(Hypertension. 1999;33:1225-1232.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Paracrine Role of Adventitial Superoxide Anion in Mediating Spontaneous Tone of the Isolated Rat Aorta in Angiotensin II-Induced Hypertension
From the Vascular Biology Unit, Department of Medicine, Boston University Medical Center, Boston, Mass; and the Department of Veterinary Molecular Biology (M.T.Q.), Montana State University, Bozeman, Mont.
Correspondence to Richard A. Cohen, MD, Vascular Biology Unit, R408, Boston University School of Medicine, 80 E Concord St, Boston, MA 02118. E-mail racohen{at}med-med1.bu.edu
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Abstract |
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Abstract—The relationship between vascular generation of superoxide anion and spontaneous tone observed in the isolated aorta was studied in hypertensive rats infused with angiotensin II. Aortic rings from hypertensive, but not from sham-operated rats, demonstrated oscillatory spontaneous tone that represented 52±5.6% of the maximal contraction to KCl. Spontaneous tone was prevented by calcium-free buffer or by blocking calcium influx through L-type calcium channels with nifedipine. The production of superoxide anion measured by lucigenin chemiluminescence was up to 15-fold higher than in sham-operated rat aorta. The adventitial site of production of superoxide anion was suggested by the fact that lucigenin chemiluminescence was 5.5-fold higher from the adventitia than from the intima. This was confirmed histochemically by demonstrating that the adventitia was the site of reduction of nitroblue tetrazolium as well as immunohistochemical staining of NAD(P)H oxidase subunit proteins. A causal link between superoxide anion production by NAD(P)H oxidase and the spontaneous tone is suggested by the fact that superoxide dismutase or the inhibitor of NAD(P)H oxidase, diphenylene iodonium, decreased both superoxide anion production and spontaneous tone. L-NAME or removal of the endothelium from the aorta had no significant effect on superoxide anion levels or spontaneous tone. However, although superoxide dismutase decreased superoxide anion levels in the presence of L-NAME or in endothelium-denuded rings, it no longer inhibited the tone. This suggests that the effect on tone of superoxide anion originating in the adventitia is mediated by inactivating endothelium-derived nitric oxide, which promotes smooth muscle calcium influx and spontaneous tone. The adventitia is not a passive bystander during the development of hypertension, but rather it may have an important role in the regulation of smooth muscle tone.
Key Words: adventitia • superoxide dismutase • nitric oxide • hypertension • NAD(P)H oxidase
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Introduction |
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Certain types of hypertension are associated with elevated circulating levels of angiotensin II (Ang II), and local production of Ang II in the vessel wall may have autocrine and paracrine effects, even though the circulating peptide level is normal or low.1 Although Ang II has been thought to mediate its effects on the vasculature by way of its direct contractile effects mediated by AT1 receptors, it has recently become apparent that vascular superoxide anion production may have a role in this mediation. In rats, hypertension caused by the infusion of Ang II, but not phenylephrine, increased superoxide anion production from aortic segments as well as increased NAD(P)H oxidase activity in aortic homogenates.2 3 Furthermore, treatment of the Ang II-infused hypertensive rats with superoxide dismutase (SOD) decreased blood pressure, indicating a role for superoxide anion in mediating the hypertension.4
It has been reported that both cultured endothelial5 6 7 8 9 and cultured vascular smooth muscle cells10 11 are capable of producing superoxide anion. However, the largest amount of superoxide anion is generated in aorta of normal rabbits12 and rats13 by way of a membrane-bound NAD(P)H oxidase which is localized in the adventitia. Furthermore, Ang II increases the production of superoxide anion and increases the activity of NAD(P)H oxidase in cultured aortic adventitial fibroblasts.14
Our preliminary studies of the isolated aorta of hypertensive rats infused for 6 days with Ang II showed that the aorta of these rats, but not those from sham-operated normotensive rats, developed oscillatory spontaneous contractile tone. We investigated whether this contractile tone occurred as a result of increased production of superoxide anion and whether the oxidative stress accompanying its production influenced the mechanisms which govern calcium homeostasis in the smooth muscle. Our results demonstrate that the adventitia of the aorta remains the primary site of superoxide anion production and that the contractile tone depends on the effect of superoxide anion to inactivate the endogenous vasodilator, nitric oxide.
