The Neuroendocrine Control of Atrial Natriuretic Peptide Release J Antunes-Rodrigues1, ALV Favaretto1, J Gutkowska2 and SM Mccann3

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REVIEW ARTICLE: A TRIBUTE TO SM McCANN The neuroendocrine control of atrial natriuretic peptide release J Antunes-Rodrigues1, ALV Favaretto1, J Gutkowska2 and SM McCann3

1Department of Physiology, School of Medicine, University of Sao Paulo, 14049 Ribeirao Preto, SP, Brazil; 2Laboratory of Biochemistry of Hypertension, Clinical Research Institute, University of Montreal, Montreal, Quebec, H2W 1R7 Canada; 3Pennington Biomedical Research Center (LSU), 6400 Perkins Road, Baton Rouge, LA 70808–4124, USA

In the initial experiments reviewed here, we show that atrial natriuretic peptide (ANP) plays an important inhibitory role in the control of sodium chloride and water intake since injections of ANP into the third ventricle (3V) caused a reduction in dehydration-induced drinking and also the drinking of salt in salt-depleted rats. Attention was then turned to the possible role of the brain ANP neurons in producing natriuresis which had earlier been shown to be caused by stimulations within the anterior ventral third ventricular region (AV3V). Stimulation in this region by carbachol produced natriuresis accompanied by a dramatic increase in plasma ANP concentrations and increased content of the peptide in medial basal hypothalamus (MBH), neurohypophysis (NH) and anterior pituitary gland (AP), without alterations in the content of ANP in lungs or atria. This suggested that the natriuresis resulting from the stimulation is brought about, at least in part, by the release of ANP from the brain. Conversely, there was a dramatic decline in plasma ANP at both 24 and 128 h after AV3V lesions had been placed. In view of the much larger quantities of the peptide stored in the atria, it is probable that the changes in the atrial release of the peptide were the main factors altering plasma ANP, but that there was concomitant alteration in the release of brain ANP as well. Blood volume expansion (BVE) by intraatrial injection of isotonic saline in the rat is a profound stimulus for ANP release. Lesions in the AV3V region, median eminence, or neurohypophysectomy blocked BVE-induced release of ANP indicating the crucial participation of the CNS in the response of ANP and natriuresis. Baroreceptor impulses from the carotid-aortic sinus regions and the kidney are important in the neuroendocrine control of ANP release since deafferentation of these regions lowered basal plasma ANP concentrations and prevented the increase after BVE. The evidence indicates that the ANP release, in response to BVE, is mediated by afferent baroreceptor impulses to the AV3V, which mediates the increased ANP release via activation of the hypothalamic ANP neuronal system. Our recent data support the hypothesis that BVE causes the release of ANP from ANPergic neurons in the hypothalamus that in turn stimulates release of oxytocin from the neurohypophysis. This oxytocin acts to release ANP from the right atrium that has negative chrono- and inotropic effects in the right atrium to reduce cardiac output, thereby reducing effective circulating blood volume. Then, the released ANP circulates to the kidneys and evokes natriuresis to return circulating blood volume to normal. This is further accomplished by reduction in intake of water and salt mediated also by brain ANP. Keywords: antero-ventral third ventricle (AV3V) region; blood volume expansion; ANP; neurons; oxytocin; vasopressin; plasma ANP; thirst; salt intake; natriuresis; kaliuresis

Introduction 1964) to do my postdoctoral studies under Professor McCann’s supervision. I am honored to participate in this satellite symposium I became interested in the central control of water of the Endocrine Society Meeting in honor of Professor and salt metabolisn in 1955 (in my second year as a SM McCann. I consider myself a very lucky person. In medical student) under the supervision of Professor all my life, I have received God’s blessing. First, my Covian, who had already shown that the hemidecorti- parents gave me important guidance; second, at the cate rat presented several endocrine changes. One of University in Medical School, I decided to work in the most evident effects was on the adrenal weight. At science because of my former Professor M Rolando that time, it was clear from the work of Fisher, Ingram Covian. And finally, I was really lucky choosing (in and Ranson,1 Andersson and McCann,2 Montemurro and Sevenson,3 and Smith and McCann,4 that the Correspondence: SM McCann, Pennington Biomedical Research hypothalamus was very important in the control of Center (LSU), 6400 Perkins Road, Baton Rouge, LA 70808-4124, water and electrolyte metabolism. Andersson and USA McCann2 were among the first to provide evidence that the central nervous system (CNS) controls salt