Carol Moreno, Almudena Lpez, Mara T. Llins, Francisca Rodrguez, Antonio Lpez-Farr, Eduardo Nava and F.

Javier Salazar
Am J Physiol Regulatory Integrative Comp Physiol 282:31-37, 2002. You might find this additional information useful... This article cites 45 articles, 27 of which you can access free at: http://ajpregu.physiology.org/cgi/content/full/282/1/R31#BIBL This article has been cited by 11 other HighWire hosted articles, the first 5 are: Angiotensin II Decreases Nitric Oxide Synthase 3 Expression via Nitric Oxide and Superoxide in the Thick Ascending Limb V. D. Ramseyer and J. L. Garvin Hypertension, February 1, 2009; 53 (2): 313-318. [Abstract] [Full Text] [PDF] Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats R. Zhang, Y.-G. Bai, L.-J. Lin, J.-X. Bao, Y.-Y. Zhang, H. Tang, J.-H. Cheng, G.-L. Jia, X.-L. Ren and J. Ma J Appl Physiol, January 1, 2009; 106 (1): 251-258. [Abstract] [Full Text] [PDF] Chronic angiotensin IV treatment reverses endothelial dysfunction in ApoE-deficient mice A. Vinh, R. E. Widdop, G. R. Drummond and T. A. Gaspari Cardiovasc Res, January 1, 2008; 77 (1): 178-187. [Abstract] [Full Text] [PDF] Chronic AT1 receptor blockade alters mechanisms mediating responses to hypoxia in rat skeletal muscle resistance arteries S. A. Phillips, I. Drenjancevic-Peric, J. C. Frisbee and J. H. Lombard Am J Physiol Heart Circ Physiol, August 1, 2004; 287 (2): H545-H552. [Abstract] [Full Text] [PDF] Alterations in Renal Endothelial Nitric Oxide Synthase Expression by Salt Diet in Angiotensin Type-1a Receptor Gene Knockout Mice K. Sato, M. Kihara, T. Hashimoto, K. Matsushita, Y.-I. Koide, K. Tamura, N. Hirawa, Y. Toya, A. Fukamizu and S. Umemura J. Am. Soc. Nephrol., July 1, 2004; 15 (7): 1756-1763. [Abstract] [Full Text] [PDF] Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl on the following topics: Oncology .. Protein Expression Physiology .. Mesenteric Arteries Physiology .. Left Ventricle Physiology .. Blood Pressure Physiology .. Renal Cortex Physiology .. Rats Updated information and services including high-resolution figures, can be found at: http://ajpregu.physiology.org/cgi/content/full/282/1/R31 Additional material and information about American Journal of Physiology - Regulatory, Integrative and Comparative Physiology can be found at: http://www.the-aps.org/publications/ajpregu

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The American Journal of Physiology - Regulatory, Integrative and Comparative Physiology publishes original investigations that illuminate normal or abnormal regulation and integration of physiological mechanisms at all levels of biological organization, ranging from molecules to humans, including clinical investigations. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2002 by the American Physiological Society. ISSN: 0363-6119, ESSN: 1522-1490. Visit our website at http://www.the-aps.org/.

Am J Physiol Regulatory Integrative Comp Physiol 282: R31R37, 2002.

Changes in NOS activity and protein expression during acute and prolonged ANG II administration
CAROL MORENO,1 ALMUDENA LOPEZ,2 MARIA T. LLINAS,1 FRANCISCA RODRIGUEZ,1 ANTONIO LOPEZ-FARRE,2 EDUARDO NAVA,3 AND F. JAVIER SALAZAR1 1 Department of Physiology, University of Murcia School of Medicine, E-30100 Murcia; 2 Fundacion Jimenez Daz, E-28040 Madrid; and 3Department of Medical Sciences, University of Castilla-La Mancha, E-02071 Albacete, Spain
Received 8 March 2001; accepted in nal form 31 August 2001

