Inhibitory effect of centrally administered atrial natriuretic polypeptide on the brain dopaminergic system in rats

European Journal of Pharmacologt', 131 (1986) 171-177

171

Elsevier EJP 00568

Inhibitory effect of centrally administered atrial natriuretic polypeptide on the brain dopaminergic system in rats K a z u w a N a k a o 1,2,*, G o r o K a t s u u r a 3, N a r i t o Morii 1, H i r o s h i Itoh 1, Shozo S h i o n o 1 T a k a y u k i Y a m a d a 1, A k i r a S u g a w a r a 1, M a k o t o S a k a m o t o 1, Y o s h i h i k o Saito 1, M a s a m i Eigyo 3, Akira Matsushita 3 and Hiroo Imura 1 1 Second Division Department of Medicine, Kvoto University School of Medicine, Kyoto 606; 2 Radioisotope Research Center, Kyoto University, Kyoto 606, and 3 Shionogi Research Laboratories, Shionogi Co. Ltd., Osaka 553, Japan Received 1 May 1986, revised MS received 6 August 1986, accepted 19 August 1986

The effects of intracerebroventricular injection of atrial natriuretic polypeptide (ANP) and angiotensin II (All) on the concentration of dopamine, noradrenaline, serotonin and their primary metabolites in the rat brain were studied using high performance liquid chromatography with electrochemical detection. ANP (2 and 5/~g) decreased the level of dopamine and its metabolite in the septum and hypothalamus. In contrast, AII (100 ng) increased their levels in these brain regions. The simultaneous administration of ANP (5 ~g) with AII (100 ng) resulted in a marked reduction of the AII-induced increase of dopamine and its metabolite. No significant changes were observed in the concentrations of noradrenaline-aiid serotonin throughout the brain. These results suggest that the central action of ANP is mediated in part via the dopaminergic system. Atrial natriuretic polypeptide; Angiotensin II; Dopamine; Noradrenaline; Serotonin; Hypothalamus; Septum

1. Introduction

Using a specific radioimmunoassay (RIA) for atrial natriuretic polypeptide (ANP) (Nakao et al., 1984; Morii et al., 1985; Kawata et al., 1985a) and immunohistochemical methods (Kawata et al., 1985a,b), we have previously shown the presence and distribution of ANP in the rat brain, with the highest concentration in the hypothalamus and septum, regions which are known to be involved in the central control of water and electrolyte balance and blood pressure (Brody and Johnson, 1980). Similar findings were reported from other laboratories (Tanaka et al., 1984; Jacobowitz et * To whom all correspondence should be addressed: Second Division, Department of Medicine, Kyoto University School of Medicine, 54 Shogoin Kawaharacho, Sakyo-ku, Kyoto 606, Japan. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.

al., 1985; Saper et al., 1985; Glembotski et al., 1985; Samson, 1985). Specific receptors for ANP were also detected in the rat central nervous system (Quirion et al., 1984). We also reported that the predominant molecular form of ANP in the brain has a low molecular weight (Morii et al., 1985; 1986) and that-'the effects of water deprivation and salt-loading on the ANP level in the brain differ from the effect in the heart (Morii et al., 1986). These findings prompted us to study the possible actions of ANP in the brain. We found that ANP injected intracerebroventricularly (i.c.v.) inhibited the water intake induced in rats by angiotensin II (All) or by water deprivation (Nakamura et al., 1985), and that antiserum for ANP administered i.c.v, potentiated the water intake in rats (Katsuura et al., 1986). We and others also reported that ANP injected i.c.v, attenuated salt appetite (Itoh et al., 1986a; Fitts et al., 1985).

172 In addition, we demonstrated the inhibitory actions of A N P administered i.c.v, on the AII-induced pressor response (Itoh et al., 1986d), AII-induced vasopressin secretion (Yamada et al., 1986) and AII-induced corticosterone secretion (Itoh et al., 1986c). These central actions of A N P suggest the antagonism of A N P and AII in the central nervous system as well as in the periphery (Nakao et al., 1986), and also indicate that A N P acts as a neurotransmitter or neuromodulator as well as a circulating hormone (Sugawara et al., 1985; 1986; Cantin and Genest, 1985; Needleman et al., 1985; De Bold, 1985). However, little has been done to define the biochemical effects of A N P on classical neurotransmitter systems in the brain. In the present study, we investigated the effects of A N P administered i.c.v, on the concentration of monoamines and their primary metabolites in various brain regions. The interaction of A N P and AII on these neurotransmitter systems was also examined.

