Proc. Nail. Acad. Sci. USA Vol. 89, pp. 11391-11395, December 1992 Physiology The role of endogenous atrial natriuretic peptide in resting and stress-induced release of corticotropin, prolactin, growth hormone, and thyroid-stimulating hormone (third ventricular I Jection/atrial natriuretic peptide antiserum) CELSO R. FRANCI*, JANETE A. ANSELMO-FRANCI*, AND SAMUEL M. MCCANNt4 *DePartamento de Fisiologia, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, 14049 - Ribeirao Preto, Sao Paulo, Brazil; and tNeuropeptide Division, Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9040 Contributed by Samuel M. McCann, August 28, 1992

ABSTRACT Our previous studies have shown that stim- play a role in thyroid-stimulating hormone release. On the ulation of the anteroventral third ventricle region increases other hand, the endogenous peptide appears to have a physi- atrial natriuretic peptide (ANP) release, whereas lesions of the ologically significant inhibitory role in suppressing ACTH anteroventral third ventricle or median eminence block the release during stress, mediated at least partly by suppression of release of ANP from blood volume expansion, suggesting a vasopressin release. Endogenous ANP has a pathophysiologic critical central nervous system participation in this response. role in augmenting the prolactin release in stress either by ANPis also produced within neurons that have cell bodies in the inhibiting release of prolactin-inhibiting factors or, alterna- rostral hypothalamus and axons that extend to the median tively, by enhancing release of prolactin-releasing factors. eminence and neural lobe. In addition to its natriuretic effect, Endogenous ANP appears to inhibit resting, without altering the peptide can inhibit the release ofcorticotropin (ACTH) and stress-induced inhibition of growth hormone release by stim- prolactin, anterior pituitary hormones that are released during ulating somatostatin release and/or inhibiting growth hor- stress. To determine the physiologic sinfca of ANP in the mone-releasing hormone release or by both actions. control ofbasal and stress-induced release ofanterior pituitary hormones, highly specific antiserum against the peptide (AB- Atrial natriuretic peptide (ANP) produced and released from ANP) was microinjected into the third cerebral ventricle of atrial myocytes induces natriuresis, diuresis, and lowering of conscious freely moving male rats to Immunoneutralize hypo- blood pressure (1, 2). In addition, it decreases water and salt thalamic ANP. In the initial experiment, the antiserum or intake (3-6). All of these actions tend to promote a decrease control normal rabbit serum (NRS) was injected into the third in body fluid volume and they appear to be homeostatic cerebral ventride to determine the effect of the antiserum on responses to increased extracellular fluid volume. Further- basal release of pituitary hormones. The antiserum had no more, the peptide suppresses vasopressin release from the effect on the concentrations of plasma ACTH, prolactin, or neural lobe (7), which can cause renal water loss and inhibits thyroid-stimulating hormone for 3 hr after the injection; release corticotropin (ACTH) (8) and prolactin (9) from the however, plasma growth hormone concentration, although anterior lobe of the pituitary gland. The decreased ACTH unchanged for 2 hr, was markedly elevated at 3 hr. These release would reduce the secretion of aldosterone from the results indicate that although ANP appears to have no effect on adrenal glomerulosa, which is further reduced by direct the basal release ofthe other hormones, it has a physiologically action of ANP (10), leading to additional renal sodium loss. significant inhibitory effect on growth hormone release. The The decreased prolactin release could further augment na- delay of the effect is probably related to the time required for triuresis since prolactin has a direct salt-retaining action on the