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Methods |
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Animal Model
Male Wistar rats (270 to 300 g) were anesthetized with an intraperitoneal injection of 80 mg/kg ketamine and 10 mg/kg xylazine, and an incision was made in the midscapular region under sterile conditions. Osmotic minipumps (Alzet, Alza Corp) containing Ang II dissolved in 0.15 mol/L NaCl and 1 mmol/L acetic acid were implanted. The delivery rate was 0.7 to 1.3 mg · kg-1 · d-1 for 6 days. Sham-operated animals underwent an identical surgical procedure, except that an osmotic minipump containing 0.15 mol/L NaCl was implanted. Systolic pressures were determined immediately before surgery and at the end of the infusion period by tail cuff plethysmography. Procedures were according to institutional guidelines.
Preparation of Rings of Rat Thoracic Aorta
The thoracic aorta was cleaned of adherent fat, and rings 5
mm long were cut and mounted on triangular stirrups for isometric
tension recording in organ chambers containing 6 mL of buffer
as described.13 The rings were maintained at 37°C and pH
7.4 with 95% O2 /5% CO2
and stretched gradually over 1 hour to optimal tension (50 mN) for
smooth muscle contraction. Sodium nitroprusside
(10-7 mol/L) or papaverine
(10-5 mol/L) was introduced for the last 30
minutes to allow adjustment under passive conditions. On the removal of
the sodium nitroprusside or papaverine by washing, aortic rings from
Ang II-infused hypertensive rats, but not from sham-operated
normotensive rats, contracted spontaneously. This is termed spontaneous
contractile tone. After being stretched to the resting tension, aortic
rings from normotensive and hypertensive rats were at similar points of
their length-tension relationship, as indicated by the fact that
contractions to KCl (120 mmol/L) were not significantly different
after treatment with SOD (see the Table).
The role of superoxide anion and nitric oxide were determined by
incubating rings with SOD (150 units [U]/mL), diphenylene iodonium
(DPI) (10 µmol/L), or L-NAME (300 µmol/L). The role of
calcium in mediating the spontaneous tone was examined in rings treated
with the L-type channel blocker nifedipine (100 nmol/L) or
by replacement of the buffer with one that was identical except that it
lacked calcium. Each of these treatments was for 
90 minutes during
the stretching of the ring to its optimal tension. In some studies,
nifedipine or calcium-free buffer was added after the
spontaneous tone had developed.
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At the conclusion of the experiments, the rings were contracted by 120 mmol/L KCl. The spontaneous tone in rings from hypertensive rats was expressed in grams of contraction as well as a percentage of the 120 mmol/L KCl contraction.
To examine the in vitro effect of Ang II on tone and superoxide anion production, aorta of normal rats were contracted with 100 nmol/L of Ang II.
Detection of Superoxide Anion by Lucigenin
Chemiluminescence
The details of this assay have been published
previously.12 Briefly, after completion of the studies in
the organ chamber, the rings were rinsed in normal buffer and
transferred to test tubes that contained 1 mL of HEPES buffer (pH 7.4)
containing lucigenin (250 µmol/L) and maintained at 37°C in
the presence or absence of the same treatments as for the tension
studies. Because measurements with lucigenin (250 µmol/L) have
been questioned because of redox cycling by the indicator and
generation of superoxide anion, some studies were done with lucigenin
(5 µmol/L) which has been shown not to be responsible for redox
cycling.15 The luminometer was set to report arbitrary
units of light emitted and integrated over a 30 second interval; after
a 20 minute equilibration, repeated measurements were collected during
5 minutes and averaged. Tiron (10 mmol/L), a cell permeant,
nonenzymatic scavenger of superoxide anion, was then added to quench
the superoxide anion-dependent chemiluminescence; readings from the
last 90 seconds of an additional 5-minute period were averaged. To
examine the adventitial and intimal production of superoxide
anion, the rings were cut longitudinally, flattened, and tied with 3-0
silk suture to a small black plastic plate, with either adventitial or
intimal surfaces facing outward.13 The rings were then
transferred to test tubes that contained 5 mL of buffer and were
maintained at 37°C and 95% O2/5%
CO2 for 1 hour. Rings were then placed into 1 mL
of HEPES buffer (pH 7.4) containing lucigenin 5 µmol/L.
