Angiotensin II stimulates the production of NO and peroxynitrite in endothelial cells

Maria E. Pueyo, Jean-François Arnal, Jacques Rami, and Jean-Baptiste Michel

Institut National de la Santé et de la Recherche Médicale U460, 75018 Paris; and Institut National de la Santé et de la Recherche Médicale U397 and Physiology Department, Faculty of Medicine, 31054 Toulouse, France

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Angiotensin II (ANG II) produces vasoconstriction by a direct action on smooth muscle cells via AT1 receptors. These receptors are also present in the endothelium, but their function is poorly understood. This study was therefore undertaken to determine whether ANG II elicits the release of nitric oxide (NO) from cultured rat aortic endothelial cells. NO production, measured by the accumulation of nitrite and nitrate, was enhanced by 10-7 M ANG II. The biological activity of the NO released by ANG II action was evaluated by measuring its guanylate cyclase-stimulating activity in smooth muscle cells. The guanosine 3',5'-cyclic monophosphate (cGMP) content of smooth muscle cells was significantly increased by exposure of supernatant from ANG II-stimulated endothelial cells. These effects resulted from the activation of NO synthase, as they were inhibited by the L-arginine analogs. These ANG II actions were mediated by the AT1 receptor, as shown by their inhibition by the AT1 antagonist losartan. The cGMP production by reporter cells was inhibited by the calmodulin antagonist W-7, suggesting that ANG II activates endothelial calmodulin-dependent NO synthase. This hypothesis is also supported by the increase of intracellular free calcium induced by ANG II in endothelial cells. ANG II also stimulated luminol-enhanced chemiluminescence in endothelial cells. This effect was inhibited by Nomega -monomethyl-L-arginine and superoxide dismutase, suggesting that this luminol-enhanced chemiluminescence reflected an increase in peroxynitrite production. Thus ANG II stimulates NO release from macrovascular endothelium, which may modulate the direct vasoconstrictor effect of ANG II on smooth muscle cells. However, this beneficial effect may be counteracted by the simultaneous production of peroxynitrite, which could contribute to several pathological processes in the vascular wall.

nitric oxide synthase; guanosine 3',5'-cyclic monophosphate; intracellular free calcium; smooth muscle cells

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ANGIOTENSIN II (ANG II), the main biologically active peptide of the renin-angiotensin system, plays an important role in the regulation of blood pressure (29). ANG II causes vasoconstriction by direct stimulation of ANG II receptors on smooth muscle cells. This action can be due to circulating ANG II but also to ANG II produced within the vascular wall (8). Experimental evidence suggests that endothelial cells may also be targets for plasma angiotensins (7).

The presence of ANG II receptors in cultured endothelial cells has been reported by several investigators (10, 14, 28, 35). In a previous study, we demonstrated that the ANG II receptors on endothelial cells belong to the AT1A and AT1B subtypes, but we found no AT2 receptors on these cells (31). The pharmacological characteristics of ANG II receptors on endothelial cells are similar to those on smooth muscle cells. The binding of ANG II to endothelial AT1 receptors leads to the activation of phospholipase C and phospholipase A2, as in other cell types (31).

Despite these observations, the physiological relevance of ANG II action in endothelial cells remains poorly understood. The effects of ANG II on endothelial cells could counterbalance its vasoconstrictive effect on smooth muscle cells. This is supported by the finding that the vasoconstrictive effect of ANG II is potentiated after endothelial removal in isolated aortic rings (12). ANG II could also elicit endothelium-dependent vascular relaxation under certain conditions or in some vascular beds (15, 34, 36). These effects could be due to the release of the endothelium-derived relaxing factor, which is nitric oxide (NO) or a nitrosothiol compound (16, 26, 27). NO is synthesized from arginine by a calcium-dependent endothelial NO synthase. Major reactions of NO are the binding to ferrous heme in guanylate cyclase to activate guanosine 3',5'-cyclic monophosphate (cGMP), the formation of oxidant species such as peroxynitrite by reacting with superoxide, and its destruction by reacting with oxyhemoglobin (2). NO has a short half-life and only diffuses locally (19), making it difficult to directly assess NO production in vivo.