Moreno, Carol, Almudena Lopez, Mara T. Llinas, Francisca Rodrguez, Antonio Lopez-Farre, Eduardo Nava, and F. Javier Salazar. Changes in NOS activity and protein expression during acute and prolonged ANG II administration. Am J Physiol Regulatory Integrative Comp Physiol 282: R31R37, 2002.The aim of this study was to assess the effects of acute or prolonged increases of ANG II on nitric oxide synthase (NOS) activities and protein expression in mesenteric resistance vessels, left ventricle, renal cortex, and renal medulla. The response of NOS activities to ANG II is compared with that induced by phenylephrine. ANG II or phenylephrine were infused over either 3 h or 3 days to conscious rats. NOS activity was examined by measuring the rate of conversion of L-[14C]arginine to L-[14C]citrulline. Protein levels of endothelial (e) and neuronal (n) NOS were determined by Western blot analysis. Arterial pressure (AP) increased (P 0.05) during acute and prolonged ANG II infusion. Ca2 -dependent NOS activity values (pmol of citrulline min 1 g wet wt 1) for control rats were 21 9 in mesenteric arteries, 13 7 in left ventricle, 14 8 in renal cortex, and 411 70 in renal medulla. Acute ANG II infusion increased (P 0.05) Ca2 -dependent NOS activity in renal cortex and renal medulla (81 18 and 611 48, respectively), but no differences were found in mesenteric arteries and left ventricle with respect to control rats. In contrast to the renal changes in NOS activity, acute ANG II infusion did not modify eNOS or nNOS expression in any of the tissues examined. Prolonged ANG II infusion increased (P 0.05) Ca2 -dependent NOS activity in mesenteric arteries (70 17), renal cortex (104 31), and left ventricle (49 8) and did not elicit changes in renal medulla. After a prolonged ANG II infusion, eNOS and nNOS levels increased in all tissues examined with the exception of eNOS in the mesenteric arteries and nNOS in the left ventricle, which were not altered. Acute and prolonged phenylephrine infusion elevated AP to a similar extent as ANG II but only elicited signicant increments of Ca2 dependent NOS activity in renal cortex. These data indicate that acute and prolonged elevations in ANG II upregulate Ca2 -dependent NOS activity and protein expression in different tissues related to the control of blood pressure. However, these ANG II effects are heterogeneous with respect to the tissue implicated, the time course of the stimulation, and the NOS isoform involved. Phenylephrine only induces a signicant elevation of Ca2 -dependent NOS activity in renal cortex.
Address for reprint requests and other correspondence: E. Nava, Dept. of Medical Sciences, Univ. of Castilla-La Mancha, School of Medicine, Benjamin Palencia Bldg., E-02071 Albacete, Spain (Email: eduardo@med-ab.uclm.es). http://www.ajpregu.org

heart; mesenteric arteries; nitric oxide synthase; phenylephrine renal cortex; renal medulla Downloaded from ajpregu.physiology.org on September 1, 2010

THE INTERACTION BETWEEN NITRIC OXIDE (NO) and ANG II has been examined in a large number of studies (68, 19, 26, 33, 42, 47). It is well accepted that NO modulates the acute ANG II effects (7, 8, 26, 33, 47) and that NO production is stimulated in response to acute rises in ANG II (8, 31, 47). However, there are contradictory results with respect to the changes in NO synthesis in response to long-term elevations in ANG II (6, 8, 19). Despite many studies that have proposed the existence of an interaction between NO and ANG II, a number of questions remain to be answered on the nature of this interaction. It is not clear whether the rise of NO production in response to acute increments in ANG II is secondary to a greater NO synthase (NOS) activity and/or to a greater expression of the different NOS isoforms. Second, it is not known if the increases in NOS activity or NOS expression that may occur in response to acute rises in ANG II levels are modied when elevated plasma ANG II levels are maintained during several consecutive days. Also, it is unclear if the elevation in NOS activity and/or expression is limited to the kidney or also occurs in other tissues related to arterial pressure regulation. Finally, we wished to clarify whether the response of NOS activity to ANG II elevations, which may occur in tissues related to blood pressure regulation, are solely due to the vasoconstrictor ANG II effects and thus reproducible with another vasoconstrictor. The rst objective of this study was to examine the effects of acute and prolonged ANG II infusion on Ca2 -dependent and -independent NOS activities, in mesenteric arteries, left ventricle, renal cortex, and renal medulla. Because all these tissues display receptors for ANG II (2, 17, 36) and express the three isoforms of NOS (3, 5, 21, 27), we also assessed by Western blot analysis which of the isoenzymes of NOS is affected by ANG II. The tissues mentioned will be