Localization of Superoxide Anion by Nitroblue Tetrazolium Reduction
and Immunohistochemistry
To examine the site of superoxide anion generation in the
tissue, aortic rings were incubated with nitroblue tetrazolium to allow
superoxide anion generated by the tissue to reduce the nitroblue
tetrazolium to blue formazan as described.13 16
To localize the source of superoxide production, we analyzed frozen sections of rat aorta by use of immunohistochemistry with monoclonal antibodies that recognize 4 NAD(P)H oxidase proteins known to be essential for NAD(P)H oxidase activity in leukocytes as described.17 18 19 20
Drugs
Lucigenin, L-NAME, nitroblue tetrazolium,
nifedipine, papaverine, SOD, sodium nitroprusside, and
Tiron were purchased from Sigma. Diphenylene iodonium was
purchased from Biomol. All drug solutions were made fresh just before
each experiment. Drugs were added in aliquots of <1% of the solution
volume. DPI and nifedipine were prepared in pure dimethyl
sulfoxide. All other drugs were prepared as stock solutions in
distilled water.
Data Analysis
Data are expressed as mean±SEM. Statistical comparisons were
made by Student t test. Significance was accepted when
P was <0.05.
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Results |
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Effect of Ang II on Systolic Blood Pressure
By the seventh day, Ang II infusion increased systolic blood pressure (188±4 mm Hg, n=12) with a range of 170 to 206 mm Hg. This blood pressure was significantly higher than in sham-operated rats (128±4 mm Hg, n=7, P<0.01) with a range of 113 to 162 mm Hg.
Spontaneous Tone in Rings From Ang II-Infused Hypertensive
Rats
In Ang II-infused hypertensive rats, spontaneous tone was evident
by the oscillatory tension generated in the absence of added
contractile agonists. In 11 of 12 hypertensive rats in which rings were
equilibrated at the optimal resting tension for contraction (50 mN) in
the presence of sodium nitroprusside (10-7
mol/L) or papaverine (10-5 mol/L), washing the
rings to remove the vasodilator resulted in either persistent (Figure 1 and 2) or intermittent (Figure 3)
spontaneous tone. Rapid oscillations in tone with an
average frequency of 3 to 6 per minute were superimposed on the
spontaneous tone in some, but not all rings. The peak tension achieved
after washout of sodium nitroprusside was 40±4.8 mN (n=11), which
represented 52±5.6% of the maximal contractions to
120 mmol/L KCl (Table). In additional experiments using
papaverine as the vasodilator, the peak spontaneous tone achieved after
washout of papaverine was 46±8.0% (n=6) of the maximal contraction to
120 mmol/L KCl, which was not significantly different from that
observed with washout of sodium nitroprusside. Rings from sham-operated
rats developed no significant tension (0.2±0.1 mN [n=9] after
washing out sodium nitroprusside; 0.3±0.3 mN [n=9] after washing out
papaverine).
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Superoxide Anion Production by Lucigenin
Chemiluminescence
Lucigenin (250 µmol/L) chemiluminescence in rings from
sham-operated normotensive rats was 34±2.9 milliunits ·
mg-1 · min-1 (n=7)
and was significantly increased in the rings from Ang II-infused
hypertensive rats (60±8.0 milliunits ·
mg-1 · min-1, n=9,
P<0.01). After Tiron was added, chemiluminescence was
reduced to 17±0.8 milliunits · mg-1
· min-1 in rings from normal rats and 13±1.9
milliunits · mg-1 ·
min-1 in rings from Ang II-infused rats,
P>0.05). After subtracting lucigenin chemiluminescence in
the presence of Tiron from that obtained in its absence, the calculated
Tiron-quenchable chemiluminescence was significantly higher in rings
from Ang II-infused rats (44±8.0 milliunits ·
mg-1 · min-1) than
in the rings from sham-operated normotensive rats (18±2.8
milliunits · mg-1 ·
min-1, P<0.01).
Chemiluminescence measured with lucigenin (5 µmol/L) in rings from sham-operated normotensive and Ang II-infused hypertensive rats was 22±4.7 and 52±7.8 milliunits · mg-1 · min-1, respectively (n=5, P<0.05). After Tiron was added, the chemiluminescence was decreased to a similar level in rings from both groups of rats (21±4.6 and 17±3.2 milliunits · mg-1 · min-1, in sham-operated and Ang II-infused rats, respectively, P>0.05). The Tiron-quenchable chemiluminescence was 15-fold higher in the rings from Ang II-infused rats compared with those from sham-operated normotensive rats (35±7.9 and 2.3±1.3 milliunits · mg-1 · min-1, respectively, P<0.01).