2. Materials and methods

2.1. Chemicals Synthetic c~-human atrial natriuretic polypeptide ( a - h A N P ) and AII were purchased from the Protein Research Foundation, Osaka, Japan. Noradrenaline (NA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were obtained from Sigma Chemical Company, St. Louis, MO, USA.

2.2. Experimental animals Male Wistar rats, weighing approximately 240 g, were used. The animals were housed three per cage and maintained on standard rat biscuits and water ad libitum. They were kept under constant room temperature (25 _+ 1°C) and a 12 h light-dark cycle (light on 07:00 h). All experiments were performed between 09:00 and 11:00 h. The rats were anesthetized with pentobarbital (50 m g / k g i.p.) and a stainless steel guide cannula was implanted stereotaxically into the skull in an

a p p r o p r i a t e position as previously reported ( N a k a m u r a et al., 1985; Katsuura et al., 1986). After a one-week recovery period from the surgery, the rats received the peptide solution (5 ~tl) injected i.c.v, at a rate of 0.5 /~l/s using a microsyringe. AII and a - h A N P were dissolved in physiological saline solution. The control animals received physiological saline of the same volume.

2.3. Brain tissues Thirty minutes after the injection of the peptides, the animals were killed by decapitation, and the brain was rapidly removed from the skull and placed on an ice-cooled paraffin plate for dissection of various brain areas according to the method of Glowinski and Iversen (1966). After weighing, the tissues were immediately frozen on dry ice and stored at - 8 0 ° C until extraction.

2.4. Tissue extraction The brain tissues were homogenized in 0.4 N perchloric acid (1 or 2 ml depending on tissue weight). The homogenates were centrifuged at 10000 x g for 20 min at 4°C. The supernatants were filtered through 0.45 /~m filters and the filtrates were injected directly into the high performance liquid chromatography with electrochemical detection system.

2.5. High performance liquid chromatography with electrochemical detection (HPLC-ECD) NA, DA, DOPAC, HVA, 5-HT and 5-HIAA were determined simultaneously using H P L C - E C D (LC-4B, Bioanalytical systems) with a glassy carbon electrode maintained at a positive potential of 0.75 V versus an Ag/AgC1 reference electrode. Compounds were separated on a 25 cm × 4.6 m m internal diameter C-18 /~Bondapak reverse phase column with a buffered mobile phase that consisted of 0.1 M citrate buffer (adjusted to p H 3.4) containing 1 m M sodium EDTA, 11.5% ( v / v ) methanol and 0.005% sodium octyl sulfate (Bioanalytical systems). The mobile phase was filtered through 0.22 ~ m filters and degassed under vacuum. All components of the H P L C - E C D sys-

173

tem were left at ambient temperature. The mobile phase flow rate was maintained at 1 m l / m i n .

levels in the septum and hypothalamus were observed after the treatments.

2.6. Statistical analysis

3.2. Effects in the cerebral cortex, striatum and other brain regions

The data were analyzed by Duncan's multiple range test following one-way analysis of variance.

3. Results

3.1. Effects in the septum and hypothalamus Tables 1 and 2 show the results for the septem and hypothalamus, respectively, a - h A N P (2,5 ttg) injected i.c.v, decreased the concentration of DA and D O P A C in these regions. The D O P A C / D A ratios were not influenced by the a - h A N P injection. In contrast, A I I (100 ng) administered i.c.v. significantly increased the DA levels in these brain regions while the D O P A C / D A ratios decreased significantly. The simultaneous administration of a - h A N P and AII resulted in a marked reduction in the AII-induced increases of the DA levels in the septum and h y p o t h a l a m u s though the D O P A C / D A ratios did not change significantly. N o significant changes in NA, 5-HT or 5-HIAA

