© 1998 American Heart Association, Inc.
Original Contribution |
Expression of Constitutive and Inducible Nitric Oxide Synthases in the Vascular Wall of Young and Aging Rats
From the Nephrology, Hypertension, and Cardiovascular Research Laboratory, Fundación Jiménez Díaz (M.R.C., L.S. de M., M.G.-D., F.G.-F., I.M., M.M., L.R., T. de F., J.A.R.-F., J.G., C.C., S.C., A.L.-F.) and Comparative Neuroanatomy Department, Instituto Cajal (J.R., P.F.), Madrid, Spain.
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Abstract |
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Abstract—Two NO synthase (NOS) isoforms have been described in vessels, an endothelial constitutive NOS (eNOS) and an inducible NOS (iNOS). The purpose of the present study was to examine the endothelium-dependent and endothelium-independent hypotensive response in aging rats, analyzing the ability of their vessels to produce NO. The studies were performed in 2 groups of euvolemic, conscious, male Wistar rats: aging rats (n=20, 18 months old) and young rats (n=20, 5 months old). The hypotensive responses to acetylcholine, bradykinin, and sodium nitroprusside were determined. Furthermore, the expression of the NOS isoforms by Western blot and the eNOS and iNOS activities, defined as Ca2+-dependent and Ca2+-independent conversion of [14C]L-arginine into [14C]L-citrulline, respectively, were also determined. In the aging rats, we found an impaired hypotensive response to acetylcholine and bradykinin (2 NO- and endothelium-dependent hypotensive agents) that was accompanied by a preserved hypotensive response to sodium nitroprusside. Aging rats also demonstrated an enhanced sensitivity response to the pressor effect of the L-arginine antagonist L-N
-nitro-L-arginine
and a reduced vasoconstrictor response to angiotensin II.
The inhibition of NO synthesis normalized the pressor effect of
angiotensin II in the aging animals. Nitrite plus nitrate
plasma levels were increased in aging rats. Furthermore, cGMP content
was also higher in the aging vessels. In the aging aortas, the
expression of both eNOS and iNOS isoforms was enhanced. However, in
aging rats, the activity of the eNOS isoform was markedly reduced, a
finding that was accompanied by the presence of iNOS activity. The
vessel wall of aging rats showed an enhanced expression of eNOS and
iNOS isoforms. However, eNOS activity was reduced in the aging animals.
These findings could explain the impaired
endothelium-dependent hypotensive response associated
with aging.
Key Words: acetylcholine • aging • arteries • endothelium-derived relaxing factor
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Introduction |
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Nitric oxide is a multifunctional molecule with an important role in the relationship between the cells that compose the microvascular environment. NO has been implicated in the regulation of different vascular functions (for review, see References 11 to 8). However, the first described effect of NO was its vasodilating property.1 NO is generated from the metabolic conversion of L-arginine into L-citrulline by the activity of NO-synthesizing enzyme (NO synthase [NOS]).9 In the vessels, 2 major classes of NOS activities have been described. One NOS isoform is constitutively expressed (eNOS) in the endothelium under basal conditions and is involved in the endothelium-dependent vasodilating response. The NO generated by the eNOS activation provokes vasodilation by stimulating soluble guanylate cyclase in the vascular smooth muscle cells.1 3 10 Nitrovasodilators cause relaxation of vascular tissue, independent of endothelium, by activating cGMP formation in adjacent smooth muscle cells and directly releasing NO.10 The other NOS isoform (iNOS) is inducibly expressed, and it has been found in cytokine-treated vessels. Whereas activation of eNOS generates small amounts of NO for short periods of time, iNOS stimulation results in a delayed and prolonged release of large amounts of NO.3 11
Several vascular disorders, including a diminished endothelium-dependent relaxation, have been demonstrated in aging humans and in experimental animals.12 13
Different considerations have been raised to explain the reduction of the endothelium-dependent vasodilation described in the aging animals: a decreased number of vasodilator receptors in the endothelium,14 a diminished capability to generate NO by the endothelium,12 and a reduction in guanylate cyclase activity in vascular smooth muscle cells.15
Based on the above described data, the first aim of the present study was to examine the endothelium-dependent and endothelium-independent vasodilator response in aging rats, determining the capability of their vessels to produce NO.
