Angiotensin II and nitric oxide: a question of balance

Regulatory Peptides 81 (1999) 1–10

Invited review

Angiotensin II and nitric oxide: a question of balance a b b, Lesley J. Millatt , Emaad M. Abdel-Rahman , Helmy M. Siragy * a

b

Department of Anesthesiology, University of Virginia, Health Sciences Center, Charlottesville, VA 22908, USA Department of Internal Medicine, University of Virginia, Health Sciences Center, Charlottesville, VA 22908, USA Received 15 January 1999

Abstract The vasoconstrictor peptide angiotensin II (Ang II) and the endogenous vasodilator nitric oxide (NO) have many antagonistic effects, as well as influencing each other’s production and functioning. In the short-term, Ang II stimulates NO release, thus modulating the vasoconstrictor actions of the peptide. In the long-term, Ang II influences the expression of all three NO synthase (NOS) isoforms, while NO downregulates the Ang II Type I (AT 1 ) receptor, contributing to the protective role of NO in the vasculature. Within the cardiovascular system, Ang II and NO also have antagonistic effects on vascular remodeling and apoptosis. In the kidney, the distribution of the NOS isoforms coincides with the sites of the components of the renin-angiotensin system. NO influences renin secretion from the kidney, and NO-Ang II interactions are important in the control of glomerular and tubular function. In the adrenal gland, NO has been shown to affect Ang II-induced aldosterone synthesis, while in the brain NO appears to influence Ang II-induced drinking behavior, although conflicting data have been reported. In this review, we focus on the diverse ways in which Ang II and NO interact, and on the importance of maintaining a balance between these two important mediators.  1999 Elsevier Science B.V. All rights reserved. Keywords: Angiotensin II; Nitric oxide; Cardiovascular system; Kidney; Adrenal; Central nervous system

1. Introduction Since the identification of nitric oxide (NO) as an endogenous vasodilator in 1987, it has become increasingly apparent that a delicate balance exists between the vasoconstrictor angiotensin II (Ang II) and NO. Within the vasculature, Ang II and NO are ideally situated to interact with each other and to influence each other’s functions. Endothelial cells generate both Ang II and NO, and vascular smooth muscle cells are important targets of both molecules. Vascular smooth muscle vasoconstricts in response to Ang II, and vasodilates in response to NO. Moreover, these two molecules appear to have antagonistic effects not only on vascular tone, but also in such diverse areas as vascular remodeling, renal function, and behavior. In this article, we will review the ways in which Ang II

*Corresponding author. Tel.: 1 1-804-924-5510; fax: 1 1-804-9823626.

and NO interact with each other in multiple tissues, including effects on each other’s production.

2. The renin-angiotensin system The traditional view of the renin-angiotensin system (RAS) is of a typical endocrine system. Circulating renin, produced predominantly by the juxtaglomerular cells of the kidney, acts on circulating angiotensinogen of hepatic origin, to produce the inactive decapeptide angiotensin I (Ang I) within the plasma (Fig. 1). This decapeptide is cleaved to the octapeptide Ang II by angiotensin-converting enzyme (ACE), present on the luminal surface of endothelial cells throughout the vasculature. In addition to this classical endocrine system, a local RAS exists within specific organs. A significant amount of tissue Ang II is also formed through non-ACE pathways (Fig. 1). The actions of Ang II are mainly mediated via two types of receptors [1] known as AT 1 and AT 2 (Table 1). The

0167-0115 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 99 )00027-0

L. J. Millatt et al. / Regulatory Peptides 81 (1999) 1 – 10

2

Fig. 1. Pathways for formation of angiotensin peptides and for non-ACE formation of angiotensin II.

Table 1 Properties of the angiotensin II receptors

Affinity Selective antagonists Structure G-protein coupled Distribution Function

AT 1

AT 2

Ang II . Ang III . Ang I losartan, valsartan 7-transmembrane domains yes cardiovascular system, kidney, adrenal gland, brain vasoconstriction, aldosterone synthesis, thirst, cardiovascular hypertrophy and remodeling

Ang III . Ang II . Ang I PD 123319, PD 123177 7-transmembrane domains possibly fetal tissues, adult brain, kidney, heart, uterus, endothelial cells apoptosis, inhibition of proliferation, regulation of NO production

AT 1 receptor has a widespread distribution, being present in such organs as the heart, kidney, adrenal gland, and brain. The receptor is also expressed by vascular smooth muscle cells, where it mediates the vasoconstrictor and proliferative actions of Ang II. Indeed, the vast majority of the physiological and pathophysiological roles of Ang II have been shown to occur via the AT 1 receptor. The AT 2 receptor was discovered more recently, and much less is known about its functions and signal transduction pathways than for the AT 1 receptor. In fetal tissues, the AT 2 receptor is highly and ubiquitously expressed, but expression in many organs is dramatically

reduced after birth [2,3]. However, expression levels are maintained in the adult adrenal gland, brain, uterus, and ovary, and the receptor is also expressed by endothelial cells [4]. Observations of increased AT 2 receptor expression in myocardial infarction [5] and skin wounds [6], as well as its much higher level of expression in fetal compared to normal adult tissues, have led to speculation as to a possible role for the AT 2 receptor in cell growth, development, and / or differentiation. The physiological roles of the AT 2 receptor remain the subject of intense scrutiny. Functions demonstrated to date include apoptosis [7], suppression of proliferation [4], stimulation of renal

L. J. Millatt et al. / Regulatory Peptides 81 (1999) 1 – 10

3

Table 2 Properties of the three major NOS isoforms nNOS

iNOS

eNOS

Prototypical Source Monomer Molecular Weight Vmax (mmol / mg / min) Amount of NO Produced Dependence on Ca 21 / calmodulin Subcellular Localization

Neuronal | 160 kDa 0.3–3.4 pmoles Yes Cytoplasm

Macrophage | 130 kDa 0.9–1.6 nmoles No Cytoplasm

Expression

Constitutive but regulated

Inducible

Endothelium | 135 kDa 0.015 pmoles Yes Plasma membrane Constitutive but regulated

NO production [8], and inhibition of NO release by neuronal cells [9].

