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Neuroendocrine Control of Body Fluid Metabolism

Departments of Physiology and Internal Medicine, School of Medicine of Ribeirao Preto, University of São Paulo, Ribeirao Preto, São Paulo, Brazil; and Pennington Biomedical Research Center, Department of Basic Sciences, Louisiana State University, Baton Rouge, Louisiana

ABSTRACT

I. INTRODUCTION

II. VASOPRESSIN AND OXYTOCIN EFFECTS ON WATER METABOLISM

A. The Hypothalamo-Neurohypophysial System

B. Osmotic Control of Vasopressin Release

C. Volume Control of Vasopressin Release

D. Vasopressin and Oxytocin Receptors

1. Vasopressin receptors

2. Oxytocin receptors

E. Actions of Vasopressin and Oxytocin

III. THE RENIN-ANGIOTENSIN SYSTEM IN THE BRAIN

A. General Aspects of the Brain Angiotensin System

B. Functions of Brain Angiotensin in Thirst

C. Functions of Brain Angiotensin in Salt Intake

D. Angiotensin Receptors

E. Interactions With Other Hormones

IV. AUTONOMIC NERVOUS SYSTEM AND BODY FLUID METABOLISM

A. Role of the Sympathetic Nervous System Innervation of the Kidney in Sodium Excretion

B. CNS Regulation of the Renal Sympathetic Nerve Activity

C. Role of {alpha}-Adrenergic and Cholinergic Receptors in Control of Natriuresis

V. THE NEUROENDOCRINE REGULATION OF ATRIAL NATRIURETIC PEPTIDE SECRETION

A. Control of Hydromineral Balance by a Brain Neural Circuit

B. CNS Neurotransmitters/Neuromodulators and Modulation of Hydromineral Homeostasis

1. Dopamine

2. Hypothalamic GABAergic neurons

3. Endothelin

4. Neurotensin

5. Substance P

6. Melanocyte-stimulating hormone

7. Neuropeptide Y

8. Opioid peptides

9. Other transmitters

C. Natriuretic Hormones and Hydromineral Balance

D. The Brain ANPergic Neurons in Water and Salt Intake

E. The Brain ANP System in the Control of Cardiovascular and Renal Functions

F. Afferent Inputs to the Brain ANPergic System

G. Role of the Brain in ANP Release in Response to Increased Extracellular [Na+] and Acute Blood Volume Expansion

H. Efferent Pathways of the CNS and the Cardiac Release of ANP

VI. INTERACTION BETWEEN NEUROHYPOPHYSIAL HORMONES AND ATRIAL NATRIURETIC PEPTIDE

A. Effects of Oxytocin on the Release of ANP From the Heart

B. Effects of Vasopressin on Cardiac Function

C. Actions of Oxytocin and Vasopressin in the Kidney

VII. CONCLUSIONS AND PERSPECTIVES

	ABSTRACT

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	I. INTRODUCTION

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The precise regulation of the volume and osmolality of body fluids is fundamental to survival. Sodium chloride (NaCl) represents an important constituent of the extracellular compartment and is the major determinant of the plasma osmolality as well as extracellular fluid volume. All vertebrates maintain plasma osmolality and extracellular volume primarily by regulating the ingestion and urinary excretion of water and electrolytes. For example, such animals develop a special behavioral sensation, thirst, that is defined as a need or desire to drink that leads the animal to increase water intake. An elevation in the plasma osmolality, and consequent cellular dehydration, is the most potent stimulus of thirst. In mammals, a minimal increase in the plasma osmolality of 1–2% induces thirst. A decrease in the extracellular fluid volume, although less effective, is also capable of generating thirst. A 10% reduction in blood volume or in arterial pressure both will induce an animal to drink water. Sodium intake occurs later, but initially the animal looks for water. Several other conditions can induce thirst, independent of changes in volume or plasma osmolality, e.g., dry mouth and breathing dry air. However, oral ingestion of water produces only a transient satiation of thirst. Because animals with esophageal or gastric fistulae are not satiated by peroral water ingestion but only by replacing the deficit through the fistula, it is apparent that absorption of water into the bloodstream also is required for satiation (for review, see Refs. 17, 18, 110, 111, 153, 254, 257, 335, 344, 371, 547, 549).

The classic studies of Verney (537) introduced the concept of effective osmolality (i.e., increased extracellular osmolality induced by solutes that do not cross the cell membrane) and the presence of osmoreceptors involved in arginine vasopressin (AVP) release in response to increased osmolality. Andersson, McCann, and co-workers (9–13) postulated that an osmoreceptor was a sodium sensor located in brain regions within the blood-brain barrier, and it could be involved in the control of sodium appetite as well as in the control of sodium excretion in response to changes in brain extracellular fluid sodium concentration.

It is accepted that in the genesis of thirst there is an important role for osmoreceptor-Na⁺ receptor cells located in the circumventricular organs (CVO) of the anterior aspect of the third ventricle. These structures contain sensory cells that respond to variations in the plasma osmotic pressure or the sodium concentration of plasma and cerebral spinal fluid (CSF). It should be pointed out that equiosmolar NaCl hypertonic solution is a more effective stimulus than nonsaline hypertonic solutions (345). Lesions in the region of the anteroventral portion of the third ventricle (AV3V), involving the ventral part of the median preoptic nuclei (MnPO), induce permanent or temporary adipsia (13, 65, 305). Sodium receptors have been demonstrated also in afferent neural terminals adjacent to the hepatic, renal, and intestinal vessels. Liver receptors are activated by an increase in [Na⁺] in the portal vein, augmenting hepatic afferent vagal inputs to the nucleus of the solitary tract (NTS) that generate efferent signals increasing renal sodium excretion, and concomitantly decreasing intestinal sodium absorption (230).

In some species, such as the rat, increased production and release of angiotensin II (ANG II) into the systemic circulation mediates thirst in response to a reduction in the extracellular volume. Central nervous system (CNS)-generated ANG II is also an important thirst inducer, acting as a neurotransmitter on ANG II-sensitive neurons in brain structures such as the subfornical organ (SFO) or organum vasculosum of the lamina terminalis (OVLT), both of which are CVO of the lamina terminalis. ANG II may be the mediator of the thirst induced by hypertonic saline (2% NaCl) microinjected into the cerebral ventricle (in rat, mouse, sheep, and rabbit), since the dipsogenic effects can be decreased by the previous administration of losartan, an AT₁ ANG II receptor antagonist (337, 340–344). In addition, Franci et al. (171) demonstrated that dehydration-induced drinking could be blocked by injection of ANG II antiserum in the third ventricle of male rats.

It is known that when water is offered to a water-deprived animal, it will begin drinking within 3–10 min and continue until the thirst is satiated. Interestingly, the process of thirst satiation commences even before plasma osmolality is normalized; thus, even while plasma osmolality has not been corrected, the volume of water drunk usually is the amount needed to return osmolality to normal. This may occur through stimuli originating in the mouth, pharynx, or stomach and conveyed by afferent impulses to CNS structures involved in the integrative response.

