Endothelial Dysfunction Coincides With an Enhanced Nitric Oxide Synthase Expression and Superoxide Anion Production
Abstract We investigated the effects of aortic banding–induced hypertension on the endothelium-dependent vasodilator responses in the aorta and coronary circulation of Sprague-Dawley rats. We studied the influence of hypertension on the endothelial nitric oxide synthase (NOS III) expression, assessed by Western blot and reverse transcription–polymerase chain reactions experiments, and on the superoxide anion (O2−) production. Two weeks after aortic banding, the endothelium-dependent relaxations were not altered. At this time, the expression of NOS III in the aorta and in confluent coronary microvascular endothelial cells (RCMECs) exhibited no marked changes, whereas O2− production was enhanced 1.9-fold in aortas from aortic-banded rats. Six weeks after aortic banding, the endothelium-dependent dilations were markedly impaired in the heart (50% decrease) and aorta (35% decrease). Analysis of NOS III protein and mRNA levels revealed marked increases in both aortas and confluent RCMECs (2.6- to 4-fold) from aortic-banded compared with sham-operated rats. There was no further increase in O2− production in both the aorta and confluent RCMECs from aortic-banded rats. An enhanced nitrotyrosine protein level was also detected in the aorta from 6-week aortic-banded rats. These findings indicate that in hypertension induced by aortic banding, an enhanced O2− production alone is not sufficient to produce endothelial dysfunction. Endothelial vasodilator hyporesponsiveness was observed only when NOS III expression and O2− production were increased and was associated with the appearance of enhanced nitrotyrosine residues. This would suggest that the development of endothelial dysfunction is linked to an overproduction of not one, but two, endothelium-derived radicals that might lead to the formation of peroxynitrite.
The endothelium plays a crucial role in the control of local vascular tone through the production of vasoconstricting and vasodilating autacoids. Among these factors, the most important appears to be NO, which is synthesized from l-arginine by the constitutive NO synthase (NOS III) under basal and stimulated conditions.1 The physiological significance of endothelium-derived NO in regulating cardiovascular homeostasis is highlighted by the findings that systemic administration of NOS inhibitors results in a marked increase in arterial blood pressure2 and that homologous recombination resulting in the lack of the gene encoding NOS III in mice results in significant hypertension.3
In various models of hypertension, impaired endothelium-dependent relaxations have been described, implying that this pathophysiological state is associated with a so-called endothelial dysfunction and an apparent decrease in the production of bioactive NO.4 5 The cellular mechanisms involved in the development of this dysfunction remain to be fully elucidated. In vessels from rats with either genetic4 or angiotensin II–induced6 hypertension, enhanced endothelial superoxide anion (O2−) production has been described. Such an increase in O2− production likely accelerates the inactivation of NO and accounts for the apparent decrease in bioactive NO. Although it is generally accepted that such an effect may explain the blunted vasodilator response observed in various forms of hypertension,7 controversy exists regarding the effect of hypertension on NOS III expression and activity (for review, see Reference 88 ). In the present study, we investigated the effects of experimental hypertension, induced by aortic banding, on the endothelium-dependent dilator responses in the aorta and heart of rats. The changes in vascular reactivity were associated with alterations in O2− production and NOS III expression in native macrovascular and cultured microvascular endothelial cells.
Bradykinin was purchased from Bachem, NG-nitro-l-arginine from Serva, and diclofenac (Voltaren injection solution) from CIBA-Geigy. Bovine recombinant SOD (Peroxinorm) was provided by Grünenthal. All other biochemicals were obtained in the highest purity available from Sigma Chemical Co or Merck. The [α-32P]dCTP was purchased from Hartmann Analytic. The cloned bovine NOS III cDNA was a gift from D.G. Harrison, Emory University, Atlanta, Ga. The mouse nitrotyrosine antibody was a gift from J.S. Beckman, University of Alabama, Birmingham. The mouse NOS III antibody was purchased from Transduction Laboratories.
