Proc. Natl. Acad. Sci. USAVol. 86, pp. 9621-9625, December 1989Physiology

Role of the hypothalamus in the control of atrial natriureticpeptide release

(medial basal hypothalamus/median eminence/pituitary)

S. BALDISSERA*, J. W. MENANI*, L. F. SOTERO DOS SANTOSt, A. L. V. FAVARETTOt, J. GUTKOWSKAt,M. Q. A. TURRINt, S. M. MCCANN§¶, AND J. ANTUNES-RODRIGUESt*Department of Physiology, School of Medicine, 14049 - Ribeirao Preto, Sao Paulo, Brazil; tDepartment of Pharmacology, Instituto Ciencias Biomedicas,University of Sao Paulo, Sao Paulo, Brazil; tLaboratory of Biochemistry of Hypertension, Clinical Research Institute of Montreal, Montreal, PQ, Canada;and §Department of Physiology, Neuropeptide Division, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,Dallas, TX 75235-9040

Contributed by S. M. McCann, August 31, 1989

ABSTRACT Stimulation of the region antero-ventral tothe third cerebral ventricle (AV3V) by a cholinergic drug,carbachol, and lesions of the AV3V have been demonstrated inprevious studies to either augment or decrease sodium excre-tion, respectively. Atrial natriuretic peptide (ANP) dramati-cally increases renal sodium excretion and has been localized tobrain areas previously shown to be involved in control ofsodium excretion. Consequently, to evaluate a possible role ofbrain ANP in evoking the changes in renal sodium excretionthat follow stimulations or lesions of the AV3V, we determinedthe effect of injection of carbachol into the AV3V of rats on theconcentration of plasma ANP and its content in several neuraltissues, the pituitary gland, lungs, and atria. Conversely, theeffect of lesions in the AV3V on plasma ANP and the contentof the polypeptide in the various organs was determined.Injection of carbachol into the AV3V produced the expectednatriuresis, which was accompanied within 20 min by a dra-matic rise in the plasma ANP concentration and a rise in ANPcontent in the medial basal hypothalamus, the neurohypophy-sis, and particularly the anterior hypophysis but withoutalterations in the content ofANP in the lungs or the right or leftatrium. Conversely, there was a dramatic decline in plasmaANP at both 24 and 120 hr after the AV3V lesions had beenplaced. This was accompanied by a slight decline in the contentof the peptide in the lungs. There was no change in its contentin the right atrium at 24 hr after lesions, but there was asignificant increase at 120 hr. There was a small decline in thecontent in the left atrium at 24 hr, followed by a rebound toslightly elevated levels at 120 hr. These small changes con-trasted sharply with the dramatic decline in content of thepeptide in the medial basal hypothalamus, median eminence,neurohypophysis, choroid plexus, anterior hypophysis, andolfactory bulb. These declines persisted or became greater at120 hr; except in the olfactory bulb in which the decline was nolonger significant. The dramatic increase in plasma ANP aftercarbachol stimulation of the AV3V that was accompanied bymarked elevations in content of the peptide in basal hypothal-amus and neuro- and adenohypophysis suggests that the natri-uresis resulting from this stimulation is brought about at leastin part by release of ANP from the brain. Conversely, thedramatic decline in plasma ANP after AV3V lesions wasaccompanied by very dramatic declines in content of ANP inthese same structures, which suggests that the previouslyshown decrease in sodium excretion obtained after these lesionsmay be at least in part due to a decrease in release ofANP fromthe brain. In view of the much larger quantities of the peptidestored in the atria, it is still possible that changes in atrialrelease may contribute to the alterations in plasma ANP

observed after stimulation or ablation of the AV3V region;however, these results suggest that the dramatic changes inplasma ANP that followed these manipulations may be due toaltered release of the peptide from brain structures as well asthe atria and lungs.

