, no [125I]a-ANP was displaced by peptides unrelated to a-ANP or BNP. In WKY, both a-ANP and BNP competed with similarly high affinities for binding sites occupied by [125I]a-ANP. This was also true for SHR. However, SHR showed a substantial reduction in the maximum number of binding sites in the subfornical organ and choroid plexus which were competed for by the peptides. Therefore, BNP may be a significant high affinity ligand for brain receptors previously thought specific for atrial natriuretic peptides, including receptors which vary between WKY and SHR.
INTRODUCTION A t r i a l natriuretic peptides ( A N P ) have important effects on cardiovascular and fluid homeostasis as physiological antagonists of the r e n i n - a n g i o t e n s i n system I" 11,16,20,24,36. The main A N P in plasma is a-ANp23; but close analogues also occur within the brain 32'37, and intracerebroventricular injections of a - A N P antagonise the effects of central angiotensin II ( A I I ) on arterial pressure and fluid balance 1"u'36. In spontaneously hypertensive rats ( S H R ) , the activity of the cerebral r e n i n angiotensin system increases, and this is implicated in the origin of their hypertension 33. The subfornical organ, which is involved in cardiovascular and fluid homeostasis 25, has m o r e A I I - b i n d i n g sites in young (prehypertensive) and adult S H R c o m p a r e d to age-matched rats from the normotensive W i s t a r - K y o t o ( W K Y ) control strain 2°. In contrast, the subfornical organ has significantly fewer A N P - b i n d i n g sites in S H R , and it has been suggested that changes in the activity of intracerebral A N P are associated with the genetic predisposition to the cardiovascular and h o r m o n a l abnormalities of adult S H R z°. Recently, however, a new class of peptides, the brain natriuretic peptides, has been discovered which, like ANP, are synthesised in the brain and cardiac atria 12' 15,18,29,31, Two peptides of this class have been isolated
from porcine brain 29'31, the 26-amino acid brain natriuretic peptide (BNP) and its N-terminally e x t e n d e d 32amino acid form (BNP-32), but o t h e r evidence suggests that peptides of this class also occur in the brains and atria of the dog 12, rat 15'22 and man 3°. Porcine BNP, like a - A N P , can antagonise the effects of intracerebral A I I TM 24,35 Its structure is strongly h o m o l o g o u s to that of a - A N P 29'31 and it is a high affinity agonist at guanylatecyclase coupled receptors for a - A N P in cultured vascular cell lines 9'26. Therefore, we have used competition displacement studies to investigate the binding of B N P to high affinity sites which are putatively specific for a - A N P in the brains of S H R and W K Y controls. MATERIALS AND METHODS Seven male WKY and six male SHR (250-300 g) were housed with free access to food (Labsure PRD, William Lillico & Co., U.K.) and water. Arterial pressures were measured by tailcuff plethysmography (rat tail plethysmograph, Ugo Basile, Italy). On the next day, the rats were killed by decapitation, and their brains were snap frozen in isopentane at -40 °C. 15/~M sections were cut in a cryostat at -21 °C, thaw-mounted onto gelatin-chrom-alum coated slides, and dried in a desiccator for 8 h at 4 °C. Sections were then preincubated in 50 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCI, 5 mM MgCI2, 0.5% bovine serum albumin and 40/~g/ml bacitracin (Sigma, U.K.) for 15 min at 17 °C. Sections were next incubated with 200 pM of 3-[I25I]iodo-28-tyrosyl rat a-ANP of specific activity 2013 Ci/mmol (Amersham International, U.K.) in the same buffer for 15 min at 17 °C. Binding was displaced in the
Correspondence: J. Brown, Physiological Laboratory, Downing Street, Cambridge, CB2 3EG, U.K.
133 presence of 1 #M unlabelled rat a-ANP or 1 #M porcine BNP, the identities of which were previously confirmed by amino acid analysis and fast atom bombardment (FAB) mass spectrometry, or in the presence of 1 #M of the unrelated peptides vasopressin, salmoncalcitonin and AII (Penninsula Laboratories, U.S.A.). After incubation, the sections were washed twice for 2.5 min in pre-incubation buffer at 4 °C and rapidly dried in a stream of cold air. Sections were then exposed to LKB Ultrofilm (LKB, Sweden) in X-ray cassettes for 5 days at 4 °C. After exposure, the sections were fixed in formalin and stained with haematoxylin and eosin for tissue iocalisation. Stability of ligands was checked in preliminary experiments. Pairs of WKY brain sections were incubated as described above with 200 pM of [t25I]a-ANP, or 1 #M unlabelled rat a-ANP or I gM porcine BNP. 100 #l aliquots of the incubation fluid were sampled within 15 s of the start of incubation and at 15 min of incubation for each pair. These samples were fractionated by high-performance liquid chromatography under identical conditions using a Waters g-Bondapak C-18 column (Millipore, U.K.) and a 10-40% linear gradient of acetonitrile in water containing 0.04% trifluoracetic acid at a flow rate of 1 ml/min. Solutes were detected at 220 nm or, in the case of radiolabelled ligand, by gamma counting of 1 mi fractions. Optical densities of autoradiograms were measured by computerised microdensitometry 7. Sets of lEVI-standards (Amersham International, U.K.) were mounted in each X-ray cassette. The scale of optical densities produced by the autoradiogram of each set of standards was used to construct a standard curve. Optical densities of specific areas from the autoradiograms of brain sections were interpolated on the standard curve to yield the activity of label bound to the tissue. Bound peptide was then calculated from the specific activity of the label corrected for any decay of 125I (ref. 10). The specific gravity of tissue blocks containing thalamus and hypothalamus from 6 further WKY rats was measured directly, and their protein content was measured by a modified Lowry method s. These measurements were used to convert values of bound peptide from fmol/mm2 into fmol/mg protein. Binding isotherms were determined using sets of serial sections, each set from one brain, incubated with a range of concentrations (10-t2-10 -6 M) of either unlabeiled rat a-ANP or unlabelled porcine BNP (Penninsula Laboratories, U.S.A.). Scatchard plots were calculated by linear regression. The standard error (S.E.) of the regression coefficient was found by analysis of variance. The concentration at which BNP displaced 50% of the bound [125I]a-ANP [ICs0] was determined from the 4-parameter logistic equation of best fit (Maximum Likelihood Program, Rothamsted Experimental Station). The ap-