antiserum to diffuse to the site of action of the peptide, the kidney (11). presumably at some distance from the ventricle. Since this Stress increases ACTH and prolactin and inhibits the effect was demonstrable after 3 in the stress release of growth hormone (GH) and thyroid-stimulating only hr, experi- hormone (TSH) in the rat (12) and the actions ofANP to alter ment, the antiserum or NRS was microinjected into the third the release of these hormones suggest that it may influence ventricle 3 hr prior to application of ether stress. The rapid the hypothalamic pituitary response to stress. These effects elevation of plasma ACTH in NRS-injected rats was markedly of ANP may be mediated via a hypothalamic ANP neuronal augmented by AB-ANP. Ether also induced a rapid increase in system with cell bodies that extend from the anteroventral plasna prolactin in the NRS-injected animals, as expected. third ventricle region to the paraventricular nucleus and have Contrary to the ACTH response, the maximal increase in axons that project to the median eminence and neural lobe of plasma prolactin after ether was attenuated in animals prein- the pituitary (13, 14). In the median eminence the peptide jected with AB-ANP. In the NRS-inJected animals, there was could enter hypophyseal portal veins and be delivered to the a significant decline in plasma growth hormone after the anterior pituitary to affect its function. Alternatively, the application of ether that was significantly accentuated by ANP neurons might alter the release of hypothalamic releas- AB-ANP, but this was probably the result of the her initial ing and inhibiting hormones via a hypothalamic action that levels of plasma growth hormone in the ANP-AB group fol- would, in turn, alter release of anterior pituitary hormones. lowed by its disappearance with a half-time similar to that of The physiologic significance of the actions of ANP on the NRS-injected group. The decline in plasma thyroid- anterior pituitary hormone release is unknown. Conse- stimulating hormone after ether stress was unaltered in the in the we evaluated the effects of animals injected with AB-ANP. The results of these immuno- quently, present study, neutralization studies suggest that endogenous ANP does not Abbreviations: ANP, atrial natriuretic peptide; ACTH, corticotropin; NRS, normal rabbit serum; TSH, thyroid-stimulating hormone; GH, The publication costs of this article were defrayed in part by page charge growth hormone; 3V, third cerebral ventricle; CRH, corticotropin- payment. This article must therefore be hereby marked "advertisement" releasing hormone. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 11391 11392 Physiology: Franci et al. Proc. Nadl. Acad. Sci. USA 89 (1992) passive immunoneutralization of brain ANP by microinject- provided with the kits. Plasma ACTH was measured with a ing highly specific antiserum developed against the peptide method (20) using commercially available antiserum (IgG into the third cerebral ventricle (3V) and measuring the effect Corp., Nashville, TN) and human ACTH provided by the on plasma pituitary hormone concentrations in conscious, NIDDK pituitary hormone program. normal, and stressed male rats. Preliminary reports of this Statistics. The values of plasma hormones were compared work have been presented (15, 16). by an ANOVA followed by the Newman-Keul test. Signif- icance of differences between two means, as for the areas values, was determined MATERIALS AND METHODS under two curves ofplasma hormone by Student's t test. Male Harlan-Sprague-Dawley rats, 200-220 g, were housed in a constant light-dark cycle (lights on between the hours of RESULTS 0500 and 1900) and temperature (23-250C)-controlled room. Laboratory chow and water were freely available. One week Table 1 shows plasma concentrations ofGH, TSH, prolactin, before the experiments, a stainless steel guide cannula was and ACTH immediately before (time 0) and 15, 30, 60, 120, implanted into the 3V as described (17) under anesthesia and 180 min after 3V microinjection of NRS or AB-ANP in induced by 2.5% tribromoethanol [Aldrich; 1 mg/100 g (body unstressed rats. Hormone values in the NRS-injected animals weight), i.p.]