3. The nitric oxide signaling pathway The endogenous formation of NO is catalyzed by the enzyme NO synthase (NOS) [10]. Nitric oxide synthase catalyzes the oxidation of one of the terminal guanidino groups of L-arginine to produce NO, with the stoichiometric formation of L-citrulline. Molecular cloning and sequence analysis has led to the identification of three broad categories of NOS isoenzymes, referred to in this review as neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) (Table 2). The neuronal NOS isoform, also known as Type I NOS, has a widespread distribution in both the central and peripheral nervous systems, as well as in a number of non-neuronal cell types. The inducible NOS isoform, also known as Type II NOS, was initially identified in activated macrophages, but has since been found to be expressed by virtually all nucleated cells if they are subjected to the appropriate stimuli. The endothelial NOS isoform, also known as Type III NOS, was first identified in vascular endothelial cells, and as such has a ubiquitous distribution, although its expression has also been demonstrated in several non-endothelial cell types. The primary cellular target for NO is the activation of the enzyme soluble guanylyl cyclase, leading to increased formation of the second messenger cyclic guanosine 39,59monophosphate (cGMP). This increased cGMP production mediates many of the biological actions of NO by activating cGMP-dependent protein kinase (PKG).

4. Ang II–NO interactions

4.1. Effect of Ang II on NO production A number of studies have shown that Ang II stimulates NO release. The first such study investigated the mechanism whereby Ang II acts within seconds to increase cGMP production in neuroblastoma cells [11]. This effect

of Ang II could be blocked by treatment of the cells with the NOS inhibitor N G -monomethyl-L-arginine ( L-NMMA), demonstrating a role for Ang II activation of NOS. Subsequently, others investigated the ability of Ang II to stimulate NO production in human proximal tubular cells, and found that this effect was not inhibited by either AT 1 or AT 2 receptor antagonists [12]. In contrast, Ang II was recently shown to act via the AT 1 receptor to activate the NO-cGMP pathway in isolated rat proximal tubules [13]. Similarly, the AT 1 receptor has been implicated as the mediator of Ang II-induced NO production in rat aortic endothelial cells [14], since this was inhibited by the AT 1 receptor antagonist losartan. It is likely that this stimulatory effect of Ang II on NOS activity occurs via increased intracellular calcium [14], although others have implicated kinin formation in the mechanism of Ang II stimulation of NO release in coronary vessels [15]. It has been suggested that the NO-releasing effect of Ang II may modulate its direct vasoconstrictor effect on smooth muscle cells, and that the vascular effects of Ang II will depend on which cell type (endothelial or vascular smooth muscle) it interacts with first [14]. Several recent in vivo studies have demonstrated Ang II induction of NO production in the rat kidney. The intravenous infusion of Ang II into anesthetized rats resulted in a significant increase in urinary nitrite and nitrate (the end-products of NO metabolism) content [16]. These findings were extended to conscious rats by Siragy and Carey [8], who showed that the infusion of Ang II resulted in a two-fold increase in renal cortex interstitial fluid cGMP content. This effect was significantly attenuated by the co-administration of either the AT 2 receptor antagonist PD123319 or the NOS inhibitor N G -nitro-L-arginine methyl ester ( L-NAME). In addition, the effect of Ang II was partially, but not completely, blocked by the nNOSspecific inhibitor 7-nitroindazole. Taken together, these results suggest that Ang II acts via the AT 2 receptor to increase renal NO production, and that this effect is mediated in part by the nNOS isoform [8]. In a recent study which directly measured NO production in the renal interstitial fluid by oxyhemoglobin-NO trapping, Ang II infusion was found to increase NO concentrations by 85% in the cortex and 150% in the medulla, and this was

4

L. J. Millatt et al. / Regulatory Peptides 81 (1999) 1 – 10

proposed to play a protective role in maintaining medullary blood flow [17].

4.2. Effects of NO on Ang II receptor expression Nitric oxide-induced down-regulation of Ang II receptors was first reported by Cahill et al. [18]. Treatment of rat vascular smooth muscle cells (VSMC) with NO donors or with lipopolysaccharide to induce iNOS expression caused an inhibition of Ang II binding to the cells, without altering receptor affinity. However, treatment of the cells with cGMP analogs had no significant effect on Ang II binding, suggesting that NO regulates Ang II receptors through a cGMP-independent mechanism [18]. Ichiki et al. [19] investigated the mechanism of this effect in rat VSMC, and demonstrated a profound inhibition of AT 1 receptor mRNA expression in cells exposed to a NO donor. The inhibitory effect of NO on AT 1 expression occurs at the transcriptional level [19]. However, the transcription factor(s) which mediate this effect remain to be identified. In the rat adrenal gland, a one-week treatment with the NOS inhibitor L-NAME enhanced both the mRNA expression and the number of AT 1 receptors, but did not influence AT 2 receptor number [20]. In contrast, one-week L-NAME treatment increased the number of both AT 1 and AT 2 receptors in the rat heart, with both receptor sub-types returning to baseline levels after 4 weeks of L-NAME treatment [21]. It therefore appears that NO may affect the expression of the Ang II receptor sub-types in a tissue-specific manner. If down-regulation of the AT 1 receptor is the predominant effect of NO, this could well be added to the growing list of anti-atherogenic and anti-hypertensive properties of NO.

4.3. Ang II effects on NOS expression 4.3.1. iNOS The effect of Ang II on the expression of the iNOS gene in vitro appears to be cell type-specific. Nakayama et al. [22] first reported an inhibitory effect of Ang II on cytokine-induced iNOS mRNA and protein expression and NO production in rat VSMC. This effect was mediated via the AT 1 receptor, and possibly involved protein kinase C [22]. In contrast, the same group reported that Ang II augments NO synthesis in cytokine-stimulated, but not in unstimulated, rat cardiac myocytes [23]. The increased NO production was accompanied by an increase in iNOS mRNA expression, and could be blocked by an AT 1 receptor antagonist or by a protein kinase C inhibitor. Similar to the results obtained with VSMC, other groups have reported an inhibitory effect of Ang II on iNOS expression in rat astroglial cultures [24], in the isolated rat

aorta [25], and in an immortalized murine proximal tubular cell line [26]. Ang II produced endogenously by the endothelium also has an inhibitory effect on iNOS expression [25]. In contrast, rats maintained on a low-salt diet (associated with increased circulating Ang II levels) demonstrated more intense immunostaining for iNOS in the kidney than rats maintained on a high-salt diet [27]. This effect of dietary salt restriction did not seem to be mediated via AT 1 receptors, since losartan treatment increased the immunostaining even further [27].