RESULTS Studies of the stability of 200 pM [125I]a-ANP in the presence of brain tissue u n d e r the conditions of incubation used for autoradiography showed a m i n o r degree of degradation by 15 min of incubation (Fig. 1). No significant degradation was detected with 1 p M unlabelled a - A N P or 1 p M BNP by 15 min of incubation. a - A N P b o u n d with high affinity to subfornical organ, choroid plexus and arachnoid mater (Figs. 2, 3; Table I). Binding was compatible with a single class of binding sites within each structure. Moreover, b o u n d [125I]a-ANP was not displaced by 1 # M vasopressin, salmon-calcitonin or AII. We found that the affinity of the subfornical organ and choroid plexus for a - A N P was increased similarly in SHR, but this difference in affinity between S H R and W K Y was not apparent for arachnoid binding sites (Table I). The affinities of a - A N P binding sites for BNP were high in WKY, and changed in parallel to those for a - A N P in SHR, so that the subfornical organ and choroid, but not the arachnoid, showed significantly higher affinities for BNP in S H R (Table I). However, the density of sites binding a - A N P with high affinity was considerably reduced in SHR. Moreover, there was no significant difference in the displacement of [125I]a-ANP by either 1 # M unlabelled a - A N P or 1 # M BNP in any structure within either strain (Table I). Thus, BNP was a high affinity ligand at all binding sites which might have been considered specific for a - A N P and which were depleted
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parent dissociation constant (Kd) and maximal binding capacity (Bmax) of particular structures for a-ANP were determined separately for each individual by the Scatchard analysis. Similarly, the apparent dissociation constants for the binding of BNP (Ki) were calculated for each rat from ICso values determined in different structures which bound [125I]a-ANp4. Results are presented as means + S.E.M. Comparisons of group data (WKY vs SHR) were by unpaired Student's t-test or by Fisher's Variance Ratio test as appropriate. Differences of means within groups were assessed by paired Student's t-tests or Fisher's Variance Ratio test as appropriate.
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in genetically hypertensive rats. The m e a n arterial pressure was 108 _+ 5 m m Hg in W K Y and 145 _+ 4 m m Hg in S H R (0.001 > P).
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The nature of the high affinity binding sites for a - A N P on subfornical organ has not been investigated directly, but a role in h a e m o d y n a m i c and fluid homeostasis is suggested by their variation in strains such as S H R 2° and Brattleboro rats 21, which have body fluid and cardiovascular abnormalities. Several cultured neural cell lines express guanylate cyclase-coupled receptors for a - A N P 6' 28, and the binding of a - A N P to subfornical organ may be
134
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135 related to such receptors. The choroid plexus has been investigated more directly and is already known to contain a guanylate-cyclase-coupled receptor for a-ANP with an activation constant of 5-10 nM 28. This is similar to the Ka for a - A N P found here on both choroid plexus and subfornical organ. There is also recent evidence, at least outside the CNS, that some of the actions of a-ANP may be mediated by second messengers other than cGMP 34. However, there is another and better defined group of receptors for a - A N P which are not coupled to guanylate cyclase and which probably clear bound pep-
tide by internalising and destroying it 17. These clearance
receptors bind ring-deleted analogues of a-ANP, such as des [Gin TM - Ser 19 - Gly 2° - Leu 21 - Gly 22] ANP(4-23) NH2, which do not bind to the guanylate cyclase-coupled receptor 17. We have recently shown that des[Gin 18Ser~9-Gly2°-Leu2LGly22]ANP(4-23)-NH2 will displace [~25I]a-ANP from the arachnoid but not from the choroid plexus or subfornical organ of the rat 3. Therefore, the binding sites on choroid plexus and subfornical organ, which have similar affinities for BNP and ANP and which vary in number between SHR and WKY may represent
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, 50
136 "FABLE I Binding constants for cx-ANP and BNP for intracerebral structures of WK Y and SHR
WKY (n = 7) Subfornical organ Choroid plexus Arachnoid mater SHR (n = 6) Subfornical organ Choroid plexus Arachnoid mater
Affinity constant (Ka) for a-ANP (nM)
Binding capacity (B,,,,Q for a-ANP (fmol/rng protein)
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2.76 _+1/.35 3.111 _+11.35 3.18_+0.42
2.99 + 0.21 2.78 _+0.08 3.92_+0.19
2.05 _+0.40 + 1.37 -+_0.29 1.10_+0.13
22.2 _+4.4 18.2 _+5.9 21.8_+2.9
*, P < 0.05 for WKY vs SHR for particular structures. ~. P < 1/.05 for comparisons of subfornical organ vs arachnoid mater and choroid plexus vs arachnoid mater within WKY and within SHR.