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examined because they are clearly involved in regulating arterial pressure, and it has been proposed (42, 44) that a regulatory balance between ANG II and NO exists in only some resistance vessels. It has also been demonstrated (32, 37) that the kidney is much more sensitive to small variations in ANG II and NO than other tissues involved in arterial pressure regulation. To differentiate between the renal cortex and renal medulla is important because blood ow to the renal medulla is affected before blood ow to the renal cortex during small variations in ANG II and NO (4, 10). It has also been reported that the ANG II-induced NO production is signicantly greater in the renal medulla than in the renal cortex (47). Finally, it has been shown that the response of NOS activity to prolonged rises in arterial pressure is different in renal cortex and renal medulla (29). On the basis of the results published, the hypothesis of this study is that the response of NOS activities and protein expression to the acute and prolonged increments in ANG II will be heterogeneous among the different tissues. The presumed increase in NOS activity would be at the expense of endothelial (e) NOS in cardiovascular tissues and mainly at the expense of neuronal (n) NOS in renal tissues. The second objective was to determine whether the effect of ANG II on NOS activity is specic for this peptide. To accomplish this objective, experiments were performed in which phenylephrine was infused for 3 h or 3 days and NOS activities were measured. Because ANG II seems to upregulate NOS via shear stress (9) and through ANG II receptors (33, 41), it is likely that, compared with another vasoconstrictor such as phenylephrine, ANG II changes NOS activity more rapidly and to a larger extent.
METHODS

Experiments were performed in male Sprague-Dawley rats (1012 wk old) bred in the animal house of the University of Murcia. Rats were anesthetized with Rompun (Bayer; 10 mg/kg) and ketamine (Rhone Merieux; 50 mg/kg), and cannulas were inserted into the right femoral vein for infusion of drugs and into the femoral artery for measurement of mean arterial pressure (MAP). Both catheters were advanced subcutaneously along the back, exteriorized at the nape of the neck, and the venous cannula was connected to a swivel for infusions. Surgery was performed under aseptic conditions. Four days were allowed for recovery of surgery. Then, rats were intravenously infused with either vehicle (NaCl, 0,9%; group 1, n 11), ANG II at 200 ng kg 1 min 1 for 3 h (group 2, n 11) or 3 days (group 3, n 11), or phenylephrine at 15 g kg 1 min 1 for 3 h (group 4, n 8) or 3 days (group 5, n 8). In the saline and ANG II-infused groups, four rats were used for Western blot analysis. The rest (n 7) were used to assess NOS activities with the citrulline assay. After ANG II or phenylephrine infusions, rats were anesthetized with pentobarbitone (Inactin; 60 mg/kg), and the kidneys, heart, and guts were removed. The left ventricle was rapidly frozen in liquid nitrogen and stored at 70C. Kidneys were processed as previously described (29). Essentially, they were immediately placed over dry ice and carefully dissected to detach the medulla from the cortex. Mesenteric arteries were obtained as previously indicated