. Animals that had regained preoperative weight were not altered except for a significant decline in plasma were submitted to an additional operation 24 hr before the TSH by 180 min. There were no significant differences in experiments during which a catheter was introduced into the hormone concentrations between the control group (3V mi- external jugular vein under tribromoethanol anesthesia (18). croinjection of NRS) and the group that received 3V micro- erimental Procedure. All experiments began between injection of AB-ANP, except that the plasma GH concentra- the hours of0830 0900 when a heparinized blood sample (0.6 tions, although unaltered earlier, were significantly higher ml) was collected from the jugular catheter immediately 180 min after microinjection of AB-ANP than NRS. before 3V microinjection of normal rabbit serum (NRS) or There was a significant elevation of plasma ACTH con- antiserum against ANP (AB-ANP). Additional blood samples centration 2 min after the onset of ether stress and a peak at (0.6 ml) were collected from unstressed animals 15, 30, 60, 5 min in the animals previously injected with NRS (Fig. 1). 120, and 180 min after the microinjections. After removal of The levels declined precipitously at 15 min and were no each blood sample, 0.6 ml of 0.91% NaCl was injected to longer elevated 30 min after ether. In contrast, the response replace the volume of blood removed. in the animals previously injected with AB-ANP was mag- Rats to be subjected to stress received microinjections of nified and significantly greater than that in the NRS-injected NRS or AB-ANP just after withdrawal of the initial blood animals 2, 15, and 30 min after the onset of ether exposure. sample (0.6 ml). They were allowed to rest for 180 min. The maximum increment in plasma ACTH in the AB-ANP- Further samples (0.6 ml) were then drawn 5 min before and treated rats was significantly greater (P < 0.01) than that in 2, 5, 15, 30, and 60 min after exposure to ether for 1 min as NRS-injected controls (Fig. 2). The area under the curve of described (19). plasma ACTH was also significantly (P < 0.01) increased in The microinjections were carried out with a Hamilton the AB-ANP-treated rats when compared to that in the microsyringe connected by PE10 polyethylene tubing to a NRS-injected controls (data not shown). needle filled with the solution to be injected. Antiserum (2 1d) As expected, ether anesthesia induced a sharp elevation in diluted 1:2 with 0.9%o NaCl or similarly diluted NRS was plasma prolactin concentrations in NRS-injected rats; the microinjected over 1 min. concentration reached a peak at 2 min and had markedly The AB-ANP (Peninsula Laboratories) crossreacts 1000% declined by 15 min (Fig. 3). Values were no longer elevated with rat ANP {human [Ile12]ANP}, human ANP, ANP48-33), at 30 and 60 min. Although they were somewhat lower in the human [IBe12]ANP-(3-29), and rat atriopeptin III. It also AB-ANP-injected group than in the NRS-injected group 2, 5, crossreacts 60% with ANP-(18-28), 10% with auriculin A, 5% and 15 min after ether, these changes were not statistically with rat atriopeptin II, and 1% with ANP-(13-28) and cross- significant at any time; however, the maximum increment in reacts negligibly with oxytocin, [Arg8]vasopressin, somato- plasma prolactin after ether was significantly lower (P < 0.01) statin, and rat atriopeptin I (H. Chang, Peninsula Laborato- in the rats preinjected with AB-ANP than in those given NRS ries, personal communication). (Fig. 4). At the end ofthe experiment, the brains were removed and Plasma GH was significantly increased 3 hr after 3V the site of microinjections was determined by microscopic