guanylate cyclase-coupled receptors or another receptor which does not bind ring-deleted analogues of a - A N P ; the binding sites on arachnoid, of generally higher affinity, and which do not vary between S H R and WKY, may represent clearance receptors. W h a t e v e r their nature, however, our results show that porcine BNP is a high-affinity ligand at all of these intracerebral sites. We have used 26-residue porcine BNP as a competitive ligand in rat brain. Both this and its N-terminally e x t e n d e d 32-amino acid form share a 17-amino acid ring closed by a disulphide b o n d between flanking cysteines 29 31. This ring is highly homologous with the 17-residue ring found in the atrial natriuretic peptides, which is essential for their activation of guanylate cyclase-coupled receptors 17"29. Consistent with this, porcine BNP, like atrial natriuretic peptides, is a powerful stimulator of guanylate cyclase in a variety of isolated or cultured rat 9'26 and bovine 26 tissues, including rat brain 26. Moreover, intracerebroventricular injections of porcine BNP given to rats mimic central effects of rat A N P 13'24"35. BNP has yet to be isolated and sequenced in animals other than the pig. H o w e v e r , the structures of BNP have recently been inferred for both the rat ~5 and man 3° by cloning and sequencing c D N A precursors. There is a considerable degree of h o m o l o g y between the apparent structures of B N P in different animals ~5"3° and, in particular, the 17-amino acid ring is highly conserved between the pig and the rat ~5. Thus, it is likely that the BNP molecules of different species will exhibit similarities of receptor binding and pharmacological effects based on their structural homology. Therefore, our results with porcine BNP point to the possibility that, in the rat, endogenous B N P may be a significant agonist at central nervous receptor sites previously thought to be related to ANP. A role for endogenous BNP at receptors previously thought specific for A N P may underlie the recent report that 125I-labelled porcine B N P is a high-affinity ligand for
the porcine subfornical organ 19. The subfornical organ is crucial in mediating the central effects of humoral A l l on arterial pressure and fluid homeostasis 25, and the altered binding capacity of the subfornical organ for a - A N P in strains of rat which cardiovascular and fluid abnormalities suggests that atrial natriuretic peptides may also modulate these homeostatic effects of this structure 2°'21. Yet, paradoxically, the rat subfornical organ receives few if any fibres which stain immunohistochemically for A N P TM 27,37. One possibility is that the organ lies outside the b l o o d - b r a i n barrier 25 so it could m e d i a t e responses to humoral a - A N P , as it does for h u m o r a l A l l ; but no such responses are known. B N P is also synthesised by the cardiac atria 15'~8 so that another interesting possibility is that humoral B N P may have central actions. Recently, however, studies with antibodies that distinguish porcine BNP from atrial natriuretic peptides have shown that the rat subfornical organ is richly innervated by fibres showing BNP-like immunoreactivity 22. Thus, the paucity of A N P immunoreactivity in the subfornical organ of the rat would be explained if e n d o g e n o u s BNP were the important ligand at the receptors in this structure which were previously thought specific for ANP, and which are related to arterial pressure and fluid homeostasis. In conclusion, therefore, we have shown that high-affinity binding sites within the central nervous system, including sites on the subfornical organ, fail to distinguish between porcine BNP and rat ct-ANP significantly. These two peptides share an amino acid structure that is essential for the binding of a - A N P to its guanylate-cyclase coupled receptors. Since the recently discovered sequence of rat BNP also shares this structure, our results open the possibility that e n d o g e n o u s BNP may be the important natural ligand at a variety of central receptors, such as those of the subfornical organ, which were previously related to ANP.
137
REFERENCES 1 Antunes-Rodrigues, J., McCann, S.M., Rogers, L.C. and Samson, W.K., Atrial natriuretic factor inhibits dehydration- and angiotensin II-induced water intake in the conscious,unrestrained rat, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 8720-8723. 2 Brown, J., Polak, J.M., Power, R.E, Salad, S.P. and Singleton, A., Differential distribution of subtypes of specific binding sites for alpha atrial natriuretic peptide (aANP) in the rat kidney, J. Physiol. (Lond.), 403 (1988) 21P. 3 Brown, J. and Czarnecki, A., Autographic localisation of the binding of porcine brain natriuretic peptide (BNP) and des (18-22) atrial natriuretic peptide(4_23)-NH2 (C-ANP4_23) in rat brain, Am. J. Hypertens., 2 (1989) 100A. 4 Cheng, Y. and Prusoff, W.H., Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (ICso) of an enzymatic reaction, Biochem. Pharmacol., 22 (1973) 3099-3108. 5 Friedl, A., Harmening, C. and Hamprecht, B., Atrial natriuretic hormones raise the level of cyclic GMP neural cell lines, J. Neurochem., 46 (1986) 1522-1527. 6 Gibson, T.R., Zysking, A.D. and Giembotski, C.C., Characterisation of solubilized atrial natriuretic peptide receptors from rat olfactory bulb and A10 smooth muscle cells, Neuroscience, 8 (1987) 3067-3073. 7 Goochee, C., Rasband, W. and Sololoff, L., Computerised densitometry and colour coding of [14C]deoxyglucoseautoradiographs, Ann. Neurol., 7 (1980) 359-370. 8 Hartree, E.E, Determination of protein: a modification of the Lowry method that gives a linear photometric response, Anal. Biochem., 48 (1972) 422-427. 9 Hirata, Y., Shichiri, M., Emori, T., Marmumo, E, Kangawa, K. and Matsuo, H., Brain natriuretic peptide interacts with atrial natriuretic peptide receptor in cultured rat vasocular smooth muscle cells, FEBS Lett., 238 (1988) 415-518. 10 Israel, A., Correa, EM.A., Niwa, M. and Saavedra, J.M., Quantitative determination of angiotensin II binding sites in rat brain and pituitary gland by autoradiography, Brain Research, 322 (1984) 341-345. 11 Itoh, H., Nakao, K., Morii, N., Yamada, T., Shiono, S., Sakamoto, M., Sugawara, A., Saito, Y., Katsuura, G., Shiomi, T., Eigyo, M., Matsushita, A. and Imura, H., Central action of atrial natriuretic polypeptide on blood pressure in conscious rats, Brain Res. Bull., 16 (1986) 745-749. 12 Itoh, H., Nakao, K., Saito, Y., Yamada, T., Shirakami, G., Nukoyama, M., Arai, H., Hosoda, K., Suga, S.-I., Minamino, N., Kangawa, K., Matsuo, H. and Imura, H., Radioimmunoassayfor brain natriuretic peptide (BNP) detection of BNP in canine brain, Biochem. Biophys. Res. Commun., 158 (1989) 120-128. 13 Itoh, H., Nakao, K., Yamada, T., Shirakami, G., Kangawa, K., Minamino, N., Matsuo, H. and Imura, H., Antidipsogenic action of a novel peptide, brain natriuretic peptide, in rat, Eur. J. Pharmacol., 150 (1988) 193-196. 14 Kawata, M., Nakao, K., Morii, N., Kiso, Y., Yamashita, H., Imura, H. and Sano, Y., Atrial natriuretic polypeptide: topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry, Neuroscience, 16 (1985) 521-546. 15 Kogima, M., Minamino, N., Kangawa, K. and Matsuo, H., Cloning and sequence analysis of cDNA encoding a precursor for rat brain natriuretic peptide, Biochem. Biophys. Res. Commun., 159 (1989) 1420-1426. 16 Maack, T., Camargo, M.J.F., Kleinert, H.D., Laragh, J.H. and Atlas, S.A., Atrial natriuretic factor: structure and functional properties, Kidney Int., 27 (1985) 607-615. 17 Maack, T., Suzuki, M., Almeida, EA., Nussenzveig, D., Scarborough, R.M., McEnroe, G.A. and Lewicki, J.A., Physiological role of silent receptors of atrial natriuretic factor, Science, 238 (1987) 675-678. 18 Minamino, N., Kangawa, K. and Matsuo, H., Isolation and identification of a high molecular weight brain natriuretic peptide in porcine cardiac atrium, Biochem. Biophys. Res. Commun., 157 (1988) 402-409.