The role played by the central nervous system (CNS) in thecontrol of renal sodium excretion has been demonstrated byseveral authors (1-4). Cholinergic or adrenergic stimulationof the medial septal area, medial preoptic area, anteriorlateral hypothalamus, and subfornical organ as well as theanterior portion of the third ventricle (AV3V) induces adose-related natriuresis accompanied by a lesser kaliuresis(5-17). Thus, considerable evidence indicates that the medialpreoptic area, anterior lateral hypothalamus, subfornicalorgan, AV3V, habenula, stria medullaris, supraoptic nu-cleus, and medial septal area are organized in a neural circuitinvolved in the regulation of water and sodium intake andexcretion (12, 13, 17). The AV3V plays a key role in centralcontrol of sodium excretion since its stimulation by carbachol(5-17), a cholinergic drug, angiotensin II (All) or hypertonicsaline (18) enhances and its destruction blocks sodium ex-cretion in goats (18) and rats (15, 16).

Atrial natriuretic peptide (ANP) has potent natriuretic anddiuretic effects by renal actions. It has been shown byimmunocytochemistry, radioimmunoassay, and radiorecep-tor techniques that ANP and its receptors are also localizedin those structures of the CNS related to the control of waterand electrolyte balance (19, 20).ANP and All appear to have opposite actions in these

structures as well as in the periphery in the control of salt andwater balance (21). For example, it has been reported thatmicroinjections of ANP into the third ventricle (3V) or thesubfornical organ inhibit water and salt intake, whereas Allinjected into these sites is a powerful stimulant to both ofthese appetitive behaviors (21-23). ANP acts within the brainto inhibit All release as well as vasopressin release inresponse to hemorrhage or water deprivation (24-26). Thispeptide blocks All-induced water intake (22). ANP also isable to inhibit aldosterone release from the adrenal gland,whereas All stimulates its release (27).The natriuresis induced by CNS cholinergic and adrenergic

stimulation was not prevented by renal denervation or hy-pophysectomy, but lesions of the median eminence (ME) inrats blocked the natriuretic response to intraventricular in-

Abbreviations: AV3V, antero-ventral region of the third cerebralventricle; ANP, atrial natriuretic peptide; CNS, central nervoussystem; AII, angiotensin II; 3V, third ventricle; ME, median emi-nence; AP, anterior pituitary; MBH, medial basal hypothalamus;OB, olfactory bulbs; NL, neurohypophysis; CP, choroid plexus.ITo whom reprint requests should be addressed.

9621

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

5, 2

021

Proc. Natl. Acad. Sci. USA 86 (1989)

jection of norepinephrine or hypertonic saline; these resultssuggest that a hormonal pathway mediates the effects ofthesestimuli on sodium excretion (7). Since we have shown that thecholinergic, adrenergic, or osmotic stimulation of tissueadjacent to the 3V induces natriuresis and kaliuresis (5-11)and since ANP is localized in these structures (19), in thepresent experiments, we determined if the effects on sodiumexcretion of stimulation and ablation of the AV3V regionmight be mediated, at least in part, by altered ANP release.

MATERIALS AND METHODSAdult male rats (Wistar strain, weighing 250-280 g) werehoused in individual cages and maintained under standardlaboratory conditions (23 ± 20C; lights on from 0700 to 1900)with food and water ad libitum.For the experiments the animals were divided into two

experimental groups: (i) Rats with chronically indwelling 3Vcannulae were subjected 7-10 days later to carbachol stim-ulation to study its effect on sodium, potassium, and waterexcretion. These rats were sacrificed just prior to (0 min) and20 or 40 min after injection of carbachol into the 3V to studythe possible correlation between its effect on natriuresis,kaliuresis, and water excretion with the ANP content inseveral brain structures, anterior and neural lobe of thepituitary, right and left atria, lungs, and plasma. (ii) Lesionswere located in the AV3V, and the rats were killed bydecapitation 24 or 120 hr later. The ANP content in severalbrain structures, anterior and neural lobe of the pituitary,right and left atria, lungs, and plasma was measured in theseanimals. Control rats with sham AV3V lesions were sacri-ficed 24 or 120 hr later, and the levels ofANP were measured.The site oflesions was verified by examining slides of serial

frontal sections (7 /Lm). Only rats with destruction of at least95% of the AV3V region were used.3V Cannulation. A chronically indwelling guide cannula