During the last four decades a number of studies have attempted to identify brain areas specifically involved in satiety, regulation of plasma osmolality, water/electrolyte ingestion, and excretion. The first studies to determine the synaptic transmitters in the CNS circuits that control body fluid homeostasis were published in the 1960s by Grossman (197, 198). He demonstrated that hypothalamic cholinergic or noradrenergic stimulation induced an increase in water or food intake. Cholinergic and angiotensinergic stimulation of the AV3V region caused a rapid increase in water intake in normal hydrated animals, as well as increased natriuresis (21, 152, 155, 197). Intracerebroventricular (icv) injection of carbachol (a cholinergic agonist) also evoked dramatic natriuretic, kaliuretic, and antidiuretic responses similar to the effects observed with central (icv) injection of hypertonic saline (121). Thus it became clear that both -adrenergic and cholinergic synapses are involved in the control of both natriuresis and kaliuresis. Microinjection of agonists and antagonists of these neurotransmitters into the septal area, AV3V, or the third ventricle altered the natriuretic, kaliuretic, and carbachol-induced antidiuretic responses (72, 73, 120, 170, 172, 351, 365, 431, 432, 443, 444). Phentolamine, an -adrenergic antagonist, abolished the natriuretic response to third ventricle injection of hypertonic saline, norepinephrine, or carbachol. Meanwhile, isoproterenol, a -adrenergic agonist, exhibited an antinatriuretic and antikaliuretic effect. In contrast, propranolol, a -receptor blocker, induced natriuresis and kaliuresis when injected alone and also potentiated the natriuretic response to carbachol. Cholinergic blockade with atropine decreased the response to norepinephrine and blocked the natriuretic response to hypertonic saline (365, 366, 399).

The lamina terminalis is a forebrain structure that contains the SFO, the MnPO, and the OVLT. The AV3V includes the ventral part of the MnPO and the OVLT. The AV3V and SFO region contains neurons that are sensitive to changes in plasma or CSF osmolality (50, 207, 340, 384, 385, 386), and these cells have direct connections with the paraventricular nucleus (PVN) (204, 356, 480, 550). Also, a direct connection from the region of the lamina terminalis to the raphé nuclei and locus ceruleus (LC) has been described (483). These connections appear to be important to induce the hormonal, sympathetic nervous system, and behavioral changes that restore the body fluid balance as described below. Sly et al. (482) demonstrated a polysynaptic pathway connecting neurons in the brain areas controlling body fluid balance to the kidney, by injecting the Bartha strain of pseudorabies virus into the kidney of rats. The application of this neurotropic virus resulted in retrograde infections, which permitted the identification of higher order neurons (putative third and fourth order) in regions of the forebrain including the OVLT, MnPO, SFO, bed nucleus of the stria terminalis, anteroventral periventricular nucleus, medial and lateral preoptic area, supraoptic nucleus (SON), retrochiasmatic nucleus, primary motor cortex, and the visceral area of the insular cortex. Renal innervation (considered to be entirely sympathetic) participates in the control of three aspects of renal function: renal blood flow, tubular reabsorption of electrolytes, and renin secretion. Thus renal sympathetic nerves regulate the function of the vasculature, the tubules, and the juxtaglomerular granular cells that, following activation of -adrenergic receptors, cause an increase in renin secretion rate and renal blood flow and a reduction in urinary sodium excretion (115, 116).

In summary, mammals control the volume and osmolality of their body fluids in response to stimuli that arise from both the intracellular and extracellular fluid compartments. These stimuli are sensed by two kinds of receptors: osmoreceptor-Na⁺ receptors (plasma osmolality or sodium concentration) and volume or pressure receptors. This information is conveyed to specific areas of the CNS responsible for an integrated response, which is dependent on the integrity of the AV3V (OVLT and MnPO) and SFO. In addition, the PVN, SON, LC, dorsal raphé nuclei (DRN), and the lateral parabrachial nuclei, among others, also represent important structures involved in hydromineral balance. Such structures, once stimulated, can determine responses that involve 1) the induction of thirst, salt appetite, or both; 2) changes in sympathetic activity; 3) activation of the renin-angiotensin-aldosterone system; or 4) secretion of AVP and oxytocin (OT) from the neurohypophysis and natriuretic peptides from the heart.

	II. VASOPRESSIN AND OXYTOCIN EFFECTS ON WATER METABOLISM

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A. The Hypothalamo-Neurohypophysial System

The hypothalamo-neurohypophysial system is located in the medial part of the anterior hypothalamus and comprises the paired PVN on each side of the dorsolateral wall of the third ventricle and the paired SON. The perikarya of the magnocellular neurons responsible for the synthesis and release of OT and AVP are located in both the PVN and SON (292). The PVN contains a preponderance of OT neurons and the SON a preponderance of AVP neurons. The axons of these neurons form the hypothalamo-hypophysial tract, which terminates in the neurohypophysis. Some of these axons terminate in the median eminence in juxtaposition to the capillaries of the hypophysial portal veins, whereas most terminate in the neural lobe (32, 66, 214, 424). The AVP and OT released in the median eminence are transported by the hypophysial portal vessels to the anterior lobe of the pituitary gland where they act to stimulate the release of ACTH and prolactin, respectively (333, 334, 336). The AVP and OT released from the neural lobe are in part transported by the short portal vessels to the anterior lobe, and the blood from both lobes empties into the hypophysial veins to return to the heart (409).

OT and AVP are synthesized and released by magnocellular neurosecretory neurons classified into AVP- and OT-producing subtypes. Recent evidence from qualitative RT-PCR experiments on single cells confirms the fact that the majority of magnocellular neurons coexpress both peptide mRNAs. Furthermore, there is some OT and AVP mRNA coexpression in virtually all of the magnocellular neurons in the SON of the hypothalamus (559). However, because PCR grossly magnifies the mRNA content, it is clear that most of these neurons express only one of these peptides at a functionally significant level.

Changes in the firing pattern and frequency of magnocellular neurons in response to relevant physiological stimuli regulate the circulating levels of their secreted hormones (196, 424). The electrophysiological profiles of OT and AVP neurons can be distinguished from each other, and from that of neurons in the immediately adjacent perinuclear zone (33). Oxytocinergic neurons possess properties that favor the production of short spike trains, which are enhanced during lactation (289, 492). In contrast, vasopressinergic magnocellular neurons in the hypothalamus exhibit phasic electrical activity that depends on intrinsic membrane properties and is influenced by extrinsic factors such as plasma osmolality, blood volume, and pressure (for review, see Ref. 32). OT and AVP, released from the soma and dendrites of neurons, bind to specific autoreceptors and induce an increase in intracellular [Ca²⁺]. In OT cells, the increase in [Ca²⁺] results from a mobilization of Ca²⁺ from intracellular stores, whereas in AVP cells, it results mainly from an influx of Ca²⁺ through voltage-dependent channels (99, 191).

A selective afferent neural input to the vasopressinergic neurons provides a mechanism for the release of AVP independently of OT in response to appropriate physiological stimuli. Two alternative models of the neural pathways and transmitters involved in the activation of the supraoptic hypophysial tract have been suggested. Some authors have suggested the existence of an excitatory relay through a cholinoceptive area on the ventral surface of the brain stem that has been termed the nicotine-sensitive area because topical application of nicotine to this area in the cat causes the release of AVP without OT (51). Afferent input from a noradrenergic projection from the NTS to the SON has also been demonstrated (102, 553). With the use of combined retrograde tracer-immunofluorescence methods, OT and AVP neurons in the SON and PVN were shown to receive noradrenergic innervation that arises mainly from A1 neurons in the ventrolateral medulla (457). Furthermore, an inhibitory relay was demonstrated through the A1 group of noradrenergic neurons on the ventral surface, which selectively innervate the AVP-secreting neurons in the SON. This model implies an inhibitory role for norepinephrine acting on - or ₂-receptors and explains the antinatriuretic effect of -adrenergic receptor activation (72, 73, 365, 443). However, most investigations suggest an excitatory, rather than inhibitory, function of the A1 noradrenergic neurons involving ₁-receptors, consistent with the previously described stimulatory role of ₁-receptors in natriuresis (72, 73, 365, 443). The posterior magnocellular division of the PVN and SON is mainly innervated by the A1 noradrenergic cell group (93), and noradrenergic afferents have been shown to have a facilitatory role in the regulation of the activity of neurohypophysial AVP neurons (101, 102). The PVN receives a dense noradrenergic innervation from the A1 cell bodies of the caudal ventrolateral medulla, A2 cell bodies of the NTS, and A6 cell bodies of the LC. Noradrenergic neurons in the LC participate in the baroreflex activation of the diagonal band of Broca (195), which has been shown to be an integral component of the pathway regulating the baroreceptor-induced inhibition of AVP release and, possibly, the stimulation of OT release (94, 251). In addition, an inhibitory GABAergic pathway from the diagonal band of Broca preferentially innervates AVP-secreting SON neurons, supporting the view that the baroreflex-induced depression of SON firing may be mediated by GABA (252).