Table 3 shows the effects of a - h A N P and AII on concentrations of monoamines and their metabolites in the cerebral cortex and striatum. a - h A N P (5 /zg) did not affect the D A levels in these brain regions, whereas AII (100 ng) markedly elevated the DA levels. The simultaneous administration of a - h A N P (5 #g) and All (100 ng) reduced the AII-induced increase in the DA level in the striatum but not in the cerebral cortex. The D O P A C / D A ratios in the cerebral cortex and striatum were significantly reduced by All. ah A N P did not affect this reduction of the D O P A C / D A ratio in the cerebral cortex but lowered it in the striatum. NA, 5-HT and 5-HIAA levels in these brain areas were not altered by a - h A N P a n d / o r All. The levels of monoamines and their metabolites in the midbrain-thalamus, pons-medulla, olfactory tubercle, hippocampus, olfactory bulb and cerebellum were also studied after the same treatments, but did not show a significant change.

TABLE 1 Effects of a - h A N P and AII on concentration ( n g / g tissue weight) of monoamines and their metabolites in rat septum. Treatment

No. of rats

NA

DA

DOPAC

DOPAC/DA

5-HT

5-HIAA

5-HIAA/5-HT

Saline

22

1017 +56.2

984 -+82.1

282 _+23.7

0.31 _+0.03

706 +41.1

369 _+23.8

0.55 _+0.03

a - h A N P 1 ~g

10

2 ~g

8

5/~g

10

1036 _+79.6 955 _+98.1 834 -+92.3

789 _+97.9 522 a --+70.5 529 a _+54.4

266 +52.1 128 b _+22.4 187 a +11.4

0.34 +0.05 0.25 _+0.02 0.38 +0.04

694 _+33.7 629 +39.5 663 _+71.4

350 _+24.2 276 _+37.3 319 -+37.3

0.52 _+0.04 0.46 +0.03 0.53 _+0.07

a - h A N P 5/~g AI I 100 ng

11

1080 _+213.3

1221 c _+91.5

254 +17.3

0.21 a +0.01

582 +_45.7

333 +26.7

0.62 _+0.03

AI I 100 ng

14

1087 _+142.5

1616 b _+198.8

326 ± 43.4

0.20 b + 0.01

605 ± 24.6

411 + 16.5

0.70 + 0.04

a p < 0.05, b p < 0.01 versus saline; c p < 0.05 versus AII alone.

174 TABLE 2 Effects of a - h A N P and AII on concentration ( n g / g tissue weight) of monoamines and their metabolites in rat hypothalamus. Treatment

No. of rats

NA

DA

DOPAC

DOPAC/DA

5-HT

5-HIAA

5-HIAA/5-HT

Saline

22

2122 +-95.2

511 +-18.1

136 +5.3

0.27 +0.01

1022 -+30.6

503 _+26.0

0.49 -+0.02

a - h A N P 1/~g

10

2~g

8

5 ~g

10

2326 +121.3 2 309 -+59.5 2329 +69.1

509 -+29.1 456 +38.2 376 a -+17.9

132 +13.1 109 -+11.7 96 a +9.3

0.26 -+0.02 0.24 -+0.01 0.26 +0.02

1 108 _+17.5 1 080 _+17.5 1065 _+25.3

473 +-32.9 406 -+32.9 576 _+55.3

0.43 -+0.03 0.38 -+0.02 0.54 +_0.04

c~-hANP 5/~g AI I 100 ng

11

2411 +--95.1

542 b +_22.6

112 b _+4.8

0.21 a +_0.01

i 031 +_51.7

522 +i6.I

0.51 +0.03

AI I 100 ng

14

2379 +_ I01.8

731 ~ +_55.5

160 +13.1

0.22 _+0.01

1 141 +34.3

577 _+15.0

0.51 +0.01

a p < 0.01 versus saline: b p < 0.01 versus AII alone.