4.3.2. nNOS Elevated plasma Ang II levels are associated with increased immunostaining for nNOS in the macula densa of the rat kidney [28]. In addition, nNOS staining was almost completely absent in rats treated with desoxycorticosterone acetate (DOCA) plus high-sodium diet, a combination that resulted in a dramatic reduction in plasma Ang II levels [28]. Rats which were treated with an AT 1 receptor antagonist alone showed no change in nNOS expression, despite markedly elevated levels of plasma Ang II, suggesting that Ang II acts via the AT 1 receptor to increase nNOS expression in the macula densa [28]. Similarly, total renal nNOS mRNA levels were significantly increased in rats fed a low-sodium diet, but not in rats treated with the AT 1 receptor antagonist losartan [29]. In contrast, Mattson and Higgins [30] reported an increase in nNOS protein levels in the inner medulla of rats fed a high-sodium diet compared to rats fed a low-sodium diet, with no change in nNOS expression in the outer medulla, the cortex, or the aorta. The reason for this discrepancy is unclear, although it may be related to the duration of salt loading (ten days [29] versus three weeks [30]).

4.3.3. eNOS Olson et al. [31] first demonstrated a stimulatory effect of Ang II on eNOS mRNA, protein, and NO production in cultured bovine pulmonary artery endothelial cells. Interestingly, no effect of Ang II on eNOS expression was found in parallel studies using bovine coronary artery endothelial cells [31]. The authors proposed that this effect might be a protective mechanism whereby low pulmonary artery pressures are maintained in systemic hypertension. In Ang II infusion studies in rats, Hennington et al. [32] reported that an acute infusion of 110 min increased kidney eNOS mRNA levels, with no effect on eNOS protein levels. In contrast, a chronic infusion of ten days increased eNOS protein levels, but had no effect on eNOS mRNA levels [32]. Since this study was performed with whole-homogenized kidneys, the localization of these changes within the kidney remains to be determined. In addition, neither of these studies [31,32] addressed the question of which Ang II receptor mediates the changes in eNOS expression observed in response to Ang II.

L. J. Millatt et al. / Regulatory Peptides 81 (1999) 1 – 10

4.4. Ang II-NO interactions in the cardiovascular system

4.4.1. Vascular remodeling Vascular remodeling is characterized by smooth muscle cell hypertrophy and extracellular matrix synthesis, leading to medial thickening and luminal narrowing, and an increase in the wall-to-lumen ratio. Within the cardiovascular system, both Ang II and NO play major roles in the vascular remodeling process. Ang II is known to be a potent stimulus for vascular smooth muscle hypertrophy. NO has been implicated as an anti-proliferative agent, although some studies suggest that it may be a mitogenic factor for vascular smooth muscle cells [33]. Nevertheless, Ang II and NO generally appear to have opposing effects on vascular remodeling. Therefore, commonly-studied models of vascular remodeling involve either elevated Ang II levels or reduced NO levels achieved by the inhibition of NO production. Acute administration of the NOS inhibitor L-NAME causes increased blood pressure. Chronic L-NAME administration additionally results in the characteristic changes associated with vascular remodeling, as well as myocardial hypertrophy and end-stage organ damage [34,35]. The acute L-NAME response has been attributed to reduced NO production, while more complex mechanisms are involved in the chronic response. In particular, the RAS is activated during chronic L-NAME administration, with increased plasma renin activity [35] and cardiac ACE activity [36] both reported. The importance of this RAS activation to the pathogenesis of chronic NOS inhibition has been shown in a number of studies. Michel et al. [37] reported that the administration of the ACE inhibitor quinapril to rats one month after commencing L-NAME treatment reversed L-NAME-induced hypertension, prevented renal damage, and significantly reduced the mortality associated with long-term L-NAME administration. Takemoto et al. [38] showed that the co-treatment of rats with L-NAME and the ACE inhibitor temocapril additionally prevented the L-NAME-associated vascular remodeling of the coronary arteries. The important role of increased ACE activity in L-NAME-induced pathologies was demonstrated by the significant positive correlation between the wall-to-lumen ratio of the aorta and aortic ACE activity, and between left ventricular weight and cardiac ACE activity [38]. In a recent study, chronic L-NAME administration induced the expression of iNOS and the adhesion molecules VCAM-1 and ICAM-1 in the aorta, indicating an inflammatory response [39]. These changes in the vascular wall were associated with inflammatory cell infiltration and fibrosis in the heart, all of which were prevented by the co-administration of an AT 1 receptor antagonist [39]. Taken together, these results suggest that L-NAME-induced vascular remodeling is associated with an increase in cardiac ACE activity, leading to increased activation of AT 1 receptors and the

5

resultant inflammatory and structural changes in the coronary vasculature and the myocardium. The mechanism by which ACE is activated during chronic L-NAME administration remains relatively unexplored. However, some insight may be gained from the recent finding that NO competitively inhibits ACE activity both in a biochemical assay and in an isolated rat carotid artery study of endothelial-derived NO [40]. Additionally, the same group demonstrated that ACE mRNA expression and activity in the rat carotid artery are rapidly increased in vivo after endothelial denudation [41]. These data suggest that one of the physiological protective roles of NO may be to reduce the expression and activity of ACE, thus preventing the formation of excess Ang II.