(28). Briey, the mesenteric arterial tree was quickly dissected ( 10 min) in ice-cold Krebs solution under a microscope, and arteries 300 m in external diameter (resistance arteries) were collected and frozen on dry ice. The time taken for the dissection of the mesenteric tree was 1020 min. All tissues were stored at 70C until analyzed. Determination of constitutive and inducible NOS activity. As previously reported (28, 29), frozen tissues were homogenized in a solution containing 320 mM sucrose, 50 mM Trizma base, 1 mM EDTA, 1 mM DL-dithiothreitol, 10 g/ml leupeptin, 100 g/ml phenylmethylsulfonyl uoride, 10 g/ml soybean trypsin inhibitor, and 2 g/ml aprotinin brought to pH 7 with HCl. Myocardial and renal tissue was homogenized in 3 vol of this buffer, and mesenteric arteries were homogenized in 4 vol. The homogenates were centrifuged at 12,000 g for 20 min (4C). NO formation was measured in the supernatant by the rate of conversion of radiolabeled L-[14C]citrulline from L-[14C]arginine (Amersham), as we earlier described (28, 29). Briey, tissue extracts were incubated in a pH 7 buffer (100 l) containing (in mM) 50 potassium dihydrogen orthophosphate, 60 L-valine, 0.12 -nicotinamide adenine dinucleotide phosphate, 1.2 L-citrulline, 1.2 magnesium chloride, 0.24 calcium chloride, 0.024 L-arginine, and 0.002 L-[U-14C]arginine (297 mCi/mmol). Because NOS is maximally active only in a narrow pH range 7.5 (13), both the homogenization and the incubation buffers were brought to pH 7 with HCl before being used. Duplicate incubations were performed for 10 min at 37C for each sample in the presence or absence of either EGTA (2 mmol/l) or EGTA plus NG-nitro-L-arginine methyl ester (L-NAME; 2 mmol/l) to determine the Ca2 -dependent and -independent NOS activities, respectively. The rst was calculated as the L-[14C]citrulline formed in the control tubes minus the L-[14C]citrulline formed in tubes incubated with EGTA. The latter was calculated as the L-[14C]citrulline formed in these tubes minus the L-[14C]citrulline formed in tubes incubated with EGTA plus L-NAME. L-[14C]citrulline in the supernatant was separated from L-[14C]arginine by addition of a Dowex-50W resin. Determination of NOS protein expression. The level of expression of the eNOS and nNOS protein was analyzed by Western blot, as previously reported (25). In brief, tissues were pulverized and solubilized in Laemmli buffer containing 2-mercaptoethanol, and proteins were separated in denaturing SDS-10% polyacrylamide gels (15 g/lane). Proteins were then blotted into nitrocellulose (Immobilon-P, Millipore). Blots were blocked overnight at 4C with 5% nonfat dry milk in TBS-T (20 mmol/l Tris HCl, 137 mmol/l NaCl, 0.1% Tween 20). Western blot analysis was performed with a monoclonal antibody against eNOS and nNOS (BD Transduction Laboratories). Blots were incubated with the rst antibody (1:250) for 1 h at room temperature and, after extensive washing, with the second antibody (horseradish peroxidase-conjugated antimouse immunoglobulin) at a dilution of 1:1,500 for another hour. Specic eNOS and nNOS proteins were detected by enhanced chemiluminescence (Amersham) and evaluated by densitometry (Molecular Dynamics). Prestained protein markers were used for molecular mass determinations. The monoclonal antibody used specifically recognizes either the eNOS and nNOS isoforms (140 and 155 kDa, respectively) and does not cross-react with the inducible NOS isoform (25). To compare NOS expression with the expression of another protein, we analyzed the expression of -actin by Western blot using a -actin monoclonal antibody (Sigma Aldrich). For this purpose, a parallel gel with identical samples was run, and after blotting onto
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nitrocellulose, the Western blot analysis was performed with the -actin monoclonal antibody (1:5,000). Statistical analysis. Data are expressed as means SE. Statistical differences were evaluated using an ANOVA and the Fishers test. A P 0.05 was considered to indicate a signicant difference.
RESULTS

Table 1. Densitometric values of nNOS and eNOS expression in mesenteric arteries, left ventricle, renal cortex and renal medulla after the acute (3 h) or prolonged (3 days) infusion of ANG II
Control Acute ANG II Prolonged ANG II

Before infusions, MAP was similar in all experimental groups, averaging 99 3 mmHg. There were no changes in this parameter after saline infusion in the control group (99 5 mmHg); however, MAP increased (P 0.05) after 3 h (138 3 mmHg) or 3 days (148 9 mmHg) of ANG II administration. MAP values after phenylephrine infusion were similar to those achieved with ANG II, that is, 140 2 mmHg after 3 h and 146 4 mmHg after 3 days of phenylephrine infusion. Changes in Ca2 -dependent NOS activity during acute or prolonged ANG II infusion are shown in Fig. 1. Values of NOS activities are expressed as picomoles of citrulline per minute per gram of tissue. In control rats, Ca2 -dependent NOS activities were 21 9 for mesenteric arteries, 13 7 for left ventricle, 14 8 for renal cortex, and 411 70 for renal medulla. When ANG II was infused for 3 h, Ca2 -dependent NOS activity increased (P 0.05) in renal cortex and medulla, to 81 18 and 611 48, respectively, whereas values in mesenteric arteries and left ventricles remained unchanged (31 11 and 13 7, respectively; Fig. 1). ANG II infusion for 3 days induced an increase

Mesenteric arteries nNOS eNOS Left ventricle nNOS eNOS Renal cortex nNOS eNOS Renal medulla nNOS eNOS

100 100 100 100 100 100 100 100

103 101 104 101 103 103 101 101

1 1 2 1 1 1 1 1

138 103 105 215 165 178 171 157

8* 2 1 7* 14* 14* 12* 4*

Values are means SE. The expressions of both isoforms are expressed as arbitrary units with respect to that found in control rats. nNOS and eNOS, neuronal and endothelial nitric oxide synthase. * P 0.05 vs. control.