Male Sprague-Dawley rats weighing 250 to 280 g were obtained from Möllegaard (Skensved, Denmark). Animals were housed in a room in which a constant temperature and humidity were maintained. Rats were fed Altromin and had free access to food and water. Animal care and treatment were conducted in conformity with institutional guidelines that are in compliance with German laws and policies. The rats were fasted for 12 hours before surgery. Anesthesia was induced by intraperitoneal injection of 200 mg/kg hexobarbital (Evipan). The abdomen was opened by a cut parallel to the linea alba; the abdominal aorta was exposed above the left renal artery and a silk thread was passed under it. A cannula (0.9×40 mm) was placed longitudinally on the aorta, and both the aorta and cannula were tied. The cannula was then removed, leaving an aortic lumen determined by the diameter of the cannula. Before the abdomen was closed with cat gut, the animals received 5.5 mg rolitetracycline (Reverin, Hoechst AG). The skin was then closed by clipping and covered with tar spray. Sham-operated animals were subjected to the same procedure, without aortic banding. During the first 5 days after surgery, the animals received tetracycline (1 g/350 mL) in the drinking water.
Isolation and Culture of RCMECs
RCMECs were isolated as described by Piper et al,9 with minor modifications. The animals were anesthetized with 200 mg/kg hexobarbital IP, and blood pressure was measured via catheters in the left carotid artery. The hearts were excised and mounted via the aorta to a Langendorff perfusion system. After 5 minutes of non-recirculating perfusion (80 mm Hg; 10 mL/min) with a modified KHB (pH 7.4, 37°C, continuously gassed with 95% O2 and 5% CO2), perfusion was switched to the recirculating mode with 40 mL protease solution (Boehringer) (dispase II from Bacillus polymyxa, 120 μg/mL; activity, 0.5 IU/mg; trypsin, 120 μg/mL; collagenase D from Clostridium histolyticum, 30 IU/mL; albumin, 270 μg/mL; CaCl2, 25 μmol/L) dissolved in KHB. After 15 to 20 minutes, atria were discarded, and the ventricles were minced and again incubated for 15 minutes in 10 mL of the recirculating protease solution. The cell homogenate was filtered through nylon mesh (200 μm). The resulting filtrate was mixed with stock Percoll (Sigma; density, 1.12 mg/mL), yielding a density of 1.01 g/mL. Isolation of RCMECs was performed by two linear Percoll gradient centrifugations. The first gradient (density, 0.498 to 0.876 g/mL) provided a separation of muscle and nonmuscle cells, and by the second gradient (density, 0.168 to 0.62 g/mL), RCMECs were separated from the nonmuscle cell fraction. The RCMEC fraction was washed once with modified HEPES buffer (pH 7, in mmol/L: NaCl 140, KCl 5, MgCl2 0.8, CaCl2 1.8, Na2HPO4 1, HEPES 25, and glucose 5) and centrifuged at 173g for 10 minutes. Thereafter, RCMECs were cultured in medium containing a 1:1 formulation of Dulbecco’s modified Eagle’s and Ham’s F-12 media supplemented with penicillin (50 IU/mL), streptomycin (50 μg/mL), l-glutamine (1 mmol/L), glutathione and l-(+)-ascorbic acid (5 μg/mL each, Biotect protection medium), heparin (8.8 IU/mL), endothelial cell growth supplement (50 μg/mL), and heat-inactivated calf serum (20%). RCMECs were seeded on six-well plates (Nunc Intermed) precoated with collagen A and were maintained in a 95% O2/5% CO2 humidified incubator at 37°C. The culture medium was changed 1 hour after seeding and the following day. Experiments were performed 1 week after seeding for confluent RCMECs. For extraction of protein and mRNA, RCMECs were frozen at −80°C in guanidinium thiocyanate solution.10
Isolated Perfused Rat Heart
Rats were anesthetized with sodium pentobarbital (60 mg/kg IP). After heparinization (500 U IV), the thorax was opened and the heart rapidly perfused in situ through a cannula inserted into the aortic stump (Langendorff preparation). After ligation of the superior and inferior venae cavae close to the right atrium, the heart was excised and perfused at a constant flow to obtain a CPP of approximately 75 mm Hg. Modified KHB (pH 7.4, 37°C, continuously gassed with 95% O2 and 5% CO2) containing the cyclooxygenase inhibitor diclofenac (1 μmol/L) was used as perfusate. CPP was monitored with a pressure transducer (Gould P2310) connected to a sidearm of the aortic perfusion cannula. Isovolumetric LVP was measured with a fluid-filled latex balloon inserted into the left ventricle via the left atrium and connected to a second pressure transducer (Gould CP-01). Balloon volume was adjusted to obtain a diastolic pressure of about 10 mm Hg; heart rate was derived from the LVP signal by a cardiotachometer. The heart was allowed to equilibrate for at least 20 minutes so a stable CPP was obtained. Vasodilator agents, bradykinin (3 to 300 pmol) or SNP (1 and 10 nmol), were administered to the coronary vascular bed as bolus injections (10 μL). Dilator responses were calculated as the percentage decrease in CPP.