4.4.2. Apoptosis Many studies have been published concerning the effects of Ang II and NO on apoptosis. The predominant view is that Ang II induces apoptotic cell death, as demonstrated in both adult [42] and neonatal [43] rat ventricular myocytes, in R3T3 mouse fibroblasts and rat pheochromocytoma cells [7], in rat vascular smooth muscle cells [44], and in mouse embryo fibroblasts [45]. In addition, p53, a pivotal regulator of apoptosis, was shown to induce myocyte apoptosis by activating the local RAS [46,47]. In myocytes, Ang II induces apoptosis via activation of the AT 1 receptor [42,43,46], while Ang II-induced apoptosis in cell types which lack the AT 1 receptor has been shown to occur via the AT 2 receptor [7,44,45]. For NO, both pro-apoptotic and anti-apoptotic effects have been reported, even within the same cell type, and the consensus of opinion is that the effect observed is very much dependent on NO concentration. In general, low concentrations of NO such as produced by eNOS and nNOS are associated with cGMP-mediated protection against apoptosis, while the pathophysiological concentrations of NO produced by iNOS tend to induce apoptosis via a cGMP-independent mechanism [48]. To date, only two publications have directly investigated the relative roles of Ang II and NO in apoptosis, and these provided apparently contradictory results. Pollman et al. [49] were the first to test the hypothesis that VSMC apoptosis is regulated by the balance between Ang II and NO. Using cultured rabbit aortic VSMC, they found that NO donors caused a dose-dependent increase in apoptosis, which was mimicked by a cGMP analog and abolished by an inhibitor of PKG, demonstrating the importance of the NO-cGMP signaling pathway in this effect. Additionally, co-incubation with Ang II was found to markedly reduced the apoptotic effect of NO donors, as well as that of a PKG agonist, suggesting that the effect of Ang II occurs downstream of cGMP [49]. In direct contrast to these findings, Dimmeler et al. [50] recently reported that Ang II induces apoptosis in human umbilical vein endothelial cells (HUVEC), an effect which could be mimicked by an AT 2 receptor agonist. Interest-

L. J. Millatt et al. / Regulatory Peptides 81 (1999) 1 – 10

6

ingly, the authors found no effect of Ang II, at the same doses, on VSMC apoptosis. However, NO donors were found to significantly reduce Ang II-induced apoptosis in HUVEC. This anti-apoptotic effect seemed to be cGMPindependent, since a cGMP analog failed to reduce Ang II-induced apoptosis [50]. Two explanations were put forward by Dimmeler et al. for the apparent discrepancy between their results [50] and those of Pollman et al. [49]. Firstly, Pollman et al. [49] used cultured VSMC, which express only AT 1 and no AT 2 receptors. Since Ang II was shown to induce endothelial cell apoptosis via the AT 2 receptor [50], it is therefore not surprising that Ang II had no apoptotic effect in VSMC. In addition, Dimmeler et al. [50] suggested that the dose of NO donors used by Pollman et al. [49] was sufficiently high to induce apoptosis via a direct DNA-damaging mechanism. However, in the absence of further studies using lower doses of NO donors, it still remains possible that the effects of Ang II and NO on apoptosis are opposite in VSMC and endothelial cells.

4.5. Ang II-NO interactions in the kidney All the components of the RAS are present within the kidney [3,51–58] (Table 3). Earlier studies failed to detect AT 2 receptor mRNA in the adult rat and mouse kidney, but detected it in embryonic renomedullary interstitial cells [3,53]. More recently, Ozono et al. [55] showed that AT 2 receptor protein, while expressed in the fetal and newborn rat kidney, diminishes in adult life, but is re-expressed in the adult rat in response to sodium depletion. In the kidney, the three NOS isoenzymes are distributed in proximity to the sites of the RAS components (Table 3), which may explain the interactions between Ang II and NO. nNOS is found in glomeruli and vasculature, as well as the macula densa, collecting duct, and inner medullary thin limb [59–61]. Both immunohistochemistry and in situ hybridization confirmed the presence of nNOS in the macula densa in rats, rabbits, and humans [61,62]. mRNA for iNOS in rats was found primarily in the thick ascending limb and intercalated cells of the inner medullary collecting ducts [61]. eNOS was found to be expressed in Table 3 Localization of RAS components and NOS isoforms within the kidney

Macula densa Renal vasculature Vasa recta Glomerulus Proximal tubule Medullary thick ascending limb Collecting duct

Renin-Angiotensin System

NOS Isoforms

Renin /AT 1 AT 1 /AT 2 AT 1 AT 1 /AT 2 Angiotensinogen /Ang I / Ang II /AT 1 /AT 2 AT 1

nNOS nNOS / eNOS / iNOS eNOS / iNOS nNOS / eNOS eNOS / iNOS

AT 1

nNOS / iNOS

eNOS / iNOS

glomeruli, interlobular arterioles, and, to a lesser extent, in arcuate vessels [61]. The presence of nNOS in the macula densa suggests that NO may influence the secretion of renin from the kidney. In most instances, NO is an enhancer of renin secretion, acting via cGMP inhibition of cyclic adenosine 39,59monophosphate (cAMP) degradation. Occasionally, the stimulatory effect of NO on renin secretion may switch to an inhibitory one, mediated via PKG activity. However, the factor(s) responsible for this switch are still not clear [63]. A major area of interaction between Ang II and NO in the kidney is the control of arteriolar and glomerular function. Ang II increases afferent and efferent arteriolar resistance as an autoregulatory response to increased systemic blood pressure. The Ang II effect is greater on the efferent arterioles, resulting in an increase in glomerular capillary pressure [64]. On the other hand, NO plays a major role in the maintenance of renal perfusion and glomerular filtration in the normal kidney, by exerting a tonic dilator influence principally on the afferent arterioles of the superficial glomeruli [59,60]. Systemic administration of NOS inhibitors produces dose-dependent and prolonged afferent arteriolar vasoconstriction [65]. This vasoconstriction causes reductions in renal plasma flow and glomerular filtration rate, and may be attributed to the unopposed vasoconstrictor actions of Ang II. Administration of the AT 1 receptor antagonist losartan prevented the effects of the NOS inhibitor L-NMMA on rat nephron plasma flow and efferent resistance, while having no effect on baseline glomerular hemodynamics [66]. Furthermore, Ito et al. [67] demonstrated that, in isolated microperfused rabbit glomeruli, L-NAME pretreatment of the glomerular vasculature augmented the vasoconstrictor action of Ang II in the afferent but not the efferent arteriole, indicating the selective modulation of afferent arteriolar Ang II actions by NO. Interactions between Ang II and NO are also important in controlling tubular function. Ang II can produce antinatriuresis by enhancing sodium reabsorption and decreasing sodium excretion [68]. NO, on the other hand, acts as a natriuretic factor. Strong evidence suggests that NO has direct tubular actions to inhibit Na 1 reabsorption in the proximal tubule and collecting duct by inhibition of Na 1 / H 1 exchange and Na 1 –K 1 –ATPase [69]. Whereas NO donors induce natriuresis, NOS inhibition can promote sodium retention, acting similarly to Ang II [70]. AT 1 receptor blockade by losartan prevented the changes in tubular reabsorption associated with L-NMMA, suggesting that the decrease in proximal tubular reabsorption during L-NMMA treatment is Ang II-dependent [66]. Local NO was also shown to antagonize Ang II actions with respect to the regulation of tubuloglomerular feedback (TGF) responsiveness. NOS inhibitors strongly potentiated both TGF-dependent and TGF-independent actions of Ang II on the preglomerular vasculature [71].