In contrast to the evidence that cytokines stimulate NO
production by the iNOS activity, several reports have
demonstrated that the hypotensive response related to the eNOS activity
could be inhibited by the same cytokines through downregulation
of the eNOS expression. In this regard, we recently demonstrated that
tumor necrosis factor-
(TNF-) reduced eNOS protein expression in
cultured bovine endothelial cells through the binding
of endothelial-cytosolic proteins to the
3'-untranslated region of eNOS mRNA.16 Recent
studies have shown that TNF- is increased in the vascular wall of
aging rats17 ; therefore, in a second set of
experiments, we analyzed the expression of both eNOS and iNOS
proteins in isolated aortas obtained from both aging and young
rats.
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Materials and Methods |
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Animal Instrumentation
The studies were performed in 2 groups of euvolemic, conscious, male Wistar rats: aging rats (18 months old, 398±44 g body wt) and young rats (5 months old, 276±38 g body wt). Before the experiments, the rats were maintained overnight without food and with water ad libitum. Animals were surgically prepared by inserting catheters in the left femoral vein and artery as described.18 19 At the end of surgery, the animals were introduced in cages with water ad libitum. The temperature was held constant at 24°C in the animal housing. Mean arterial pressure (MAP) and heart rate were continuously monitored with a pressure transducer (Power Laboratory System, Cibertec). The experiments were started after the animals were fully awakened from anesthesia (
3 hours) and MAP and
heart rate were stabilized.
Experimental Studies
Study I
To analyze the pressure-lowering effect of 2
endothelium-dependent vasorelaxing agents,
acetylcholine (ACh) and bradykinin, and an
endothelium-independent agent, sodium nitroprusside
(SNP), a dose-response curve was made with each agent. The agonists
were administered as intravenous bolus, and an interval of
enough time (10 minutes) was allowed between each dose to fully recover
baseline MAP. For each agent, a different animal was used.
Changes in the hypotensive response to each agonist were calculated as absolute MAP decrease and percent MAP decrease from the basal MAP level in young and aging rats, respectively.
Study II
To further assess the role of the
L-arginine/NO–mediated vasodilator system in the aging
animals, we studied the effect of intravenous cumulative
doses of the L-arginine antagonist
L-N-nitro-L-arginine
(L-NwNLA) on MAP. For this purpose, a dose-response curve of the effect
of L-NwNLA on MAP was constructed. Enough time (10 minutes) was allowed
between each dose to stabilize MAP.
Study III
To study the effects of the inhibition of NOS activity in
relation to the pressure-increasing effect of a vasoconstrictor agent,
angiotensin II (Ang II), a dose-response curve of
Ang II was constructed in either the presence or the absence of a
previous subpressor dose of L-NwNLA (0.65 mg/kg body wt). Ang II was
administered as an intravenous bolus.
Study IV
In groups of young and aging rats that previously were not used
in studies I, II, and III, we analyzed the activity and the
expression of the NOS isoforms in tissue and cytosolic extracts and the
cGMP content in their vascular walls. For this purpose, animals were
first anesthetized and exsanguinated, and the blood samples
were immediately centrifuged (3500 rpm) for plasma separation.
Plasma samples were used for creatinine and nitrite
measurement.
The aortas were perfused at a pressure of 100 mm Hg with 200 mL of isotonic saline. Then, the aorta, from the iliac bifurcation to the heart, was removed, cut into 3 portions, and immediately frozen in liquid nitrogen. It is important to note that catheters had not been previously implanted in these animals. After the aortas were extracted, aging and young rats were autopsied for macroscopic examination in search of pulmonary infection and the presence of tumors.
Each of the aforementioned studies was performed in at least 6 different young and aging rats.