In the present study, we used cultured endothelial cells to determine whether ANG II elicits the production of NO. This production was evaluated by measuring the accumulation of nitrite and nitrate, the stable degradation products of NO. We also studied the capacity of ANG II-induced NO to stimulate the soluble guanylate cyclase in smooth muscle cells. The possible mechanisms of ANG II-induced NO production were investigated, particularly the effects of ANG II on intracellular calcium concentration, one of the major mechanisms of activation of endothelial NO synthase. Finally, we measured the production of radical oxygen intermediates (ROI) in response to ANG II, with the use of luminol-enhanced chemiluminescence.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. Dulbecco's modified Eagle's medium (DMEM), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), horse serum, and trypsin-EDTA were obtained from Boehringer Mannheim. ANG II, penicillin, streptomycin, collagen, endothelial cell growth supplement, 3-isobutyl-1-methylxanthine (IBMX), superoxide dismutase (SOD), catalase, N G-nitro-L-arginine methyl ester (L-NAME), N omega -monomethyl-L-arginine (L-NMMA), sodium nitroprusside (SNP), and n-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) were from Sigma Chemical (St. Louis, MO). Collagenase was obtained from Eurobio, rat endothelial cell antibody was purchased from Medac Diagnosika (Hamburg, Germany), and antibody against alpha -actin was from Dako (Glostrup, Denmark). Losartan was kindly provided by Merck Sharp & Dohme, PD-123177 by Dr P. Janiack (Servier, Paris), and HOE-140 by Prof. B. Schoëlkens (Hoechst, Frankfurt, Germany).

Cell culture. Endothelial cells (RAEC) and smooth muscle cells (RSMC) were isolated from rat aorta as previously described (1). Briefly, the thoracic aortas were excised and rinsed, and fat and collateral vessels were removed. The adventitia was separated, and the remaining media plus intima were sliced into fine rings and incubated in DMEM containing collagenase (1,248 IU/ml) for 40 min at 37°C. The rings were flushed and filtered to dislodge the endothelial cells. The cells were centrifuged, resuspended in culture medium, and plated by incubation for 40 min in a plastic flask coated with rat fibronectin. Only endothelial cells adhere during this short period. RSMC were isolated by placing the aortic rings in collagenase (1,248 IU/ml) plus elastase (17.5 IU/ml) for 60 min at 37°C. The cells were centrifuged, resuspended in culture medium, and plated out in plastic flasks coated with 0.1% collagen. This method gives pure cell preparations (>95% of endothelial or smooth muscle cells), as shown by immunostaining with a specific antibody against rat endothelial cells (9) or with a monoclonal antibody for smooth muscle alpha -actin.

Confluent RAEC were detached using trypsin-EDTA and were propagated in DMEM supplemented with 15% horse serum, 5% fetal calf serum, 75 µg/ml endothelial cell growth supplement, 20 mM HEPES, 2 mM glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin. RSMC were cultured in DMEM supplemented with 15% fetal calf serum. Cells from passages 1-4 were used in these studies.

Evaluation of NO production by measuring nitrite and nitrate. NO production was evaluated by measuring the accumulation of nitrite and nitrate, the stable degradation products of NO (nitrogen oxides), as previously described (25). RAEC were grown to confluence in six-well dishes, washed gently three times with modified Krebs-HEPES buffer (in mM: 99 NaCl, 4.69 KCl, 1.87 CaCl2, 1.2 MgSO4, 25 NaHCO3, 1.2 K2HPO4, 20 HEPES, and 11.1 D-glucose), and incubated in 1 ml of Krebs-HEPES buffer with or without 10-7 M ANG II at 37°C for 1 h. When used, antagonists were preincubated for 30 min. An aliquot of the supernatant (200 µl) was then injected into a reflux chamber containing vanadium III dissolved in HCl heated to 85°C. These conditions reduce both nitrite and nitrate stoichiometrically to NO. The released NO was purged with a stream of nitrogen gas directed via a vacuum into the reaction chamber of a chemiluminescence NO analyzer (Cosma, Igny, France). The chemiluminescence analyzer was calibrated daily using nitrate standards. The amount of nitrogen oxides released was normalized with respect to the protein content in each culture dish.