L. J. Millatt et al. / Regulatory Peptides 81 (1999) 1 – 10

Overall, these data clearly suggest an important interaction between Ang II and NO in controlling both glomerular and tubular functions.

4.6. Ang II–NO interactions in the adrenal gland One of the most important physiological roles of Ang II is the stimulation of the adrenal cortex to synthesize and secrete aldosterone, a major regulator of renal salt and water excretion. In addition, supraphysiological levels of Ang II increase catecholamine release from the adrenal medulla by depolarizing chromaffin cells. Within the rat adrenal cortex, 80–90% of Ang II receptors are of the AT 1 sub-type, and 10–20% the AT 2 sub-type, while in the medulla the reverse is true. However, in the human adrenal, AT 1 receptors predominate in both the cortex and the medulla, and the AT 1 receptor mediates all the known functions of Ang II in the adrenal gland. In addition to circulating Ang II, a local RAS has been found within the adrenal gland. Renin and Ang II are located predominantly in the zona glomerulosa steroid-producing cells, indicating a possible role for local Ang II as a regulator of aldosterone secretion [72]. The expression of the eNOS and nNOS isoforms has been identified within the adrenal gland. The adrenal is a highly vascularized organ, and it is likely that endothelialderived NO is responsible for the tonic vasodilatory effect of NO within the gland [73]. In addition, eNOS expression has been identified in freshly isolated adrenal cortical glomerulosa cells [74]. Nitric oxide has been shown to have an inhibitory effect on the basal release of catecholamines from the adrenal gland, although the NOS isoform responsible was not identified [75]. A number of studies have examined the effect of either endogenous or exogenous NO on Ang II-stimulated aldosterone synthesis in the adrenal cortex. The NOS inhibitor L-NAME was found to markedly inhibit Ang II-induced aldosterone secretion, without affecting the basal aldosterone secretion rate, in nephrectomized rats [76]. This study therefore suggested that endogenous NO plays a role in mediating Ang II-induced aldosterone secretion. Similar results were reported by others using the NOS inhibitor L-NMMA in isolated perfused rat adrenal glands stimulated with Ang II [77]. However, these results have not been confirmed by in vitro studies. A number of NO donors were shown to inhibit Ang II-induced, but not basal, levels of aldosterone synthesis in cultured rat and human adrenal glomerulosa cells [74]. Surprisingly, LNMMA had no effect on Ang II-induced aldosterone synthesis, despite strong eNOS expression in these cells, indicating that endogenous NO may not play an inhibitory role under normal physiological conditions [74]. In the most recent study, the NO donor DETA / NONOate caused a dose dependent increase in cGMP production in bovine adrenal glomerulosa cells, and attenuated Ang II-induced aldosterone synthesis over the

7

same concentration range [78]. However, the inhibitory effect of DETA / NONOate could not be blocked by ODQ, a selective inhibitor of soluble guanylyl cyclase, despite the complete inhibition of cGMP production [78]. The authors therefore suggested that NO inhibits aldosterone synthesis by a direct effect on the activity of the enzymes involved in steroidogenesis, similar to a previously observed effect of NO on testosterone synthesis [79]. Therefore, there seems to be a discrepancy between results obtained from in vivo and in vitro studies concerning the effect of NO on Ang II-induced aldosterone synthesis. Further work will be necessary to determine the reason for this discrepancy.

4.7. Ang II–NO interactions in the central nervous system Circulating Ang II is unable to cross the blood-brain barrier, meaning that its actions in the central nervous system (CNS) are limited to sites outside this barrier, such as the circumventricular organs. However, in addition to circulating Ang II, the CNS may be exposed to Ang II formed by the local RAS. The expression of genes for renin, angiotensinogen, and ACE have all been localized within the CNS, and Ang I, II, and III have been extracted from the brains of nephrectomized animals. In the brain, the highest concentrations of AT 1 receptors are present in regions associated with the classical functions of Ang II, such as the regulation of blood pressure and fluid and electrolyte homeostasis [80]. The mammalian brain also contains AT 2 receptors, although little is known about their function. In NOS enzyme activity assays, the brain is found to express higher levels of activity than any other tissue. Indeed, NOS expression has been identified in all areas of the brain, with nNOS (expressed by neurons and astrocytes) and eNOS (expressed by endothelial cells) being the predominant isoforms under normal conditions. Within the cerebral circulation, NO generated by eNOS acts as a vasodilator, and is believed to play a protective role in maintaining blood flow during cerebral ischemia [81]. NO produced by the nNOS isoform acts as a neurotransmitter, and plays a role in processes such as learning, memory, and nociception. Due to the rapidly emerging role of NO in the CNS, the effect of central NO on Ang II-induced drinking behavior has recently been examined, but contradictory results were obtained. One study investigated the effect of intracerebroventricular (i.c.v.) injection of the NO precursor L-arginine on drinking induced by either water deprivation or i.c.v. injection of Ang II [82]. L-arginine was found to dose-dependently reduce water intake in both cases, and this effect could be reversed in water-deprived rats by co-treatment with the NOS inhibitor L-NAME. However, another study showed the opposite effect, namely that i.c.v. injection of L-NAME antagonized Ang II-induced drink-

8

L. J. Millatt et al. / Regulatory Peptides 81 (1999) 1 – 10

ing, and that this effect was reversed by co-injection of L-arginine [83]. Additionally, this study demonstrated that i.c.v. injection of methylene blue, a non-specific inhibitor of soluble guanylyl cyclase, inhibited Ang II-induced drinking, suggesting the involvement of cGMP. It is difficult to reconcile the opposing viewpoints concerning the role of NO in Ang II-induced drinking which are supported by these two reports. Clearly, further in-depth studies are required to elucidate the role of NO, and to delineate the pathways involved. It was recently shown that the excitatory amino acid N-methyl-D-aspartate (NMDA) stimulates the release of NO from neuronal PC12W cells, and that Ang II reduces this effect of NMDA [9]. This effect of Ang II could be attenuated by pre-treatment of the cells with the AT 2 receptor antagonist PD123319, indicating a role for the AT 2 receptor in modulating NMDA-induced NO production [9]. In this way, the AT 2 receptor may be a mediator of protection from NMDA-induced cytotoxicity and excessive NO production.