B. Osmotic Control of Vasopressin Release

The hypothalamo-neurohypophysial system plays a fundamental role in the maintenance of body fluid homeostasis by secreting AVP and OT in response to osmotic and nonosmotic stimuli (461). Microinjections of hypertonic saline into the AV3V area, the major central site for the regulation of body fluid composition, cardiovascular, and renal function, were first shown to induce an increase in water intake in goats by Andersson, McCann, and co-workers (9–13). These results were confirmed in rats by Antunes-Rodrigues and McCann (21). Electrical stimulation of this structure also induced water intake together with natriuresis (12). In addition, lesions of the AV3V had several important effects, including adipsia and hypernatremia (10, 13), impaired drinking responses and AVP secretion in response to hypertonic saline and ANG II (280), impaired recovery of arterial pressure in response to hypertonic saline in rats submitted to hemorrhagic shock (41), decreased osmotic- and volume-induced atrial natriuretic peptide (ANP) release (25, 417), a decrease in the number of Fos-like immunoreactive neurons in the MnPO, PVN, and SON in response to intravenous infusion of hypertonic saline (223, 561), and an interruption of neuronal inputs that trigger AVP secretion from the posterior pituitary as well as AVP release into the extracellular compartment of the SON (315).

Other evidence indicates that, besides the AV3V, other structures such as the MnPO, SFO, medial septal area, anterior lateral hypothalamus, SON, PVN, medial habenula, and stria medullaris are organized into a neural circuit involved in the regulation of water/sodium intake and excretion (90, 170, 172, 432). Neurons in the PVN, MnPO, preoptic and hypothalamic periventricular nuclei, median eminence, and OVLT also contain -ANP as determined by immunocytochemistry (245, 267, 268, 363, 401, 573), which suggests that ANP neurons may be one of the effectors involved in control of water and salt intake. It was also shown that the SFO and OVLT send ANP immunoreactive fibers to the PVN and SON (318). Therefore, osmoresponsive neurons located in the OVLT project to magnocellular and parvicellular neurosecretory neurons and are likely candidates for cerebral osmoreceptors (8, 339, 340, 384). There is evidence for the involvement of other brain areas, such as the area postrema (AP) and NTS in the osmotic response (75).

The most important physiological osmotic regulation of AVP release takes place in the CNS within the regions listed above, although it has been suggested that peripheral osmoreceptors in the liver, mouth, and stomach detect the early osmotic impact of foods and fluid intake (51, 230, 429). Indeed, intragastric hypertonic saline infusion increases portal venous but not systemic plasma osmolality and increases Fos-like immunoreactivity in the AP, NTS, lateral parabrachial nuclei, the SON, and PVN (391). Osmoreceptors are highly specialized neurons capable of transducing changes in external osmotic pressure into electrical signals that activate CNS areas involved in the control of water and salt intake and excretion by the release of acetylcholine or angiotensin at synapses in the SON (51). Patch-clamp studies using isolated rat SON magnocellular neurosecretory cells demonstrated that these neurons are, respectively, depolarized and hyperpolarized by increases and decreases in extracellular osmolality and that these responses result from changes in the activity of mechanosensitive cation channels (59).

Osmoreceptors are located in the OVLT and SFO, structures that lie outside the blood-brain barrier and, therefore, are in contact with plasma ionic concentrations and hormones, such as ANP and ANG II (256, 346). Small changes in plasma osmolality within the physiological range can rapidly stimulate AVP transcription in the SON and PVN, suggesting that stored AVP released into the blood circulation is rapidly replaced by de novo synthesis, processing, and transport of AVP (29).

Fos protein expression, a marker of neuronal activity, has been used to investigate the hypothalamic activation following systemic osmotic stimulation (207). Water deprivation for 24 h resulted in the expression of the c-Fos protein in the PVN and SON (448). Transient expression of c-Fos protein was also detected in magnocellular neurons of the SON and PVN of the rat hypothalamus after injection of hypertonic saline. This activation of the SON and PVN was maintained by a chronic osmotic stimulus induced by the drinking of hypertonic NaCl solution or by water deprivation, and was reversed by water intake for 24 h after the chronic osmotic stimulation. Chronic stimulation provides sustained activation of these neurons, presumably accompanied by increased synthesis and release of AVP and, possibly, OT (358). Fos immunoreactivity was also observed in many neurons of the MnPO, OVLT, and, to a lesser extent, in the SFO of rats submitted to water deprivation for 24 or 48 h, confirming that these structures play a role in homeostatic responses to dehydration (187, 347). It was also demonstrated that lesions within the AV3V region (including the MnPO) suppressed water intake after 24 h of water deprivation, as well as c-Fos expression in the SON and, less completely, in the PVN nuclei, indicating that the cellular response of supraoptic neurons to osmotic stimuli requires inputs from the AV3V region, while the PVN is less dependent on this (561).

As expected, chronic osmotic stimulation also increases AVP mRNA expression in the SON and PVN (69, 303). Hyperosmolality causes a 1.5- to 2-fold increase in AVP mRNA expression (474). On the other hand, longterm hyposmolality reduces AVP mRNA expression in the hypothalamus to only 10–15% of the control level (430, 534). Recently, using in situ hybridization histochemistry techniques, Glasgow et al. (188) confirmed earlier studies reporting an increase in the AVP, OT, and AVP-binding protein (neurophysin) mRNAs during hypernatremia, as well as a decrease in these mRNAs during hyponatremia. In addition, these authors demonstrated that the magnocellular neurons of the SON responded to hypernatremia with an increase in the expression of a variety of genes including cytochrome oxidase, tubulin, Na⁺-K⁺-ATPase, spectrin, PEP-19, calmodulin, GTPase, DnaJ-like, clathrin-associated protein, and synaptic glycoprotein, a regulator of GTPase activation. This analysis suggests that adaptation to chronic osmotic stress results in global changes in gene expression in the magnocellular neurons of the SON.

The brain stem has also been implicated in the control of body fluid homeostasis. For example, ascending projections from the caudal region of the ventrolateral medulla have been implicated based on the expression of Fos protein and AVP mRNA in the SON after electrical stimulation of this region (475). Furthermore, intravenous infusions of hypertonic saline increase c-fos gene activity in neurons of the caudal ventrolateral medulla (224).