balance. CNS control of ANP J Antunes-Rodrigues et al 360 With Dr Covian, we started to investigate the role saline.8 Our group in Brazil, and McCann with Orias played by the CNS in the control of salt intake.5 In our and Mariana Morris in the USA, carried out inde- laboratories, systematic studies were undertaken to pendent studies to investigate the effects of chol- determine the effects of bilateral localized lesions of inergic, adrenergic and hypertonic saline injections the rat hypothalamus on the free choice ingestion of into the 3V of conscious male rats made diuretic by tap water and 2% NaCl solution. After several experi- intragastic water load. Both natriuretic and kaliuretic ments were performed, it was possible to demonstrate responses and an increase in the Na+/K+ ratio were the existence of a neural circuit that controls the induced by intraventricular injection of noradrenalin sodium intake and/or excretion. This circuit seems to and carbachol, whereas dopamine had no effect. The involve the septal area and particularly the AV3V, beta receptor stimulator, isoproterenol, induced an amygdaloid complex and hypothalamus. The role antinatriuretic and antikaliuretic effect. To determine played by the circuit in sodium intake was demon- the nature of the receptors involved, we injected adre- strated by electrolytic lesions and by electrical and nergic blockers and found that the alpha adrenergic chemical stimulation. Lesions of the anterior medial blocker, phentolamine, abolished the natriuretic hypothalamus caused a decrease in the intake of saline response to intraventricular injection of either hyper- solution, while lesions in the anterior lateral hypo- tonic saline, noradrenaline or carbachol. In contrast, thalamus caused an increase in saline intake. Lesions the beta adrenergic blocker, propranolol, induced a of the septal area also increased salt intake. Lesions of natriuresis and kaliuresis when injected alone. Pro- the corticomedial region of the amygdala increased Na+ pranolol also potentiated the natriuretic response to intake and lesions of the basolateral region decreased carbachol. Cholinergic blockade with atropine dimin- it. When the same sites of the septum and amygdala ished the response to norepinephrine and blocked the that had altered Na+ intake were stimulated electri- natriuretic response to hypertonic saline.9 At that time cally, stimulation of the septal area was specific for with McCann, we suggested that sodium receptors in intake of this ion. Interaction studies provided evi- the ventricular wall modify renal sodium excretion by dence that the hypothalamus is the principal structure a stimulatory pathway involving cholinergic and alpha in the circuit regulating saline ingestion/excretion and adrenergic receptors and that it can inhibit sodium that the septum and amygdala have modulating influ- excretion by a tonically active beta receptor pathway. ences on its activity in this regard.6 Much later, these receptors were described by Petrovic To study the synaptic transmitters involved in these et al.10 effects, carbachol, noradrenaline and hypertonic saline Our group in Ribeirao Preto (Brazil) mapped the vari- solution (NaCl 2%) were microinjected into the septal ous pathways within the CNS controlling salt intake area or AV3V regions. They did not increase salt and excretion. Cholinergic and adrenergic stimulation intake, as occurs with the application of ANG-II; how- of the medial septal area, medial preoptic area, anterior ever, cholinergic and alpha-adrenergic pathways lateral hypothalamus, subfornical organ as well as the appear to be involved in the natriuretic and kaliuretic AV3V region induced a dose-related natriuresis effects obtained by microinjection of these neuro- accompanied by a lesser kaliuresis. Thus, considerable transmitters into the septal, AV3V or 3rd ventricle evidence indicates that the medial preoptic area, areas. Therefore, it was clear to us at that time that anterior, lateral hypothalamus, subfornical organ, structures controlling the intake of sodium were also AV3V, habenula, stria medullaris, supraoptic nucleus, implicated in the control of its urinary output. medial septal area and paraventricular nucleus are On the basis of McCann’s work on neural control of organized in a neural circuit involved in the regulation body fluids, I joined him in Philadelphia in 1964. We of water and sodium intake.11 evaluated the effect of various transmitters on water, The role played by the CNS in the control of renal sodium chloride and food intake and reported that the sodium excretion has also been demonstrated by sev- intraventricular (ICV) injection of carbachol induced a eral other laboratories.12–21 dramatic, rapid increase in water intake with no At this time, we were looking for the mechanism changes in food intake. This was in agreement with which coulld explain the observed effects from