Effect of Inhibitors of Enzymatic Sources of Superoxide
Anion Production on Spontaneous Tone and Superoxide Anion
Production
In the rings from sham-operated rats, SOD (150 U/mL) reduced
Tiron-quenchable lucigenin (250 µmol/L) chemiluminescence from
18±2.8 to 2.8±2.0 milliunits ·
mg-1 · min-1 (n=8,
P<0.01), and there was no observable effect on tone.
In the rings from Ang II-infused hypertensive rats, SOD (150 U/mL) reduced spontaneous tone from 39±5 mN (n=9) to 7±3 mN (n=8, P<0.01), which represents 12±4.1% of the maximum contraction to KCl (Figure 2B). Tiron-quenchable lucigenin (250 µmol/L) chemiluminescence production in the rings from hypertensive rats was also significantly decreased from 44±8.0 to 6.5±1.6 milliunits · mg-1 · min-1 in the presence of SOD (n=8, P<0.01, Figure 2C). The possibility that the inhibitory effect of SOD is due to its contamination by NH4Cl was considered by determining the effect of SOD, which was heated by autoclave for three 1-hour cycles. The autoclaved SOD had a significantly reduced inhibitory effect on spontaneous tone of aorta from Ang II-induced hypertensive rats (33±5.3% KCl, n=6, P>0.05 compared with untreated aorta). Autoclaved SOD retained some activity to reduce Tiron-quenchable chemiluminescence, but this effect was also statistically smaller compared with control SOD (19±1.3 milliunits · mg-1 · min-1, n=6, P<0.01 compared with control SOD).
The effects of various inhibitors of enzymatic sources of superoxide anion generation were also tested. DPI (10 µmol/L), an NAD(P)H oxidase inhibitor, effectively inhibited both spontaneous tone and superoxide production in rings from Ang II-infused hypertensive rats (spontaneous tone, 13±13% of the KCl contraction, n=4, P<0.01, Figure 2C; Tiron-quenchable lucigenin (250 µmol/L) chemiluminescence, 2.4±0.9 milliunits · mg-1 · min-1, n=4, P<0.05). In contrast, neither the NADH dehydrogenase inhibitor, rotenone (10 µmol/L), nor the xanthine oxidase inhibitor, oxypurinol (300 µmol/L), decreased spontaneous tone or superoxide production in the rings from Ang II-infused hypertensive rats (spontaneous tone: oxypurinol, 28.5±7.5%, n=6; rotenone, 54±5.6%, n=5, P>0.05; Tiron-quenchable chemiluminescence: oxypurinol, 55±5.2 milliunits · mg-1 · min-1, n=6, P>0.05 versus control; rotenone, 48±8.7 milliunits · mg-1 · min-1, n=5, P>0.05).
Role of Extracellular Calcium in Spontaneous Tone and Superoxide
Anion Production
Spontaneous tone was abolished in calcium-free buffer or by the
L-type calcium channel blocker, nifedipine (100 nmol/L,
n=6, not shown). The blockade was reversible; replacement with buffer
containing calcium (2.5 mmol/L) or washing out the
nifedipine restored tone. Spontaneous tone was also
prevented in rings pretreated with nifedipine from the
beginning of the experimental protocol (3±3% KCl contraction, n=6,
P<0.001 versus control). In rings treated with
nifedipine, Tiron-quenchable chemiluminescence was not
significantly different compared with rings without
nifedipine (32±10 milliunits ·
mg-1 · min-1, n=6,
P>0.05).
Effect of L-NAME or Endothelium Denudation on
Spontaneous Tone and Superoxide Anion Production
The role of endogenous nitric oxide in the modulation
of spontaneous tone and superoxide anion levels was tested by the
measurement of spontaneous tone in rings treated with L-NAME (Figure 3) or in rings from which the endothelium was
denuded (Figure 4).
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In the rings from sham-operated normotensive rats, L-NAME (300 µmol/L) did not result in significant spontaneous tone or increase in superoxide anion production (17±6.8 in control, 19±4.1 milliunits · mg-1 · min-1 in L-NAME-treated rings, n=4, P>0.05). In endothelium-denuded rings of sham-treated rats, a small increase in spontaneous tone developed after washout of sodium nitroprusside (2.3±0.7% of the KCl contraction compared with 0.3±0.2% of the KCl contraction in endothelium-intact rings, P>0.05, n=8 and 5, respectively).