Nitric oxide (NO) has been proposed as a local modulator of magnocellular neuron activity. NO is a neuronal messenger produced from L-arginine by neuronal NO synthase (nNOS). The presence of nNOS in the PVN and SON vasopressinergic and oxytocinergic neurons and its increase in these cells after osmotic stimulation or dehydration suggest a role for NO in the regulation of AVP and OT (61, 213, 263, 528, 538, 564). In addition, nNOS has been detected by immunocytochemistry in other neural structures involved in AVP secretion, such as the SFO, OVLT, and MnPO (539). However, the role of NO in OT and AVP release is still not clearly defined. Ota et al. (392) showed that the intracerebroventricular injection of S-nitroso-N-acetylpenicillamine (SNAP), which spontaneously breaks down to form NO, caused a transient, dose-related increase in plasma AVP concentration. In addition, when D-arginine, which cannot be used as a substrate by NO synthase, was injected intracerebroventricularly, there was only a slight, delayed increase in the plasma AVP concentration. Thus NO can act centrally to stimulate AVP release and may serve as a neuromodulator controlling its release. However, intracerebroventricular injection of N^G-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, increased plasma OT and AVP above basal levels, suggesting that NO tonically inhibits both hormones. On the other hand, L-NAME did not change the AVP response to an osmotic stimulus (260). Furthermore, studies using rat hypothalamic explants showed that L-arginine reduced the KCl-evoked AVP release, and this effect was reversed by the inhibition of NOS (571); thus NO appears to directly and specifically inhibit the stimulated release of AVP from rat hypothalamic explants in vitro. In another study, discharges of SON neurons from slices of rat hypothalamus were inhibited by sodium nitroprusside, a spontaneous releaser of NO; furthermore, preincubation of the slices with hemoglobin, an inactivator of NO, prevented this inhibition (261, 310). These data demonstrate that NO exerts a predominantly inhibitory effect on SON neurons.

Endogenous NO may be involved in the regulation of magnocellular functions, especially when the internal environment is disturbed. Chronic salt loading upregulates the expression of nNOS mRNA in the SON and PVN of the hypothalamus, and this is accompanied by an increase in NOS activity in the posterior pituitary. The effects of NO on AVP and OT regulation were summarized in a recent review, as follows: NO tonically inhibits the basal release of AVP and OT into plasma, but the NO inhibition of AVP secretion is removed during water deprivation, hypovolemia, moderate osmotic stimulation, and after injection of ANG II, while the inhibition of OT is enhanced. NO facilitates drinking behavior stimulated by water deprivation, osmotic stimulation, hemorrhage, and ANG II (261).

C. Volume Control of Vasopressin Release

The maintenance of body fluid homeostasis requires autonomic and endocrine responses and activation of specific behaviors. Changes in blood volume or pressure lead to appropriate changes in renal fluid and electrolyte excretion through neural and endocrine adaptive responses. Hypovolemia induces AVP release from magnocellular neurons, which acts by increasing reabsorption of water in the distal nephron by opening aquaporin-2. The threshold for stimulation of AVP release in hypovolemia is generally reported to be between 10 and 20% of the blood volume in several different species (471). In normal, standing human subjects, a reduction in blood volume of 6% or a reduction in plasma volume of 10% induced by furosemide injection was sufficient to increase the plasma AVP concentration (272). On the other hand, isotonic expansion of blood volume results in a reduction in plasma AVP concentration (206, 258, 295, 298, 470, 471).

The release of AVP from the neurohypophysial terminals of hypothalamic magnocellular neurosecretory neurons is regulated by peripheral baroreceptors, cardiopulmonary volume receptors, and the circulating ANG II concentration (522). Information from these sources is transmitted through afferent pathways with differential effects on the excitability of the AVP-secreting cells (423). A brief increase in arterial pressure, sufficient to activate baroreceptors, is associated with a transient and selective GABAergic inhibition of these neurosecretory neurons, achieved through a multisynaptic pathway that involves ascending catecholaminergic projections from neurons in the diagonal band of Broca (DBB). Baroreceptor activation induces a consistent increase in firing of DBB neurons, which project to the hypothalamic supraoptic neurosecretory neurons, indicating that baroreceptor-induced inhibition of hypothalamic vasopressinergic neurons may be mediated through DBB neurons (81, 251, 252, 271, 393).

Afferent nerve impulses from stretch receptors in the left atrium, aortic arch, and carotid sinus tonically inhibit AVP secretion, and a reduction in their discharge leads to AVP release (51). Baroreceptors in the atrium and ventricles signal changes in blood volume, and the receptors in the aortic arch and carotid sinuses signal changes in arterial blood pressure. These data are relayed through, respectively, the vagal and glossopharyngeal nerves to the NTS in the brain stem, from which postsynaptic pathways connect with the magnocellular neurons of the SON and PVN (125, 471). Indeed, stimulation of the cervical vagus induces Fos expression in noradrenergic A1 neurons of the caudal ventrolateral medulla and excites AVP cells (103). Low-pressure receptors in the atrium tonically inhibit AVP release via a pathway involving the NTS, and AVP release induced by hypovolemia occurs through a reduction in the activity of this inhibitory input (51, 471).

Although there is abundant evidence to support the role of the AV3V and the low-pressure receptors in the regulation of AVP release, the afferent pathways controlling AVP release appear to be more complex, and it has been suggested that other mechanisms might also be involved in this regulation. A decrease in arterial pressure activates peripheral low-volume receptors in the great veins, atria, and lungs, which give rise to neural inputs that result in an increase in the excitability of AVP-secreting neurons, achieved via pathways that include direct projections from caudal ventrolateral medulla A1 neurons. The AVP response to an acute reduction in central blood volume, such as that produced by hemorrhage, depends on the A1 projection only if the stimulus is of moderate intensity. Severe stimuli appear to involve activation of both the A1 projection and an additional AVP-stimulatory pathway that bypasses the A1 region (483). There is evidence that the area postrema, the most caudal circumventricular organ located on the dorsal surface of the medulla, is also involved in several physiological control mechanisms, including the regulation of AVP synthesis and release. Lesions of the AP decreased AVP mRNA levels in the PVN and SON as well as plasma AVP levels in the basal state and after hyperosmolality or hypovolemic stimulation (30).

The neurosecretory system contains an elaborate array of neural inputs, including a catecholaminergic innervation that is predominantly noradrenergic, but which also has a dopaminergic component (105, 562, 563). The precise role of hypothalamic norepinephrine in the control of AVP release remains unclear, due to reports of both inhibitory and excitatory effects of norepinephrine (NE) and only a few studies with direct hypothalamic manipulation (298). The excitatory effect of central noradrenergic stimulation on serum AVP is highly site-specific and localized to the PVN and SON (297, 367, 373, 413). Activation of the locus ceruleus-PVN ascending noradrenergic pathway accounts for the increase in NE release in rat PVN induced by systemic hemorrhage (367). However, NE has also been reported to inhibit AVP and OT release from cells in the PVN of lactating rats (228). Adrenergic receptors may be differentially distributed in vasopressinergic neurons allowing excitatory or inhibitory impulses (298) or, alternatively, the results may be accounted for by a bell-shaped response of AVP to NE.

Vasopressin release under conditions of hypovolemia involves stimulation by ANG II/III. Hypotension causes renal renin release and leads to the formation of ANG II; binding of this hormone to AT₁ receptors in the SFO neurons promotes activation of a central angiotensinergic input that, in turn, has a predominantly excitatory effect on AVP neurons. In support of this role for ANG II, injection of ANG II or III into the SON or PVN increases magnocellular activity and AVP release into the bloodstream (28, 476, 578).