central earlier findings of SP Grossman who injected these nervous system manipulation. The median eminence drugs into hypothalamic tissue.7 Both carbachol and lesions temporarily blocked the natriuresis, kaliuresis isoproterenol, a beta adrenergic agonist, evoked large and antidiuresis which followed the injection of hyper- increases in salt intake. Hypertonic saline injected into tonic saline, carbachol or noradrenaline into the 3V. the 3V produced a delayed increase in both water and Sham lesions did not interfere with the responses. food intake, but did not alter sodium intake. We inter- Hypophysectomy did not block but delayed the preted the results to mean that there was a cholinergic responses which ruled out the participation of anterior synapse in the pathways, which mediate water intake, pituitary hormones.21 The responses still occurred in whereas both cholinergic and adrenergic synapses rats with diabetes insipidus which lacked AVP.22 might be involved in the mediation of salt and food During the sixties, much attention was paid to the intake. possibility of the existence of a natriuretic hormone. With Dorn, we showed that intraventricular injection Cort and his coworkers23 even reported the purification of carbachol evoked a natriuretic response which mim- of a hypothalamic natriuretic factor. At this time, Orias icked the response to intraventricular hypertonic and McCann started working on the problem and were CNS control of ANP J Antunes-Rodrigues et al 361 able to demonstrate that AVP, OT and ␣-MSH had of ANP did not block responses to larger doses of natriuretic activities in water-loaded rats.24–26 Until ANGII ranging from 96 to 956 pmol.35 Similar results now, it was not clear that these hormones had a physio- were described by Massoto and Negro-Vilar36 and logical role in the induction of natriuresis. Orias and Inagami’s groups.37 McCann obtained natriuretic activity in median emi- Since ANGII also increases salt intake, we speculated nence extracts and, at this time, they thought that they that ANP should have the opposite effect and would had ruled out the possible participation of AVP and inhibit saline intake when injected into the 3V of con- OT in this activity since the treatment of the extracts scious, salt-depleted rats. The animals were salt- with thioglycollate, an agent which opens the disulfide depleted by 4 days of salt restriction followed by intra- bridge in the neurophyophyseal hormones, thereby peritoneal dialysis with 5% glucose solution to pro- inactivating them, did not interfere with the natriur- duce hyponatria. Intake of 1.5% sodium chloride sol- etic activity. ution was reduced dramatically by a minimal effective De Wardener and Clarkson27 presented evidence dose of 0.2 nmol of ANP.38 showing that natriuresis could occur following blood volume expansion, even though factors such as Brain ANPergic neuronal system and release of increased glomerular filtration rate, or changes in aldo- ANP sterone secretion were eliminated. Davis and Freeman obtained evidence for a circulating natriuretic factor in We next addressed the question of the possible role of volume-expanded dogs by cross-circulation experi- the brain ANPergic neuronal system in CNS-induced ments.28 Gauer and Henry29 showed that dilation of the natriuresis brought on by intraventricular injection of atria could induce diuresis. At that time, it was thought hypertonic saline or activation of brain cholinergic and that distension of the atria activated impulses which adrenergic circuits by carbachol or norepinephrine. In traveled up the vagus to inhibit the release of AVP. the early days, we thought that these effects were Immersion in thermal baths had been known to evoke brought about by the release of a hypothalamic natriur- diuresis since the mid-19th century. Immersion prob- etic hormone. Alternatively, they might be brought ably increased venous return to the heart and dilated about by neural activation of the release of ANP from the atria. the atria. Therefore, we evaluated the possible role of Therefore, it was very important to know if the brain ANP in evolving the changes in renal sodium diuresis and natriuresis which follows the distension excretion that followed stimulations or lesions of the of the atria was not due to a reflex activation of the AV3V, a region previously implicated in control of brain but instead to the release of atrial natriuretic pep- sodium excretion by earlier experiments.20 tide (ANP), discovered by De Bold’s group,30 which cir- Injection of carbachol into the AV3V produced natri- culated to the kidneys and evoked natriuresis. uresis as expected on the basis of our earlier experiments, which was accompanied by a dramatic increase in the plasma ANP concentration and an increase in The brain ANPergic neurons ANP content in the medial basal hypothalamus (MBH), In 1985, Samson demonstrated the presence of ANP in the