Lesions of the hypothalamus and pituitary inhibit volume-expaansininduced release of atrial natriuretic peptide

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 88, pp , April 1991 Physiology/Pharmacology Lesions of the hypothalamus and pituitary inhibit volume-expaansininduced release of atrial natriuretic peptide (anteroventral ventral third ventricular lesions/wedian eminence lesions/posterior lobectomy/hypophysectomy/plasma atrial natriuretic peptide) J. ANTUNES-RODRIGUES*, M. J. RAMALHO*, L. C. REIS*, J. V. MENANIt, M. Q. A. TURRINt, J. GUTKOWSKA, AND S. M. MCCANN* II *Department of Physiology, School of Medicine, Ribeirao Preto, S.P. Brazil; tdepartment of Pharmacology, Instituto de Ciencias Biomedicas, Universidad SAo Paulo 05508A, S.P., Brazil; tdepartment of Physiology, School of Dentistry-UNESP-Araraquara, S.P., Brazil; Laboratory of Biochemistry of Hypertension, Clinical Research Institute, University of Montreal, Montreal, PQ, Canada H2W 1R7; and IDepartment of Physiology, Neuropeptide Division, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX Contributed by S. M. McCann, December 31, 1990 ABSTRACT Ension of the blood volume causes a release of atrial natriuretic peptde (ANP) that is believed to be important in induction of the subsequent natriuresis and diuresis which, in turn, acts to reduce the increase in blood volume. Since stimulation of the anteroventral portion of the third cerebral ventricle (AV3V) induced a rapid elevation of plasna ANP, whereas lesions of the AV3V were followed by a marked decline in plasmaconcentration ofthe peptide, we hypothesized that release ofanp fm the brain ANP Ceuronal system might be important to the control of plasma ANP. The perikarya of the ANP-containing neuron are densely distributed in the AV3V and their axons project to the median emience and neural lobe. To test the hypothesis that these neurons are involved in v n -induced ANP release, by using electrolysis we destroyed the AV3V, the site of the perikarya, in male rats. Other lesions were made in the median eminence and posterior pituitary, sites of termination of the axons of these neurons, and also hypophysectomy was performed in other animals. In conscious freely moving animals, volume expanson and stimulatin of plated ium receptors in the hypothalamus were induced by iijection of hypertonic NaCI solution [0.5 or 0.3 M NaCl; 2 mi/100 g (body weight)]. Volum ex n alone was induced with the same volume of an isotonic solution (NaCl or glucose). In the sham-operated rats, volume expansion with hypertonic or istonic solutions caused equivalent rapid increases in plasma ANP that peaked at 5 min and returned nearly to control values by 15 min. Lesions caused a decrease in the initial levels of plasna ANP on comparison with values from the sham-operated rats, and each type of lesion induced a highly s cant suppres sion of the response to volume expansion on sting 1-5 days after lesions were made. Because a common denominator of the lesions was emition of the brain ANP neuronal system, these results suggest that the brain ANP plays an important role in the mediation ofthe release ofanp that occurs after volume expnsio. Since the content of ANP in this system is much less than that in the atria, there must be a remarkable incase in synthesis and release of brain ANP assocated with this stimulus. It is also possible that blockade of volume-expansion-induced release of other neurohypophyseal hormones, such asendothelin, may block release ofanp from atrial myocytes. It is probable that volume ex io detete by stretch of atrial and carotid-aortic baroreceptors causes afferent input to the brain ANP system, thereby causing increased release of the peptide from the median eminence and mural lobe. Our results A the importance of brain ANP to the control of ANP release to the blood. Atrial natriuretic peptide (ANP) plays an important role in control of body fluid homeostasis by promoting decreased The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. salt and water intake and increased salt and water excretion (1-3). This peptide is released into the circulation after expansion of blood volume and induces the ensuing natriuresis in part by a direct action on the kidneys (4, 5). It directly suppresses the release of aldosterone from the adrenal glomerulosa, which also promotes sodium excretion (1). ANP inhibits the release of renin from the juxtaglomerular apparatus of the kidneys (6, 7), thereby decreasing secretion ofangiotensin II leading to decreased water and salt intake. ANP has a direct effect in the brain to inhibit water and NaCl intake and to antagonize the dipsogenic action of angiotensin II in dehydration-induced water intake (8, 9). Thus, ANP reduces the increased blood volume by decreasing intake and increasing output of water and sodium. One of the most effective stimuli for release of ANP into the circulation is expansion of the circulating blood volume. Such expansion induces increased venous return to the right atrium, which causes additional stretch of the atrial myocytes. This is thought to evoke directly