was surgically implanted into the 3V, as described (28), using2.5% (vol/vol) tribromoethanol [Aldrich, 1 ml/100 g (bodyweight), i.p.] to induce anesthesia. After surgical implanta-tion of the guide cannula (23 gauge, stainless steel), the ratsreceived a prophylactic dose of penicillin G procaine (60,000units, i.m.) and were handled daily for 7-10 days before beingutilized in the experiments. The criteria for selection of theanimals for the experiments were the presence of cerebro-spinal fluid in the 3V guide cannula and the return of theirbody weight to preoperative values.AV3V Lesions. Rats were anesthetized with ether and

placed in a David Kopf stereotaxic instrument. After alongitudinal incision in the scalp, the bregma and lambdawere placed at the same horizontal level. An insulatednichrome electrode (26 gauge) with 0.5 mm of the tip exposedwas inserted into the brain in the midline, according to thefollowing stereotaxic coordinates: anterior posterior (AP) =0.3 mm caudal to begma; lateral = 0 mm; vertical = 7.0 mmbelow the level of the dura mater. The lesion was producedby passing an anodal current of 2 mA for 15 sec. Control ratsunderwent the same surgical procedures, except that theelectrodes were lowered only 6mm below the dura mater andno current was passed (28).Experiments Measuring Sodium and Potassium Excretion

and Urinary Volume. Animals utilized in these studies werefasted overnight and given water ad libitum. Just prior to theexperiment, the rats were weighed, received a water loadequivalent to 5% of their body weight by stomach tube, andplaced in individual metabolic cages. One hour later, asecond water load, equal in volume to the first, was admin-istered. A total of three urine samples was then collected at20-min intervals (-20 to 0, 0 to 20, and 20 to 40 min). Aftercollection of the first urine sample, 1 1Ld of 0.15 M NaCI

(saline) or 0.1 pug of carbachol (carbamylcholine chloride) in1.0 A.l of saline was injected into the 3V.The rats of this experiment were sacrificed by decapitation

at 0 min (after collection ofthe first urine sample and injectionof 1.0 1.l of saline solution into the 3V) or at 20 or 40 min aftercholinergic stimulation or intraventricular saline injection.Trunk blood, brain tissues, AP, neural lobe, left and rightatria, and lungs were quickly removed and treated as de-scribed below for ANP determination.ANP Determination. Plasma ANP levels. The animals were

decapitated and trunk blood (2 ml) was collected into tubescooled in crushed ice. The tubes contained the followingproteolytic enzyme inhibitors: 2 mg of EDTA, 20 1.d of 1 mMphenylmethylsulfonyl fluoride (Sigma, P-7626), and 20 ,ul of500 ,uM pepstatin A (Sigma, P-4265). The collected sampleswere centrifuged at low speed for 20 min, and the plasma wasstored at -200C. Immunoreactive ANP was extracted from 1ml of plasma by heat-activated Vycor glass (Corning no.7930, Mesh 140) as described by Gutkowska et al. (29), andthe lyophylized residue was stored frozen at -20'C. Eachsample was resuspended in 500 ,Al of ANP buffer [0.05 Mpotassium phosphate, pH 7.4, containing 0.15 M NaCl, 0.1%bovine serum albumin (Sigma no. A-7888), 0.1% TritonX-100, and 0.02% NaN3], and aliquots of 50 and 100 ,ul weretaken in duplicate for radioimmunoassay (29).

Tissue ANP content. After decapitation and blood collec-tion from the trunk as described above, the following tissueswere quickly removed and placed in 0.5 ml of 0.1 M aceticacid containing the proteolytic enzyme inhibitors used forplasma: AP, medial basal hypothalamus (MBH), olfactorybulbs (OB), choroid plexus (CP), and left and right atria. Thetissues were homogenized with a Polytron. Whole lungs werehomogenized in 5 ml of ice-cold 0.1 M acetic acid. Thehomogenates were centrifuged at high speed at 4°C for 10min. The supernatant was lyophilized and stored at -20°Cuntil assayed for ANP. Each sample was reconstituted with500 ,A ofANP buffer, and aliquots of50 and 100 ,ul were takenfor radioimmunoassay (29). The sensitivity and specificity ofthe ANP radioimmunoassay have been described in detail byGutkowska et al. (30).