Vascular Reactivity Studies
The descending thoracic aorta was removed from anesthetized (60 mg/kg sodium pentobarbital IP) rats, cleaned of connective tissue, and dissected into three sections. The upper section (15 mm) was immediately frozen in liquid nitrogen for Western blot analysis. The lower section (10 mm) was used for measurement of superoxide anion production, and the remaining middle part was cut into four rings of about 3 mm in length that were mounted in a thermostated (37°C) organ bath (Schuler-Organbad, Hugo Sachs Elektronik) for isometric measurement of contractile tone. The rings were equilibrated for 30 minutes under a resting tension of 2 g in carbogenated (95% O2, 5% CO2) KHB, pH 7.4, in the presence of diclofenac (1 μmol/L). Rings were repeatedly contracted by phenylephrine (1 μmol/L) until reproducible contractions were obtained. Thereafter, the relaxant response to acetylcholine was assessed in the presence or absence of SOD (100 nmol/L). After a washout period, rings were contracted by phenylephrine (1 μmol/L) in the presence of NG-nitro-l-arginine (a NOS inhibitor, 100 μmol/L), and the relaxant response to SNP was assessed.
Analysis of NOS III Expression by RT-PCR
Total RNAs were extracted from confluent RCMECs according to the method of Chomczynski and Sacchi.10 For the RT step, 2 μg total RNA was incubated with 200 U reverse transcriptase (GIBCO), dNTP (125 μmol/L), oligo(dT) (200 ng), and reaction buffer in a final volume of 20 μL at 37°C for 60 minutes. In some reaction mixtures, reverse transcriptase or total RNA was omitted to determine the amplification of contaminating genomic DNA or cDNA. After a final denaturation step at 94°C for 7 minutes, 6 μL cDNA was subjected to PCR consisting of denaturation at 94°C for 1 minute, followed by 90 seconds of annealing at 52°C and 90 seconds of elongation at 72°C for 30 cycles. The last cycle was ended with 7 minutes of elongation at 72°C. In each PCR, the cDNAs for NOS III and GAPDH were coamplified. The primers used to amplify NOS III were derived from the sequence of the partially cloned rat NOS III cDNA (Genbank accession number, U02534) (sense primer: 5′-CGTGCGCCAGGCTCTCACTTAC-3′) and from the sequence of the cloned human NOS III cDNA11 12 (antisense primer: 5′-GGCTGCAGCCCTTTGCTCTCA-3′), allowing the amplification of a 550-bp fragment. The primers used to amplify GAPDH were derived from the cloned rat GAPDH cDNA13 (sense primer: 5′-TATGACAACTCCCTCAAGAT-3′; antisense primer: 5′-AGATCCACAACGGATACATT-3′), allowing the amplification of a 320-bp fragment. The PCR contained 0.4 μmol/L of each primer, dNTP (200 μmol/L), MgCl2 (1 mmol/L) reaction buffer, and 2.5 U Taq polymerase (Promega) in a final volume of 50 μL. The amplified cDNAs were size-fractionated by agarose gel electrophoresis, visualized under UV light with ethidium bromide staining, transferred to a nylon membrane (Hybond-N), and hybridized with a 32P-labeled NOS III fragment obtained from the cloned bovine NOS III cDNA and with a 32P-labeled GAPDH fragment isolated by PCR. The NOS III cDNA and GAPDH cDNA were quantified after autoradiography by scanning densitometry. The NOS III cDNA was normalized by comparison with GAPDH cDNA.