Evaluation of NO bioactivity by measuring cGMP production. The bioactivity of NO was evaluated by measuring the guanylate cyclase activity. Previously, this guanylate cyclase activity was evaluated in RAEC and in RSMC by measuring the cGMP production in response to different concentrations of SNP. RAEC and RSMC were cultured in six-well plates. After reaching confluence, the culture medium was removed, and cells were washed twice with phosphate-buffered saline and preincubated with 1 ml of buffer containing 10-4 M IBMX for 20 min at 37°C. The composition of the buffer was (in mM) 150 NaCl, 4.8 KCl, 1.8 Ca2Cl, 1.2 MgCl2, 3.6 NaHCO3, 5.5 glucose, and 20 HEPES, pH 7.4. Cells were exposed to different concentrations of SNP in the presence of SOD (100 U/ml) for 4 min.

For the experiments with both cell types, RAEC were preincubated with SOD (100 U/ml) and antagonists (losartan, PD-123177, HOE-140, W-7, L-NAME), when used, for 20 min. RSMC were preincubated with 10-4 M IBMX for 20 min. RAEC were then exposed to ANG II and/or antagonists for 3 min in the presence of SOD (100 U/ml), and an aliquot (800 µl) of the conditioned medium was transferred to the RSMC dishes and incubated for 4 min. Reactions were stopped by removing the incubation buffer and placing the cells in 0.01 N HCl for 1 h. The cells were scraped off with a rubber policeman and the cell suspension centrifuged at 15,000 g for 20 min. cGMP was measured in supernatants by radioimmunoassay (Amersham, UK). Cell pellets were resuspended in NaOH and the protein content measured by the method of Bradford (Bio-Rad).

Measurements of calcium transients. Intracellular calcium was measured as described elsewhere (33). RAEC were grown to confluence on coverslips coated with fibronectin. Before experiments, cells were loaded with fura 2 by incubation for 2 h with 5 µM fura 2-acetoxymethyl ester in Hanks' solution with 0.01% Pluronic at room temperature. They were then washed with buffer containing (in mM) 127 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 K2HPO4, 1 Mg2Cl, 2 CaCl2, 5 glucose, 10 sodium acetate, and 20 HEPES, pH 7.4. The cells on the coverslips were placed on the stage of an inverted microscope and were continuously superfused at 37°C with buffer at a flow rate of 0.8 ml/min. Fura 2 was alternatively excited at wavelengths of 340 and 380 nm with the use of a 75-W xenon light source, filters, and a chopper (Photon Technology International Photoscan II System, Kontron). Intracellular calcium values were calculated by the Grynkiewicz formula (13).

Luminol-enhanced chemiluminescence. The production of ROI in response to ANG II was measured by chemiluminescence in the presence of a chemiluminogenic probe, luminol. The medium was removed from confluent cells in 20-cm2 petri dishes, and the cells were washed three times with a modified Krebs buffer (in nM: 2.5 CaCl2, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 118 NaCl, 25 NaHCO4, and 11 glucose). When antagonists were used, they were added 30 min before the experiments. Cells were then scraped off using a rubber policeman in 400 µl of buffer and placed in a luminometer cuvette. Any large cell aggregates were dissociated by gentle pipetting. Luminol was then added to a final concentration of 6.6 × 10-5 M, and the cuvette was placed in a thermostated (37°C) luminometer 1251 LKB (18). Chemiluminescence was triggered by adding 10-7 M ANG II and was continuously monitored for 10 min; the peak (expressed in mV) and the area under the curve (expressed in mV · s) were calculated using a Hewlett-Packard 85 computer.

Statistical analysis. Data are expressed as means ± SE. Groups were compared by a Mann-Whitney U test. Differences were considered significant at P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Evaluation of NO production. NO production by RAEC in response to ANG II was evaluated by measuring the nitrite and nitrate by chemiluminescence (Fig. 1). Nitrite and nitrate production was significantly higher in ANG II-stimulated cells (0.83 ± 0.04 pmol · µg protein-1 · h-1) than in control cells (0.53 ± 0.02 pmol · µg protein-1 · h-1; P < 0.001). This effect was similar to that observed after stimulation with 10-7 M bradykinin (data not shown).