neurophypophysis (NH) and particularly in the extracts from various hypothalamic regions.31 Phil- anterior pituitary gland (AP), but without alterations in lips32 reviewed the immunocytochemical studies dem- the content of ANP in the lungs or the right or left onstrating that the ANP neurons were located in the atrium.39 same regions of the CNS which are related to ANG-II Conversely, there was a dramatic decline in plasma and blood pressure regulation. These findings gave us ANP at both 24 and 120 h after the AV3V lesions had the idea that ANP could be also involved in control of been placed. This was accompanied by a slight decline water intake. The ANPergic neurons are localized in in the content of the peptide in the lungs. There were the region extending from the paraventricular nucleus no changes in its content in the right atrium at 24 h rostrally to the organum subfornicalis and ventrally to after lesions, but there was a significant increase in the organum vasculosum lamina terminalis, areas 120 h. There was a dramatic decline in the ANP con- known to be involved in thirst, and their axons also tent in the MBH, NH, AP, choroid plexus and olfactory project caudally and ventrally to the median eminence bulbs (OB). These reductions persisted or became more and neural lobe, terminating in proximity to the long evident at 120 h, except in the olfactory bulb in which or short portal vessels, so that the peptide could be the decline was no longer significant.40 released into the anterior pituitary (AP) sinusoids and Since the dramatic increase in plasma ANP after car- also into the general circulation.33,34 bachol stimulation of the AV3V was accompanied by marked elevation in content of the peptide in the basal hypothalamus and the neuro- and adenohypophysis, The ANPergic neurons in water and salt intake we suggested that the natriuresis resulting from this With McCann, we demonstrated that ANP induces a stimulation is brought about, at least in part, by the dose-related blockade of dehydration and ANGII- release of ANP from the brain. induced drinking.35 This inhibitory response was Along the same line of reasoning, the decline of ANP present at doses of intraventricularly injected ANGII in the plasma and in these same tissues (MBH, OB, ranging from 4.8 to 25 pmol; however, the 2-nmol dose choroid plexus, NH and AP) which was accompanied CNS control of ANP J Antunes-Rodrigues et al 362 by a decrease in sodium excretion in rats with AV3V 100 g body weight, over 1 min) in conscious freely lesions, may be explained at least in part by decreased moving rats. In sham-operated rats, the BVE induced a release of ANP from the brain. In view of the much rapid increase in plasma ANP as before. The concen- larger quantities of the peptide stored in the atria, it is tration peaked at 5 min and remained elevated at probable that changes in atrial release contribute in a 15 min after saline injection. One week after deaffer- major way to the alterations in plasma ANP observed entation of the carotid-aortic baroreceptors, basal after the stimulation or ablation of the AV3V region. plasma ANP concentrations were highly significantly However, these results suggest that the dramatic decreased on comparison with values of sham-oper- changes in plasma ANP that followed these manipu- ated rats. Plasma ANP levels, at 5 min after BVE in the lations may be due to altered release of the peptide deafferented rats, were greatly reduced. Unilateral right from the brain structures as well as the atria and vagotomy reduced resting levels of plasma ANP, but lungs.40 not the response to BVE. The resting levels and BVE response were normal in bilateral vagotomized rats. In the rats that had undergone renal deafferentation, the Role of the ANPergic neurons in volume resting levels were normal, but the response to BVE expansion-induced release of ANP was significantly reduced. These data indicated that It was well known at this time that expansion of the the afferents impulses via right vagus nerve may be blood volume (BVE) causes a release of ANP that is important under basal conditions, but they are not believed to be important in induction of the subsequent required for the ANP release induced by BVE. In con- natriuresis and diuresis which, in turn, acts to reduce trast, baroreceptor impulses from the carotid-aortic the increase in blood volume. Since stimulation of the sinus regions and the kidney are important pathways AV3V region induced a rapid elevation of plasma ANP, involved in the neuroendocrine control of ANP release. whereas lesions of the AV3V are followed by a marked The evidence from these experiments and our previous decrease in plasma concentration of the peptide, we stimulation and lesion studies indicates that the ANP hypothesized that release of ANP from the brain ANP release in response to volume expansion is mediated neuronal system might be important to the