5. Conclusions The Ang II and NO signaling pathways interact with each other in many ways (Fig. 2). Ang II acts at both AT 1 and AT 2 receptors to stimulate the production of NO. The NO thus produced has several actions which oppose those of Ang II acting via the AT 1 receptor. In addition, NO down-regulates the expression of the AT 1 receptor, such that a greater proportion of Ang II acts via the AT 2 receptor. In this way, the multiple functions of Ang II and NO are finely balanced, with NO apparently produced as a protective mechanism to counteract Ang II action at the AT 1 receptor. Clearly, any disease process which disrupts the delicate balance between Ang II and NO, for example by causing changes in NOS expression or Ang II receptor number,

Fig. 2. Schematic representation of angiotensin II–NO interactions (see text).

will potentially affect such wide-ranging functions as blood pressure control, renal function, and cellular proliferation. Therefore, a major challenge for the next century will be to develop a deeper understanding of the multiple interactions which occur between Ang II and NO, and of the consequences of such interactions in health and disease.

Acknowledgements This work was supported by American Heart Association Fellowship F98266V (to LJM), and by grants HL47669 and HL-57503 from the National Institutes of Health (to HMS). Dr. Siragy is the recipient of Research Career Development Award K04-HL-03006 from the National Institutes of Health.

References [1] Clauser E, Curnow KM, Davies E, Conchon S, Teutsch B, Vianello B, Monnot C, Corvol P. Angiotensin II receptors: protein and gene structures, expression and potential pathological involvements. Eur J Endocrinol 1996;134:403–11. [2] Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE, Dzau VJ. Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors. J Biol Chem 1993;268:24539–42. [3] Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo T, Inagami T. Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J Biol Chem 1993;268:24543–6. [4] Stoll M, Steckelings UM, Paul M, Bottari SP, Metzgei R, Unger T. The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest 1995;95:651–7. [5] Nio Y, Matsubara H, Murasawa S, Kanasaki M, Inada M. Regulation of gene transcription of angiotensin II receptor subtypes in myocardial infarction. J Clin Invest 1995;95:46–54. [6] Viswanathan M, Saavedra JM. Expression of angiotensin II AT2 receptors in the rat skin during experimental wound healing. Peptides 1992;13:783–6. [7] Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci USA 1996;93:156–60. [8] Siragy HM, Carey RM. The subtype 2 (AT2) angiotensin receptor mediates renal production of nitric oxide in conscious rats. J Clin Invest 1997;100:264–9. [9] Schelman WR, Kurth JL, Berdeaux RL, Norby SW, Weyhenmeyer JA. Angiotensin II type-2 (AT2) receptor-mediated inhibition of NMDA receptor signalling in neuronal cells. Mol Brain Res 1997;48:197–205. [10] Michel T, Feron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest 1997;100:2146–52. [11] Zarahn ED, Ye X, Ades AM, Reagan LP, Fluharty SJ. Angiotensininduced cyclic GMP production is mediated by multiple receptor subtypes and nitric oxide in N1E-115 neuroblastoma cells. J Neurochem 1992;58:1960–3. [12] McLay JS, Chatterjee PK, Mistry SK, Weerakody RF, Jardine AG, McKay NG, Hawksworth GM. Atrial natriuretic factor and angiotensin II stimulate nitric oxide release from human proximal tubular cells. Clin Sci 1995;89:527–31.

L. J. Millatt et al. / Regulatory Peptides 81 (1999) 1 – 10 [13] Zhang C, Mayeux PR. Angiotensin II signaling activates the NOcGMP pathway in rat proximal tubules. Life Sci 1998;63:L75–80. [14] Pueyo ME, Arnal JF, Rami J, Michel JB. Angiotensin II stimulates the production of NO and peroxynitrite in endothelial cells. Am J Physiol 1998;274:C214–20. [15] Seyedi N, Xu X, Nasjletti A, Hintze TH. Coronary kinin generation mediates nitric oxide release after angiotensin receptor stimulation. Hypertension 1995;26:164–70. [16] Deng X, Welch WJ, Wilcox CS. Role of nitric oxide in short-term and prolonged effects of angiotensin II on renal hemodynamics. Hypertension 1996;27:1173–9. [17] Zou AP, Wu F, Cowley AWJ. Protective effect of angiotensin II-induced increase in nitric oxide in the renal medullary circulation. Hypertension 1998;31:271–6. [18] Cahill PA, Redmond EM, Foster C, Sitzmann JV. Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells. Eur J Pharmacol 1995;288:219–29. [19] Ichiki T, Usui M, Kato M, Funakoshi Y, Ito K, Egashira K, Takeshita A. Downregulation of angiotensin II type I receptor gene transcription by nitric oxide. Hypertension 1998;31:342–8. [20] Usui M, Ichiki T, Katoh M, Egashira K, Takeshita A. Regulation of angiotensin II receptor expression by nitric oxide in rat adrenal gland. Hypertension 1998;32:527–33. [21] Katoh M, Egashira K, Usui M, Ichiki T, Tomita H, Shimokawa H, Rakugi H, Takeshita A. Cardiac angiotensin II receptors are upregulated by long-term inhibition of nitric oxide synthesis in rats. Circ Res 1998;83:743–51. [22] Nakayama I, Kawahara Y, Tsuda T, Okuda M, Yokoyama M. Angiotensin II inhibits cytokine-stimulated inducible nitric oxide synthase expression in vascular smooth muscle cells. J Biol Chem 1994;269:11628–33. [23] Ikeda U, Maeda Y, Kawahara Y, Yokoyama M, Shimada K. Angiotensin II augments cytokine-stimulated nitric oxide synthesis in rat cardiac myocytes. Circulation 1995;92:2683–9. [24] Chandler U, Kopnisky K, Richards E, Crews FT, Sumners C. Angiotensin II decreases inducible nitric oxide synthase expression in rat astroglial cultures. Am J Physiol 1995;268:C700–7. [25] Monton M, Lopez-Farre A, Mosquera JR, Sanchez de Miguel L, Garcia-Duran M, Sierra MP, Bellver T, Rico L, Casado S. Endogenous angiotensin II produced by endothelium regulates interleukin1b-stimulated nitric oxide generation in rat isolated vessels. Hypertension 1997;30:1191–7. [26] Wolf G, Ziyadeh FN, Schroeder R, Stahl RAK. Angiotensin II inhibits inducible nitric oxide synthase in tubular MCT cells by a posttranscriptional mechanism. J Am Soc Nephrol 1997;8:551–7. [27] Tojo A, Madsen KM, Wilcox CS. Expression of immunoreactive nitric oxide synthase isoforms in rat kidney. Effects of dietary salt and losartan. Jpn Heart J 1995;36:389–98. [28] Murakami K, Tsuchiya K, Naruse M, Naruse K, Demura H, Arai J, Nihei H. Nitric oxide synthase I immunoreactivity in the macula densa of the kidney is angiotensin II dependent. Kidney Int Suppl 1997;33:S208–10. [29] Schricker K, Potzl B, Hamann M, Kurtz A. Coordinate changes of renin and brain-type nitric-oxide-synthase (b-NOS) mRNA levels in rat kidneys. Pflugers Arch 1996;432:394–400. [30] Mattson DL, Higgins DJ. Influence of dietary sodium intake on renal medullary nitric oxide synthase. Hypertension 1996;27:688– 92. [31] Olson SC, Dowds TA, Pino PA, Barry MT, Burke-Wolin T. ANG II stimulates endothelial nitric oxide synthase expression in bovine pulmonary artery endothelium. Am J Physiol 1997;273:L315–21. [32] Hennington BS, Zhang H, Miller MT, Granger JP, Reckelhoff JF. Angiotensin II stimulates synthesis of endothelial nitric oxide synthase. Hypertension 1998;31:283–8. [33] Hassid A, Arabshahi H, Bourcier T, Dhaunsi GS, Matthews C. Nitric oxide selectively amplifies FGF-2-induced mitogenesis in primary rat aortic smooth muscle cells. Am J Physiol 1994;267:H1040–8.