the release of ANP into the circulation from storage granules in the myocytes (1); however, it has yet to be shown that the amount of stress induced by expansion of the circulating blood volume is sufficient to release ANP directly (10). A role for the central nervous system in the control of renal sodium excretion was proposed long before the discovery of ANP. For example, microinjection of hypertonic saline into the third cerebral ventricle evokes natriuresis (11, 12). This central neural mechanism is stimulated by excitatory cholinergic and a-adrenergic and inhibited by B-adrenergic synapses (13, 14). This central mechanism of sodium control is believed to reside in the anteroventral third ventricle (AV3V) region since stimulation of this region enhances, and its destruction inhibits, excretion of sodium (15, 16). ANP-containing neurons have been demonstrated in the hypothalamus with their perikarya particularly in the AV3V (17, 18). Indeed, ANP neurons with cell bodies in the paraventricular nucleus project axons to the external layer of the median eminence (ME) (19) and also to the neural lobe (19, 20). These ANP neurons appear to play an important role in mediation of the effects of the AV3V on natriuresis since stimulation of the AV3V resulted in a rapid elevation of plasma ANP concentration, whereas its destruction resulted in a dramatic decrease in plasma ANP (21). Since there was little or no change in ANP content of the atria but rapid changes in the central nervous system and pituitary content of the peptide after stimulation of the AV3V by carbachol, we hypothesized (21) that release of ANP from the central Abbreviations: ANP, atrial natriuretic peptide; AV3V, anteroventral third ventricle; ME, median eminence. '[To whom reprint requests should be addressed. 2956

2 Physiology/Pharmacology: Antunes-Rodrigues et al. nervous system by way of the ME and/or neural lobe of the pituitary gland might play a role in the carbachol-induced elevation of plasma ANP. In support of this concept, early experiments from this laboratory before the discovery of ANP demonstrated that lesions of the ME of the tuber cinereum blocked the natriuresis that followed microinjection of hypertonic saline or norepinephrine into the third ventricle of rats (22). Consequently, to determine whether or not the brain mediates the release of ANP in response to the physiological stimulus of volume expansion, we evaluated the effects of lesions in the brain and pituitary on volume-expansion-induced ANP release. Lesions were made in the AV3V region or the ME. Posterior lobectomy and complete hypophysectomy were also performed. The results indicate that all of these lesions were effective in largely inhibiting volume-expansioninduced release of ANP. A preliminary report of the initial studies with ME lesions has appeared (23). MATERIALS AND METHODS Adult male Sprague-Dawley derived rats were housed individually in a temperature-controlled (23 ± 20C) and lightcontrolled (hours lights on from 0700 to 1900) room with free access to rat chow and water. Animals were anesthetized with ether for all cranial operations. By using a Stoelting stereotaxic instrument, bilateral electrolytic lesions were made in the ME by a cathodal current of 2.5 ma for 20 s from a flattened 26-gauge nichrome wire insulated except at the tip as described (22, 23). Sham operations were performed similarly except that the electrodes were not lowered into the brain. Lesions were also made in the AV3V region as described (21) using a David Kopf stereotaxic instrument. At sacrifice the location of AV3V and ME lesions was confirmed by microscopic examination of serial frontal sections (7 am) stained with Luxol fast blue. Posterior pituitary lobectomy and complete hypophysectomy were carried out by the parapharyngeal approach. The completeness of lesions was ascertained by examination under a dissecting microscope. Hypophysectomized rats had free access to a corticosterone solution (4 mg/liter) as drinking water. Proc. Natl. Acad. ScL USA 88 (1991) 2957 At the end of the operation, all animals were injected s.c. with penicillin (60,000 units). Since all of these lesions influence water intake, it was measured daily in all rats after brain or hypophyseal lesions. Plasma sodium and potassium concentrations were measured by flame photometry in sham-operated animals and in those with AV3V lesions. Rats with lesions and sham-operated controls were used for experiments 1 or 5 days after operation in the first experiment with ME lesions. In subsequent experiments, rats with ME lesions were used 5 days after lesions. Other rats were used 3 days after operation. On the morning prior to the experiment, a catheter was inserted into the right external jugular vein and advanced to the right atrium as described (24). Plastic tubing filled with a heparinized saline solution (20 units/ml in isotonic saline) was attached to the jugular catheter and the rats were allowed to rest for at least 60 min prior to removal of an initial control blood sample. Blood (1 ml) was removed each time and replaced immediately with 