control of by afferent baroreceptor impulse input to the AV3V plasma ANP. To test this hypothesis, we destroyed the region, which mediates the increased ANP release via AV3V, the site of the perikarya, in male rats by electro- activation of the hypothalamic ANP neuronal system.42 lytic lesions as just described. Other lesions were made In another group of experiments, we determined the in the median eminence and posterior pituitary sites necessity of the brain ANP neuronal system for BVE of termination of the axons of these neurons, and also ANP release induced by hypertonic or isotonic saline, hypophysectomy was performed in other animals.41 by injecting an antiserum directed against ANP into the In conscious, freely moving rats, volume expansion 3rd ventricle prior to inducing BVE. The antiserum had and stimulation of postulated sodium receptors in the no effect on resting levels of ANP; however, it partially hypothalamus were induced by intra-atrial injection of blocked the increase in ANP and natriuresis which fol- hypertonic saline (NaCl 0.3 M; 2 ml per 100 g body lowed the BVE.43 Other experiments in sheep had weight, over 1 min). Volume expansion was also given similar results.44 induced with the same volume of isotonic saline or Finally, we evaluated the essentiality of cholinergic glucose 5%. In the sham-operated rats, BVE with and adrenergic synapses within the brain on the hypertonic or isotonic solution induced equivalent response of ANP to BVE by injecting the receptor- rapid increases in plasma ANP that peaked at 5 min blocking agents into the 3rd ventricle 30 min before the and returned to control values by 15 min. These lesions BVE. These blockers had no effect on resting levels of (AV3V, ME, NH, or Hypox) caused a decrease in the hormone; however, a highly significant blockade of the basal levels of plasma ANP in comparison with those response was induced by prior injection of the muscar- of the sham-operated rats. Each type of lesion induced inic cholinergic receptor blocker, atropine sulfate a highly significant decrease of the response to BVE on (5 nmol in 2 ␮l 0.9% NaCl). Similar results were testing 1 or 5 days after lesions were made. Because a obtained with micro-injection of the alpha receptor common denominator of the lesion was the elimination blocker, phentolamine (5 nmol in 2 ␮l saline).45 of the brain ANP neuronal system, these results suggest We have illustrated the putative pathway of acti- that the brain ANP plays a role in the mediation of the vation of ANP release and natriuresis via BVE release of ANP that occurs after volume expansion. (Figure 1). It involves distension of baroreceptors in the Thus, the results at this point indicated the crucial right atria, carotid and aortic sinuses and in the kidney participation of the CNS in the response of ANP and which alters their afferent input to the brain stem in the natriuresis to volume expansion. We considered the nucleus tractus solitarius (NTS). Impulses from there possibility that the baroreceptors, when they were activate the locus coeruleus (LC), since lesions of the stretched by BVE, would activate the brain ANP neu- LC, a major source of noradrenergic axons to the hypo- rons which would then produce the release of ANP and thalamus lowered resting ANP levels (J Anselmo- the ensuing natriuresis. We set out to determine the Franci and J Antunes-Rodrigues, unpublished). The role of baroreceptors in affecting the increase in plasma axons of these noradrenergic neurons projecting to the ANP from the BVE induced by i.v. (intra-atrial) injec- AV3V region activate the cholinergic interneurons tion of hypertonic saline solution (0.3 M NaCl, 2 ml per there, which in turn, stimulate the hypothalamic CNS control of ANP J Antunes-Rodrigues et al 363 released from the brain plays a minor role in the response. Rather, we would suggest that these ANPergic neurons activate descending pathways which then activate efferent pathways to the heart with consequent release of ANP from the cardiac myocytes. The efferent pathway to the heart might be completely neural; however, it cannot be cholinergic since bilateral section of both vagi does not block the response to BVE. It is unlikely that it is a sympathetic efferent pathway, since BVE by elevating blood pressure should, if anything, diminish sympathetic outflow. Rather, we suspect that ANP may be released by an unknown efferent pathway reaching the atria. It could be peptidergic in nature. Alternatively, ANP neurons may stimulate release of other brain peptides from the neurohypophysis such as endothelin,46 AVP or OT.47 AVP and particularly OT are natriuretic peptides and can be released in large quantities from the neurohypophysis. Therefore, we considered the possibility that they might be released by BVE, circulate to the right atrium and directly release ANP. As indicated above, the axons of ANP neurons terminate in the