In the rings from Ang II-infused hypertensive rats, L-NAME (300 µmol/L)had no significant effect on spontaneous tone (58±6.8% KCl, n=6, P>0.05, Figure 3C) or on superoxide anion production (39±4.0 milliunits · mg-1 · min-1, n=6, P>0.05) compared with the untreated rings. In the presence of L-NAME, SOD (150 U/mL) failed to significantly inhibit the spontaneous tone (62±7.9% of KCl contraction, P>0.05, Figure 3D). SOD did however significantly decrease Tiron-quenchable superoxide anion production in rings treated with L-NAME to 6.5±1.6 milliunits · mg-1 · min-1 (P<0.01).
Endothelium denudation had no significant effect on the magnitude of spontaneous tone in rings from Ang II-infused hypertensive rats (69±8.6% of KCl contraction, n=5, P>0.05 compared with endothelium-intact rings, Figure 4B). Tiron-quenchable lucigenin chemiluminescence also was not significantly different in rings of Ang II-infused hypertensive rats from which the endothelium was removed (31±5.8, n=6, P>0.05 compared with intact rings). In endothelium-denuded rings of Ang II-infused hypertensive rats, the maximal spontaneous tone in rings treated with SOD (150 U/mL) was not significantly different from the values of untreated, endothelium-denuded rings (50±10% KCl contraction, n=5, P>0.05, Figure 4C). The reduction in Tiron-quenchable chemiluminescence caused by SOD was not significantly affected by removal of the endothelium (7.2±2.5 milliunits · mg-1 · min-1, n=5, P<0.05 compared with in the absence of SOD).
Contractions to KCl
The contractions of aortic rings to 120 mmol/L KCl are shown
in the Table. The contractions to KCl of the rings from Ang
II-infused hypertensive rats were significantly greater than the rings
from sham-operated normotensive rats. Removal of
endothelium or L-NAME treatment did not have a
significant effect on the contractions to KCl in the rings from either
sham-operated normotensive rats or Ang II-infused hypertensive rats
(Table).
SOD decreased the KCl-induced contractions in endothelium-intact, but not in endothelium-denuded rings from Ang II-infused hypertensive rats. SOD had no significant effect on KCl-induced tone in the sham-operated normotensive rats. After treatment with SOD, there was no significant difference in the contractions caused by KCl of endothelium-intact aortic rings from sham-operated normotensive and Ang II-infused hypertensive rats (Table).
Neither nifedipine nor DPI significantly decreased contraction to 120 mmol/L KCl (Table). Neither oxypurinol (300 µmol/L) nor rotenone (10 µmol/L) had any significant effect on the maximal contraction to 120 mmol/L KCl (Table).
Adventitial Superoxide Anion Production Measured by
Lucigenin Chemiluminescence
Lucigenin (5 µmol/L) chemiluminescence in aortic rings in
which the adventitia was facing outward was significantly higher than
in those in which the intimal surface was facing outward (Figure 5). The chemiluminescence measured from
the adventitial side of aortic rings from Ang II-infused rats was
2.6-fold higher than in those from sham-operated rats (13±2.9 and
4.9±1.1 milliunits · mg-1 ·
min-1 in Ang II-infused and in sham-operated
rats, respectively, n=4 each, P<0.05). The
chemiluminescence detected from the intimal side of aortic rings from
hypertensive as well as sham-operated rats was significantly lower than
that from the adventitial side (Figure 5).
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P<0.05
compared with sham. Each group contains rings from 4 rats.
Localization of Tissue Site of Superoxide Anion Production
by Reduction of Nitroblue Tetrazolium and by Immunohistochemistry of
NAD(P)H Protein Subunits
Incubation of rings from 5 Ang II-infused rats with nitroblue
tetrazolium resulted in blue staining predominantly of the adventitial
layer. Figure 6 shows an example of a
cross section of an Ang II-treated rat aortic ring that had been
incubated with nitroblue tetrazolium and counterstained with eosin. It
was not possible to discriminate any difference in the intensity of
nitroblue tetrazolium staining between the adventitia of Ang II-infused
rats and that observed in normal rats (not shown).13
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Immunohistochemistry performed with monoclonal antibodies against p67phox (Figure 7, panel B), p47phox (panel C), and p22phox (panel D) showed localization of each of these protein subunits of NAD(P)H oxidase to the adventitial layer. As with nitroblue tetrazolium staining, there was no evident difference between the intensity of staining observed in hypertensive versus normal rat aortic adventitia (not shown).13 Nonimmune serum (panel A) showed no staining.