9

[34] Baylis C, Mitruka B, Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest 1992;90:278–81. [35] Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension 1992;20:298–303. [36] Arnal JF, el Amrani A, Chatellier G, Menard J, Michel JB. Cardiac weight in hypertension induced by nitric oxide synthase blockade. Hypertension 1993;22:380–7. [37] Michel JB, Xu Y, Blot S, Philippe M, Chatellier G. Improved survival in rats administered NG-nitro L-arginine methyl ester due to converting enzyme inhibition. J Cardiovasc Pharmacol 1996;28:142–8. [38] Takemoto M, Egashira K, Usui M, Numaguchi K, Toriita H, Tsutsui H, Shimokawa H, Sueishi K, Takeshita A. Important role of tissue angiotensin-converting enzyme activity in the pathogenesis of coronary vascular and myocardial structural changes induced by long-term blockade of nitric oxide synthesis in rats. J Clin Invest 1997;99:278–87. [39] Luvara G, Pueyo ME, Philippe M, Mandet C, Savoie F, Henrion D, Michel JB. Chronic blockade of NO synthase activity induces a proinflammatory phenotype in the arterial wall: prevention by angiotensin II antagonism. Arterioscler Thromb Vasc Biol 1998;18:1408–16. [40] Ackermann A, Fernandez-Alfonso MS, Sanchez de Rojas R, Ortega T, Paul M, Gonzalez C. Modulation of angiotensin-converting enzyme by nitric oxide. Br J Pharmacol 1998;124:291–8. [41] Fernandez-Alfonso MS, Martorana PA, Licka I, van Even P, Trobisch D, Scholkens BA, Paul M. Early induction of angiotensin I-converting enzyme in rat carotid artery after balloon injury. Hypertension 1997;30:272–7. [42] Kajstura J, Cigola E, Malhotra A, Li P, Cheng W, Meggs LG, Anversa P. Angiotensin II induces apoptosis of adult ventricular myocytes in vitro. J Mol Cell Cardiol 1997;29:859–70. [43] Cigola E, Kajstura J, Li B, Meggs LG, Anversa P. Angiotensin II activates programmed myocyte cell death in vitro. Exp Cell Res 1997;231:363–71. [44] Yamada T, Akishita M, Pollman MJ, Gibbons GH, Dzau VJ, Horiuchi M. Angiotensin II type 2 receptor mediates vascular smooth muscle cell apoptosis and antagonizes angiotensin II type I receptor action: an in vitro gene transfer study. Life Sci 1998;63:L289–95. [45] Li W, Ye Y, Fu B, Wang J, Yu L, Ichiki T, Inagami T, Ichikawa I, Chen X. Genetic deletion of AT2 receptor antagonizes angiotensin II-induced apoptosis in fibroblasts of the mouse embryo. Biochem Biophys Res Commun 1998;250:72–6. [46] Pierzchalski P, Reiss K, Cheng W, Cirielli C, Kajstura J, Nitahara JA, Rizk M, Capogrossi MC, Anversa P. p53 induce myocyte apoptosis via the activation of the renin-angiotensin system. Exp Cell Res 1997;234:57–65. [47] Len A, Claudio PP, Li Q, Wang X, Reiss K, Wang S, Malhotra A, Kajstura J, Anversa P. Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell. J Clin Invest 1998;101:1326–42. [48] Shen YH, Wang XL, Wilcken DE. Nitric oxide induces and inhibits apoptosis through different pathways. FEBS Lett 1998;433:125–31. [49] Pollman MJ, Yamada T, Horiuchi M, Gibbons GH. Vasoactive substances regulate vascular smooth muscle cell apoptosis. Countervailing influences of nitric oxide and angiotensin II. Circ Res 1996;79:748–56. [50] Dimmeler S, Rippmann V, Weiland U, Haendeler J, Zeiher AM. Angiotensin II induces apoptosis of human endothelial cells. Protective effect of nitric oxide. Circ Res 1997;81:970–6. [51] Navar LG, Imig JD, Zou L, Wang CT. Intrarenal production of angiotensin II. Semin Nephrol 1997;17:412–22. [52] Sexton PM, Zhuo J, Mendelsohn FA. Localization and regulation of