19 Niwa, M., Shigematsu, K., Kurihara, M., Kataoka, Y., Maeda, T., Nakao, K., Imura, H., Matsuo, H., Tsuchiyama, H. and Ozaki, M., Receptor autoradiographic evidence of specific brain natriuretic peptide binding sites in the porcine subfornical organ, Neurosci. Lett., 95 (1988) 113-118. 20 Saavedra, J.M., Correa, EM.A., Plunkett, L.M., Israel, A., Kurihara, M. and Shigematsu, K., Binding of angiotensin and atrial natriuretic peptide in brain of hypertensive rat, Nature (Lond.), 320 (1986) 758-760. 21 Saavedra, J.M., Israel, A., Correa, F.M.A. and Kurihara, M., Increased atrial natriuretic peptide (6-33) binding sites in the subfornical organ of water deprived and brattleboro rats, Proc. Soc. Exp. Biol. Med., 182 (1986) 559-563. 22 Saper, C.B., Hurley, K.M., Moga, M.M., Holmes, H.R., Adams, S.A., Leahy, K.M. and Needleman, P., Brain natriuretic peptide differential localisation of a new family of neuropeptides, Neurosci. Lett., 96 (1989) 29-34. 23 Schwartz, D., Geller, D.M., Manning, P.T., Siegel, N.R., Fok, K.F., Smith, C.E. and Needleman, P., Ser-Leu-Arg-Argatriopeptin III: the major circulating form of atrial peptide, Science, 229 (1985) 397-400. 24 Shirakami, G.K., Nakao, T., Yamada, H., Itoh, K., Mori, K., Kangawa, K., Minamino, N., Matsuo, H. and Imura, H., Inhibitory effect of brain natriuretic peptide on central angiotensin II-stimulated pressor response in conscious rats, Neurosci. Lett., 91 (1988) 77-83. 25 Simpson, J.B., The circumventricular organs and the central actions of angiotensin, Neuroendocrinology, 32 (1981) 248-256. 26 Song, D.L., Kohse, K.P. and Murad, E, Brain natriuretic factor. Augmentation of cellular cyclic GMP, activation of particulate guanylate cyclase and receptor binding, FEBS Lett., 232 (1988) 125-129. 27 Standaert, D.G., Needleman, P. and Saper, C.B., Organisation of atriopeptin-like immunoreactive neurones in the central nervous system of the rat, J. Comp. Neurol., 253 (1986) 315-341. 28 Steardo, L. and Nathanson, J.A., Brain barrier tissues: end organs for atriopeptins, Science, 235 (1987) 470-473. 29 Sudoh, T., Kangawa, K., Minamino, N. and Matsuo, H., A new natriuretic peptide in porcine brain, Nature, 332 (1988) 78-81. 30 Sudoh, T., Maekawa, K., Kogima, M., Minamino, N., Kangawa, K. and Matsuo, H., Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide, Biochem. Biophys. Res. Commun., 159 (1989) 1427-1434. 31 Sudoh, T., Minamino, N., Kangawa, K. and Matsuo, H., Brain natriuretic peptide-32: N-terminal sex amino acid extended form of brain natriuretic peptide identified in porcine brain, Biochem. Biophys. Res. Commun., 155 (1988) 726-732. 32 Ueda, S., Sudoh, T., Fukuda, K., Kangawa, K., Minamino, N. and Matsuo, H., Identification of a-atrial natriuretic peptide (4-28 and 5-28) in porcine brain, Biochem. Biophys. Res. Comrnun., 149 (1987) 1055-1062. 33 Unger, T., Badoer, E., Ganten, D., Lang, R.E. and Rettig, R., Brain angiotensin: pathways and pharmacology, Circulation, 77 (1988) 1-40-I-54. 34 Willenbrock, R.C., Tremblay, J., Garcia, R. and Hamet, P., Dissociation of natriuresis and diuresis and heterogenity of the effector system of atrial natriuretic factor in rats, J. Clin. Invest., 83 (1989) 482-289. 35 Yamada, T., Nakao, K., Itoh, H., Shirakami, G., Kangawa, K., Minamino, N., Matsuo, H. and Imura, H., Intracerebroventricular injection of brain natriuretic peptide inhibits vasopressin secretion in conscious rats, Neurosci. Lett., 95 (1988) 223-228. 36 Yamada, T., Nakao, K., Mouii, N., Itoh, H., Shiono, S., Sakamoto, M., Sugawara, A., Saito, Y., Ohno, H., Kanai, A., Katsuura, G., Eigyo, M., Matsushita, A. and Imura, H., Central effects of atrial natriuretic polypeptide on antiotensin IIstimulated vasopressin secretion in conscious rats, Eur. J. Pharmacol., 125 (1986) 453-456. 37 Zamir, N., Skofitsch, G., Eskay, R.L. and Jacobowitz, D.M., Distribution of immunoreactive atrial natriuretic peptides in the central nervous system of the rat, Brain Research, 365 (1986) 105-111.