TABLE 3 Effects of a - h A N P and AII on concentration ( n g / g tissue weight) of monoamines and their metabolites in rat cerebral cortex and striatum. Treatment

No. of rats

NA

DA

DOPAC

DOPAC/DA

HVA

5-HT

5-HIAA

5-HIAA/5-HT

18

336 +29.2

350 _+31.4

87 +_5.1

0.27 +_0.02

63 +8.0

534 +11.4

206 +10.2

0.39 +_0.02

Cerebral cortex

Saline

a - h A N P 5/xg

8

422 +_140.1

245 +_19.9

73 _+2

0.31 +_0.03

49 +5.8

540 +28.4

239 +9.9

0.45 ±0.04

c~-hANP 5/Lg AI I 100 ng

7

452 +_140.1

529 b +26.0

101 +16.8

0.19 b +0.03

61 +_8.8

509 +34.7

224 +6.4

0.45 +0.03

10

389 +108.6

612 b _+32.6

94 -+9.7

0.16 b +_0.02

59 +10.9

545 +_25.1

216 +-9.6

0.41 +0.03

18

303 +_24.1

13177 +646.1

2248 +166.3

0.17 +-0.01

911 +81.2

932 +106.0

634 -+48.3

0.73 +0.04

a - h A N P 5/~g

8

365 +17.81

13048 +959.3

2144 +_189.7

0.18 _4-0.01

1 112 +95.4

1 200 +_179.4

731 +43.0

0.72 +_0.11

a - h A N P 5 ~g A I I 100 ng

7

404 +_175.1

12150 _+518.6

2009 +141.3

0.17 c +0.01

659 +60.7

595 +55.0

472 +16.2

0.82 +0.05

10

348 +_73.66

19 378 b +_1269.7

1 410 ~ + 227.3

0.08 a + 0.02

927 +_72.5

793 + 44.5

693 + 52.4

0.87 + 0.02

AI I 100 ng

Striatum

Saline

A I I 100 ng

P < 0.05, u P < 0.01 versus saline; c p < 0.05 versus AII alone.

175

4. Discussion The present study demonstrated that ANP injected i.c.v, preferentially decreased the DA levels in the septum and hypothalamus, where the endogenous brain ANP level is highest and ANPcontaining cell bodies and varicose fibers are most prevalent (Morii et al., 1985; Kawata et al., 1985a,b). This inhibitory effect of ANP on the dopaminergic system in these brain regions is compatible with the distribution of A N P receptors in the rat brain (Quirion et al., 1984). All has several central actions such as dipsogenic, pressor, vasopressin-releasing, ACTH-releasing and salt appetite-stimulating actions (Fitzsimons, 1980; Lang et al., 1983). The site of these actions is known to be the anteroventral third ventricle region (AV3V) located at the septum and anterior hypothalamus where specific All binding sites or receptors are distributed predominantly (Brody and Johnson, 1980). These central actions of All are at present thought to be mediated mainly via the dopaminergic or noradrenergic systems (Fitzsimons, 1980; Lang et al., 1983; Camacho and Phillips, 1981; Gordon et al., 1985; Summers and Phillips, 1983; Fitzsimons and Setler, 1975; Gordon et al., 1979; Summers et al., 1979; Dourish, 1983). The present study indicated clearly that All injected i.c.v, increased the DA levels in the septum, hypothalamus, cerebral cortex and striatum. This finding is consistent with previous reports that All-induced drinking was reduced by the pretreatment with DA antagonists and 6-hydroxydopamine (Fitzsimons and Setler, 1975; Gordon et al., 1979; Summers et al., 1979). We have recently studied the structure-activity relationship using a-rat ANP, a-rat ANP(4-28), a-rat ANP(5-28) and a-hANP, because we found that brain ANP is composed of a-rat ANP(4-28) and a-rat ANP(5-28) (Shiono et al., 1986). No significant difference in antidipsogenic action was observed among these peptides (Itoh et al., 1986b). We therefore used a-hANP in the present experiments. Also no difference in the binding affinity of a-rat ANP and a-hANP for receptors in the brain was reported (Quirion et al., 1986). The doses of a-hANP (1-5 ~g) and All (100 ng) used in this study corresponded to those tested in our