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(P 0.05) of Ca2 -dependent NOS activity in mesenteric arteries (71 18), left ventricle (49 8), and renal cortex (104 31). However, this activity was not elevated in renal medulla (493 98) with respect to that found in the control group (Fig. 1). Table 1 summarizes the densitometric values found in all tissues studied both in acute and chronic experiments. eNOS and nNOS expression did not change signicantly in any of the examined tissues after acute infusion of ANG II, compared with the expression found in vehicle-treated rats. Long-term ANG II-infused rats exhibited an increased (P 0.05) eNOS protein expression in renal cortex, renal medulla, and left ventricle. In this group of animals, nNOS protein levels were also elevated (P 0.05) in renal cortex, renal medulla, and mesenteric arteries. eNOS expression in mesenteric arteries and nNOS protein levels in left ventricle were not altered after the 3-day infusion of ANG II (Table 1). Figures 2 and 3A show a representative blot for left ventricle and renal medulla, respectively. No changes in the expression of -actin protein were observed between tissues obtained from control, acute, and prolonged ANG IItreated rats (Fig. 3B). When phenylephrine was infused for 3 h, Ca2 -dependent NOS activity was elevated only in the renal

Fig. 1. Ca2 -dependent nitric oxide synthase (NOS) activity in mesenteric resistance vessels, left ventricle renal cortex, and renal medulla from control rats (open bars) and rats infused with ANG II for 3 h (hatched bars) or 3 days (solid bars). *P 0.05.

Fig. 2. Representative Western blots of endothelial (e) NOS and neuronal (n) NOS in left ventricle of normotensive control rats and hypertensive rats in which ANG II was infused over 3 h (acute) or 3 days (chronic).
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Fig. 3. A: representative Western blots of eNOS and nNOS in renal medulla of normotensive control rats and hypertensive rats in which ANG II was infused over 3 h (acute) or 3 days (chronic). B: representative Western blots of -actin in renal medulla of normotensive and ANG II-infused rats.

cortex (65 10, P 0.05) when compared with the activity found in vehicle-treated rats (Fig. 4). Ca2 dependent NOS activity was not signicantly changed in renal medulla (358 41), left ventricle (15 6), or mesenteric vessels (42 17) after the acute phenylephrine administration (Fig. 4). A similar result was found after the 3-day infusion of phenylephrine, because Ca2 -dependent NOS activity was found elevated only in the renal cortex (to 82 26, P 0.05). Ca2 -dependent NOS activity was not altered in renal medulla (384 64), left ventricle (16 7), and mesenteric arteries (19 6; Fig. 4). Ca2 -independent NOS activities (pmol citrulline min 1 g tissue 1) in control rats were 4 2 for mesenteric arteries, 14 5 for left ventricle, 120 33 for renal cortex, and 47 20 for renal medulla. Neither acute nor prolonged ANG II infusion elicited changes of Ca2 -independent NOS activity in mesenteric arteries (5 3 and 11 4), left ventricle (10 6 and 3 1), renal cortex (141 33 and 147 33), and renal medulla (49 19 and 28 8). In the same line, neither acute nor prolonged phenylephrine infusion induced signicant changes of Ca2 -independent NOS activity in any of the tissues examined.
DISCUSSION

an increase in shear stress (9) or via receptor stimulation (33, 41). We failed to observe an increase in eNOS expression, but instead noted an elevation in nNOS expression. Recent studies show that nNOS is normally expressed throughout the systemic vasculature, being located in smooth muscle cells (40) and perivascular nerves (11). Indeed, vascular nNOS has been reported to express on ANG II stimulation (3). The fact that the increase of Ca2 -dependent NOS activity in mesenteric arteries was signicant with the prolonged ANG II infusion but not with that of phenylephrine suggests that ANG II effect on NOS activity is receptor mediated and not due to an increase in MAP. The notion that the increase in NOS activity is receptor mediated is supported by the results reported by Seyedi et al. (41). Nevertheless, it is possible that other mechanisms are also involved in the elevation in NOS activity after prolonged elevation in ANG II levels. In this context, the higher NO available probably plays a compensatory role against the ANG II-induced vasoconstriction. Another long-term effect of ANG II is cell proliferation, which has been described specically in mesenteric arteries (43). Because NO is known to inhibit smooth muscle cell proliferation (15), it might compensate the mitogenic effects of ANG II. The left ventricle also showed an increase in Ca2 dependent NOS activity after the 3-day infusion but not after the acute infusion of ANG II. The increase appeared to be endothelial in origin, as no changes in nNOS expression were detected. This is consistent