Statistical Analysis. Using the STATPACK computer pro-gram, the Kolmogorov test was applied to define the datadistribution. The immunoreactive ANP values for lungs, rightand left atrial, plasma, and CNS structures followed a normaldistribution. For the CP, hypophysis, and OB, ANP valueswere normalized after logarithmic transformation since thedistribution was not normal. Thereafter, the results from eachstructure were analyzed by one-way analysis of varianceusing the Newman-Keuls test to determine significance ofdifferences between treatments (31).

RESULTSExperiment 1: Effects of the 3V Injections of Carbachol (0.1

,ug) or Isotonic Saline (1 ja) on Sodium, Potassium, and WaterExcretion as Well as ANP Content in Several CNS Structures,AP, Neurohypophysis (NL), and Plasma. 3V injection ofcarbachol (0.1 ,g in 1 ,l) induced a significant increase insodium and potassium excretion (Fig. 1). The natriureticresponse was greater than the kaliuretic response so that thesodium/potassium ratio increased. Since there was also asignificant decrease in urine volume (Fig. 1), the concentra-tions of both ions increased in the urine samples. Theresponse was maximal during the first 20 min after injectionand was still significant between 20 and 40 min. Controlinjection ofintraventricular isotonic saline did not induce anysignificant changes in the values for urine volume or sodiumand potassium excretion.Plasma ANP concentrations increased significantly and

maximally at 20 min after 3V injection of carbachol and

%22 Physiology: Baldissera et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

5, 2

021

Proc. Natl. Acad. Sci. USA 86 (1989) 9623

Ea)E0

a)rc

2.0 - 40T.. (8)

.? " " - ....(8)*4 A.?1.0 F

6.0c

E0rw

=L

+

CU

z

h-.).E0*

w

U)C

4.0

2.0

2.0

1.0

a

2' 300

,L 20z

F-I

10 -

(X8)... (8

,' (5) (5)

,,(8) (8)

I,

(5)

l :

0 20Time (min)

40

FIG. 1. Effect of intraventricular (3V) carbachol (1 Ag) (o) or thesaline diluent (e) on excretion of sodium and of potassium and onurine volume. Numbers in parentheses are number of measurementsmade. Vertical bars indicate SEM. Eq, equivalents. *, P < 0.05versus controls; **, P < 0.01 versus controls.

remained elevated at 40 min (Fig. 2). The intraventricularinjection of isotonic saline did not alter plasma levels ofANP.Since these values and the initial levels just prior to injectionwere unchanged, they were pooled as the control value (Fig.2).By contrast with the dramatic changes in plasma ANP

concentration, the basal values for ANP content in the lungwere not significantly changed at 20 or 40 min after 3V

3000l

2500-

J 2000__ _EM 1 500

z<:1000_

500 t(n'17) (nl10) (n-8)

FIG. 2. Effect of intraventricular (AV3V) injections of carbacholon plasma ANP. Values for ANP in animals injected with saline justprior to (control, open bar) and at the two times [T1 (solid bar) andT2 (hatched bar), 20 and 40 min, respectively] after intraventricularinjection ofcarbachol are shown. *, P < 0.05 versus pooled controls.The control value was 172 ± 35 pg/ml (mean ± SEM). The standarderror was too small to illustrate on the figure.

0 N'17

T-Ii---

LUNGS

FIG. 3. Effect of AV3V carbachol injections on ANP content inthe lung. Bars are as in Fig. 2.

injection of carbachol (Fig. 3). Similarly, there was nosignificant alteration in the ANP content in either the right orleft atria (Fig. 4). In contrast, the ANP content of the MBH,NL, and AP increased significantly and maximally at 20 min(Fig. 5). Values remained elevated at 40 min in the MBH, butthey were no longer significantly altered from the controlcontent in the NL and AP. In the other CNS structuresanalyzed, there were no changes between the control andexperimental groups.Experiment 2: Effects of AV3V Lesions on ANP Content in