1.0 ml of isotonic saline. Sixty minutes after the removal of the initial blood sample, the rats were injected i.v. during 60 s with 2 ml/100 g (body weight) containing either hypertonic NaCl (0.3 or 0.5 M) to produce a hypertonic expansion of blood volume or 0.15 M NaCl or 0.3 M glucose to produce isotonic expansion of blood volume. Additional blood samples were removed at 5, 15, and 30 min after the injection. Plasma ANP was measured after extraction by radioimmunoassay as described (2). Data were analyzed statistically by a two-way analysis of variance with repeated measures and the significance of differences between group means was determined by the Newman-Keuls test. RESULTS In the first experiment, blood volume expansion was induced by i.v. injection of hypertonic saline [0.5 M, 2 ml/100 g (body weight)j to cause not only volume expansion but also hypernatremia, which might stimulate sodium receptors in the hypothalamus (25). Since the results from sham-operated rats did not differ whether the rats were used 1 or 5 days after operation, data were pooled for presentation (Fig. 1). This E the A4 200 * S is Time, min 30 FIG. 1. Effect of ME lesions on ANP release induced by hypertonic blood volume expansion [2 ml/100 g (body weight), 0.50 M NaCl]. In this and subsequent figures, data are mean ± SEM and the number of rats is given in parentheses. o, Sham-operated control rats; *, rats with ME lesions after 24 hr; and A, rats 120 hr after lesion. *, P < 0.01 compared to sham-operated control.

3 2958 Physiology/Pharmacology: Antunes-Rodrigues et al. stimulus induced a highly significant increase in plasma ANP concentration within 5 min after the injection of hypertonic saline. Plasma ANP concentrations declined markedly 15 min after injection and had decreased further by 30 min, so that they were no longer significantly above baseline. In rats with ME lesions, initial concentrations of plasma ANP were 70% less than those of sham-operated controls 1 day after surgery (Fig. 1). By 5 days after lesions, initial values were still 50% lower than those of Volume expansion with hypertonic saline on the day after the lesions produced within 5 min a much smaller increase in plasma ANP, which was significantly lower than that observed in the sham-operated controls (Fig. 1). The concentration of ANP declined in these rats by 15 and 30 min after injection and was no longer significantly elevated above the initial value. They remained significantly depressed when compared with plasma ANP levels in the sham-operated Results were similar in the rats with ME lesions in which volume expansion was performed 5 days, instead of 1 day, after operation. There was also a significant increase in plasma ANP concentrations at 5 min in these rats, but the rise was not greater than that observed in animals tested at 1 day and again was much lower than that observed in shamoperated Again, the responses of rats 5 days after lesions were no longer different from basal values at 15 and 30 min. Plasma ANP concentrations remained significantly lower than those of control rats. In the second experiment, volume expansion was induced by isotonic (0.15 M NaCl) instead of hypertonic saline injected in the same volume as before. In the sham-operated animals, this resulted in an increment in plasma ANP concentrations that was similar to the increase observed with the hypertonic expansion, and the response again peaked at 5 min. In the animals with ME lesions, again plasma ANP was initially significantly less than that of the sham-operated rats. There was a significant enhancement at 5 min in levels of plasma ANP in the rats with lesions; however, the response was significantly less than that of the control animals at all times (Fig. 2). In the third experiment, the expansion was obtained by isotonic glucose given in the same volume. This produced a lower elevation of plasma ANP in the sham-operated control animals; however, the response was not significantly less than that in the saline-injected rats. In this experiment, the animals with ME lesions had lowered initial levels of plasma ANP and had a significantly lower elevation of plasma ANP after volume expansion than did the control animals (data not shown). 600 a 400 I, E CZ Time. min FIG. 2. Effect of ME lesions on ANP release induced by isotonic [2 ml/100 g (body weight), 0.15 M NaCl] blood volume expansion (BVE) in 11 sham-operated rats and 7 rats with lesions. Open bars, sham-operated rats; hatched bars, rats with ME lesions. *, P < 0.05; **, P < Both values are compared to values in sham-operated E ' a- z (/) (U Si Proc. Natl. Acad. Sci. USA 88 (1991) BVE 'I TiME 1 5 emmin FIG. 3. Effect of AV3V lesions on ANP release induced by the blood volume expansion (BVE) in sham-operated controls (seven or eight rats) and rats with lesions (six or seven rats). Open bars, sham-operated; hatched bars, AV3V lesions. *, P < 0.05 versus AV3V Lesions. In this experiment, blood volume expansion was accomplished by injecting 0.3 M NaCl in the same volume. This caused a marked increase in plasma ANP in the sham-operated animals