microinjection of AB-ANP, S min before application ofether examination of frozen coronal sections. stress (Fig. 5), as in the first experiment. After ether stress, Radlonimunoassay. Plasma prolactin, GH, and TSH were plasma GH decreased in both groups, but it remained signif- measured using RIA kits provided by the National Institute icantly higher in the group injected with AB-ANP. The of Diabetes, Digestive and Kidney Disease (NIDDK). Re- decline in plasma GH in the AB-ANP-treated group was sults were expressed in terms ofthe RP-2 reference standards greater (P < 0.01) than that in the NRS-treated group; Table 1. Plasma GH, TSH, prolactin, and ACTH in normal rats immediately before (0 min) and 15, 30, 60, 120, and 180 min after microinjection of NRS or AB-ANP into the 3V Hormone Serum 0 min 15 min 30 min 60 miin 120 miin 180 min GH, ng/ml NRS (9) 44.8 ± 6.8 43.8 ± 4.0 45.0 ± 3.9 35.0 ± 4.3 34.4 ± 3.6 36.3 ± 3.4 AB-ANP (9) 45.2 ± 4.2 35.0 ± 7.1 40.7 ± 4.2 37.1 ± 3.5 37.7 ± 4.2 66.9 ± 8.2* TSH, ng/ml NRS (8) 163.8 ± 30.0 143.4 ± 19.7 110.8 ± 10.2 105.3 ± 10.0 89.9 ± 8.8 81.2 ± 6.8 AB-ANP (11) 162.2 ± 20.3 98.1 ± 24.4 95.5 ± 15.3 103.0 ± 31.5 79.5 ± 10.3 97.2 ± 13.5 Prolactin, ng/ml NRS (10) 11.6 ± 1.6 10.9 ± 1.4 9.5 ± 1.4 8.1 ± 1.3 8.3 + 1.0 8.6 ± 1.5 AB-ANP (10) 10.8 ± 3.1 11.4 ± 2.2 10.2 ± 2.0 9.4 ± 1.7 9.8 + 2.0 9.4 ± 1.3 ACTH, pg/ml NRS (9) 8.4 + 1.1 7.6 ± 0.9 7.3 ± 0.8 7.7 ± 1.1 8.1 ± 1.7 7.8 ± 1.2 AB-ANP (8) 8.9 1.2 6.2 ± 1.4 7.0 ± 1.2 9.2 1.5 8.3 ±2.3 7.6 ± 1.9 Values are mean ± SEM. Number in parentheses indicates group size. *P < 0.01 vs. control value (NRS). Physiology: Fmnci et al. Proc. Nati. Acad. Sci. USA 89 (1992) 11393

150o 40G j lNRS -- NR. 1 C. 130 [ I U D0-c--~~~(9) L l AB-ANP 301 i_ i,r'. I 1 0F DE ABANPo /' \ n 0- HT- 90 v C) T' 0 rt 70 h T/ ; MU20-o Mim) CL 50 i- K \ 10- 30 r

- 3 5 02 15 30 5 60 a022 5 15 30 60 Tirne. !-tS Time, min Time, rein FIG. 1. Effects of 3V microinjection of NRS and AB-ANP on FIG. 3. Effects of 3V microinjection of NRS and AB-ANP on plasma ACTH in rats exposed to ether stress. In this and subsequent plasma prolactin in rats submitted to ether stress. figures, numbers in parentheses indicate the size of the group. *, P < 0.05; **, P < 0.02 vs. control values (NRS). hypothalamus and thereby relieves an inhibition of stress- however, the half-time of its disappearance (7 min) was not induced vasopressin release produced by ANP. Because altered from that of the NRS-injected rats. vasopressin is released into the hypophyseal portal vessels After ether stress, plasma TSH levels were reduced to near during stress and directly stimulates ACTH release by the minimal values at 5 min and remained low for the 60-min corticotrophs of the anterior pituitary (21, 22), this could duration of the experiment in the NRS-injected animals (Fig. account for the inhibitory action of ANP on stress-induced 6). There was no difference between the results in the ACTH release. Vasopressin also appears to increase release AB-ANP and NRS-injected groups. of corticotropin-releasing hormone (CRH) and to potentiate stimulation ofACTH release by CRH (21-24). ANP may also directly inhibit CRH release but this has not been demon- DISCUSSION strated. Although intraventricular injection ofAB-ANP had no effect ANP has also been reported to suppress ACTH release via on resting ACTH levels, it markedly augmented the ACTH a direct action on the pituitary (8), and ifthe antisera reached release caused by ether stress. This result indicates that the pituitary in sufficient concentrations in the present ex- endogenous central ANP plays an inhibitory role in the periments, it might have blocked this inhibition as well. Our control of ACTH release in response to stress. We speculate results indicate that ANP has a physiologically significant that the antiserum immunoneutralizes the ANP within the action to suppress stress-induced ACTH release by hypo- thalamic and/or pituitary action. * * ANP has been shown to act centrally to suppress prolactin release (9) via stimulation ofdopamine release into the portal 140 vessels, which then inhibits release of prolactin from the lactotrophs. The inhibitory action of centrally injected ANP on prolactin release appears not to be physiologically signif- 120 icant in the control of resting prolactin release since the AB-ANP did not alter resting prolactin values. However, 40 NRS AB-ANP