Western Blot Analysis
Crude protein extracts were obtained after alcoholic precipitation of the phenol phase obtained after the guanidinium isothiocyanate/phenol/chloroform extraction method.10 A total of 100 μg of the extracts was subjected to SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) as previously described.14 Prestained molecular weight marker proteins (Bio-Rad) were used as standards for SDS-PAGE. Ponceau staining was performed to verify the quality of the transfer and equal amounts of protein in each lane. Proteins were detected using their respective antibodies as described in “Results” and were visualized by enhanced chemiluminescence using a commercially available kit (Amersham). Autoradiographs were analyzed by scanning densitometry.
Measurement of Reactive Oxygen Species
The O2− generation of the rings was assessed by lucigenin-enhanced chemiluminescence as described previously.15 For measurement of H2O2, confluent primary cultures of RCMECs on quartz coverslips were loaded with the fluorescent dye 5- (and 6-) carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCHF, Molecular Probes) by incubation with 20 μmol/L DCHF bis(acetoxymethyl) ester at 37°C for 40 minutes. Thereafter, the coverslips were washed in HEPES-modified Tyrode’s solution of the following composition (mmol/L): NaCl 132, KCl 4, CaCl2 1, MgCl2 0.5, HEPES 9.5, and glucose 5; the rate of intracellular H2O2 formation was determined fluorometrically (Deltascan, Photon Technology Inc) over 30 minutes using an excitation wavelength set at 490 nm and emission wavelength of 520 nm.
Data are expressed as mean±SEM. Statistical analysis was performed by one-way ANOVA followed by a Bonferroni t test or two-tailed Student’s t test for unpaired data when appropriate. Values of P<.05 were considered statistically significant.
Mean Arterial Blood Pressure and Heart Weight
Mean blood pressure and left ventricular weight were significantly enhanced in rats 2 weeks after aortic banding compared with sham-operated rats, whereas right ventricular weight remained unchanged (Table 1⇓). No further increase in either blood pressure or ventricular weight was observed in animals 6 weeks after aortic banding. The weight gain in aortic-banded animals did not differ significantly from that in the sham-operated animals. Mean blood pressure in sham-operated animals 2 and 6 weeks after surgery did not differ (Table 1⇓).
Vasodilator Responses in Isolated Perfused Heart
No differences in basal CPP, heart rate, and flow were observed between sham-operated rats and rats subjected to aortic banding (Ab-rats) (Table 2⇓). LVP was moderately increased in the hearts from Ab-rats, although the difference failed to attain statistical significance. Bolus injections of the endothelium-dependent vasodilator bradykinin (3 to 300 pmol) induced a dose-dependent decrease in CPP in the hearts from sham-operated rats and Ab-rats (Fig 1A⇓ and 1B⇓). Although slightly attenuated, no significant changes in the amplitude of the bradykinin-induced dilation were apparent 2 weeks after aortic banding (Fig 1A⇓). However, the dilator response to bradykinin was markedly reduced in the hearts from Ab-rats after 6 weeks (Fig 1B⇓), the maximal dilator response being only 50% of that observed in hearts from sham-operated animals (n=5, P<.05). The endothelium-independent vasodilator SNP elicited a dose-dependent decrease in CPP without affecting LVP or heart rate. Two weeks after aortic banding, the SNP-induced dilation was decreased, although the difference failed to attain statistical significance (Fig 1A⇓). The decrease in the SNP-induced maximal dilator response was statistically significant 6 weeks after aortic banding (48% decrease, P<.05) (Fig 1B⇓).