10

[53]

[54]

[55]

[56]

[57]

[58]

[59] [60] [61]

[62]

[63] [64]

[65]

[66]

[67]

L. J. Millatt et al. / Regulatory Peptides 81 (1999) 1 – 10 renal receptors for angiotensin II and atrial natriuretic peptide. Tohoku J Exp Med 1992;166:41–56. Kakinuma Y, Fogo A, Inagami T, Ichikawa I. Intrarenal localization of angiotensin II type 1 receptor mRNA in the rat. Kidney Int 1993;43:1229–35. Mendelsohn FA, Millan M, Quirion R, Aguilera G, Chou ST, Catt KJ. Localization of angiotensin II receptors in rat and monkey kidney by in vitro autoradiography. Kidney Int Suppl 1987;20:540– 54. Ozono R, Wang ZQ, Moore AF, Inagami T, Siragy HM, Carey RM. Expression of the subtype 2 angiotensin (AT2) receptor protein in rat kidney. Hypertension 1997;30:1238–46. Grone HJ, Simon M, Fuchs E. Autoradiographic characterization of angiotensin receptor subtypes in fetal and adult human kidney. Am J Physiol 1992;262:F326–31. Goldfarb DA, Diz DI, Tubbs RR, Ferrario CM, Novick AC. Angiotensin II receptor subtypes in the human renal cortex and renal cell carcinoma. J Urol 1994;151:208–13. Matsubara H, Inada M. Molecular insights into angiotensin II type 1 and type 2 receptors: expression, signaling and physiological function and clinical application of its antagonists. Endocr J 1998;45:137–50. Kone BC, Baylis C. Biosynthesis and homeostatic roles of nitric oxide in the normal kidney. Am J Physiol 1997;272:F561–78. Kone BC. Nitric oxide in renal health and disease. Am J Kidney Dis 1997;30:311–33. Star RA. Intrarenal localization of nitric oxide synthase isoforms and soluble guanylyl cyclase. Clin Exp Pharmacol Physiol 1991;24:607– 10. Mundel P, Bachmann S, Bader M, Fischer A, Kummer W, Mayer B, Kriz W. Expression of nitric oxide synthase in kidney macula densa cells. Kidney Int 1992;42:1017–9. Kurtz A, Wagner C. Role of nitric oxide in the control of renin secretion. Am J Physiol 1998;275:F849–62. Blantz RC, Gabbai FB. Effect of angiotensin II on glomerular hemodynamics and ultrafiltration coefficient. Kidney Int Suppl 1987;20:S108–11. Granger JP, Alberola AM, Salazar FJ, Nakamura T. Control of renal hemodynamics during intrarenal and systemic blockade of nitric oxide synthesis in conscious dogs. J Cardiovasc Pharmacol 1992;20(Suppl 12):S160–2. De Nicola L, Blantz RC, Gabbai FB. Nitric oxide and angiotensin II. Glomerular and tubular interaction in the rat. J Clin Invest 1992;89:1248–56. Ito S, Arima S, Ren YL, Juncos LA, Carretero OA. Endotheliumderived relaxing factor / nitric oxide modulates angiotensin II action

[68] [69] [70]

[71]

[72] [73]

[74]

[75]

[76] [77]

[78]

[79] [80]

[81]

[82] [83]

in the isolated microperfused rabbit afferent but not efferent arteriole. J Clin Invest 1993;91:2012–9. Navar LG, Carmines PK, Huang WC, Mitchell KD. The tubular effects of angiotensin II. Kidney Int Suppl 1987;20:581–8. Majid DS, Navar LG. Nitric oxide in the mediation of pressure natriuresis. Clin Exp Pharmacol Physiol 1997;24:595–9. Dijkhorst-Oei LT, Koomans HA. Effects of a nitric oxide synthesis inhibitor on renal sodium handling and diluting capacity in humans. Nephrol Dial Transplant 1998;13:587–93. Braam B, Koomans HA. Nitric oxide antagonizes the actions of angiotensin II to enhance tubuloglomerular feedback responsiveness. Kidney Int 1995;48:1406–11. Mulrow PJ. Renin-angiotensin system in the adrenal. Horm Metab Res 1998;30:346–9. Breslow MJ, Tobin JR, Bredt DS, Ferris CD, Snyder SH, Traystman RJ. Nitric oxide as a regulator of adrenal blood flow. Am J Physiol 1993;264:H464–9. Natarajan R, Lanting L, Bai W, Bravo EL, Nadler J. The role of nitric oxide in the regulation of aldosterone synthesis by adrenal glomerulosa cells. J Steroid Biochem Mol Biol 1997;61:47–53. Ward LE, Hunter LW, Grabau CE, Tyce GM, Rorie OK. Nitric oxide reduces basal efflux of catecholamines from perfused dog adrenal glands. J Auton Nerv Syst 1996;61:235–42. Simmons JC, Freeman RH. L-arginine analogues inhibit aldosterone secretion in rats. Am J Physiol 1995;268:R1137–42. Nakayama T, Izumi Y, Soma M, Kanmatsuse K. A nitric oxide synthesis inhibitor prevents the ACTH-stimulated production of aldosterone in rat adrenal gland. Endocr J 1996;43:157–62. Hanke CJ, Drewett JG, Myers CR, Campbell WB. Nitric oxide inhibits aldosterone synthesis by a guanylyl cyclase-independent effect. Endocrinology 1998;139:4053–60. Del Punta K, Charreau EH, Pignataro OP. Nitric oxide inhibits Leydig cell steroidogenesis. Endocrinology 1996;137:5337–43. Lenkei Z, Corvol P, Llorens-Cortes C. The angiotensin receptor subtype ATIA predominates in rat forebrain areas involved in blood pressure, body fluid homeostasis and neuroendocrine control. Mol Brain Res 1995;30:53–60. Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, Moskowitz MA. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. J Cereb Blood Flow Metab 1996;16:981–7. Calapai G, Caputi AP. Nitric oxide and drinking behaviour. Regul Pept 1996;66:117–21. Zhu B, Herbert J. Angiotensin II interacts with nitric oxide-cyclic GMP pathway in the central control of drinking behaviour: mapping with c-fos and NADPH-diaphorase. Neuroscience 1997;79:543–53.