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Fig. 1.   Stimulation of NO production in endothelial cells by ANG II, evaluated by measuring nitrogen oxides [nitrite and nitrate (NOx)] by chemiluminescence. Cells were incubated with 10-7 M ANG II alone or with 10-4 M Nomega -monomethyl-L-arginine (L-NMMA) or 10-6 M losartan for 1 h. Data are means ± SE of 4-6 experiments. ** P < 0.01, *** P < 0.001, compared with control cells. ## P < 0.01, ### P < 0.001, compared with ANG II-stimulated cells. prot, Protein.

ANG II-induced NO production was inhibited by losartan, suggesting that this effect is mediated by the AT1 receptor. Incubation of cells with L-NMMA decreased basal levels of NO production (0.17 ± 0.04 pmol · µg protein-1 · h-1; P < 0.001 vs. control cells) and prevented the ANG II-induced increase in NO production (Fig. 1).

The bioactivity of NO was evaluated by measuring the stimulation of guanylate cyclase activity. Guanylate cyclase activity was previously evaluated in RAEC and in RSMC by measuring the cGMP production in response to different concentrations of SNP. The stimulated cGMP production was much higher in RSMC than in RAEC. Only high concentrations of SNP (10-7 M) doubled cGMP production in RAEC, whereas cGMP production in RSMC was increased 2.5-fold by 10-9 M SNP and 7-fold by 10-7 M SNP.

ANG II-induced NO in RAEC was therefore evaluated by incubating RSMC with supernatant from treated and untreated RAEC. The cGMP content of RSMC was significantly increased by supernatant from unstimulated RAEC (0.63 ± 0.03 vs. 1.08 ± 0.13 pmol/mg protein; P < 0.01), and it was further enhanced by the supernatant from ANG II-stimulated RAEC (1.79 ± 0.1 pmol/mg protein; Fig. 2). The cGMP content of RSMC tended to be decreased by direct ANG II stimulation: 0.63 ± 0.03 vs. 0.48 ± 0.04 pmol/mg protein, although this decrease was at the limit of significance (P = 0.07).


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Fig. 2.   Production of cGMP in smooth muscle cells. Cells were equilibrated in buffer containing 10-4 M 3-isobutyl-1-methylxanthine for 20 min and then incubated with medium ±10-7 M ANG II or with the supernatant from endothelial cells incubated ±10-7 M ANG II in the presence of superoxide dismutase (SOD; 100 U/ml). Data are means ± SE of 5 experiments performed in duplicate. ** P < 0.01 vs. basal smooth muscle cells (SMC); ### P < 0.001 vs. basal SMC + endothelial cell (EC) supernatant.

The type of endothelial receptor implicated in the ANG II-induced NO production was determined using selective antagonists for each ANG II receptor type. cGMP production, which was induced by medium from RAEC stimulated by ANG II, was completely abolished by treatment of RAEC with the AT1 antagonist losartan, whereas the AT2 receptor antagonist PD-123177 had no effect (Fig. 3). These results suggest that only AT1 receptors mediate the stimulation of NO production in response to ANG II in these cells.


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Fig. 3.   Effect of ANG II receptor antagonists on ANG II-induced NO release. Endothelial cells were incubated for 20 min with 10-6 M losartan or 10-6 M PD-123177 and then with 10-7 M ANG II for 3 min. The supernatant was removed and added to SMC for 4 min. Data are means ± SE of 4 experiments performed in duplicate. ** P < 0.01 vs. control.

A pharmacological approach was used to determine whether the increase in cGMP production by RSMC was due to an activation of NO synthase by ANG II. L-NAME, an NO synthase inhibitor, completely blocked the ANG II-induced NO release (Fig. 4) as did W-7, a calmodulin antagonist. In contrast, the B2-receptor antagonist HOE-140 did not alter this effect.