:---.-. * . :---- .- -. _ .---.- < 80 :----. * . 0) .---.-.- -. E . * . 30 :----. .. qco :---.- *.-..*.... * . 60 F-_ :---.- * :.: 0.L- E :--- ** .---.-. * . :---- E 20 ::-:. ..*. E 40 l_ CE :---- 0cut .- -. -.1 .---.- *....*.... * . :---.- * :.: 20 :--- 10_ .---.-. * . E :---- :---.-. . :----. * . *.- - NRS AB-ANP

FIG. 2. Maximal increase in plasma ACTH after inhalation of FIG. 4. Maximal increase in plasma prolactin after inhalation of ether by NRS- and AB-ANP-injected rats. **, P < 0.01 vs. NRS- ether by NRS- and AB-ANP-injected rts. **, P < 0.01 vs. NRS- injected rats. injected rats. 11394 Physiology: Franci et al. Proc. Natl. Acad. Sci. USA 89 (1992)

-7 NRS C) peptide that is suppressing GH secretion. This suppression 130 ,.. (10) could be mediated either by stimulation of somatostatin AB-ANP ____ release into portal vessels, which would then block release of LiF--. (12) GH from the somatotrophs, by inhibition of GH-releasing hormone release into the portal vessels, or by a combination 90- ofthese two actions. Alternatively, ifANP has a direct effect U:D to suppress GH release at the pituitary (8), the antiserum might immunoneutralize the peptide and block this inhibitory (5 70i ~~~~~~~~~~~~~~~------\ lr action after its uptake by portal veins and transport to the =% In 50j pituitary. Our studies indicate that % ofANP injected into C- the 3V reaches the anterior pituitary (J. Antunes-Rodrigues, J. Gutkowska, and S.M.M., unpublished data). The long time 30 ¢ course for the antiserum to be effective is consistent with the possibility that it might be immunoneutralizing ANP, which 10 F is inhibiting GH release from the pituitary directly. Thus, it 3 502 5 15 30 60 is clear that ANP exerts a tonic inhibitory control over GH Time. hr Time, min release, probably at the hypothalamus, but possibly also directly at the pituitary. FIG. 5. Effects of 3V microinjection of NRS and AB-ANP on Although the lowering of plasma GH that followed stress plasma GH in rats submitted to ether stress. *, P < 0.001; **, P < was significantly greater in the animals injected 3 hr before 0.01 vs. control values (NRS). with AB-ANP than in the NRS-injected animals, we believe that this greater decline in GH in AB-ANP-injected rats is although the ANP antiserum had no effect on resting prolac- simply a reflection oftheir higher prestress levels ofGH since tin levels, the elevation of plasma prolactin after ether stress the half-time of disappearance ofthe peptide was not altered. was decreased below that in NRS-injected animals. This is Thus, stress suppresses GH release similarly in both groups. consistent with a physiologically significant stimulatory ac- Consequently, we conclude that endogenous ANP does not tion of ANP on prolactin release during stress. During stress, alter the inhibitory effect of stress on GH release, which is this action is presumably mediated by its ability to stimulate mediated by increased somatostatin release (28). the release of a prolactin-releasing factor, such as oxytocin There have been few studies of the effects of ANP on GH (25), or to inhibit the release of a prolactin release inhibitory release. In one report, intraventricular injection of ANP factor, such as dopamine (26). increased plasma GH probably by promoting a decrease of Our results demonstrate a clear elevation in plasma GH, somatostatin release rather than an increase ofGH-releasing which was present only at 3 hr after 3V injection ofAB-ANP. hormone release (29). Thus, in that