at 5 min after injections, which dissipated by 15 min. The response (Fig. 3) was not significantly less than that obtained with the more hypertonic NaCl solution used in the first experiment (Fig. 1). In the rats with AV3V lesions, the initial values were significantly lower than those of sham-operated animals but were similar to those found in the rats with ME lesions. The response to volume expansion was significantly less than that in the shamoperated controls at all times (Fig. 3). Posterior Lobectomy and Hypophysectomy. Volume expansion was also accomplished in this experiment by injecting the same volume of 0.3 M NaCI as used in the preceding experiment. There was a highly significant elevation of plasma ANP at 5 min after expansion in the sham-operated control animals (Fig. 4). Initial values in the posterior lobectomized animals were significantly less than those of the control sham-operated rats, whereas those in hypophysectomized animals were smaller than those of controls, but not significantly so. Both of these groups of animals showed a markedly impaired response to volume expansion that was indistinguishable from that in the animals with either AV3V or ME lesions (Fig. 4). Water Intake in Animals with Lesions. ME lesions in general resulted in diabetes insipidus and an augmented water intake [118 ± 22 ml/day (mean ± SEM), 5 days after operation]; however, there was no relationship between the degree of diabetes insipidus, as indicated by daily water consumption, and initial plasma ANP or the response to stimulation. AV3V lesions caused a slight suppression of water intake on the day prior to the experiment (24.2 ± 3.2 ml/day) that 400 BVE E 300 CD 0. z Time, min FIG. 4. Effect of hypophysectomy (hatched bars) or posterior lobectomy (open bars) on the response to blood volume expansion (BVE). Solid bars, sham-operated control rats. *, P < 0.05; **, P < Both values are versus control. 30

4 Physiology/Pharmacology: Antunes-Rodrigues et al. was below that of sham-operated rats (35.5 ± 2.8 ml/day). The AV3V lesions were accompanied by marked hypernatremia and hyperkalemia as reported (24) (plasma [Na'] = 177 ± 5; [K+] = 5.6 ± 0.9,uequiv/ml). Plasma [Na'] and [K+] were not significantly altered 5 min after volume expansion with 0.3 M NaCl in sham-operated rats or rats with AV3V lesions. After posterior lobectomy, there was a very mild diabetes insipidus, as indicated by slight increases in water consumption, whereas the hypophysectomized rats had increased water intake only during the first 24 hr after the operation. Location of Lesions. Serial sections through the ME in animals with these lesions revealed that the region was completely destroyed in most rats; there was a rough correlation between the amount of the ME destroyed and the degree of diabetes insipidus. The AV3V lesions were complete and were similar to those reported to be effective to produce elevated plasma sodium (24). The posterior lobectomies and hypophysectomies were complete as determined by inspection under a stereoscopic microscope. DISCUSSION These results indicate that lesions of the AV3V, which contains large populations of ANP neuronal cell bodies, and of the regions of their axon terminals in the ME or neural lobe largely prevented the ANP response to volume expansion. Complete hypophysectomy that included the neural lobe and thereby the axonal terminals of ANP neurons was also effective. Since posterior lobectomy and hypophysectomy blocked the ANP response to volume expansion without damaging the overlying brain, it is clear that descending pathways from the AV3V region do not mediate the response. These results support the concept that the hypothalamic-pituitary ANP neuronal system may play an important role in the release of ANP induced by volume expansion by release of the peptide from terminals in the ME and neural lobe into the circulation, as illustrated in Fig. 5. For release from the ME, the peptide presumably traverses the hypophyseal portal vessels and pituitary sinusoids before exiting into the circulation, whereas for release from the neural lobe, there may be direct release to venous channels. This view is supported by the dramatic reduction in content of ANP in the ME, neural lobe, and anterior lobe after AV3V lesions, whereas after stimulation of the AV3V with carbachol, there is a marked increase in the content in the neural lobe and anterior pituitary gland, consistent with increased release through these channels (19). The content of ANP in these brain structures is very small compared to that in the atria (21). Consequently, if this mechanism is to play a significant role in the increase in ANP release that occurs in volume expansion, there would have to be a rapid increase in synthesis and release of the peptide from the brain ANP neuronal system. The stimulus for the ANP release apparently was largely plasma volume expansion since there was no significant difference between the response to isotonic volume expansion and that to various degrees of hypertonic volume expansion that was expected to produce a greater response by stimulation of putative hypothalamic sodium receptors (25). Measurement of plasma sodium 5 min after hypertonic volume expansion revealed no change, so it is possible that the threshold for activation of the central sodium receptors was not reached by this brief increase in sodium concentration. Since stimulation of the AV3V regions by either carbachol (21) or hypertonic saline (J.A.