Brain Research. s 12 (b190) 132- 137
BRES 15301
Binding of atrial and brain natriuretic peptides in brains of hypertensive rats John Brown and Andrzej Czarnecki Physiological Laboratory, Cambridge (U. K.) (Accepted 15 August 1989)
Key words: Brain natriuretic peptide; Atrial natriuretic peptide; Receptor; Subfornical organ; Cboroid plexus; Arachnoid mater; Hypertension
Displacement of bound [125I]a-atrialnatriuretic peptide (a-ANP) by brain natriuretic peptide (BNP) was used to map receptors common to both peptides in rat brain by in vitro autoradiography. Both spontaneously hypertensive rats (SHR) and the normotensive Wistar-Kyoto control strain (WKY) were studied. In both strains, [125I]a-ANP bound densely to subfornical organ, choroid plexus and arachnoid mater. Binding at these sites in either strain was displaced similarly by 1 #M unlabelled a-ANP or BNP. However, no [125I]a-ANP was displaced by peptides unrelated to a-ANP or BNP. In WKY, both a-ANP and BNP competed with similarly high affinities for binding sites occupied by [125I]a-ANP. This was also true for SHR. However, SHR showed a substantial reduction in the maximum number of binding sites in the subfornical organ and choroid plexus which were competed for by the peptides. Therefore, BNP may be a significant high affinity ligand for brain receptors previously thought specific for atrial natriuretic peptides, including receptors which vary between WKY and SHR.
INTRODUCTION A t r i a l natriuretic peptides ( A N P ) have important effects on cardiovascular and fluid homeostasis as physiological antagonists of the r e n i n - a n g i o t e n s i n system I" 11,16,20,24,36. The main A N P in plasma is a-ANp23; but close analogues also occur within the brain 32'37, and intracerebroventricular injections of a - A N P antagonise the effects of central angiotensin II ( A I I ) on arterial pressure and fluid balance 1"u'36. In spontaneously hypertensive rats ( S H R ) , the activity of the cerebral r e n i n angiotensin system increases, and this is implicated in the origin of their hypertension 33. The subfornical organ, which is involved in cardiovascular and fluid homeostasis 25, has m o r e A I I - b i n d i n g sites in young (prehypertensive) and adult S H R c o m p a r e d to age-matched rats from the normotensive W i s t a r - K y o t o ( W K Y ) control strain 2°. In contrast, the subfornical organ has significantly fewer A N P - b i n d i n g sites in S H R , and it has been suggested that changes in the activity of intracerebral A N P are associated with the genetic predisposition to the cardiovascular and h o r m o n a l abnormalities of adult S H R z°. Recently, however, a new class of peptides, the brain natriuretic peptides, has been discovered which, like ANP, are synthesised in the brain and cardiac atria 12' 15,18,29,31, Two peptides of this class have been isolated
from porcine brain 29'31, the 26-amino acid brain natriuretic peptide (BNP) and its N-terminally e x t e n d e d 32amino acid form (BNP-32), but o t h e r evidence suggests that peptides of this class also occur in the brains and atria of the dog 12, rat 15'22 and man 3°. Porcine BNP, like a - A N P , can antagonise the effects of intracerebral A I I TM 24,35 Its structure is strongly h o m o l o g o u s to that of a - A N P 29'31 and it is a high affinity agonist at guanylatecyclase coupled receptors for a - A N P in cultured vascular cell lines 9'26. Therefore, we have used competition displacement studies to investigate the binding of B N P to high affinity sites which are putatively specific for a - A N P in the brains of S H R and W K Y controls. MATERIALS AND METHODS Seven male WKY and six male SHR (250-300 g) were housed with free access to food (Labsure PRD, William Lillico & Co., U.K.) and water. Arterial pressures were measured by tailcuff plethysmography (rat tail plethysmograph, Ugo Basile, Italy). On the next day, the rats were killed by decapitation, and their brains were snap frozen in isopentane at -40 °C. 15/~M sections were cut in a cryostat at -21 °C, thaw-mounted onto gelatin-chrom-alum coated slides, and dried in a desiccator for 8 h at 4 °C. Sections were then preincubated in 50 mM Tris-HCl buffer, pH 7.4, containing 100 mM NaCI, 5 mM MgCI2, 0.5% bovine serum albumin and 40/~g/ml bacitracin (Sigma, U.K.) for 15 min at 17 °C. Sections were next incubated with 200 pM of 3-[I25I]iodo-28-tyrosyl rat a-ANP of specific activity 2013 Ci/mmol (Amersham International, U.K.) in the same buffer for 15 min at 17 °C. Binding was displaced in the
Correspondence: J. Brown, Physiological Laboratory, Downing Street, Cambridge, CB2 3EG, U.K.
133 presence of 1 #M unlabelled rat a-ANP or 1 #M porcine BNP, the identities of which were previously confirmed by amino acid analysis and fast atom bombardment (FAB) mass spectrometry, or in the presence of 1 #M of the unrelated peptides vasopressin, salmoncalcitonin and AII (Penninsula Laboratories, U.S.A.). After incubation, the sections were washed twice for 2.5 min in pre-incubation buffer at 4 °C and rapidly dried in a stream of cold air. Sections were then exposed to LKB Ultrofilm (LKB, Sweden) in X-ray cassettes for 5 days at 4 °C. After exposure, the sections were fixed in formalin and stained with haematoxylin and eosin for tissue iocalisation. Stability of ligands was checked in preliminary experiments. Pairs of WKY brain sections were incubated as described above with 200 pM of [t25I]a-ANP, or 1 #M unlabelled rat a-ANP or I gM porcine BNP. 100 #l aliquots of the incubation fluid were sampled within 15 s of the start of incubation and at 15 min of incubation for each pair. These samples were fractionated by high-performance liquid chromatography under identical conditions using a Waters g-Bondapak C-18 column (Millipore, U.K.) and a 10-40% linear gradient of acetonitrile in water containing 0.04% trifluoracetic acid at a flow rate of 1 ml/min. Solutes were detected at 220 nm or, in the case of radiolabelled ligand, by gamma counting of 1 mi fractions. Optical densities of autoradiograms were measured by computerised microdensitometry 7. Sets of lEVI-standards (Amersham International, U.K.) were mounted in each X-ray cassette. The scale of optical densities produced by the autoradiogram of each set of standards was used to construct