neurohypophysis and these could contribute to the ANP release from BVE. However, as stated before, these neurons are unlikely to provide enough ANP to account for the BVE-induced release since their ANP content is 1000 times less than that of the right atrium. Decreases in blood volume, such as occur with hemorrhage, stimulate vasopressin release via baroreceptor input to the brain stem. Therefore, one would predict that BVE would not elevate, and perhaps would suppress, vasopressin release. Therefore, we hypothesized that hypothalamic ANP neurons cause release of oxytocin which triggers the release of ANP from the atria. We found that BVE induced concurrent oxytocin and ANP release which was followed by natriuresis. Figure 1 Schematic diagram of the ANP neuronal control of Isotonic BVE decreased plasma vasopressin concen- ANP release. For explanation see the text. AIIn = angiotensin trations. Our experiments (described below) support II neuron; Achn = acetylcholinergic neuron; NEn = norepi- the hypothesis that volume expansion-induced ANP = nephrinergic neuron; ANPn ANPergic neuron; release and natriuresis is caused by release of oxytocin = = On oxytocinergic neuron; 5HTn 5HTergic neuron; which stimulates ANP release that in turn induces OC = optic chiasm; A = artery; PV = portal vessel; natriuresis.47 AL = anterior lobe of the pituitary gland; NL = neural lobe of the pituitary gland; v = vein; Rn = raphe` nuclei; LC = locus ceruleus; NTS = nucleus tractus solitarius; IC = internal carotid artery; CS = carotid sinus; AS = aortic sinus; A = aorta; RA = right atrium; V = ventricles; Bra = baroreceptor afferents; Effect of i.p. injections of OT on urinary sodium, rBr = renal baroreceptors; K = kidney (From Reis et al, with potassium, osmolality and volume permission).46 In water-loaded rats undergoing diuresis, oxytocin induced a significant (PϽ0.01), dose-related increase in ANPergic neurons. These neurons would activate effer- sodium and potassium excretion, as well as in urine ent neurohumoral or neuronal pathways which induce osmolality. A dose-response relationship was evident the release of ANP from the brain and the atria. What by 20 min with sodium and by 40 min with potassium. are the efferent pathways involved in the brain control The natriuresis was maximal at 40 min. The kaliuretic of ANP release? Some of the ANP neurons terminate response was smaller than that of sodium. There was in the median eminence and neural lobe of the also a dose-related decrease in urine volume which hypophysis. It is probable that their activation leads to was maximal at 40 min with the 1-nmol (1.0 ␮g) dose release of the peptide into the vasculature draining the and at 80 min for the 10-nmol dose. Control injections median eminence or the neural lobe.39–41 Since the of isotonic saline did not induce any significant quantity of the peptides is 1000-fold less in these struc- changes in the values for urine volume, sodium and tures than in the atria,39–41 we believe that ANP potassium excretion and urine osmolality. CNS control of ANP J Antunes-Rodrigues et al 364 Effect of oxytocin on plasma ANP effect of Atripeptin II on force and rate of contractions of isolated, spontaneously beating rat atria in vitro in In water-loaded rats, plasma ANP concentrations × −8 Ͻ concentrations of up to 4 10 M. They also described increased significantly (P 0.01) and maximally at that at this concentration, ANP produced a maximal 20 min after i.p. injection of OT (10 nmol) which relaxation in the aorta and carotid artery. Presumably, induced the greatest natriuretic response. In the saline- they would have found effects with the higher concen- injected controls, there were no significant changes in trations used in our experiments. Oxytocin had a simi- plasma ANP. In rats which were not water-loaded, i.v. lar negative ino- and chronotropic effect on the atria at injection of OT induced a dose-related increase a slightly higher dose and its effects were slightly (PϽ0.001) in plasma ANP concentrations at 5 min after reduced in atropinized atria to eliminate any effects of i.v. injection. The minimal effective dose was 1 nmol. acetyl choline. Thus, OT by releasing ANP, may act as The plasma ANP levels remained elevated for 15 min a cardiac muscle relaxant and exert a negative chrono- only with the highest dose (10 nmol) of OT. No changes tropic action as well. in plasma ANP levels were observed in the control rats. We thought that these effects of OT were mediated by the ANP released by this polypeptide, but the con- Effect of BVE on plasma oxytocin concentrations centrations of ANP in this relatively large tissue bath (25 ml) precluded measurement of ANP by RIA. Conse- BVE is a well-known stimulus for ANP release. In this quently, in another set of experiments, we incubated experiment, we wished to determine the effect of BVE quartered atria singly in 1 ml of incubation medium. on