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The results of the present study demonstrate that there is a heterogeneous response of both NOS activity and NOS expression in renal cortex, renal medulla, mesenteric arteries, and left ventricle to acute and prolonged increases in ANG II levels. The response of Ca2 -dependent NOS activity to ANG II seems to be specic for this peptide in every tissue examined except for the renal cortex. Ca2 -dependent NOS activity in mesenteric arteries increased 3.5-fold after a 3-day ANG II infusion but did not change in response to the infusion of ANG II during three consecutive hours. According to the current knowledge on the effects of vasoconstriction on the vasculature, we expected to detect a rise in eNOS expression after a prolonged exposure to ANG II due to

Fig. 4. Ca2 -dependent NOS activity in mesenteric resistance vessels, left ventricle renal cortex, and renal medulla from control rats (open bars) and rats infused with phenylephrine for 3 h (hatched bars) or 3 days (solid bars). *P 0.05.
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with our previous ndings in cardiac NOS activity, which practically disappeared after endocardial and coronary endothelial denudation (30). Indeed, myocardial tissue does not express nNOS, which is relegated to the conduction tissue (21). As with mesenteric arteries, phenylephrine did not induce signicant changes in NOS activity. Thus the changes in the NO pathway in the heart appear also to be specic for ANG II working via receptors. The presumable increase in NO availability in the heart after ANG II infusion may have a protective effect against the deleterious actions of this vasoactive peptide. ANG II is known to have inotropic and proliferative actions on the myocardium (2), and it has been demonstrated that the ventricular hypertrophy seen after myocardial infarction appears to be related to a local release of ANG II (2). Indeed, angiotensin-converting enzyme inhibition has a protective effect, reducing ischemic areas of infarcted muscle (2). The hypothesis that the long-term cardiac actions of ANG II may be counteregulated by NO is supported by the results obtained by Heeneman et al. (18), showing that the prolonged infusion of ANG II elicits a decrease in cardiac index and an increase of the aortic cGMP concentration. In the same line, Takizawa et al. (45) demonstrated that NO donors affect cardiac broblast growth by inhibiting the thymidine incorporation caused by ANG II. Several studies have indicated that NO counteracts the renal actions of acute ANG II infusion, protecting against the ANG II-induced vasoconstriction (8, 26), and there is indirect evidence that ANG II stimulates NO production in the kidney (8, 47). This increase in renal NO production could be due to an activation of the different NOS isoforms and/or to an increase in NOS protein synthesis. In this regard, Hennington et al. (19) found an increased eNOS mRNA production in the whole kidney after an acute ANG II infusion, without changes in renal eNOS expression. We also found that an acute exposure to ANG II fails to elicit NOS overexpression in renal cortex and medulla but causes an increase in Ca2 -dependent NOS activity. This suggests that a 3-h stimulation with ANG II is enough to initiate mRNA production but not new protein synthesis. There is indeed an increase in NO production, but it is mainly secondary to an activation of the existing Ca2 -dependent NOS in both renal cortex and renal medulla. Unlike in the renal medulla, the response of Ca2 -dependent NOS activity in the renal cortex was not specic for ANG II, because a similar rise was observed when an equipressor dose of phenylephrine was infused. It is remarkable that the acute infusion of ANG II enhances NOS activity in the renal cortex and renal medulla without affecting NOS expression. We do not have an explanation for this, but there are several possibilities to be considered taking into account that ANG II can affect NOS activity by acting on various regulatory mechanisms, namely, calmodulin, caveolins, and Akt-dependent phosphorylation. The rst possibility implies an activation of calmodulin by ANG II. Saito et al. (38) reported that ANG II can activate