a standard curve. Optical densities of specific areas from the autoradiograms of brain sections were interpolated on the standard curve to yield the activity of label bound to the tissue. Bound peptide was then calculated from the specific activity of the label corrected for any decay of 125I (ref. 10). The specific gravity of tissue blocks containing thalamus and hypothalamus from 6 further WKY rats was measured directly, and their protein content was measured by a modified Lowry method s. These measurements were used to convert values of bound peptide from fmol/mm2 into fmol/mg protein. Binding isotherms were determined using sets of serial sections, each set from one brain, incubated with a range of concentrations (10-t2-10 -6 M) of either unlabeiled rat a-ANP or unlabelled porcine BNP (Penninsula Laboratories, U.S.A.). Scatchard plots were calculated by linear regression. The standard error (S.E.) of the regression coefficient was found by analysis of variance. The concentration at which BNP displaced 50% of the bound [125I]a-ANP [ICs0] was determined from the 4-parameter logistic equation of best fit (Maximum Likelihood Program, Rothamsted Experimental Station). The ap-
RESULTS Studies of the stability of 200 pM [125I]a-ANP in the presence of brain tissue u n d e r the conditions of incubation used for autoradiography showed a m i n o r degree of degradation by 15 min of incubation (Fig. 1). No significant degradation was detected with 1 p M unlabelled a - A N P or 1 p M BNP by 15 min of incubation. a - A N P b o u n d with high affinity to subfornical organ, choroid plexus and arachnoid mater (Figs. 2, 3; Table I). Binding was compatible with a single class of binding sites within each structure. Moreover, b o u n d [125I]a-ANP was not displaced by 1 # M vasopressin, salmon-calcitonin or AII. We found that the affinity of the subfornical organ and choroid plexus for a - A N P was increased similarly in SHR, but this difference in affinity between S H R and W K Y was not apparent for arachnoid binding sites (Table I). The affinities of a - A N P binding sites for BNP were high in WKY, and changed in parallel to those for a - A N P in SHR, so that the subfornical organ and choroid, but not the arachnoid, showed significantly higher affinities for BNP in S H R (Table I). However, the density of sites binding a - A N P with high affinity was considerably reduced in SHR. Moreover, there was no significant difference in the displacement of [125I]a-ANP by either 1 # M unlabelled a - A N P or 1 # M BNP in any structure within either strain (Table I). Thus, BNP was a high affinity ligand at all binding sites which might have been considered specific for a - A N P and which were depleted
I t25 I ~ - A N P
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parent dissociation constant (Kd) and maximal binding capacity (Bmax) of particular structures for a-ANP were determined separately for each individual by the Scatchard analysis. Similarly, the apparent dissociation constants for the binding of BNP (Ki) were calculated for each rat from ICso values determined in different structures which bound [125I]a-ANp4. Results are presented as means + S.E.M. Comparisons of group data (WKY vs SHR) were by unpaired Student's t-test or by Fisher's Variance Ratio test as appropriate. Differences of means within groups were assessed by paired Student's t-tests or Fisher's Variance Ratio test as appropriate.
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in genetically hypertensive rats. The m e a n arterial pressure was 108 _+ 5 m m Hg in W K Y and 145 _+ 4 m m Hg in S H R (0.001 > P).
30
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The nature of the high affinity binding sites for a - A N P on subfornical organ has not been investigated directly, but a role in h a e m o d y n a m i c and fluid homeostasis is suggested by their variation in strains such as S H R 2° and Brattleboro rats 21, which have body fluid and cardiovascular abnormalities. Several cultured neural cell lines express guanylate cyclase-coupled receptors for a - A N P 6' 28, and the binding of a - A N P to subfornical organ may be
134
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135 related to such receptors. The choroid plexus has been investigated more directly and is already known to contain a guanylate-cyclase-coupled receptor for a-ANP with an activation constant of 5-10 nM 28. This is similar to the Ka for a - A N P found here on both choroid plexus and subfornical organ. There is also recent evidence, at least outside the CNS, that some of the actions of a-ANP may be mediated by second messengers other than cGMP 34. However, there is another and better defined group of receptors for a - A N P which are not coupled to guanylate cyclase and which probably clear bound pep-
tide by internalising and destroying it 17. These clearance
receptors bind ring-deleted analogues of a-ANP, such as des [Gin TM - Ser 19 - Gly 2° - Leu 21 - Gly 22] ANP(4-23) NH2, which do not bind to the guanylate cyclase-coupled receptor 17. We have recently shown that des[Gin 18Ser~9-Gly2°-Leu2LGly22]ANP(4-23)-NH2 will displace [~25I]a-ANP from the arachnoid but not from the choroid plexus or subfornical organ of the rat 3. Therefore, the binding sites on choroid plexus and subfornical organ, which have similar affinities for BNP and ANP and which vary in number between SHR and WKY may represent
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, 50
136 "FABLE I Binding constants for cx-ANP and BNP for intracerebral structures of WK Y and SHR
WKY (n = 7) Subfornical organ Choroid plexus Arachnoid mater SHR (n = 6) Subfornical organ Choroid plexus Arachnoid mater
Affinity constant (Ka) for a-ANP (nM)
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[1251]c~-ANP displacedby lO° M unlabelled a-ANP (fmol/mgprotein)
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4.87 + (I.53" ~ 5.12 _+0.40 *~ 1.63 + 0.33
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2.99 + 0.21 2.78 _+0.08 3.92_+0.19
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22.2 _+4.4 18.2 _+5.9 21.8_+2.9
*, P < 0.05 for WKY vs SHR for particular structures. ~. P < 1/.05 for comparisons of subfornical organ vs arachnoid mater and choroid plexus vs arachnoid mater within WKY and within SHR.