Several Brain Structures, AP, NL, and Plasma. There was adramatic decline in plasma ANP levels within 24 hr afterAV3V lesions; this decline was maintained at 120 hr (Fig. 6).The ANP content of the lungs was also significantly de-creased within 24 hr, and this decrease was maintained at 120hr, but the degree of decline was only -50% in contrast to the8-fold decline in plasma concentrations (Fig. 7). Although theANP content of the right atrium was not significantly changedat 24 hr after lesions compared to values in sham-operatedcontrols, at 120 hr-the content was significantly increased(Fig. 8). On the other hand, the values ofANP content of theleft atrium were significantly reduced 24 hr after lesion buthad significantly increased by 120 hr (Fig. 8).There was a marked decline in the content of ANP in the

MBH, ME, CP, AP, and OB within 24 hr after AV3V lesions(Fig. 9). The only structure in which-ANP content did notdecline was the NL. ANP values at 120 hr after lesions werealso significantly reduced in all ofthese structures except thatthe decrease in the OB was no longer significant statistically(Fig. 9).

12

10

0)CD0cm

::.

a-z 4

to T

2

N=13 N=7 N:5RIGHT ATRIUM

N10 Nu7 N=5LEFT ATRIUM

FIG. 4. Effect of AV3V carbachol injections on ANP content inthe atria. Bars are as in Fig. 2.

Physiology: Baldissera et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

5, 2

021

9624 Physiology: Baldissera et al.

T

4

64-

5KI~~~imxr uJ3ziO

MBH NL ME CP AP OB

FIG. 5. Effect of AV3V carbachol injections on ANP content inthe MBH, NL, ME, CP, AP, and OB. ANP is presented as pg x 10-3per organ. Numbers above bars are number of tissues analyzed. Barsare as in Fig. 2. *, P < 0.01 versus control; **, P < 0.0004 versuscontrol.

DISCUSSIONAlthough it has been known for a number of years that CNSstimulation by carbachol could evoke natriuresis (5-11),whereas lesions of the AV3V conversely inhibited salt ex-cretion (3, 15, 16, 21), the mechanism by which these effectswere accomplished was unknown. It is now clear from theresults of these experiments that a major factor in evokingthese changes in natriuresis is the level of blood ANP. Inaddition to ANP, recent evidence indicates that there is alsoa brain natriuretic peptide that has partial sequence homologywith ANP (32, 33). Therefore, the question arises as towhether the changes in ANP that we found actually reflectchanges in ANP or possibly reflect brain natriuretic peptidethat might crossreact in our immunoassay. There is little orno crossreactivity of the antiserum employed in this studywith brain natriuretic peptide (J.G., unpublished data). Con-sequently, we believe that the changes that we have foundreflect changes in ANP and not brain natriuretic peptide.

This study does not prove the origin of the ANP found inplasma after these manipulations. It would appear on thebasis of the rapid increase in ANP present in brain structures,in the neural and anterior lobes of the pituitary, and in theME, that carbachol may stimulate its release from these sites,which then results in an increase in plasma levels of thepeptide. This scenario appears quite likely because thechanges in atrial content of the peptide were minimal; how-ever, some change in the release of the peptide from the atria

FIG. 7. Effect of AV3V lesions on ANP content of the lungs. P< 0.05 in lesioned versus control. Bars are as in Fig. 6.

cannot be ruled out because the stores of the peptide in theatria are so much larger than those in any brain structure.

Since the total content of ANP in the hypothalamic-pituitary region at the onset of carbachol stimulation is lessthan the content in the total circulating blood volume andsince this content increases dramatically after carbacholstimulation, it is apparent that, if the increase in plasma ANPis mediated by increased release of peptide from the brain,then the turnover of brain ANP must increase manyfold.

Conversely, after lesions of the AV3V that lowered plasmaANP levels, there was a concomitant reduction in the contentof the peptide in a variety of brain structures and also inneural and anterior lobe of the pituitary. In contrast, therewas no change in the content of the peptide in the right atria,its principal source. These results suggest that the majorcause of the decrease in plasma ANP after these lesions maybe the decrease in release of the peptide from brain sites.Again because of the greater stores of the peptide present inthe atria and lungs and the fact that there was an actualtransient decline in the content in the left atria and lungs, wecannot rule out participation of decreased release of peptidefrom these organs as a contributor to the decline in plasmaANP after the lesions.The results therefore lead us to the following hypothesis.