The SFO exhibits functional segregation, which may be observed through the distinct patterns of c-Fos expression in this area induced by hypovolemic or osmotic stimuli. Hypertonic saline induces c-Fos expression in the peripheral SFO only, while hypovolemia induced by subcutaneously administered polyethylene glycol (PEG) induces c-Fos in the central region of the SFO (484). In addition, c-Fos protein is rapidly induced in hypothalamic magnocellular nuclei following hemorrhage. AVP and OT neurons express c-Fos in a graded response to hypovolemic stimuli, which was correlated with stimulus intensity and also with the amount of hormone released into the peripheral blood. A differential pattern of activation of AVP neurons occurs in response to hemorrhagic stimuli. AVP neurons in the SON had a lower response threshold than those in the PVN (472). OT neuron activation requires a greater hypovolemic stimulus than that for AVP, revealing functional heterogeneity among magnocellular neurons (428). Lesions of the hypothalamic SON blunted the increase in plasma AVP to below levels attained in normal rats submitted to hemorrhage, indicating that these nuclei are primary regulatory sites for AVP release in response to hemorrhage and that lack of adequate AVP release significantly retards blood pressure recovery after bleeding (146). In addition to AVP release, AVP gene transcription in the SON and PVN is increased in the hypothalamus of conscious rats submitted to hemorrhage or normovolemic hypotension, as determined by intronic in situ hybridization (264).

D. Vasopressin and Oxytocin Receptors

1. Vasopressin receptors

The actions of AVP are mediated by plasma membrane receptors, which belong to the G protein-coupled receptor family characterized by the presence of seven transmembrane helices connected by three extracellular and three intracellular loops. Three different subtypes of AVP receptors, V_1a, V_1b, and V₂, have been cloned (312, 360, 507). V_1a receptor expression has been described in smooth muscle and liver, with the V_1b receptor in the anterior pituitary and the V₂ receptor in the kidney (270, 311, 517). V_1a receptors are involved in blood pressure control and in all other known functions of AVP, except for the stimulation of corticotropin secretion by the adenohypophysis, which is mediated via the V_1b receptor. The presence of V_1a receptors has been described in structures of the limbic system (septum, amygdala, bed nucleus of the stria terminalis, accumbens nucleus), in the suprachiasmatic and dorsal tuberal region of the hypothalamus, and in the area of the nucleus of the solitary tract, suggesting that V_1a is the main receptor responsible for the central effects of AVP (525). Recently, V_1b receptors have been detected by RT-PCR and in situ hybridization not only in pituitary corticotrophs but also in the hypothalamus, amygdala, cerebellum, and in those areas close to the CVO (medial habenula, SFO, OVLT, median eminence, and nuclei lining the third and fourth ventricles), as well as in the external zone of the median eminence. These data suggest that V_1b receptors may also mediate different functions of AVP in the brain (220).

V₂ AVP receptors are responsible for the antidiuretic effect of AVP. The expression of V₂ receptors has been described in some of the thick ascending limbs and all of the principal and inner medullary collecting duct cells, not only in the basolateral membrane but also in the luminal membrane (382, 378). Vasopressin regulates transcription of the aquaporin-2 gene through a cAMP regulatory element located in the 5'-flanking region (232, 327, 330).

X-linked nephrogenic diabetes insipidus is a disease that results from mutations in the V₂ receptor gene (46) that map to chromosome region Xq28 (48, 434). Functional characterization of V₂ AVP receptor gene mutations identified in patients has brought insight into the residues that are critical for V₂ receptor expression and function (552). Natural mutation of Arg113Trp in the V₂ receptor significantly reduced receptor expression in transfected cells, receptor-ligand binding affinity, and G_s coupling (47). A similar reduction in binding affinity and the inability to concentrate the urine after the administration of the antidiuretic hormone AVP was found in association with the deletion of Arg-202, which is located in the second extracellular loop of the human V₂ receptor (2), suggesting that this domain is also important for ligand binding.

Vasopressin receptor subtypes are coupled to different G proteins (136). V_1a and V_1b receptors are coupled to G proteins of the G_q/11 family, which mediate the breakdown of phosphatidylinositol (64, 517), whereas V₂ receptors are coupled to the G_s protein, which activates adenylate cyclase (48). Different single intracellular domains determine the G protein-coupling selectivity profile of the different AVP receptor subtypes.

Liu and Wess (309) created and analyzed a series of V_1a and V₂ hybrid receptors in which distinct intracellular domains were systematically exchanged between the two wild-type receptors. cAMP assays showed that all mutant receptors that contained the V₂ receptor sequence in the third intracellular loop were able to stimulate adenylate cyclase activity with high efficacy, whereas all mutant receptors in which the third intracellular loop was derived from the V_1a receptor had little or no effect on intracellular cAMP levels. These data strongly suggest that the third intracellular loop of the V₂ receptor plays a key role in the proper recognition and activation of G_s. On the other hand, all hybrid constructs in which the second intracellular loop consisted of the V_1a receptor sequence were able to activate the phosphoinositide (PI) cascade in a fashion very similar to the wild-type V_1a receptor, whereas all mutant receptors that contained the V₂ sequence in this receptor region displayed only residual PI activity, similar to that of the wild-type V₂ receptor, indicating that the second intracellular loop of the V_1a receptor is critically involved in the selective activation of G_q/11.

2. Oxytocin receptors

In a recent paper, Gimpl and Fahrenholz (186) comprehensively reviewed the current knowledge of the physiological effects of OT and the distribution of OT receptors throughout the body, focusing mainly on the research of the past decade.

The OT receptor has been cloned from human (273), rat (440), and other species (43, 190, 284, 427, 451). The OT receptor is highly conserved across species and has been found in a variety of tissues, particularly in uterus but also in mammary gland, pituitary, brain, kidney, thymus, ovary, testis, heart, and blood vessels. The OT receptor density in the uterus increases through pregnancy (477, 535). In the brain, Tribollet et al. (525) showed that the anatomical localization of OT receptors in the olfactory tubercle, the ventromedial hypothalamic nucleus, the central amygdaloid nucleus, and the ventral hippocampus was markedly different from that of the binding sites for AVP.

The OT receptor is a member of the class I protein-coupled receptor superfamily that activate G_q and G_i, which in turn stimulate phospholipase C-mediated hydrolysis of PI. The cleavage of PI generates inositol 1,4,5-trisphosphate, which mobilizes calcium from the sarcoplasmic reticulum, and 1,2-diacylglycerol, which activates protein kinase C, resulting in the phosphorylation of several target proteins. Oxytocin causes a rapid increase in intracellular free calcium, activates mitogen-activated protein (MAP) kinase, and stimulates prostaglandin E₂ synthesis by activating cyclooxygenase (157, 362). In addition, an increase in Ca²⁺-bound calmodulin leads to the activation of a kinase primarily responsible for the phosphorylation of myosin light chain, which regulates myometrial contractility (487).

The existence of OT receptor subtypes remains to be established (186, 477, 535). The presence of such subtypes has been suggested in the rat uterus, kidney, and brain, to explain differential pharmacological profiles or immunoreactivity patterns. Oxytocin-binding sites in the macula densa and thin Henle's loop, detected in the rat kidney, may represent two subtypes of OT receptors that could mediate distinct effects of OT on kidney function (34). On the other hand, it should be pointed out that high concentrations of OT can interact with V₁ and V₂ AVP receptors, since these are closely related to the OT receptor (518). PCR and Southern analysis in several tissues known to have OT binding activity failed to identify a gene encoding a second OT receptor, making the existence of OT receptor subtypes unlikely (274).