previous experiments showing that ANP injected i.c.v, inhibits the All-induced water intake (Nakamura et al., 1985; Katsuura et al., 1986), pressor response (Itoh et al., 1986d), vasopressin secretion (Yamada et al., 1986) and corticosterone secretion (Itoh et al., 1986c). Therefore, it is likely that the antagonistic action of ANP against All in the central nervous system is explainable partly by their antagonistic effects on dopaminergic activity. It is also known that nigrostriatal, mesolimbic and periventricular DA neurons play important roles in the initiation of drinking and in the maintenance of an ongoing drinking response (Dourish, 1983). The different effects of ANP on the All-induced changes of the DA levels in the cerebral cortex and striatum suggest different actions of ANP on mesocortical and nigrostriatal DA systems. In the present study, we observed no substantial changes in the NA level. Because ANP tended to decrease the N A level in the septum, and because the central action of All is considered to be mediated in part via the noradrenergic system (Camacho and Phillips, 1981; Gordon et al., 1985; Summers and Phillips, 1983), further studies are necessary to elucidate the implication of the noradrenergic system in the central action of ANP. In conclusion, these results indicate that the action of ANP in the brain is mediated in part via the dopaminergic system and that ANP and All induce opposite effects on the dopaminergic system in the central nervous system.

Note added in proof While this manuscript was under review, there appeared two reports showingopposite changes in central ANP and All receptors in hypertensiverats (Saavedra et al., 1986a,b).

Acknowledgements We thank Mrs. H. Tabata, Miss K. Horii, Miss Y. Uchida and Miss A. Furu for secretarial assistance. This work was supported in part by researchgrants from the Japanese Ministry of Education, Science and Culture, the Japanese Ministry of Health and Welfare'Disorders of Adrenal Hormone' Research Committee, Japan, 1985, Japan Tobacco Inc. and the Yamanouchi Foundation for Research on Metabolic Dis-

176 orders, by a research grant for cardiovascular diseases (60A-3) from the Japanese Ministry of Health and Welfare and by the Japan Heart Foundation Research Grant for 1985.

References Brody, M.J. and A.K. Johnson, 1980, Role of the anteroventral third ventricle region in fluid and electrolyte balance, arterial pressure regulation, and hypertension, in: Frontiers in Neuroendocrinology, Vol. 6, eds. L. Martini and W.F. Ganong (Raven Press, New York) p. 249. Camacho, A. and M.I. Phillips, 1981, Separation of drinking and pressor responses to central angiotensin by monoamines, Am. J. Physiol. 240, R106. Cantin, M. and J. Genest, 1985, The heart and the atrial natriuretic factor, Endocr. Rev. 6, 107. De Bold, A.J., 1985, Atrial natriuretic factor: a hormone produced by the heart, Science 230, 770. Dourish, C.T., 1983, Dopaminergic involvement in the control of drinking behaviour: a brief review, Prog. Neuro-Psychopharmacol. Biol. Psychiat. 7, 487. Fitts, D.A., R.L. Thunhorst and J.B. Simpson, 1985, Diuresis and reduction of salt appetite by lateral ventricular infusion of atriopeptin II, Brain Res. 328, 118. Fitzsimons, J.T., 1980, Angiotensin stimulation of the central nervous system, Rev. Physiol. Biochem. Pharmacol. 87, 117. Fitzsimons, J.T. and P.E Setler, 1975, The relative importance of central nervous catecholaminergic and cholinergic mechanisms in drinking in response to angiotensin and other thirst stimuli, J. Physiol. 250, 613. Glembotski, C.C., G.M. Wildey and T.R. Gibson, 1985, Molecular forms of immunoactive atrial natriuretic peptide in the rat hypothalamus and atrium, Biochem. Biophys. Res. Commun. 129, 671. Glowinski, J. and L.L. Iversen, 1966, Regional studies of catecholamines in the rat brain - I, J. Neurochem. 13,655. Gordon, J.J., M.J. Brody, G.D. Fink, J. Buggy and A.K. Johnson, 1979, Role of central catecholamines in the control of blood pressure and drinking behavior, Brain Res. 178, 161. Gordon, J.J., M.J. Brody and A.M. Johnson, 1985, Regional depletion of central nervous system catecholamines: effects on blood pressure and drinking behavior, Brain Res. 345, 285. Itoh, H., K. Nakao, G. Kastuura, N. Morii, S. Shiono, M. Sakamoto, A. Sugawara, T. Yamada, Y. Saito, A. Matsushita and H. Imura, 1986a, Centrally infused atrial natriuretic polypeptide attenuates exaggerated salt appetite in spontaneously hypertensive rats, Circ. Res. (in press). Itoh, H., K. Nakao, G. Katsuura, N. Morii, S. Shiono, T. Yamada, A. Sugawara, Y. Saito, M. Sakamoto, A. Matsushita and H. Imura, 1986b, Antidipsogenic action of intracerebroventricularly administered atrial natriuretic polypeptides - potencies and sensitivities, in: The Proceedings of The First World Congress on Biologically Active