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Fig. 4.   Effect of treating rat aortic endothelial cells (RAEC) with L-NAME, W-7, or HOE-140 on ANG II-induced NO release. Endothelial cells were incubated with 10-4 M L-NAME, 10-5 M W-7, or 10-6 M HOE-140 for 20 min and then with 10-7 M ANG II for 3 min. The supernatant was removed and added to SMC for 4 min. Data are means ± SE of 3 experiments performed in duplicate. # P < 0.05, ## P < 0.01 vs. ANG II-stimulated cells in the absence of antagonists.

Measurements of calcium transients. ANG II induced a transient increase in intracellular free calcium concentration in RAEC (Fig. 5A), which was blocked by the AT1 receptor antagonist losartan (Fig. 5B).


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Fig. 5.   Effect of ANG II on intracellular calcium concentration of RAEC. A: RAEC were exposed to 10-7 M ANG II for 5 min. B: RAEC were exposed to 10-6 M losartan, and 10-7 M ANG II was added 3 min later. These tracings are representative of at least 3 experiments.

Luminol-enhanced chemiluminescence. The luminol-enhanced chemiluminescence of postconfluent RAEC was immediately increased by stimulation with 10-7 M ANG II (Fig. 6A). The chemical nature of the ROI detected by luminol-enhanced chemiluminescence was identified using SOD, catalase, and an NO synthase inhibitor. SOD (100 U/ml) inhibited 57% of the luminol-enhanced chemiluminescence evoked by ANG II, whereas catalase (100 U/ml) had no effect. The NO synthase inhibitor L-NMMA (10-4 M) inhibited 63% of the chemiluminescent signal from RAEC stimulated with 10-7 M ANG II (Fig. 6B). Addition of L-NMMA and SOD do not cause further inhibition. The ANG II effect on the luminol-enhanced chemiluminescence was mediated by the AT1 receptor, as shown by its inhibition with losartan.


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Fig. 6.   A: representative trace of luminol-enhanced chemiluminescence in RAEC stimulated with 10-7 M ANG II. B: effect of different pharmacological agents on ANG II-stimulated luminol chemiluminescence. Cells were preincubated for 30 min with 10-4 M L-NMMA, 100 U/ml SOD, 100 U/ml catalase, or 10-6 M losartan, and luminol-enhanced chemiluminescence was evaluated after stimulation with 10-7 M ANG II. Data are expressed as percentage of area under the curve obtained after stimulation with ANG II alone. Data are means and SE of 4-6 experiments. *** P < 0.001 vs. ANG II-stimulated cells in the absence of antagonists.

Altogether, these results strongly suggest that the luminol-enhanced chemiluminescence produced by stimulated RAEC was due to the enhanced production of NO and superoxide, indicating that peroxynitrite was the oxidant species responsible for the signal.

    DISCUSSION
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Methods
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Discussion
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The major finding reported in this paper is that ANG II directly stimulates NO synthase activity in endothelial cells and that the NO released increases cGMP production in smooth muscle cells. Several studies have shown that the endothelium can modulate the ANG II-induced contraction of blood vessels (3, 5, 12, 39) and that ANG II even elicits vasodilatation in some vascular beds (15, 34). These studies suggested that ANG II stimulates the release of vasodilators such as NO or prostaglandins. Gimbrone and Alexander (11) showed that ANG II stimulates the release of prostaglandins by cultured endothelial cells. However, the present work is the first direct demonstration that ANG II stimulates NO production by cultured endothelial cells. Endothelium-derived NO activates smooth muscle soluble guanylate cyclase, resulting in an increase of cGMP content in these cells. We did not detect any change in the cGMP content in endothelial cells in response to ANG II (data not shown), probably due to the small amount of endothelial soluble guanylate cyclase, as suggested by the low cGMP production induced by SNP in these cells. It has been shown that ANG II stimulates cGMP production in isolated arterial rings (4). Our results indicate that the cGMP produced in the arterial wall by ANG II stimulation is due to activation of the soluble guanylate cyclase in smooth muscle cells by endothelium-derived NO.