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Discussion |
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The major finding in this study is that the spontaneous tone of the isolated rat aorta of Ang II-infused hypertensive rats is dependent on an increased generation of superoxide anion radical. Localization of the adventitial site of generation of superoxide anion by lucigenin chemiluminescence and nitroblue tetrazolium, and the immunohistochemical localization of NAD(P)H subunit proteins in the adventitia suggests a paracrine mechanism between cells of the adventitia with cells in the remainder of vascular wall that contributes to the regulation of smooth muscle tone.
Tone of resistance arteries, termed myogenic tone, is thought to normally mediate autoregulation of blood flow in response to changes in transmural pressure.21 22 Spontaneous tone, such as that observed in this study, has been observed in isolated hypertensive resistance arteries and has been attributed to the increased transmural pressure present in vivo.22 23 24 Spontaneous tone has also been noted in larger arteries of hypertensive animal models, including the hypertensive rat aorta which normally does not display such tone,23 24 and parallels have been drawn between the mechanisms responsible for myogenic and spontaneous tone.21 The spontaneous tone noted in the present study of the hypertensive rat aorta was observed after the artery was stretched to the optimal resting tension for contraction. The active nature of the tone was made evident by the fact that it was suppressed by sodium nitroprusside or papaverine. The tone depends critically on increased levels of superoxide anion as indicated by finding that (1) SOD, a specific scavenger of superoxide anion, and DPI, an inhibitor of NAD(P)H oxidase, prevented the tone; and (2) increased generation of superoxide anion was measured with lucigenin and suppressed by SOD. Initial observations made in this study with a higher concentration of lucigenin (250 µmol/L) were confirmed with a much lower concentration (5 µmol/L) that has been shown not to be associated with redox cycling.15 In fact, the magnitude of the difference in superoxide anion production between sham-operated and Ang II-infused hypertensive rat aorta measured with the lower concentration of lucigenin was greater, perhaps because of redox cycling with the higher concentration.
Work by the groups of Griendling and Harrison2 3 4 has shown that superoxide anion generation is increased in the aorta of Ang II-infused rats by way of increased activity of NAD(P)H oxidase. Our findings with DPI, an NAD(P)H oxidase inhibitor, are consistent with their results. The lack of effect of rotenone or oxypurinol suggests that mitochondrial NADH dehydrogenase or xanthine oxidase, respectively, were not the major source of superoxide anion in the aorta of Ang II-infused rats. Our results agree with earlier results that indicate that the majority of the superoxide anion produced is by an NAD(P)H oxidase.2 3 4 Because Harrison and colleagues also showed that a superoxide anion scavenger decreased the elevated blood pressure,4 they were able to implicate vascular superoxide anion generation at the level of resistance vessels as a significant mechanism that contributes to Ang II-induced hypertension. Because superoxide anion has been linked in this way to the mechanism of hypertension in this model, as well as in the SHR rat,25 in which isolated arteries also display spontaneous tone,23 24 26 it is important to determine the cellular mechanism by which the tone is mediated.
Influx of extracellular calcium through smooth muscle L-type calcium channels appears to be responsible for the tone in the aorta as indicated by the fact that nifedipine or removal of extracellular calcium prevented the tone. This finding agrees with previous work on myogenic tone in normal22 24 27 and hypertensive23 resistance arteries, which shows the critical role of calcium influx through smooth muscle L-type calcium channels. As suggested by its effect on potassium-induced contractions (Table), DPI may have inhibited spontaneous tone, in part by its ability to inhibit L-type calcium channels.28 However, even expressed as a percentage of the reduced contraction to KCl, DPI effectively inhibited the spontaneous tone. Although it is quite clear that calcium influx is responsible for the tone observed in the Ang II-infused hypertensive rat aorta, the generation of superoxide anion was not acutely affected by nifedipine. This suggests that the increased superoxide anion is responsible for the increased smooth muscle calcium influx and is consistent with our hypothesis that the increase in adventitial superoxide anion is causally related to the smooth muscle tone observed.