calcium-calmodulin via AT1 receptors and subsequently activate constitutive NOS activity. The recent discovery that AT1 receptors possess a binding domain specic for calcium-calmodulin supports this possibility (46). Another mechanism involves a possible action on caveolin-bound NOS. NOS is concentrated in membrane caveolae bound to caveolins that keep NOS inactivated in basal conditions (35). It has been observed that when a stimulus for NOS is present, caveolar NOS activity increases without recruiting additional enzyme to the caveolae (35). Caveolins, which are present in the kidney (22), could underlie a mechanism of NOS activation without increasing its expression. Finally, ANG II could activate NOS without increasing its expression by promoting its phosphorylation. It is known that constitutive NOS can be activated in a calcium-independent manner by a phosphorylation that is dependent on protein kinase B or Akt (12). Interestingly, ANG II is a well known activator of Akt (14). The role of NO in protecting the renal vasculature from the effects induced by prolonged elevations in ANG II levels is controversial, because contradictory results have been reported (7, 8, 19, 39, 42). In our study, the response of Ca2 -dependent and -independent NOS activities to acute and prolonged elevations in ANG II levels has been evaluated. Changes of the eNOS and nNOS expression in response to ANG II were also examined. Inducible NOS expression was not evaluated, because we found no signicant changes in the Ca2 -independent activity during ANG II infusion. The results obtained in the renal cortex are in line with those previously reported by other groups (6, 7, 19). Hennington et al. (19) found that chronic ANG II infusion results in an increase of renal eNOS protein levels, but it was not determined whether this increment occurred primarily in the cortex or the medulla. In their study, Chin et al. (6) found that Ca2 -dependent NOS activity increases in response to the ANG II infusion during 13 days and that this change seems to be secondary to an overexpression of eNOS and nNOS. These results reported by Chin et al. (6) have been conrmed in the present study. However, it was unknown whether the rise in Ca2 -dependent NOS activity was specic for ANG II or also occurs in response to the prolonged administration of another vasoconstrictor. The results of our study have shown that the increase in NO production that seems to modulate the vasoconstrictor effect of ANG II on the renal cortex is not specic for this vasoactive peptide, because Ca2 dependent NOS activity also increases after a prolonged infusion of phenylephrine. The increase of NOS activity and both eNOS and nNOS expression during the prolonged ANG II administration suggests that there is an elevation in NO production that may compensate the vasoconstriction elicited by ANG II. This hypothesis is supported by one study showing that ANG II elicits a greater reduction in cortical blood ow when NO synthesis is reduced (7), but other functional studies have shown that NOS blockade does not potentiate the renal hemodynamic
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response to prolonged elevations in ANG II (8, 39). There is no easy explanation to integrate these ndings. However, it may occur that ANG II induces not only an overexpression of eNOS and nNOS, but also a stimulation of free radical production that may inactivate NO (20, 34). One possible explanation of the results obtained in studies showing that NOS blockade does not potentiate the ANG II renal vasoconstriction (8, 39) is that the dose of ANG II infused enhances the production of free radicals to a level high enough to inactivate the NO produced as a consequence of the NOS overexpression. Our results also show that Ca2 -dependent NOS activity is not enhanced in the renal medulla during prolonged ANG II infusion. However, eNOS and nNOS expression was elevated in the renal medulla after the 3-day infusion of ANG II. It remains to be elucidated why this overexpression of both NOS isoforms is not accompanied by an increase in NOS activity. Many factors can account for the downregulation of NOS activity in the renal medulla after a prolonged ANG II infusion. NOS can be inactivated by superoxide anion, which is enhanced during elevations of ANG II. This has been observed in vessels (34), tubular cells (16), and mesangial cells (20). NOS can be inactivated as well by inadequate levels of tetrahydrobiopterin (23) or by NO in excess (1). Taken together with those reported by Chin et al. (6, 7), our results suggest that NO synthesis does not increase in the renal medulla during prolonged elevations in ANG II. Chin et al. (7) found that the decrease in medullary blood ow in response to N -nitro-L-arginine is not enhanced in the ANG II-infused rats and that Ca2 -dependent NOS activity does not change after the administration of a pressor dose of ANG II over 13 days (6). Our results suggest the possibility of a differential regional regulation of NOS activity in the renal circulation in response to acute and chronic increases in ANG II levels. It is also proposed that overproduction of NO in the renal medulla, in response to ANG II, is transitory. Whether NO is relayed by other vasodilators on a long-term basis remains to be elucidated. In summary, our results show that ANG II is capable of upregulating Ca2 -dependent but not Ca2 -independent NOS activity in a variety of tissues. This interaction between ANG II and Ca2 -dependent NOS activity is heterogeneous both chronologically and anatomically. Comparing the results obtained during acute and prolonged elevations in ANG II with those found after administration of phenylephrine, it can be proposed that the upregulation of Ca2 -dependent NOS activity in renal medulla, mesenteric arteries, and left ventricle elicited by ANG II is specic for this vasoactive peptide. Perspectives The present study offers a descriptive perspective of NO-related events in various organs when ANG II is elevated in conscious animals over 3 h and 3 days. This approach inevitably leaves questions behind on every