E. Actions of Vasopressin and Oxytocin

The AVP and OT amino acid sequences differ only in the 3 and 8 positions. However, in both hormones disulfide bond formation between cysteine residues at the 1 and 6 positions results in a peptide, consisting of a 6-amino acid cyclic part and a 3-amino acid COOH-terminal part, which exerts various hormonal effects.

The antidiuretic action of AVP is the main physiological effect of this hormone, involving increased permeability to water of the renal collecting duct cells, allowing more water to be reabsorbed from urine to blood. Circulating AVP activates the AVP V₂ receptor on the luminal tubular membrane, leading to an increase in intracellular cAMP and phosphorylation of the COOH-terminal of the water channel protein aquaporin-2 in the tubular cells of the distal nephron (568). The number and distribution of aquaporin channels in the collecting duct cells are regulated by AVP V₂ receptors as shown by the decreased urine osmolality and increased aquaporin-2 expression in apical membranes and subapical cytoplasm of collecting duct cells of the inner medulla in dehydrated rats treated with a V₂ receptor antagonist (216). AVP appears to stimulate the synthesis of aquaporin-2 mRNA and also to regulate the insertion of aquaporin-2 into the luminal membrane of the collecting tubules through fast exocytosis to the plasma membrane (281, 378, 449, 455). The presence of aquaporin-2 in the apical membrane causes an increase in water permeability allowing the movement of free water from the collecting duct into the tubular cell and, thereafter, the transport of water across the basolateral membrane is facilitated by the constitutively expressed aquaporins-3 and -4 (127, 279, 516).

Oxytocin is also involved in vascular and cardiac relaxation and hydromineral homeostasis (88, 200, 202, 206, 233, 249, 486). It has long been recognized that OT increases renal electrolyte excretion in various species and that its natriuretic and kaliuretic effects are AVP independent. OT and AVP are secreted simultaneously in response to hyperosmolality and hypovolemia (38, 39, 206, 496, 498) and when systemically administered (iv or ip), they induce natriuresis (173, 458). OT is a more potent natriuretic hormone than AVP. These effects can be explained by a direct action of both peptides on specific receptors already shown to be present in the tubular cells of the kidney (494, 525). The different potencies of these hormones can be attributed to a relative affinity of OT for its own receptor or to its lower affinity for V₂ and V1 AVP receptors.

Studies have suggested a synergistic effect of AVP and OT in the inner medullary collecting duct, where both peptides induce an increase in cAMP accumulation and natriuresis (38, 160). Oxytocin binds to the AVP V₂ receptor because of its structural similarity to AVP. The urinary sodium excretion induced by OT is completely blocked by pretreatment with an OT receptor antagonist, but not affected by an AVP V₁ antagonist. However, this effect was partially blocked by the combination of AVP V₁ and V₂ antagonist treatment (536). To clarify the effect of OT as an agonist of the V₂ receptor, Terashima et al. (515) investigated the influence of acute elevation of plasma OT levels on the expression of V₂ and aquaporin-2 mRNAs in the rat. They found that OT can downregulate the former and upregulate the latter in the collecting duct, by acting as an agonist of the V₂ receptor in the same manner as AVP. However, other studies employing an inner medullar collecting duct cell line showed that both AVP and OT elicited dose-dependent increases in cAMP generation, although OT was less potent than AVP (EC₅₀ = 1.6 x 10^–8 M vs. 7.4 x 10^–10 M) (544). AVP-induced cAMP accumulation was blocked in the presence of a V₂ receptor antagonist but not by an OT receptor antagonist. On the other hand, OT-induced cAMP accumulation was reduced by the addition of an OT antagonist, while coincubation with the V₂ receptor antagonist had no effect. These results indicate that AVP and OT induce cAMP accumulation from a common ATP pool in inner medullar collecting duct cells and that separate AVP V₂ and OT receptor systems are involved, perhaps coupled to a common adenylate cyclase system (544). In addition to their peripheral effects, these peptides may also produce other effects that could complement their physiological action. Indeed, when injected into the CNS, AVP increases water intake (511). In contrast, the central administration of OT decreases salt intake (496, 500). The inhibitory role of OT in the control of sodium appetite has been supported by studies in OT knock-out mice showing that OT –/– mice display an enhanced salt appetite compared with OT +/+ mice after water deprivation (7, 411).

	III. THE RENIN-ANGIOTENSIN SYSTEM IN THE BRAIN

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A. General Aspects of the Brain Angiotensin System

Brain ANG II increases blood pressure, thirst, sodium appetite, AVP, and ACTH release. It causes sympathetic activation and decreases the baroreceptor sensitivity. Circulating ANG II has its access to the brain limited to the blood-brain barrier-free circumventricular organs. Thus the brain angiotensin system is separate and independent of blood-borne ANG II (445). The brain renin-angiotensin system acts on blood pressure regulation independently of the systemic renin-angiotensin system (RAS) by influencing the secretion of AVP and ACTH and by modulating the baroreceptor reflex and the sympathetic output. Indeed, all components of the RAS, including the precursor and enzymes required for the production and degradation of angiotensins, as well as the specific angiotensin receptors type 1 and type 2 have been identified in the brain (299, 445).

Ganten et al. (181) reported the first evidence for the presence of renin in the CNS of dogs, in tissue from the caudate nucleus. They examined renin activity and showed that aldosterone administration decreased the conversion of angiotensinogen to ANG II in the caudate nucleus; the authors further demonstrated that brain renin is not altered by bilateral nephrectomy (181, 182). Thereafter, the presence of renin in the brain was confirmed by radioimmunoassay (185) and by immunocytochemistry (70, 177). The very low levels of renin in the brain have made localization of its mRNA very difficult (126, 509); however, Lippoldt et al. (307) recently succeeded in demonstrating this in rat brain by in situ hybridization.

Other components of the RAS have also been localized to the brain. Thus ANG II has been demonstrated by immunocytochemistry and measured directly by radioimmunoassay, while angiotensinogen has been detected by Northern blotting (126, 180, 317, 406). Angiotensinogen is a critical component of the RAS, since it is the only known precursor of ANG II (361). Angiotensinogen is widely distributed in the brain, with a marked presence in the hypothalamus and midbrain (302, 316, 495), and its presence correlates with the distribution of angiotensin receptors (184, 488) and ANG II (403, 468).

There is disagreement over the cell types that express angiotensinogen (469). Angiotensinogen gene expression in astroglia has been reported in several studies (67, 240, 355, 495). Colocalization of angiotensinogen and glial fibrillary acidic protein, a glial-specific protein, further indicated the presence of angiotensinogen in the astrocytes (112, 495). However, other studies have shown that the angiotensinogen gene is expressed in both astrocytes and neurons, a finding consistent with multiple functions for brain angiotensinogen, including as a precursor for neuronal ANG II (238, 469, 570). For example, both astrocytes and neurons express angiotensinogen mRNA and secrete angiotensinogen in primary cell culture (286, 468, 519). It is not completely clear whether ANG II is formed intracellularly in neurons from endogenous angiotensinogen or if ANG II is synthesized outside the cell by angiotensin converting enzyme (ACE, an exoenzyme) and then taken up by neurons, thus forming a paracrine RAS (112, 405, 446, 469, 502). Brain levels of ANG II determined by radioimmunoassay and high-pressure liquid chromatography were found to be increased in rats submitted to bilateral nephrectomy, mainly in the hypothalamus and brain stem, suggesting that brain RAS has paracrine and autocrine functions independent of the endocrine function of circulating plasma angiotensin (526).