The spontaneous tone observed in the isolated rat aorta after 6 days of Ang II infusion contrasts with the contractions caused by Ang II in a normal rat aorta, because the latter are insensitive to SOD and are also not accompanied by a measurable increase in superoxide anion (H.D.W., unpublished observations, 1998). This is undoubtedly related to the fact that the increase in NAD(P)H oxidase activity and superoxide anion generation caused by Ang II requires significantly more time than was allowed in the acute exposure to Ang II. It is possible that a prolonged exposure to elevated levels of Ang II in vivo as in our studies results in the production of a number of growth and/or contractile factors that contribute to the regulation of superoxide anion production and might also be important in directly increasing smooth muscle cell tone.
The observation that increased superoxide anion generation in the Ang II-induced hypertensive rat aorta was responsible for abnormal endothelium-dependent relaxation to acetylcholine,2 suggests that superoxide anion generation inactivates endothelium-derived nitric oxide in this model. It was therefore of interest to determine the role of nitric oxide in the spontaneous tone in these arteries. The fact that endothelium denudation or L-NAME did not increase the magnitude of the tone suggests that nitric oxide is either made in inadequate amounts under basal conditions, or it is not sufficiently active under these conditions to inhibit the tone of the hypertensive aorta. However, the fact that SOD did not inhibit tone in endothelium-denuded rings or in the presence of L-NAME suggests that the ability of SOD to decrease tone in endothelium-intact rings depends on the ability of the superoxide anion scavenger to increase the biological activity of endothelium-derived nitric oxide. This observation indicates that nitric oxide is inactivated by superoxide anion in the aorta of Ang II-infused rats, and is the reason that the spontaneous tone develops. Thus, superoxide anion appears to modulate smooth muscle tone in the hypertensive rat aorta predominantly by inactivating endothelium-derived nitric oxide, rather than by direct effects on the smooth muscle.
In our previous studies of the normal rat13 and rabbit12 aorta, it became evident that superoxide anion was generated to a major extent in the adventitia. A larger production of superoxide anion from the adventitial aspect of the vascular wall was demonstrated by lucigenin chemiluminescence in aorta of both normotensive,13 and in this study, hypertensive rats. The endothelium apparently does not contribute a significant amount to the measured vascular superoxide anion production because endothelial denudation did not affect the levels. The adventitial source of superoxide anion was also evident in nitroblue tetrazolium staining of the aorta of Ang II-infused hypertensive rats, which suggests the importance of induction by Ang II of NAD(P)H oxidase activity in adventitial fibroblasts, a fact that has been confirmed in cell culture.14 Indeed, p22phox, p47phox, and p67phox components of neutrophil NAD(P)H oxidase were shown to be present in the aortic adventitia by immunohistochemical techniques in both normotensive,13 and in this study, Ang II-infused rats. The absence of immunohistochemical staining in medial smooth muscle cells also indicates that the lower levels of superoxide anion detected by reduction of nitroblue tetrazolium is not likely caused by greater scavenging of superoxide anion by endogenous SOD but rather a paucity of the same enzymatic source of superoxide anion that is present in the adventitia of the intact aorta.
These observations suggest that increased quantities of superoxide anion and/or growth and contractile factors arising in the hypertensive aortic wall are responsible for contractile tone. However, the spontaneous tone in the hypertensive rat aorta is observed as a result of an interesting paracrine relationship between adventitial superoxide anion and the endothelium-derived nitric oxide that it inactivates. This is made possible by the diffusion distance for superoxide anion, which is estimated to be at least several microns,29 and that of nitric oxide, whose diffusion distance is on the order of 30 µm and is capable of pervading the entire vascular wall.29 These observations suggest that novel paracrine relationships may exist between different components of the vascular wall of resistance arteries that have a role in hypertension. They also indicate that the adventitia is not a passive bystander during the development of hypertension but rather may play an important role in the regulation of smooth muscle tone.
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Acknowledgments |
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We would like to acknowledge the expert technical assistance of Gayle Callis (Department of Veterinary Molecular Biology, Montana State University) for our immunohistochemistry results. This work was supported by National Institutes of Health R01 HL55620 (R.A.C.), SCOR HL55993 (R.A.C.), R29 55425 (P.J.P.), R29 51875 (A.J.C.) and RO1 AR42426, USDA NRICGP9502274, an Arthritis Foundation Biomedical Science grant, and an Established Investigator Award from the American Heart Association (M.T.Q.).
Received November 30, 1998; first decision December 16, 1998; accepted January 6, 1999.
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