Atrial Peptides, eds. B.M. Brenner and J.H. Laragh (Raven Press, New York) (in press). Itoh, H., K. Nakao, G. Katsuura, N. Morii, T. Yamada, S. Shiono, M. Sakamoto, A. Sugawara, Y. Saito, M. Eigyo, A. Matsushita and H. Imura, 1986c, Possible involvement of central atrial natriuretic polypeptide in regulation of hypothalamo-pituitary-adrenal axis in conscious rats, Neurosci. Lett. 69, 254. Itoh, H., K. Nakao, N. Morii, T. Yamada, S. Shiono, M. Sakamoto, A. Sugawara, Y. Saito, G. Katsuura, T. Shiomi, M. Eigyo, A. Matsushita and H. Imura, 1986d, Central action of atrial natriuretic polypeptide on blood pressure in conscious rats, Brain Res. Bull. 16, 745. Jacobowitz, D.M., G. Skofitsch, H.R. Keiser, R.L. Eskay and N. Zamir, 1985, Evidence for the existence of atrial natriuretic factor-containing neurons in the rat brain, Neuroendocrinology 40, 92. Katsuura, G., M. Nakamura, M. Inouye, K. Kono, K. Nakao and H. Imura, i98& Regulatory_ role of atrial natriuretic polypeptide in water drinking in rats, European J. Pharmacol. 121,285. Kawata, M., K Nakao, N. Morii, Y. Kiso, H. Yamashita, H. Imura and Y. Sano, 1985a, Atrial natriuretic polypeptide: topographical distribution in the rat brain by radioimmunoassay and immunohistochemistw, Neuroscience 16, 521. Kawata, M., S. Ueda, K. Nakao, N. Morii, Y. Kiso, H. Imura and Y. Sano, 1985b, Immunohistochemical demonstration of a-atrial natriuretic polypeptide-containing neurons in the rat brain, Histochemistry 83, 1. Lang, R.E., T. Unger, W Rascher and D. Ganten, 1983, Brain angiotensin, in: Handbook of Psychopharmacology, eds. L. Iversen, S.D. Iversen and S.H. Snyder (Plenum Press, New York) p. 307. Morii, N., K. Nakao, M. Kihara, M. Sakamoto, A. Sugawara, M. Shimokura, Y. Kiso, Y. Yamori and H. Imura, 1986, Atrial natriuretic polypeptide in rat brain: effects of waterdeprivation and sodium-load, Hypertension 8 (Suppl. I), 1-61. Morii, N., K. Nakao, A. Sugawara, M. Sakamoto, M. Suda, M. Shimokura, Y. Kiso, M. Kihara, Y. Yamori and H. Imura, 1985, Occurrence of atrial natriuretic polypeptide in brain, Biochem. Biophys. Res. Commun. 127, 413. Nakamura, M., G. Katsuura, K. Nakao and H. Imura, 1985, Antidipsogenic action of a-human atrial natriuretic polypeptide administered intracerebroventricularly in rats, Neurosci. Lett. 58, 1. Nakao, K., N. Morii, H. Itoh, A. Sugawara, M. Sakamoto, T. Yamada, S. Shiono, Y. Saito, G. Katsuura, M. Eigyo, A. Matsushita, M. Kawata, Y. Sano and H. lmura, 1986, Atrial natriuretic polypeptide in brain: Implication in water and electrolyte balance and blood pressure control, in: Brain and Blood Pressure Control, Int. Congr. Ser. No. 695, ed. K. Nakamura (Elsevier Science Publishers B.V., Amsterdam) p. 195. Nakao, K., A. Sugawara, N. Morii, M. Sakamoto, M. Suda, J. Soneda, T. Ban, M. Kihara, Y. Yamori, M. Shimokura, Y.