Vasodilator Responses in Aorta
In phenylephrine-preconstricted aortic rings, acetylcholine induced a concentration-dependent relaxation that was not different in rings from sham-operated rats and 2-week Ab-rats (Fig 2A⇓). However, 6 weeks after aortic banding, the relaxant response to acetylcholine was significantly blunted (Fig 2B⇓, Table 3⇓). At no time was a difference observed in the endothelium-independent relaxation to SNP (Figs 2C⇓ and 2D⇓). To determine whether enhanced production of O2− accounts for the attenuated vasodilator responses observed in the heart and aorta of Ab-rats, we studied the acetylcholine-induced relaxation in aortic rings in the presence of SOD. SOD (100 nmol/L) did not modify the relaxant response of rings from 2-week Ab-rats (data not shown), whereas the attenuated relaxation of aortic rings from 6-week Ab-rats was completely restored (Table 3⇓, n=5).
Effect of Aortic Banding on Production of Reactive Oxygen Species
The production of O2− by aortic rings from Ab-rats, as assessed by lucigenin-enhanced chemiluminescence, was significantly increased above that observed in rings from sham-operated rats 2 and 6 weeks after aortic banding (1.9- and 1.5-fold increase, respectively; P<.05) (Fig 3⇓). O2− production was already maximal in rings from 2-week Ab-rats. In the presence of NG-nitro-l-arginine (100 μmol/L), the O2− production detected in the aorta from 2- and 6-week Ab-rats was not significantly modified (data not shown).
Since O2− production determines the intracellular H2O2 concentration, we monitored H2O2 production fluorometrically with DCHF in confluent RCMECs isolated from sham-operated rats and Ab-rats. The rate of intracellular H2O2 formation was increased 2.2-fold in RCMECs from 6-week Ab-rats compared with those from sham-operated rats (708±130 versus 329±114 photons per second, respectively; n=4, P<.05).
NOS III Expression in Heart and Aorta
To determine the effect of aortic banding on NOS III expression, we assessed NOS III protein and mRNA levels in aortic segments and in RCMECs. No difference in NOS III protein content was detected between the aortic segments from sham-operated rats and 2-week Ab-rats (Fig 4A⇓). However, 6 weeks after aortic banding, NOS III expression was enhanced fourfold over the level detected in aortas from sham-operated animals (n=4, P<.05) (Fig 4B⇓). No marked change was observed in NOS III expression in confluent RCMECs from 2-week Ab-rats compared with cells from sham-operated rats (Fig 5A⇓); NOS III expression was strongly increased in cells from 6-week Ab-rats (fourfold increase, P<.05) (Fig 5B⇓). The effects of aortic banding on NOS III expression in RCMECs were reflected in the steady-state level of NOS III mRNA. In RT-PCR experiments, performed on the same cell extracts, the amount of the amplified NOS III cDNA was not markedly changed in confluent RCMECs from 2-week Ab-rats (Fig 6A⇓) but was strongly increased in cells from 6-week Ab-rats (2.6-fold increase, P<.05) (Fig 6B⇓).
Effect of Aortic Banding on Peroxynitrite Production
To investigate whether the increase in NOS III expression together with enhanced O2− production 6 weeks after aortic banding was associated with peroxynitrite formation and nitration of tyrosine residues, we performed Western blot analysis of aortic proteins. The tyrosine nitration of proteins was taken as an index of peroxynitrite formation in the vascular wall. Two weeks after aortic banding, a low level of protein tyrosine nitration was detected, but no difference in the pattern of nitrated proteins was observed between sham-operated rats and Ab-rats (data not shown). However, 6 weeks after aortic banding, the tyrosine nitration of a protein corresponding to approximately 50 kD was found to be increased twofold over the level observed in the aorta from sham-operated animals (Fig 7⇓).