The mechanisms leading to the production of NO by ANG II are controversial. We have demonstrated that ANG II stimulates inositol phosphate production (31), and the present study indicates that ANG II increases free intracellular calcium concentration. This increase in intracellular calcium induced by ANG II might be responsible for the activation of NO synthase. This is supported by the finding that W-7, a calmodulin antagonist, inhibited the release of NO by ANG II from the endothelium. However, other mechanisms could also be implicated in ANG II-induced NO release. It has been suggested that the generation of kinins is responsible for NO production in response to ANG II in isolated coronary arteries (32). In this study, we did not observe any changes in ANG II-induced NO release when endothelial cells were incubated with the bradykinin B2 receptor antagonist HOE-140. This suggests that kinin production is not involved in ANG II-induced NO production by cultured RAEC. These differences in the ANG II pathways leading to NO release could be due to the diversity of ANG II receptor types or intracellular pathways coupled to these receptors in endothelial cells from different organs and different species.

Our results indicate that ANG II-induced NO release is AT1 mediated, as this response was blocked by the AT1 antagonist, losartan. These results are consistent with a study suggesting that ANG II stimulates NO release via the AT1 receptor on isolated arteries (3). However, these data do not exclude that NO synthesis and release could also be mediated by the AT2 receptor in some vascular beds (37) or could be stimulated by other angiotensin fragments, such as ANG III, ANG IV, or ANG-(1-7) (21, 30, 32).

We have also observed that ANG II increases luminol-enhanced chemiluminescence in endothelial cells. The interaction between NO and superoxide, in addition to causing their reciprocal inactivation, generates peroxynitrite. Our results are in agreement with a previous study showing that luminol-enhanced chemiluminescence mainly detects peroxynitrite in cultured endothelial cells (18). The superoxide:NO stoichiometry in the present model of cultured RAEC is thus probably within the critical range needed to generate peroxynitrite. Indeed, luminol-enhanced chemiluminescence was strongly reduced when NO synthase activity was inhibited by L-NMMA or when superoxide was dismuted by SOD.

Peroxynitrite is a potent oxidant that directly oxidizes a wide range of biological molecules. Although some authors have indicated that peroxynitrite has physiological activities (20, 23, 38), this compound is widely recognized to be a mediator of NO toxicity (2, 17). However, the action of peroxynitrite seems to depend on the biological environment. It has been reported recently that peroxynitrite causes apoptosis in transformed cell lines but not in normal endothelial cells (22), suggesting that the latter cells have a more efficient defense mechanism. It is also important to point out that peroxynitrite can also generate NO under certain conditions, i.e., in the presence of thiols (24, 38). Finally, peroxynitrite has recently been demonstrated to activate prostaglandin synthesis, suggesting that superoxide serves as a biochemical link between NO and prostaglandin synthesis (20). Further studies are needed to determine the physiological and/or pathological relevance of ANG II-induced peroxynitrite production in the vascular wall.

ANG II is known to be a powerful vasoconstrictor and a hypertrophic agent. In contrast, NO is a vasodilator and inhibits smooth muscle cell proliferation (6). The ANG II-induced release of NO by endothelial cells could thus contribute to modulation of the tone and proliferation of the underlying smooth muscle cells. Thus ANG II could lead to opposite effects, depending on the cell type with which it first interacts. ANG II formed locally within the vascular media could be bound preferentially to smooth muscle ANG II receptors, whereas plasma ANG II could bind mostly to endothelial receptors, because little of the ANG II generated in the media passes into the lumen and vice versa (7). Thus the action of ANG II may depend, at least in part, on its plasma or interstitial location. ANG II action is further complicated by the generation of peroxynitrite, as demonstrated in the present study. The importance of these effects in the physiology of the vessel wall and in the pathological changes due to elevated circulating ANG II remains to be elucidated.

    ACKNOWLEDGEMENTS

We are indebted to A. Pizard and Dr. J. Marchetti for help with the calcium measurements. We thank M. Philippe for technical assistance and A. Depardieu for artwork.

    FOOTNOTES

This work was supported by Grant 314002 from Merck Sharp & Dohme.

Address for reprint requests: M. E. Pueyo, INSERM U460, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France.

Received 28 March 1997; accepted in final form 26 August 1997.

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Abstract
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Discussion
References

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AJP Cell Physiol 274(1):C214-C220
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