-R., unpublished data) evoked release of ANP, whereas lesions of the structure decreased basal and volume-expansion-induced release of the peptide, these data provide strong evidence for an important role of Proc. Natl. Acad. Sci. USA 88 (1991) 2959 ME ANP n. Ach n. '0-,AP 'ANP FIG. 5. Schematic diagram of the proposed mechanism of ANP release induced by hypertonic volume expansion. Afferent input (Afferent n.) to the ANP neurons (ANP n.) arises from atrial and aortic-carotid baroreceptors and from putative Na' receptors in the OVLT (organum vasulosum lamina terminalis) and SFO (organum subfornicalis). ANP neurons are also driven by cholinergic neurons (Ach n.). ANP release occurs from ANP neuronal terminals in the ME and reaches the anterior pituitary (AP) by the portal vessels (PV). ANP is also released in the neural lobe of the posterior pituitary (PP). ANP in the posterior pituitary is released into veins (V) draining the posterior pituitary and into the short portal vessels (SPV) further increasing the ANP concentration in the anterior pituitary sinusoids. ANP leaves the anterior pituitary by its veins. OC, optic chiasm; MB, mammillary bodies. the brain ANP system in control of both resting and volumeexpansion-induced ANP release. There is one alternative explanation for the results with lesions. They all induced altered states of water balance. For example, the ME lesions induced diabetes insipidus, which could possibly be associated, at least in certain animals, with diminished body fluid volumes. Arguing against this possibility is the fact that there was no correlation between the intensity of diabetes insipidus and the blockade of ANP release. Furthermore, there was only mild diabetes insipidus in the posterior lobectomized animals and only a very transient diabetes insipidus in the completely hypophysectomized animals that also exhibited blockade. The converse situation existed in the rats with AV3V lesions. These rats had decreased water intake; however, a decrease in sodium excretion in these rats with AV3V lesions (26) may result in the maintenance of nearly normal blood volumes. Preliminary determination of blood volume in these animals revealed results consistent with only a small change in blood volume (J.A.-R., unpublished data). It is our view that changes in blood volume associated with these various types of lesions that could alter the thresholds for induction of ANP release after volume expansion, in all probability, are not responsible for the blockade of volume-expansion-induced ANP release by these lesions. One other possibility to explain the results should be considered: decreased release of other neural lobe hormones, such as vasopressin and/or oxytocin or the newly discovered peptide endothelin (27), might result in diminished release of ANP by loss of direct stimulation of ANP release from the atria. The fact that AV3V lesions that should augment release of vasopressin by the hypertonicity of body fluids were also associated with diminished resting and volume-expansioninduced release of ANP argues against the possibility that vasopressin is involved. PP n.

5 2960 Physiology/Pharmacology: Antunes-Rodrigues et al. Endothelin is a recently discovered peptide found in magnocellular neurons of the paraventricular nucleus, which project their axons to the neural lobe (27). Furthermore, it has been reported to increase ANP release from the atria (28). Its release would certainly be impaired by ME lesions, posterior lobectomy, and hypophysectomy but may not be altered by AV3V lesions. Similarly, calcitonin-gene-related peptide, which occurs in and is released from the ME, directly releases atrial ANP (29) and the release of ANP would be impaired by all but AV3V lesions. Consequently, interference with release of calcitonin-gene-related peptide leading to decreased release of atrial ANP may partly explain our results. The release of anterior pituitary hormones was altered by these various types of lesions as well. Little is known of the effect of AV3V lesions, but release is deranged by posterior lobectomy (30), ME lesions (31), or hypophysectomy. There is clearly no relation between these effects and the blockade of ANP release observed with each of these diverse lesions. For example, the effect of ME lesions is to increase prolactin release (31), whereas posterior lobectomy (30) and hypophysectomy reduce release of prolactin and yet the effect on ANP release was the same. What are the afferent pathways involved in inducing ANP release by volume expansion? Vagotomy, which should block afferents coming from the atria, failed to interfere with the response (J.A.-R., unpublished data). 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