As in the periphery, brain ANG II is generated by sequential cleavage of the precursor angiotensinogen by renin, producing the inactive decapeptide ANG I, which is then converted to ANG II by ACE. Thereafter, ANG II is metabolized into ANG III, which is converted to ANG IV by aminopeptidases (445). In addition to its other functions, brain ANG II may possibly influence cognitive functions by acting on the specific angiotensin receptor type 1. ANG II and ANG III have the same affinity for type 1 (AT₁) and type 2 (AT₂) angiotensin receptors (401, 445). The distribution of ANG II-containing fibers has been demonstrated by immunohistochemical studies in the hypothalamus, SFO, limbic system, the medulla oblongata, sympathetic lateral column, caudate nucleus and putamen, as well as the spinal cord (176, 179, 217, 259, 306).

It has been suggested that ANG III may have an important role in the brain RAS (419), and it may be the case that ANG III is the effector of the biological actions of ANG II (28, 210, 558). Using a selective aminopeptidase A inhibitor that blocks the metabolism of ANG II, Zini et al. (577) showed that ANG III may exert a tonic control on the basal firing level of vasopressinergic neurons. ANG III has also been shown to have a positive influence over blood pressure as seen by the reduced pressor response to ANG II in rats pretreated with microinjections into the right lateral ventricle of a selective aminopeptidase A inhibitor (418). The dipsogenic response to ANG II is decreased by immunoneutralization of aminopeptidase A, suggesting that drinking behavior is also dependent on the action of ANG III (489, 557). Angiotensin IV has been shown to be involved in memory retention and neuronal development (359, 556). ANG IV binding sites in the human brain are similar to those found in guinea pig and monkey, and their distribution supports a role for ANG IV in the facilitation of memory retention and retrieval (76). Interestingly, in contrast to ANG II, ANG IV acts as a vasodilator and increases cerebral blood flow through an ANG IV (AT₄) receptor (283, 375). Angiotensin-(1–7), an endogenous bioactive peptide constituent of the RAS, is nondipsogenic in rats even in large doses and has an inhibitory effect on angiogenesis (322). It has been demonstrated that angiotensin-(1–7) has an excitatory action on some glial cells, increasing prostaglandin synthesis in astrocytes and glioma cells (145).

B. Functions of Brain Angiotensin in Thirst

A decrease in circulating blood volume due to hemorrhage or dehydration stimulates renin release from the kidney, which results in increased circulating levels of ANG II that acts on ANG II receptors in the SFO (497). Access of circulating ANG II to the brain is limited to those CVO structures, which lack the blood-brain barrier and interact with other nuclei in the maintenance of several homeostatic processes by neural and humoral mechanisms. Neuronal structures sensitive to ANG II were identified electrophysiologically and are present in the lamina terminalis, CVOs, and limbic areas. Furthermore, these regions contain immunoreactive neurons and binding sites for ANG II (153), and studies have shown that the area postrema, SFO, and OVLT are the sites of action for angiotensin within the brain. The area postrema is involved in the pressor action of angiotensin, whereas the SFO is concerned with drinking behavior, the pressor effect, and AVP secretion (325). The OVLT and adjacent tissue have also been suggested as a site for these three central effects of angiotensin (481). Lesions of the SFO and AV3V regions (OVLT, ventral median preoptic nucleus, periventricular preoptic nuclei, and periventricular nuclei) decrease the angiotensin-induced drinking and AVP release (133, 357, 481). Access of ANG II to the anterior ventral third ventricle appears to be essential for drinking (107, 225). Circulating ANG II derived from renal renin contributes to hypovolemic thirst and sodium appetite, acting with the mineralocorticoids and other hormones. Autoradiographic studies have identified ANG II receptors within the SFO (352, 353, 447), suggesting a role for circulating ANG II in regulating activity of the SFO. Dipsogenic responses to systemic administration of ANG II are observed in normovolemic rats (257); however, blood pressure affects the dipsogenic potency of ANG II (138, 493). There is a relationship between blood pressure control and drinking behavior, as shown by the inhibition of drinking stimulated by ANG II, hyperosmolality, or hypovolemia, in the presence of increased arterial pressure (139, 493). It is likely that pressure-induced inhibition of drinking in response to ANG II is mediated by cardiopulmonary and arterial baroreceptors (278, 285, 425).

The SFO is not the only site for the dipsogenic action of ANG II in the brain. Several other structures involved in thirst and sodium appetite are located inside the blood-brain barrier and cannot be stimulated directly by circulating ANG II, including the MnPO nucleus in the lamina terminalis, the PVN (250), preoptic area (134, 154), and the central gray of the midbrain (510) which receives projections from the preoptic area (153).

The role of ANG II formed locally in the brain in thirst and salt appetite was well described by Fitzsimons (153). In the presence of a hypovolemic stimulus there is an increase in circulating ANG II due to the low pressure in the renal artery, which stimulates renin secretion. Concomitantly, changes in blood volume influence inputs to the brain through the NTS, which causes ANG II release in the brain leading to the stimulation of thirst. However, chronic lesions of the NTS do not prevent the stimulation of thirst and salt appetite during plasma volume deficits induced by polyethylene glycol (PEG) treatment in rats (459). The authors suggested that hypovolemia-induced thirst may involve pathways that bypass the NTS or, alternatively, these afferent neural signals from the heart and circulating ANG II may together stimulate hypovolemic thirst in rats. Indeed, rats submitted to a chronic NTS lesion present an increased dipsogenic response to systemic ANG II compared with control rats (460).

Angiotensin II plays a critical role in dehydration-induced drinking in male rats, since microinjection of ANG II antiserum, but not normal rabbit serum, completely blocked this drinking. A lesser, delayed effect on dehydration-induced drinking was obtained with antisera against ANP, AVP, and OT. On the other hand, ANG II antiserum had no effect on the so-called prandial drinking that occurs with feeding (171).

C. Functions of Brain Angiotensin in Salt Intake

Several hormones (ANG II, AVP, OT, ANP, and mineralocorticoids) applied to the anterior hypothalamus of the rat modify neuronal activity and appear to be involved in the regulation of fluid and electrolyte balance (23, 381, 520, 521).

Angiotensin II-induced sodium appetite presents a longer latency than water intake, and this does not result from ANG II-induced natriuresis (71). It has been suggested that an active inhibitory system may exist that restrains NaCl intake until water has been replenished. Peptides or hormones with the opposite effect to that of ANG II on fluid and electrolyte balance, such as ANP, may attenuate the response to ANG II (23). It has also been demonstrated that intracerebroventricular injection of ANG II, preceded by an OT receptor antagonist, increases NaCl intake without significant changes in water intake, suggesting an inhibitory action of OT (53).

Lesions of the SFO have been shown to reduce sodium depletion-induced salt appetite, which is largely dependent on ANG II (368, 442, 523, 548). On the other hand, infusions of ANG II in the OVLT increase salt appetite without significant changes in the blood pressure. Disconnection of afferent and efferent fibers of the rostroventral region of the SFO abolished water intake during the infusion of ANG II into the femoral vein but failed to reduce salt appetite during an infusion of ANG II into the OVLT. These data suggest that the role of the OVLT in salt appetite induced by ANG II is independent of the SFO (151).

Mechanisms of action of ANG II on sodium intake involve not only neuron depolarization but also protein synthesis, cell growth, and long-term potentiation (153). There is an interaction between central ANG II and desoxycorticosterone acetate in sodium intake as revealed by the greater sodium appetite and shorter latency than with separate applications of each hormone (14, 71, 158, 231).