release of OT, the putative stimulator of ANP OT (10−6 M) stimulated ANP release (PϽ0.01). This release. BVE either with isotonic or hypertonic saline action was blocked by an OT antagonist (Ferring, induced a rapid increase in plasma OT concentration Malmo, Sweden), which by itself at 10−6 or 10−9 M also that was highly significant at 5 min. Although the lowered basal release of ANP significantly. These release of OT was greater with the hypertonic saline results indicate that in this in vitro preparation, OT is solution, it was not significantly greater than that acting physiologically to stimulate ANP release.50 caused by isotonic saline. By 15 min, OT levels Thus we have demonstrated direct actions of OT on returned to the basal levels in the case of isotonic atrial functions, presumably mediated by ANP since saline, but were still significantly elevated after hyper- similar effects were obtained by 8-monobutyry-cGMP, tonic saline-induced BVE. Isotonic or hypertonic an analog of cGMP which mimics the actions of cGMP. saline-induced BVE was associated with equivalent Therefore, we believe that OT has a physiologically sig- increases in plasma ANP with a similar time-course as nificant effect to release ANP in vivo and in vitro. The that found for plasma OT concentrations observed in released ANP has a negative ino- and chronotropic this experiment. effect in the heart and also dilates blood vessels to pro- The magnitude of the release of OT following BVE duce a rapid decrease in effective circulatory blood was even greater than that which followed suckling in volume following blood volume expansion. This is fol- lactating rats, the classical stimulus for OT release. The lowed by natriuresis induced by the renal action of OT release induced by suckling also was associated ANP, returning blood volume to normal. with an increase in plasma ANP and this increase was prevented by prior injection of an OT antagonist which supports the hypothesis that the ANP-releasing action Effect of i.v. injection of AVP on plasma ANP 47 of OT is physiologically significant. In rats which were not water-loaded, there was an increase (PϽ0.001) in plasma ANP concentrations at Direct effect of OT in the atria to release ANP and 5 min after i.v. injection of AVP (100 ng per rat). No to alter cardiac functions changes in plasma ANP levels were observed in the control rats. There was also an increase (PϽ0.03) in Since our in vivo experiments indicated that OT stimu- plasma ANP concentrations at 5 min after i.v. injection lated ANP release from the heart, experiments were of the V2 agonist dDAVP (50 ng per rat). Values were undertaken to investigate possible direct effects of OT no longer significantly elevated at 15 min. Thus, by V2 on the atria. Besides the well-known OT effects on receptors, AVP can also stimulate ANP release. It does uterus and mammary gland, it was also described that not mediate isotonic BVE-induced ANP release since OT increased blood pressure in vivo. We hypothesized plasma AVP levels decline, but could be involved in that OT might release ANP which would decrease atr- hypertonic BVE since AVP concentrations rise.47 ial function, thus rapidly reducing effective circulating blood volume. In a series of experiments, right atria Lack of role renal nerves in natriuresis evoked by were mounted in a tissue bath, and the effects of OT hypothalamic stimulation to modify the force or rate of spontaneously beating right atria was examined by cumulative addition. At a It is well known that electrical stimulation of parts of concentration of 3 × 10−7 to 10−5 M, a decrease in the the CNS, lesions of the hypothalamus (AV3V) and force and rate of atrial contraction was observed with injection or infusion of certain neurotransmitters, cer- ANP.48 The results are not in agreement with those tain drugs or hypertonic solutions into discrete areas described by Cohen and Schenck49 who tested the of the CNS produce natriuresis in several mammalian CNS control of ANP J Antunes-Rodrigues et al 365 species. Our group and that of McCann found that injection of the cholinergic agent (carbamylcholine (carbachol)), adrenergic (norepinephrine) or hypertonic saline solution (NaCl 2%), into the AV3V of rats causes a reproducible natriuresis. Silva-Netto et al18 studied the effect of carbachol stimulation of the lateral hypothalamic area (LHA) in rats with innervated or denervated kidneys. As expected, the control diuresis and natriuresis was significantly higher from the denervated kidneys, indicating ‘denervation diuresis’. Carba- chol stimulation led to a highly significant increase in urine volume and sodium excretion from both the innervated or denervated kidneys. Therefore, the natriuresis induced by cholinergic stimulation of the LHA does not require presence of the renal nerves and is presumably mediated hormonally as already described.18 Other clearance and micropuncture