In the present study, we have demonstrated that the attenuated endothelium-dependent vasodilator responsiveness observed in the aorta and heart from rats with an experimentally induced hypertension is associated with an increased rather than a decreased NOS III expression. Although oxygen-derived free radicals have been proposed to play a crucial role in the development of the endothelial dysfunction, our results show that the enhancement in O2− production alone, which preceded the upregulation of NOS III expression, was not associated with an endothelial vasodilator hyporesponsiveness. The finding that an enhanced tyrosine nitration of proteins was detected in vessels with an apparent endothelial dysfunction suggests that peroxynitrite, the product of the interaction between both NO and O2−, rather than O2− itself might be involved in the impairment of the endothelial-dependent relaxation.
In a departure from the widely held belief that endothelial dysfunction can be attributed to the decreased production of endothelium-derived NO, recent evidence suggests that impaired endothelium-dependent relaxation in hypertension and atherosclerosis is due neither to decreased NOS activity nor to a deficiency in the availability of l-arginine but to an accelerated inactivation of NO by O2−.16 An enhanced vascular O2− production has already been reported in a number of different pathophysiological conditions associated with endothelial dysfunction including hypercholesterolemia17 and various models of hypertension.4 6 This apparent increase in O2− levels may reflect either a reduced O2− scavenging capacity or an enhanced O2− formation. In fact, experimental evidence has been provided to support both hypotheses. In two different experimental rat models of hypertension,18 19 SOD activity has been reported to decrease, a finding that appears to be in contrast with a report of increased SOD protein levels in aortas from rabbits with coarctation-induced hypertension.20 The possibility cannot be excluded that despite enhanced levels of SOD, a decrease in its activity may be one of the major factors contributing to the increased levels of O2− in the vascular wall. In support of the concept that an elevated O2− generation occurs during the development of hypertension, enhanced NADH and NADPH activity has been described in the aortas of rats with an angiotensin II–induced hypertension.6 Since the renin-angiotensin system is rapidly activated after aortic banding,21 22 it is conceivable that an angiotensin II–coupled mechanism may be responsible for the increase in O2− production observed following aortic banding.
Our data suggest that in the early stages of aortic banding–induced hypertension, the endothelium is the source of the increased O2−. Indeed, endothelium removal markedly reduces O2− formation in aortic rings from hypertensive rats (J. Bauersachs, unpublished observations, 1997). A greater amount of O2− production was also found in endothelial cells cultured from spontaneously hypertensive rats.4 An endothelial source of O2− may also account for the observed decrease in SNP responsiveness in the isolated hearts from Ab-rats. NO generated from intracoronary applied SNP would be expected to be partially inactivated in the endothelium before it is able to activate soluble guanylyl cyclase in vascular smooth muscle cells. In contrast in organ bath experiments using aortic rings from Ab-rats, where SNP is able to directly access vascular smooth muscle cells, NO is generated in a compartment that is only marginally within the vascular sphere of influence of endothelium-derived O2− and is therefore less likely to be rapidly inactivated.
Despite an increase in O2− production 2 weeks after aortic banding, endothelium-dependent dilation was not impaired, suggesting that increased levels of O2− alone may not be sufficient to affect vascular responsiveness. The endothelium-dependent dilation was found to be impaired 6 weeks after aortic banding, whereas at this time, a marked upregulation of NOS III expression was observed in the aorta and in RCMECs. An increase in NOS III activity has already been reported in hearts from spontaneously hypertensive rats using techniques that allowed NO detection and measurement of NOS III activity independently of O2− formation.23 24 The cellular mechanisms leading to the enhanced NOS III expression under such conditions remain to be defined. NOS III is, however, known to be upregulated by growth factors and cytokines, such as transforming growth factor-β25 and basic fibroblast growth factor,26 which are increased in aortas from hypertensive rats.27 Thus, growth factors are potential candidates involved in the upregulation of NOS III expression. NOS III expression is also sensitive to mechanical forces.28 It is therefore likely that not only shear stress but also other hemodynamic determinants, such as distension and pulsatile stretch, might regulate NOS III expression. An unexpected finding was that this enhanced NOS III expression as well as an increased H2O2 production could also be detected in endothelial cells cultured from hearts of 2- and 6-week Ab-rats. This would tend to suggest that hypertension induces a stable and transferable alteration within endothelial cells such as the chronic activation of transcription factors or other mechanisms involved in the regulation of NOS III expression.