Circulating ANG II also contributes to the sodium appetite in rats as demonstrated by the decrease in sodium appetite after bilateral nephrectomy (155, 156). In addition, it has been found that there is an increase in plasma renin activity and c-Fos expression in the SFO of rats submitted to a water deprivation-induced sodium appetite even after rehydration, although before sodium intake (108). These results are consistent with a role for circulating ANG II in the control of sodium appetite. The effect of circulating ANG II on sodium appetite may occur by a direct action on CNS structures outside the blood-brain barrier (for review, see Ref. 153) or through stimulation of mineralocorticoid secretion, which could act on the basolateral or medial region of the amygdala to induce sodium appetite (148, 574). It should be pointed out that the effect of systemic ANG II on sodium intake shows species differences (153).

D. Angiotensin Receptors

Angiotensin II acts through specific cell membrane receptors termed AT₁ and AT₂ receptors (106). These are seven transmembrane domain G protein-coupled receptors that belong to the rhodopsin subclass. Angiotensin receptors have been cloned from several species, including humans (45, 372, 456, 512, 513). Most, if not all, of the central and peripheral actions of ANG II, such as vasoconstriction, stimulation of aldosterone secretion, facilitation of sympathetic transmission, and promotion of cell growth are mediated by the AT₁ receptor (142, 144). The function of the AT₂ receptor has not been well established, but it may play a role in cellular differentiation, apoptosis, and vasodilatation (92). Two other receptors, AT₃ and AT₄, have been proposed that may recognize other angiotensin peptide fragments, but their transduction mechanisms are unknown (63).

In humans, rabbits, and dogs, AT₁ appears to be a single receptor with no subtypes (106). However, in rat and mouse two highly homologous AT₁ receptor subtypes have been identified, termed AT_1A and AT_1B, which are 95% identical in their amino acid sequences and possess similar binding characteristics and mRNA tissue expression (244). Both AT_1A and AT_1B mRNAs have been reported to be widely expressed in rat tissues including the adrenal gland, kidney, heart, aorta, lung, liver, testis, pituitary gland, cerebrum, and cerebellum with a predominance of AT_1A mRNA expression, except in the adrenal and pituitary glands where AT_1B mRNA predominates (277). It appears that the increase in blood pressure induced by centrally administered ANG II occurs via the AT_1A receptor, whereas the drinking response requires the presence of AT_1B receptors (98). There is a high correlation between the distribution of AT₁ receptors and that of ANG II immunoreactive nerve terminals, suggesting that ANG II may be released from nearby synapses to activate AT₁ receptors (5).

In the adult human CNS, the distribution of AT₁ receptors has been determined using quantitative in vitro autoradiography. AT₁ receptors were found in the forebrain, midbrain, pons, medulla, and spinal cord as well as in the small and large arteries in the adjacent meninges and in the choroid plexus. Both AT₁ and AT₂ receptors are present in the molecular layer of the cerebellum (321). This distribution pattern of AT₁ receptors suggests that angiotensin may act as a neuromodulator or neurotransmitter in the human CNS to influence fluid and electrolyte homeostasis, pituitary hormone release, and autonomic control of cardiovascular function (3). A dense population of AT₁ receptors is present in the circumventricular organs allowing neural inputs from these organs to inform the brain of circulating levels of ANG II (338, 404, 488). ANG II binding sites also occur in the NTS, which has connections with vagal afferent terminals in the dorsal motor nucleus of the vagus, the rostral and caudal ventrolateral medulla, and intermediolateral cell column of the spinal cord. The presence of ANG II receptors in the NTS supports the involvement of this peptide in the modulation of cardiovascular control and autonomic function (5, 329).

Expression of AT₂ receptor mRNA has been detected by in situ hybridization in the lateral septum, several thalamic nuclei, the subthalamic nucleus, the medial geniculate nuclei, the nucleus of the optic tract, the interposed nucleus of the cerebellum, and the inferior olive in adult rat brain (259, 299). The distribution of AT₂ receptors is very restricted in the human brain, but they are present in the molecular layer of the cerebellum. Although the physiological role of this receptor in the adult CNS is unclear, some of the AT₁-mediated effects may be enhanced by blockade of AT₂ receptors in the brain, suggesting that the central AT₂ receptor can exert an inhibitory control on AT₁ receptor-mediated actions in the brain (227). Deletion of the mouse gene for the AT₂ receptor subtype led to hypersensitivity to pressor and antinatriuretic effects of ANG II in vivo, reinforcing the suggestion that the AT₂ receptor subtype may counter-act some of the biological effects of AT₁ receptor signaling (60).

E. Interactions With Other Hormones

The interaction between the central ANG II and AVP systems is well established (269, 369). ANG II activates AVP neurons, as demonstrated by in vivo studies using c-Fos and AVP mRNA expression and AVP secretion (100, 226, 314, 402). Moreover, disturbances of hydromineral balance, such as dehydration or osmotic stimuli, have been shown to increase ANG II receptor density, ANG II AT_1A receptor mRNA, and AVP mRNA in the central nervous system (377, 454).

Intracerebroventricular injection of ANG II has been shown to elicit an increase in plasma AVP (323). The results obtained by Antunes et al. (16) demonstrated that both AT₁ and V₁ receptors within the SON might be involved in water and sodium intake induced by the activation of ANG II receptors within the medial septal area. Intracerebroventricular injection of ANG II also induces OT secretion, and it appears to depend on the activation of cyclooxygenase and production of prostaglandins (262).

As stated above, the actions of ANG II on water and salt intake and excretion are antagonized by ANP and OT. Within the brain ANP inhibits ANG II- and dehydration-induced drinking (22). -Adrenergic agonists block ANG II-induced drinking by stimulating the release of ANP from ANP-secreting (ANPergic) neurons within the brain (42). Previous injection of phenylephrine (an ₁-adrenergic agonist) or clonidine (an ₂-adrenergic agonist) into the anterior portion of the third ventricle significantly reduced ANG II-induced water intake (96). Injection of adrenergic agonists into the AV3V region also induces a significant increase in plasma ANP concentration and in ANP content of the olfactory bulb, AV3V, medial basal hypothalamus, and median eminence (42). These results suggest that the inhibitory effect of both -adrenergic agonists on ANG II-induced water intake can be explained, at least in part, by the increase in ANP content and its presumed release from these neural structures.

It has been shown that central OT administration decreases ANG II-induced NaCl intake (53). In addition, systemic and central administration of ANG II stimulates pituitary release of OT (143, 291). On the other hand, intracerebroventricular injections of an OT receptor antagonist enhance ANG II-induced NaCl intake without modifying ANG II-induced water intake, suggesting that OT inhibits NaCl intake in this experimental condition (53, 501, 499). Thus, within the brain, ANP and OT act together to inhibit drinking and sodium intake. They also act in concert on the heart and vasculature to produce a rapid decrease in circulating blood volume. Finally, blood volume is returned to normal by the combined natriuretic action of circulating OT on the kidney, via activation of NO production with consequent cGMP release, and by circulating ANP acting on its receptors to also release cGMP. cGMP, in turn, closes sodium channels, thereby having a natriuretic effect and finally returning blood volume to normal. At least with regard to water and salt intake, but possibly also with regard to the other actions of OT and ANP, these effects may be opposed by ANG II (486).

	IV. AUTONOMIC NERVOUS SYSTEM AND BODY FLUID METABOLISM

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A. Role of the Sympathetic Nervous System Innervation of the Kidney in Sodium Excretion