specic organ we studied. In mesenteric arteries, for instance, nNOS but not eNOS was elevated in the chronic situation. It would be important to determine by immunohistochemistry where in the mesenteric arteries nNOS is expressed. Presumably it is located in the nerve endings and not in the endothelial cells. If this were really the case, it would open a debate, because it does not t with some of the accepted aspects of vascular physiology. The accepted sequence of events happening when a vasoconstrictor is present in the blood, in this case ANG II, is that NOS is activated in the endothelial cell by specic receptors or by a shear stress mechanism. NOS then generates NO, which will travel to the smooth muscle cell to compensate the vasoconstriction. Now, if the vasoconstrictor activates nNOS instead of eNOS, what kind of signal is traveling from the endothelial surface to the nerve ending NOS? A different point of discussion is the receptor-dependent NO-activating mechanism that ANG II appears to have in most of the organs we presently study. In this regard, it is of high interest to further investigate which ANG II receptors are involved. This is particularly important in the heart, where NO may prevent cardiac hypertrophy in ischemic heart disease. If we could apply the right ANG II antagonist that could prevent the deleterious effects of ANG II and at the same time leave free the ANG II-NO pathway, this would be of high therapeutic interest.
C. P. Moreno was supported by a grant from Ministerio de Educacion y Ciencia of Spain (Ref. PN-95). M. T. Llinas was supported by a grant from University of Murcia (Spain). F. Rodrguez was sup ported by a grant from the Fondo de Investigaciones Sanitarias (FIS 98/1309). This study was supported by grants from FIS (98/1309, 99/1024, and 00/0280) of Spain. REFERENCES 1. Abu-Soud HM, Wang J, Rousseau DL, Fukuto JM, Ignarro LJ, and Stuehr DJ. Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis. J Biol Chem 270: 2299723006, 1995. 2. Baker KM, Booz GW, and Dostal DE. Cardiac actions of angiotensin II: role of an intracardiac renin-angiotensin II system. Annu Rev Physiol 54: 227241, 1992. 3. Boulanger CM, Heymes C, Benessiano J, Geske RS, Levy BI, and Vanhoutte PM. Neuronal nitric oxide synthase is expressed in rat vascular smooth muscle cells: activation by angiotensin II in hypertension. Circ Res 83: 12711278, 1998. 4. Brezis M, Heyman SN, Dinour D, Hepstein FH, and Rosen S. Role of nitric oxide in renal medullary oxygenation. J Clin Invest 88: 390395, 1991. 5. Cernadas MR, de Miguel LS, Garca-Duran M, and Lopez Farre A. Expression of constitutive and inducible nitric oxide synthases in the vascular wall of young and aging rats. Circ Res 83: 279286, 1998. 6. Chin SY, Pandey KN, Shi SJ, Kobori H, Moreno C, and Navar LG. Increased activity and expression of Ca2 -dependent NOS in renal cortex of ANG II-infused hypertensive rats. Am J Physiol Renal Physiol 277: F797F804, 1999. 7. Chin SY, Wang CT, Majid DS, and Navar LG. Renoprotective effects of nitric oxide in angiotensin II-induced hypertension in the rat. Am J Physiol Renal Physiol 274: F876F882, 1998. 8. Deng X, Welch WJ, and Wilcox CS. Role of nitric oxide in short-term and prolonged effects of angiotensin II on renal hemodynamics. Hypertension 27: 11731179, 1996. 9. Endlich K, Muller C, and Helwig JJ. Role of shear stress in nitric oxide-dependent modulation of renal angiotensin vasoconstriction. Br J Pharmacol 127: 19291935, 1999.
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