Activation of central natriuretic pathways by carbachol orpossibly physiologically by an increase in sodium concen-tration in the region of the circumventricular organs activatesthe release of ANP from the brain and possibly also reflexlyfrom the atria; this is the major cause of the natriuresis thatensues. Conversely, lesions in the central pathways, epito-

N=12 N-16 N-7RIGHT ATRIUM

N-10 N=14 N=5LEFT ATRIUM

FIG. 6. Effect of AV3V lesions on plasma ANP. Open bar,control; solid bar, 24-hr lesion; hatched bar, 120-hr lesion. **, P <0.01 versus pooled controls.

FIG. 8. Effect ofAV3V lesions on the content ofANP in the rightand left atria. Bars are as in Fig. 6. **, P < 0.05 in lesioned versuscontrol.

7

6

5

4

3

2

z

50

c 400)0m 3

2-Z 20

I-

10 -

0'N-18

LUNGS

200

150-JE

m 100a-z

50

0(n-17) (n=20) (n-7)

I I

Proc. Natl. Acad. Sci. USA 86 (1989)

1

Dow

nloa

ded

by g

uest

on

Mar

ch 2

5, 2

021

Proc. Natl. Acad. Sci. USA 86 (1989) 9625

C 7

C 200

1m 00

00

MBH NL ME CP AP OB

FIG. 9. Effect ofAV3V lesions on the content ofANP in variousneural structures and the pituitary. Bars are as in Fig. 6. **, P < 0.01or better (significant).

mized here by the AV3V, decrease the release ofcentral ANPand may also reflexly decrease the atrial release of the ANP.

Earlier work in which ME lesions abolished the natriueticresponse to intraventricular hypertonic saline or norepineph-rine (7) are consistent with this concept, but at that time,although a humoral mechanism was postulated, ANP had yetto be discovered. There was then evidence for a hypotha-lamic natriuretic peptide and on the basis of the present workit would appear that the major hypothalamic natriureticpeptide is ANP.

Further work will be needed to determine the relativecontribution of brain ANP to the natriuretic response tovolume expansion. Preliminary experiments in our labora-tory indicate that lesions of the ME can produce a significantsuppression of the increase in plasma ANP that followsvolume expansion with intravenous isotonic glucose.We are grateful to S. Zanon, M. Holanda, Rogerio Azevedo, and

Maria Valci for technical assistance. Drs. Gloria D. Colletto and M.Maestrelli provided the statistical and computer support. We thankJudy Scott for typing the manuscript. We are also indebted to SaoPaulo State Foundation, Conselho Nacional de Pequisas, Researchand Project Financing, CAPES, BID/USP Project, and GrantDK10073 from the National Institutes ofHealth forfinancial support.

1. Andersson, B., Jobin, M. & Olsson, K. (1966) Acta Physiol.Scand. 67, 127-128.

2. Andersson, B. (1977) Annu. Rev. Physiol. 39, 185-200.3. Brody, M. J. & Johnson, A. K. (1980) in Frontiers ofNeuroen-

docrinology, eds. Martini, L. & Ganong, W. F. (Raven, NewYork), Vol. 6, pp. 249-292.

4. Dorm, J. & Porter, J. C. (1970) Endocrinology 8, 1112-1117.5. Dorm, J., Antunes-Rodrigues, J. & McCann, S. M. (1970) Am.

J. Physiol. 219, 1292-1298.6. Saad, W. A., Camargo, L. A. A., Silva-Netto, C. R., Gentil,

C. G., Antunes-Rodrigues, J. & Covian, M. R. (1975) Phar-macol. Biochem. Behav. 3, 985-992.

7. Morris, M., McCann, S. M. & Orias, R. (1976) Proc. Soc. Exp.Biol. Med. 152, 95-98.

8. Saad, W. A., Camargo, L. A. A., Graeff, F. G., Silva-Netto,C. R., Antunes-Rodrigues, J. & Covian, M. R. (1976) Gen.Pharmacol. 7, 145-148.

9. Camargo, L. A. A., Saad, W. A., Silva-Netto, C. R., Gentil,C. G., Antunes-Rodrigues, J. & Covian, M. R. (1976) Can. J.Physiol. Pharmacol. 54, 219-228.