Determination of the cGMP Content in the Isolated Blood
Vessels
Aortic segments obtained from young and aging rats were isolated
and stored immediately in liquid nitrogen as mentioned above. The
frozen segments were pulverized and resuspended in an ice-cold solution
containing 200 mmol/L perchloric acid. After they were mixed, the
homogenates were centrifuged (12 000 rpm, 5
minutes at 4°C), and the supernatants were recovered and washed out 4
times with 5 vol diethyl ether. The remaining aqueous phase was dried
under a stream of N2.
cGMP was measured as described20 in acetylated samples with a kit from Amersham International. The sensitivity of the assay was 0.5 fmol. The intra-assay and interassay variations were <8.9% and <16%, respectively.
Determination of eNOS and iNOS Protein Expression
Aortic segments were isolated, cut into 3 portions, and
immediately stored in liquid nitrogen. Only one of the portions was
used for NOS determination. The frozen segments were pulverized and
solubilized in Laemmli buffer21 containing
2-mercaptoethanol. Proteins were separated on denaturing SDS–10%
polyacrylamide gels. Equal amounts of proteins (20 µg/lane)
estimated by bicinchoninic acid reagent (Pierce) were loaded. To verify
the equal amounts of proteins loaded in the gel, it was stained with
Coomassie, and the intensities of the protein bands were examined.
The separated proteins were blotted onto nitrocellulose (Immobilon-P, Millipore). Blots were blocked overnight at 4°C with 5% nonfat dry milk in TBS-T (20 mmol/L Tris-HCl, 137 mmol/L NaCl, and 0.1% Tween 20). Western blot analysis was performed with monoclonal antibodies against eNOS or iNOS proteins (Transduction Laboratories). Blots were incubated with the first monoclonal antibody (1:2500) for 1 hour at room temperature and, after extensive washing, with the second antibody (horseradish peroxidase–conjugated anti-mouse immunoglobulin antibody) at a dilution of 1:1500 for a further 1 hour. To compare NOS expression with the expression of another protein, we analyzed the expression of ß-actin by Western blot using a ß-actin monoclonal antibody (Sigma-Aldrich). For this purpose, a parallel gel with identical samples was run, and after blotting onto nitrocellulose, the Western blot analysis was performed with the ß-actin monoclonal antibody (1:5000).
Specific NOS protein was detected by enhanced chemiluminescence (ECL, Amersham International) and evaluated by densitometry (Molecular Dynamics). Prestained protein markers (Sigma) were used for molecular mass determinations. As previously reported,22 23 these monoclonal antibodies specifically recognize the iNOS (130-kDa) or eNOS (140-kDa) isoforms.
Determination of NOS Activity
Frozen vessels were pulverized and solubilized in a buffer
containing (mmol/L) sucrose 320, Tris base 50, EDTA 1,
DL-dithiothreitol 1, leupeptin 0.019, and
phenylmethylsulfonyl fluoride 0.57, along with 10 mg/mL
aprotinin, brought to pH 7 with HCl. The homogenates were
centrifuged at 12 000 rpm at 4°C for 30 minutes. NO
formation was determined in the supernatant by the conversion of
[14C]L-arginine to
[14C]L-citrulline (Amersham
International). Aortic extracts were incubated in a buffer containing
(mmol/L) KPO4H2 50,
L-valine 60, NADPH 0.12, L-citrulline 1.2,
L-arginine 0.024, MgCl2 1.2, and
CaCl2 0.24, along with 150 000 dpm
[14C]L-arginine (pH 7). Incubations
were performed for 10 minutes at 37°C in the presence or in the
absence of the Ca2+-chelating agent EGTA (1
mmol/L) to determine both the Ca2+-dependent NOS
activity (eNOS) and the Ca2+-independent NOS
activity (iNOS), respectively. The nonspecific conversion of
[14C]L-arginine to
[14C]L-citrulline was determined in
samples containing L-NwNLA (1 mmol/L).