experiment, the peptide The time required to observe effects after intraventricular itself had the same effect as the antiserum directed against it injection of antisera to various peptides has ranged from 10 in our experiments. The explanation for the apparent dis- min to 4 hr (19, 27). Presumably, the variability reflects crepancy between their results and ours (nay be related to differences in the time required for the different antisera to injection of the peptide into the right lateral ventricle, rather penetrate into hypothalamic tissue and to diffuse to the site than the 3V as we used. Lateral ventricle injection would lead of action of the immunoneutralized peptide; the longer the to asymmetric delivery of the peptide to other than hypotha- time, presumably, the further the distance ofthe site from the lamic and, particularly, cortical structures. ventricular wall. We hypothesize that the site of action of Our experiments appear to indicate that the increased AB-ANP is within the hypothalamus at some distance from release of ANP during stress dampens a number ofresponses the ventricular wall to inactivate the action ofthe endogenous to the stressful stimulus. For example, the inhibition of vasopressin secretion stimulated by stress induced by ANP would increase renal water loss and the diminished ACTH go release induced by the peptide would lead to reduction in NRS aldosterone secretion thereby diminishing the sodium reten- 160[ U~(9) tion, which characterizes stress retention. The ANP released AB-ANP C) in stress probably acts intrahypothalamically to suppress 140 L (10) vasopressin secretion, and since vasopressin augments ACTH secretion during stress, this diminution in vasopressin 120 L release would lead to the decreased ACTH secretion as o illustrated in Fig. 7. The increased prolactin release in stress S induced by the increased endogenous ANP release would not r: 1 c00[ augment sodium excretion since, at least under certain cir- cumstances, it appears to have a direct antinatriuretic effect Me 80L __-a.A------~ - ~~ on the kidney (11). ------. - 7- iS ACTH, via its stimulation of adrenal glucocorticoid secre- (-'0 :040 tion, has immunosuppressive action (12). Therefore, the action of ANP in stress would reduce this immunosuppres- 60 sive effect. Contrariwise, prolactin enhances immune re- sponses and the increased prolactin release induced by ANP would enhance immunity (12). Thus it would appear that the !,re h secretion of ANP within the hypothalamus during stress tends to alter the stress response. 5 02 5 15 30 In contrast to these results with GH, ACTH, and prolactin, Time. mir there was not only no effect of the antiserum on resting TSH values but also no alteration in the suppression of TSH FIG. 6. Effects of 3V microinjection of NRS and AB-ANP on release induced by ether stress. In other studies we have plasma TSH in rats submitted to ether stress. indicated (19) the physiological significance of the suppres- Physiology: Franci et al. Proc. Natl. Acad. Sci. USA 89 (1992) 11395 1. DeBold, A. J., Borenstein, H. B., Veress, A. T. & Sonnen- berg, H. (1981) Life Sci. 28, 89-94. 2. DeBold, A. J. (1982) Proc. Soc. Exp. Biol. Med. 170, 133-138. 3. Antunes-Rodrigues, J., McCann, S. M., Rogers, L. C. & Sam- son, W. K. (1985) Proc. Natl. Acad. Sci. USA 82, 8720-8723. 4. Antunes-Rodrigues, J., McCann, S. M. & Samson, W. K. (1986) Endocrinology 118, 1726-1729. 5. Franci, C. R., Kozlowski, G. P. & McCann, S. M. (1989) Proc. Nati. Acad. Sci. USA 86, 2952-2956. 6. Masoto, C. & Negro-Vilar, A. (1985) Brain Res. Bull. 15, 523-526. 7. Samson, W. K. (1985) Endocrinology 117, 1279-1281. 8. Shibasaki, T., Naruse, M., Yamauchi, N., Masuda, A., Imaki, T., Naruse, K., Demura, H., Ling, N., Inagami, T. & Shizume, K. (1986) Biochem. Biophys. Res. Commun. 135, 1035-1040. 9. Samson, W. R. & Bianchi, R. (1988) Can. J. Physiol. Pharma- col. 66, 301-305. 10. Goodfriend, T. L., Elliott, M. E. & Atlas, S. A. (1984) Life Sci. 35, 1675-1682. 