10. Pillar, A. X., Silva-Netto, C. R., Saad, W. A., Camargo,L. A. A., Antunes-Rodrigues, J. & Covian, M. R. (1977) Phar-macol. Biochem. Behav. 6, 561-576.

11. Camargo, L. A. A., Saad, W. A., Silva-Netto, C. R., Antunes-Rodrigues, J. & Covian, M. R. (1979) Pharmacol. Biochem.Behav. 11, 141-144.

12. Franci, C. R., Silva-Netto, C. R., Saad, W. A., Camargo,L. A. A. & Antunes-Rodrigues, J. (1980) Physiol. Behav. 25,801-806.

13. Franci, C. R., Antunes-Rodrigues, J., Silva-Netto, C. R., Ca-margo, L. A. A. & Saad, W. A. (1983) Physiol. Behav. 30,65-71.

14. Rocha, M. J. A., Franci, C. R. & Antunes-Rodrigues, J. (1985)Physiol. Behav. 34, 23-28.

15. Ramsay, D. J., Thrasher, T. N. & Keil, L. C. (1983) Prog.Brain Res. 60, 91-98.

16. Bealer, S. L., Phillips, M. I., Johnson, A. K. & Schmid, P. G.(1979) Am. J. Physiol. 236, E610-E615.

17. Covian, M. R., Antunes-Rodrigues, J., Gentil, C. G., Saad,W. A., Camargo, L. A. A. & Silva-Netto, C. R. (1975) inCentral Control of Salt Balance in Neural Integration ofPhysiological Mechanisms and Behavior, eds. Mogenson,G. J. & Calaresu, F. R. (Univ. of Toronto, Toronto), pp. 267-282.

18. McKinley, M. J., Denton, D. A., Coghlan, J. P., Harvey,R. B., McDougall, J. G., Rundgren, M., Scoggins, B. A. &Weisinger, R. S. (1985) Can. J. Physiol. Pharmacol. 65, 1724-1729.

19. Quirion, R., Dalpe, M., DeLeon, A., Gutkowska, J., Cantin,M. & Genest, J. (1984) Peptides 5, 1167-1172.

20. Zamir, N., Skofitsch, G., Eskay, R. L. & Jacobowitz, D. M.(1986) Brain Res. 365, 105-111.

21. Phillips, M. I. (1987) Annu. Rev. Physiol. 49, 413-435.22. Antunes-Rodrigues, J., McCann, S. M., Rogers, L. C. & Sam-

son, W. K. (1985) Proc. Natl. Acad. Sci. USA 82, 8720-8723.23. Antunes-Rodrigues, J., McCann, S. M. & Samson, W. K.

(1986) Endocrinology 118, 1726-1728.24. Samson, W. K. & Eskay, R. L. (1986) in Neural Endocrine

- Peptides and Receptors, ed. Moody, T. W. (Plenum, NewYork).

25. Samson, W. K. (1985) Neuroendocrinology 40, 277-279.26. Samson, W. K. (1985) Endocrinology 117, 1279-1281.27. Goodfriend, T. L., Elliot, M. E. & Atlas, S. A. (1984) Life Sci.

35, 1675-1682.28. Antunes-Rodrigues, J. & McCann, S. M. (1970) Proc. Soc.

Exp. Biol. Med. 133, 1464-1470.29. Gutkowska, J., Horky, K., Thibault, G., Januszewicz, P.,

Cantin, M. & Genest, J. (1984) Biochem. Biophys. Res. Com-mun. 125, 315-323.

30. Gutkowska, J., Thibault, G., Januszewicz, P., Cantin, M. &Genest, J. (1984) Biochem. Biophys. Res. Commun. 122, 593-601.

31. Zar, R. (1984) in Biostatistical Analysis, eds. McElroy, W. D.& Swanson, C. P. (Prentice-Hall, Englewood Cliffs, NJ).

32. Sudoh, T., Kangawa, K., Minamino, N. & Matsuo, H. (1988)Nature (London) 332, 78-81.

33. Fregoneze, J. B., Gutkowska, J., St.-Pierre, S. & Antunes-Rodrigues, J. (1989) Braz. J. Med. Biol. Res. 22, 765-768.

Physiology: Baldissera et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

5, 2

021