guanylate cyclase-coupled receptors or another receptor which does not bind ring-deleted analogues of a - A N P ; the binding sites on arachnoid, of generally higher affinity, and which do not vary between S H R and WKY, may represent clearance receptors. W h a t e v e r their nature, however, our results show that porcine BNP is a high-affinity ligand at all of these intracerebral sites. We have used 26-residue porcine BNP as a competitive ligand in rat brain. Both this and its N-terminally e x t e n d e d 32-amino acid form share a 17-amino acid ring closed by a disulphide b o n d between flanking cysteines 29 31. This ring is highly homologous with the 17-residue ring found in the atrial natriuretic peptides, which is essential for their activation of guanylate cyclase-coupled receptors 17"29. Consistent with this, porcine BNP, like atrial natriuretic peptides, is a powerful stimulator of guanylate cyclase in a variety of isolated or cultured rat 9'26 and bovine 26 tissues, including rat brain 26. Moreover, intracerebroventricular injections of porcine BNP given to rats mimic central effects of rat A N P 13'24"35. BNP has yet to be isolated and sequenced in animals other than the pig. H o w e v e r , the structures of BNP have recently been inferred for both the rat ~5 and man 3° by cloning and sequencing c D N A precursors. There is a considerable degree of h o m o l o g y between the apparent structures of B N P in different animals ~5"3° and, in particular, the 17-amino acid ring is highly conserved between the pig and the rat ~5. Thus, it is likely that the BNP molecules of different species will exhibit similarities of receptor binding and pharmacological effects based on their structural homology. Therefore, our results with porcine BNP point to the possibility that, in the rat, endogenous B N P may be a significant agonist at central nervous receptor sites previously thought to be related to ANP. A role for endogenous BNP at receptors previously thought specific for A N P may underlie the recent report that 125I-labelled porcine B N P is a high-affinity ligand for
the porcine subfornical organ 19. The subfornical organ is crucial in mediating the central effects of humoral A l l on arterial pressure and fluid homeostasis 25, and the altered binding capacity of the subfornical organ for a - A N P in strains of rat which cardiovascular and fluid abnormalities suggests that atrial natriuretic peptides may also modulate these homeostatic effects of this structure 2°'21. Yet, paradoxically, the rat subfornical organ receives few if any fibres which stain immunohistochemically for A N P TM 27,37. One possibility is that the organ lies outside the b l o o d - b r a i n barrier 25 so it could m e d i a t e responses to humoral a - A N P , as it does for h u m o r a l A l l ; but no such responses are known. B N P is also synthesised by the cardiac atria 15'~8 so that another interesting possibility is that humoral B N P may have central actions. Recently, however, studies with antibodies that distinguish porcine BNP from atrial natriuretic peptides have shown that the rat subfornical organ is richly innervated by fibres showing BNP-like immunoreactivity 22. Thus, the paucity of A N P immunoreactivity in the subfornical organ of the rat would be explained if e n d o g e n o u s BNP were the important ligand at the receptors in this structure which were previously thought specific for ANP, and which are related to arterial pressure and fluid homeostasis. In conclusion, therefore, we have shown that high-affinity binding sites within the central nervous system, including sites on the subfornical organ, fail to distinguish between porcine BNP and rat ct-ANP significantly. These two peptides share an amino acid structure that is essential for the binding of a - A N P to its guanylate-cyclase coupled receptors. Since the recently discovered sequence of rat BNP also shares this structure, our results open the possibility that e n d o g e n o u s BNP may be the important natural ligand at a variety of central receptors, such as those of the subfornical organ, which were previously related to ANP.
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REFERENCES 1 Antunes-Rodrigues, J., McCann, S.M., Rogers, L.C. and Samson, W.K., Atrial natriuretic factor inhibits dehydration- and angiotensin II-induced water intake in the conscious,unrestrained rat, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 8720-8723. 2 Brown, J., Polak, J.M., Power, R.E, Salad, S.P. and Singleton, A., Differential distribution of subtypes of specific binding sites for alpha atrial natriuretic peptide (aANP) in the rat kidney, J. Physiol. (Lond.), 403 (1988) 21P. 3 Brown, J. and Czarnecki, A., Autographic localisation of the binding of porcine brain natriuretic peptide (BNP) and des (18-22) atrial natriuretic peptide(4_23)-NH2 (C-ANP4_23) in rat brain, Am. J. Hypertens., 2 (1989) 100A. 4 Cheng, Y. and Prusoff, W.H., Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (ICso) of an enzymatic reaction, Biochem. Pharmacol., 22 (1973) 3099-3108. 5 Friedl, A., Harmening, C. and Hamprecht, B., Atrial natriuretic hormones raise the level of cyclic GMP neural cell lines, J. Neurochem., 46 (1986) 1522-1527. 6 Gibson, T.R., Zysking, A.D. and Giembotski, C.C., Characterisation of solubilized atrial natriuretic peptide receptors from rat olfactory bulb and A10 smooth muscle cells, Neuroscience, 8 (1987) 3067-3073. 7 Goochee, C., Rasband, W. and Sololoff, L., Computerised densitometry and colour coding of [14C]deoxyglucoseautoradiographs, Ann. Neurol., 7 (1980) 359-370. 8 Hartree, E.E, Determination of protein: a modification of the Lowry method that gives a linear photometric response, Anal. Biochem., 48 (1972) 422-427. 9 Hirata, Y., Shichiri, M., Emori, T., Marmumo, E, Kangawa, K. and Matsuo, H., Brain natriuretic peptide interacts with atrial natriuretic peptide receptor in cultured rat vasocular smooth muscle cells, FEBS Lett., 238 (1988) 415-518. 10 Israel, A., Correa, EM.A., Niwa, M. and Saavedra, J.M., Quantitative determination of angiotensin II binding sites in rat brain and pituitary gland by autoradiography, Brain Research, 322 (1984) 341-345. 11 Itoh, H., Nakao, K., Morii, N., Yamada, T., Shiono, S., Sakamoto, M., Sugawara, A., Saito, Y., Katsuura, G., Shiomi, T., Eigyo, M., Matsushita, A. and Imura, H., Central action of atrial natriuretic polypeptide on blood pressure in conscious rats, Brain Res. Bull., 16 (1986) 745-749. 12 Itoh, H., Nakao, K., Saito, Y., Yamada, T., Shirakami, G., Nukoyama, M., Arai, H., Hosoda, K., Suga, S.-I., Minamino, N., Kangawa, K., Matsuo, H. and Imura, H., Radioimmunoassayfor brain natriuretic peptide (BNP) detection of BNP in canine brain, Biochem. Biophys. Res. Commun., 158 (1989) 120-128. 13 Itoh, H., Nakao, K., Yamada, T., Shirakami, G., Kangawa, K., Minamino, N., Matsuo, H. and Imura, H., Antidipsogenic action of a novel peptide, brain natriuretic peptide, in rat, Eur. J. Pharmacol., 150 (1988) 193-196. 14 Kawata, M., Nakao, K., Morii, N., Kiso, Y., Yamashita, H., Imura, H. and Sano, Y., Atrial natriuretic polypeptide: topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry, Neuroscience, 16 (1985) 521-546. 15 Kogima, M., Minamino, N., Kangawa, K. and Matsuo, H., Cloning and sequence analysis of cDNA encoding a precursor for rat brain natriuretic peptide, Biochem. Biophys. Res. Commun., 159 (1989) 1420-1426. 16 Maack, T., Camargo, M.J.F., Kleinert, H.D., Laragh, J.H. and Atlas, S.A., Atrial natriuretic factor: structure and functional properties, Kidney Int., 27 (1985) 607-615. 17 Maack, T., Suzuki, M., Almeida, EA., Nussenzveig, D., Scarborough, R.M., McEnroe, G.A. and Lewicki, J.A., Physiological role of silent receptors of atrial natriuretic factor, Science, 238 (1987) 675-678. 18 Minamino, N., Kangawa, K. and Matsuo, H., Isolation and identification of a high molecular weight brain natriuretic peptide in porcine cardiac atrium, Biochem. Biophys. Res. Commun., 157 (1988) 402-409.