studies were performed by Silva-Neto et al17 before and after hypothalamic injection of carbachol. Some of these rats received pharmacological doses of AVP and mineral- ocorticoids (DOCA) throughout the experiment to eliminate their effect on the results. Glomerular filtration rate and renal plasma flow increased by 33% and 50% respectively following injection of carbachol. Frac- tional excretion of sodium increased significantly from 0.2% to 0.26%. Similar effects were obtained in rats given pharmacological doses of AVP and DOCA throughout the experiments. Injection of carbachol resulted in a significant inhibition of sodium reabsorp- tion in both proximal and distal tubules and this effect persisted for at least 90 min. Other experiments were performed in rats with abdominal aorta constriction above the kidneys, so as to maintain renal perfusion pressure constant and slightly lower than systemic arterial blood pressure. In spite of these adverse circumstances, urine volume and sodium excretion increased after the cholinergic stimu- Figure 2 Schematic diagram of the mechanism of natriuresis following BVE by injection of isotonic saline into the right lation of the hypothalamus. = = The role of the renal nerves in the excretory atrium. OXYn oxytocinergic neuron; Achn acetylcholinergic neuron; NEn = norepinephrinergic neuron; responses after the hypothalamic stimulation was ANPn = ANPergic neuron; OC = optic chiasm; A = artery; further studied by measuring the impulse frequency of PV = portal vessel; AP = anterior lobe of the pituitary gland; efferent renal nerves before and after the cholinergic NL = neural lobe of the pituitary gland; v = vein; LC = locus stimulation of the LHA. A dramatic decrease in renal ceruleus; NTS = nucleus tractus solitarius; IC = internal nerve activity occurred immediately after carbachol carotid artery; A = aorta; RA = right atrium; V = ventricles; stimulation (mainly during the first 9 min), which Br = baroreceptor afferents; KBR = renal baroreceptor affer- coincided with a fall in blood pressure (during the first ents; K = kidney (From Haanwinckel et al, with permission).48 6 and 9 min). Subsequently, blood pressure increased significantly. This increase in blood pressure may account for the depressed renal nerve activity observed baroreceptor input to the brain stem in the nucleus from 9 to 30 min after cholinergic stimulation of the tractus solitarius (NTS) (Figure 2). These neurons send LHA. projections to the locus coeruleus (LC) which activate From all of these results, it is clear that the natriur- the noradrenergic neurons located there. These send esis induced by intrahypothalamic injection of carba- axons to the AV3V and stimulate cholinergic interneu- chol occurs completely independently of the alter- rons which activate the ANPergic neurons. These neu- ations in renal efferent nerve activity induced by rons stimulate the release of OT from the neurohypo- intrahypothalamic actions of carbachol. physis. The released OT circulates to the atria and triggers ANP release which then induces the natriur- Conclusions esis. The fact that OT antagonists block the ANP release Our hypothesis is that BVE induces stretch of sino- from suckling and basal and OT-induced release of aortic and renal baroreceptors which increases afferent ANP from atria in vitro strongly supports our hypoth- CNS control of ANP J Antunes-Rodrigues et al 366 esis. Since our previous experiments showed that neur- 13 Camargo LAA, Saad WA, Silva-Netto CR, Antunes-Rodrigues J, olobectomy, which would largely abolish the release of Covian MR. Effect of beta-adrenergic stimulation of the septal area of renal excretion of electrolytes and water in the rat. Pharmacol oxytocin and vasopressin following BVE, nearly com- Biochem Behav 1979; 11: 141–144. pletely blocked the BVE-induced release of ANP, we 14 Franci CR, Silva-Netto CR, Saad WA, Camargo LAA, Antunes-Rod- believe that neurohypophyseal hormones are the major rigues J. Interaction between the lateral hypothalamic area (LHA) mediator of CNS-induced release of ANP. and the medial septal area (MSA) in the control of sodium and The evidence is clear that direct release of ANP can potassium excretion in rats. Physiol Behav 1980; 25: 801–806. 15 Franci CR, Antunes-Rodrigues J, Silva-Netto CR, Camargo LAA, occur by atrial stretch, but this does not contribute Saad WA. Identification of pathways involved in the natriuretic, appreciably to the response to blood volume expansion kaliuretic and diuretic responses induced by cholinergic stimu- under our conditions since it can be almost completely lation of the medial septal area (MSA). Physiol Behav 1983; 30: blocked by denervation of baroreceptors or lesions of 65–71. the AV3V and caudal projections of ANPergic, oxytoci- 16 Saad WA, Camargo LAA, Silva-Netto CR, Gentil CG, Antunes-Rod- rigues J, Covian MR. 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