Although an enhanced NO formation might be expected to be protective, we observed that an increase in NOS III expression correlated with a depression rather than improvement in endothelial function in aortas from Ab-rats. It has been described that NOS, particularly NOS I, under conditions of l-arginine and/or tetrahydrobiopterin depletion,29 30 might generate O2−. The generation of O2− by NOS III was also shown to be inhibited by the NOS inhibitor Nω-nitro-l-arginine methyl ester.31 Since O2− production was not increased in aortas from 6-week compared with 2-week Ab-rats and was not inhibited in the presence of NG-nitro-l-arginine, it appears that NOS III is not the source of the O2− production. Moreover, the NOS III substrate does not seem to be rate-limiting, as the attenuated endothelium-dependent dilation in aortas from 6-week Ab-rats was restored in the presence of SOD.
In vitro, equimolar concentrations of O2− and NO react rapidly to generate the potent oxidant peroxynitrite, which is able to oxidize thiols,32 lipids,33 and desoxyribose34 as well as to nitrate aromatic amino acid residues. The reaction rate for the formation of peroxynitrite is approximately six times faster than the scavenging of O2− by SOD, implying that peroxynitrite formation can occur in vivo.35 Although in certain biological systems bolus administration of peroxynitrite elicits effects that can be attributed to the formation of intermediate NO-releasing compounds,36 more prolonged exposure to peroxynitrite is likely to have deleterious effects, a phenomenon that may be due to depletion of intracellular thiols. Indeed, in the isolated perfused rat heart, high concentrations of peroxynitrite induced a concentration-dependent vasodilator response that was sensitive to oxyhemoglobin and rapidly exhibited tachyphylaxis.37 Repeated administration of peroxynitrite induced vascular dysfunction and attenuated the endothelium-dependent dilation to other vasodilator compounds. How peroxynitrite is able to alter endothelial function is unclear, but it is possible that nitration of tyrosine residues plays a central role in this process. Peroxynitrite-mediated tyrosine nitration of specific proteins attenuates their tyrosine phosphorylation38 and thus is able to inactivate proteins whose activity depends on the phosphorylation of tyrosine residues. For example, agonist-induced calcium signaling in endothelial cells is regulated by tyrosine phosphorylation,14 39 and peroxynitrite is able to inhibit this response.40 As a marked increase in the nitration of a tyrosine-containing protein was detected in aortas from 6-week Ab-rats, it is likely that peroxynitrite was formed in the aorta. However, in-depth studies are required to investigate the involvement of peroxynitrite and subsequent tyrosine nitration in the development of endothelial dysfunction.
In summary, in aortic banding–induced hypertension, impaired endothelium-dependent relaxations were associated with an enhanced O2− production, an upregulation of NOS III expression, and an increase in the nitrotyrosine protein content. Although the evidence is indirect, we suggest that the elevated intracellular levels of O2− and NO react to produce peroxynitrite, which might be involved in the initiation of endothelial dysfunction.
Selected Abbreviations and Acronyms
|CPP||=||coronary perfusion pressure|
|LVP||=||left ventricular pressure|
|NOS||=||nitric oxide synthase|
|PCR||=||polymerase chain reaction|
|RCMEC||=||rat coronary microvascular endothelial cell|
|SDS-PAGE||=||sodium dodecyl sulfate–polyacrylamide gel electrophoresis|
This study was supported in part by the Deutsche Forschungsgemeinschaft (Bu 43616-1) and the Commission of European Communities (BMH4-CT96-0979). Anne Bouloumié is a recipient of a fellowship from “Association Française pour la Recherche Thérapeutique.” The expert technical assistance of Michaela Stächele, Isabel Winter, and Andreas Schäfer is gratefully acknowledged.
- Received November 7, 1996.
- Revision received January 3, 1997.
- Accepted March 10, 1997.
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