19 Niwa, M., Shigematsu, K., Kurihara, M., Kataoka, Y., Maeda, T., Nakao, K., Imura, H., Matsuo, H., Tsuchiyama, H. and Ozaki, M., Receptor autoradiographic evidence of specific brain natriuretic peptide binding sites in the porcine subfornical organ, Neurosci. Lett., 95 (1988) 113-118. 20 Saavedra, J.M., Correa, EM.A., Plunkett, L.M., Israel, A., Kurihara, M. and Shigematsu, K., Binding of angiotensin and atrial natriuretic peptide in brain of hypertensive rat, Nature (Lond.), 320 (1986) 758-760. 21 Saavedra, J.M., Israel, A., Correa, F.M.A. and Kurihara, M., Increased atrial natriuretic peptide (6-33) binding sites in the subfornical organ of water deprived and brattleboro rats, Proc. Soc. Exp. Biol. Med., 182 (1986) 559-563. 22 Saper, C.B., Hurley, K.M., Moga, M.M., Holmes, H.R., Adams, S.A., Leahy, K.M. and Needleman, P., Brain natriuretic peptide differential localisation of a new family of neuropeptides, Neurosci. Lett., 96 (1989) 29-34. 23 Schwartz, D., Geller, D.M., Manning, P.T., Siegel, N.R., Fok, K.F., Smith, C.E. and Needleman, P., Ser-Leu-Arg-Argatriopeptin III: the major circulating form of atrial peptide, Science, 229 (1985) 397-400. 24 Shirakami, G.K., Nakao, T., Yamada, H., Itoh, K., Mori, K., Kangawa, K., Minamino, N., Matsuo, H. and Imura, H., Inhibitory effect of brain natriuretic peptide on central angiotensin II-stimulated pressor response in conscious rats, Neurosci. Lett., 91 (1988) 77-83. 25 Simpson, J.B., The circumventricular organs and the central actions of angiotensin, Neuroendocrinology, 32 (1981) 248-256. 26 Song, D.L., Kohse, K.P. and Murad, E, Brain natriuretic factor. Augmentation of cellular cyclic GMP, activation of particulate guanylate cyclase and receptor binding, FEBS Lett., 232 (1988) 125-129. 27 Standaert, D.G., Needleman, P. and Saper, C.B., Organisation of atriopeptin-like immunoreactive neurones in the central nervous system of the rat, J. Comp. Neurol., 253 (1986) 315-341. 28 Steardo, L. and Nathanson, J.A., Brain barrier tissues: end organs for atriopeptins, Science, 235 (1987) 470-473. 29 Sudoh, T., Kangawa, K., Minamino, N. and Matsuo, H., A new natriuretic peptide in porcine brain, Nature, 332 (1988) 78-81. 30 Sudoh, T., Maekawa, K., Kogima, M., Minamino, N., Kangawa, K. and Matsuo, H., Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide, Biochem. Biophys. Res. Commun., 159 (1989) 1427-1434. 31 Sudoh, T., Minamino, N., Kangawa, K. and Matsuo, H., Brain natriuretic peptide-32: N-terminal sex amino acid extended form of brain natriuretic peptide identified in porcine brain, Biochem. Biophys. Res. Commun., 155 (1988) 726-732. 32 Ueda, S., Sudoh, T., Fukuda, K., Kangawa, K., Minamino, N. and Matsuo, H., Identification of a-atrial natriuretic peptide (4-28 and 5-28) in porcine brain, Biochem. Biophys. Res. Comrnun., 149 (1987) 1055-1062. 33 Unger, T., Badoer, E., Ganten, D., Lang, R.E. and Rettig, R., Brain angiotensin: pathways and pharmacology, Circulation, 77 (1988) 1-40-I-54. 34 Willenbrock, R.C., Tremblay, J., Garcia, R. and Hamet, P., Dissociation of natriuresis and diuresis and heterogenity of the effector system of atrial natriuretic factor in rats, J. Clin. Invest., 83 (1989) 482-289. 35 Yamada, T., Nakao, K., Itoh, H., Shirakami, G., Kangawa, K., Minamino, N., Matsuo, H. and Imura, H., Intracerebroventricular injection of brain natriuretic peptide inhibits vasopressin secretion in conscious rats, Neurosci. Lett., 95 (1988) 223-228. 36 Yamada, T., Nakao, K., Mouii, N., Itoh, H., Shiono, S., Sakamoto, M., Sugawara, A., Saito, Y., Ohno, H., Kanai, A., Katsuura, G., Eigyo, M., Matsushita, A. and Imura, H., Central effects of atrial natriuretic polypeptide on antiotensin IIstimulated vasopressin secretion in conscious rats, Eur. J. Pharmacol., 125 (1986) 453-456. 37 Zamir, N., Skofitsch, G., Eskay, R.L. and Jacobowitz, D.M., Distribution of immunoreactive atrial natriuretic peptides in the central nervous system of the rat, Brain Research, 365 (1986) 105-111.