11. Stier, C. T., Cowden, E. A., Friesen, H. G. & Allison, M. E. M. (1984) Endocrinology 115, 362-367. 12. McCann, S. M., Rettori, V., Milenkovic, L., Jurcovicova, J. & Gonzalez, M. C. (1990) in Circulating Regulatory Factors and Neuroendocrine Function, eds. Porter, J. C. & Jezova, D. (Plenum, New York), Vol. 274, pp. 315-329. 13. Jacobowitz, D. M., Skofitsch, G., Keiser, H. R., Eskay, R. L. & Zamir, N. (1985) Neuroendocrinology 40, 92-94. 14. Nakamaru, M., Takayanagi, R. & Inagami, T. (1986) Peptides 7, 373-375. 15. McCann, S. M., Rettori, V., Wenger, T. & Dalterio, S. (1987) in Neuro. Endocrinologia, Proceedings ofthe National Acad- emy ofSciences ofArgentina, ed. Arguilles, A. (National Acad. Press, Buenos Aires, Argentina), pp. 81-98. 16. McCann, S. M. (1990) in Neuroendocrinology: New Frontiers, eds. Gupta, D., Woolmann, E. & Ranke, W. (Brain Research FIG. 7. Schematic diagram of the presumed influence of ANP Promotion, Tubingen, F.R.G.), pp. 1-17. released from hypothalamic neurons on suppression ofthe release of 17. Antunes-Rodrigues, J. & McCann, S. M. (1970) Proc. Soc. vasopressin, and consequently, the release of ACTH from the Exp. Biol. Med. 133, 1464-1470. anterior lobe of the pituitary gland. The decrease in stress-induced 18. Harms, P. G. & Ojeda, S. R. (1974) J. Appl. Physiol. 36, vasopressin release would decrease the antidiuresis that occurs in 391-392. stress, whereas the decrease of ACTH release would lead to de- 19. Franci, C. R., Anselmo-Franci, J. A. & McCann, S. M. (1989) creased aldosterone release and diminished salt retention. Vasopres- Neuroendocrinology 51, 683-687. sin released into the long portal veins (LPV) and into the short portal 20. Nicholson, W. E., Davis, D. R., Sherrell, B. J. & Orth, D. N. vessels (SPV) stimulates ACTH release from the corticotropes (1984) Clin. Chem. 30, 259-265. directly and amplifies the response to CRH. ANPn, ANP neuron; 21. McCann, S. M., Lumpkin, M. D. & Samson, W. K. (1982) in VPn, vasopressin n; OC, optic chiasm; ME, median eminence of the Neuroendocrinology of Vasopressin, Corticoliberin and Opi- tuber cinereum; MB, mammillary bodies; S, pituitary stalk; V, vein; omelanocortins, eds. Baertsche, A. J. & Dreifuss, J. J. (Aca- NL, neural lobe; AC, adrenal cortex; K, kidney; Aldo, aldosterone; demic, London), pp. 319-329. Na', Na+ excretion rate; UV, urine excretion rate; -, inhibition; +, 22. Ono, N., Lumpkin, M. D., Samson, W. K., McDonald, J. K. excitation; , increased; j, decreased. & McCann, S. M. (1984) Life Sci. 35, 1117-1123. 23. Ono, N., Bedran de Castro, J. & McCann, S. M. (1985) Proc. sive action of ANP on basal leuteinizing hormone but not Natl. Acad. Sci. USA 82, 3528-3531. follicle-stimulating hormone release. 24. Ono, N., Samson, W. K., McDonald, J. K., Lumpkin, M. D., Bedran de Castro, J. C. & McCann, S. M. (1985) Proc. Natl. In conclusion, these immunoneutralization experiments Acad. Sci. USA 82, 7787-7790. support the concept that ANP acts centrally during stress to 25. Samson, W. K., Lumpkin, M. D. & McCann, S. M. (1986) dampen the stress-induced stimulation of ACTH and aug- Endocrinology 119, 554-560. ment the stress-induced prolactin release that characterize 26. McCann, S. M., Lumpkin, M. D., Mizunuma, H., Khorram, the hypothalamic pituitary stress response in the male rat. O., Ottlecz, A. & Samson, W. K. (1984) Trends Neurosci. 7, The stress-induced inhibition of TSH and GH release is 127-131. 27. Ottlecz, A., Snyder, G. D. & McCann, S. M. (1988) Proc. Natl. unmodified, but ANP released tonically inhibits GH release Acad. Sci. USA 85, 9861-9865. but not that of the other hormones. 28. Aguila, M. C., Pickle, R. L., Yu, W. H. & McCann, S. M. (1991) Neuroendocrinology 54, 515-520. This work was supported by National Institutes of Health Grants 29. Murakami, Y., Kato, Y., Tojo, K., Inoue, T., Yanaihara, N. & HD09988 and DK10073. Imura, H. (1988) Endocrinology 88, 2103-2108.