177 Kiso and H. Imura, 1984, Radioimmunoassay for a-human and rat atrial natriuretic polypeptide, Biochem. Biophys. Res. Commun. 124, 815. Needleman, P., S.P. Adams, B.R. Cole, M.G. Currie, D.M. Geller, M.L. Michener, C.B. Saper, D. Schwartz and D.G. Standaert, 1985, Atriopeptins as cardiac hormone, Hypertension 7, 469. Quirion, R., M. Dalpe, A. De Lean, J. Gutkowska, M. Cantin and J. Genest, 1984, Atrial natriuretic factor (ANF) binding sites in brain and related structures, Peptides 5, 1167. Quirion, R., M. Dalpe and Than-Vinh Dam, 1986, Characterization and distribution of receptors for the atrial natriuretic peptides in mammalian brain, Proc. Natl. Acad. Sci. U.S.A. 83, 174. Saavedra, J.M., F.M.A. Correa, L.M. Plunkett, A. Israel, M. Kurihara and K. Shigematsu, 1986a, Binding of angiotensin and atrial natriuretic peptide in brain of hypertensive rats, Nature 320, 758. Saavedra, J.M., A. Israel, M. Kurihara, and E. Fuchs, 1986b, Decreased number and affinity of rat atrial natriuretic peptide (6-33) binding sites in the subfornical organ of spontaneously hypertensive rats, Circ. Res. 58, 389. Samson, W.K., 1985, Dehydration-induced alterations in rat brain vasopressin and atrial natriuretic factor immunoreactivity, Endocrinology 117, 1279. Saper, C.B., D.C. Standaert, M.G. Currie, D. Schwartz, D.M. Geller and P. Needleman, 1985, Atriopeptin-immunoreactive neurons in the brain: presence in cardiovascular regulatory area, Science 227, 1047. Shiono, S., K. Nakao, N. Morii, T. Yamada, H. Itoh, M. Sakamoto, A. Sugawara, Y. Saito, G. Katsuura and H.

Imura, 1986, Nature of atrial natriuretic polypeptide in rat brain, Biochem. Biophys. Res. Commun. 135, 734. Sugawara, A., K. Nakao, N. Morii, M. Sakamoto, K. Horii, M. Shimokura, U. Kiso, K. Nishimura, T. Ban, M. Kihara, Y. Yamori, K. Kangawa, H. Matsuo and H. Imura, 1986, Significance of a-human atrial natriuretic polypeptide as a hormone in man, Hypertension 8 (Suppl. I), 1-151. Sugawara, A., K. Nakao, N. Morii, M. Sakamoto, M. Suda, M. Shimokura, Y. Kiso, M. Kihara, Y. Yamori, K. Nishimura, J. Soneda, T. Ban and H. Imura, 1985, a-Human atrial natriuretic polypeptide is released from the heart and circulates in the body, Biochem. Biophys. Res. Commun. 129, 439. Summers, C. and M.I. Phillips, 1983, Central injection of angiotensin II alters catecholamine activity in rat brain, Am. J. Physiol. 244, R257. Summers, C., G.N. Woodruff, J.A. Poat and K.A. Munday, 1979, The effect of nenroleptic drugs on drinking induced by central administration of angiotensin or carbachol, Psychopharmacology 60, 291. Tanaka, I., K.S. Misono and T. Inagami, 1984, Atrial natriuretic factor in rat hypothalamus, atria and plasma: determination by specific radioimmunoassay, Biochem. Biophys. Res. Commun. 124, 663. Yamada, T., K. Nakao, N. Mofii, H. Itoh, S. Shiono, M. Sakamoto, A. Sugawara, Y. Saito, H. Ohno, A. Kanai, G. Katsuura, M. Eigyo, A. Matsushita and H. Imura, 1986, Central effect of atrial natriuretic polypeptide on angiotensin II-stimulated vasopressin secretion in conscious rats, European J. Pharmacol. 125, 435.