[14C]L-Citrulline was separated from [14C]L-arginine by a Dowex AG 50WX8 (Na+ form) column as previously described.24
Immunohistochemistry
Aortic tissue was collected from aging and young Wistar rats.
Rats were perfused through the left ventricle with 50 mL of saline,
followed by 500 mL of fixative solution containing 4%
paraformaldehyde in 0.1 mol/L PBS at pH 7.4. The aortas
were removed and postfixed for 4 hours (4%
paraformaldehyde in 0.1 mol/L PBS) at room temperature.
The aortas were then rinsed by immersion overnight at 4°C in a
solution of 0.1 mol/L PBS, containing 30% sucrose, with
continuous stirring.
Free-floating 40-µm-thick sections from aortic rings were incubated first with PBS containing 3% normal goat serum (ICN Biochemicals Ltd) and 0.1% Triton X-100 for 30 minutes and subsequently with the monoclonal antibodies against eNOS or iNOS proteins (Transduction Laboratories) at a 1:500 dilution overnight at 4°C. After they were washed in PBS, the sections were incubated in a biotinylated anti-rabbit IgG (1:200 dilution, Vector Laboratories Ltd) for 1 hour and in a solution of peroxidase-linked ABC (ABC kit, Vector Laboratories Ltd) for a further 1 hour. To reveal the peroxidase activity, the nickel-enhanced diaminobenzidine procedure was used.
Nitrite Plus Nitrate Measurement
Nitrite plus nitrate (NOx) plasma levels were assessed as
nitrite concentration. Plasma was ultrafiltered through an Ultrafree-MC
microcentrifuge filter (Millipore) to remove hemoglobin
resulting from cell lysis. NOx was measured by a commercial kit (Cayman
Chemical), based in the Griess reaction, after conversion of nitrate to
nitrite with nitrate reductase. NOx concentrations were determined at
an optical density of 554 nm by comparison with standard
solutions of sodium nitrite and corrected by the corresponding plasma
creatinine levels. Plasma creatinine was
measured by an automatic method (Astra IV, Beckmann).
Statistical Methods
Results are expressed as mean±SEM. Unless otherwise stated,
each of the 4 studies was performed in a minimum of 6 rats. Comparisons
were performed by ANOVA or paired and unpaired Student t
tests when appropriate. The Bonferroni correction for multiple
comparisons was used to determine the level of significance of the
P value.
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Results |
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Hypotensive Effects of Endothelium-Dependent and Endothelium-Independent Agents
The basal MAP levels were significantly higher in aging than in young rats (128±3 versus 100±2 mm Hg, P<0.05), whereas the basal heart rate was not different between the 2 groups (young rats, 252±4 bpm; aging rats, 249±6 bpm; P=NS).
The endothelium-dependent agent ACh produced a
dose-related decrease in arterial pressure in both groups
of animals (Figure 1A
). Both the
threshold dose and the EC50 for ACh were
significantly higher in aging than in young rats (threshold doses:
young rats, 0.8±0.02 µg/kg body wt; aging rats, 3±0.01 µg/kg body
wt; EC50: young rats, 15.2±0.6 µg/kg body wt;
aging rats, 20.1±0.8 µg/kg body wt; P<0.05). The percent
MAP decrease calculated from the basal level was also significantly
less in aging than in young rats (Figure 2A). ACh administration up to 150 µg/kg
body wt slightly increased the heart rate in both young and aging rats
(percent increase: young rats, 14±4%; aging rats, 12±3%;
P<0.05 with respect to each corresponding basal value)
(Figure 3A).
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Another endothelium-dependent vasodilator agent,
bradykinin, also decreased MAP at a lesser magnitude in aging than in
young rats (Figure 1B
). A significant increase in both the threshold
and EC50 for bradykinin was observed in aging
rats with respect to young rats (threshold dose: young rats, 0.3±0.09
ng/100 g body wt; aging rats, 4±0.3 ng/100 g body wt;
EC50: young rats, 9.5±0.9 ng/100 g body wt;
aging rats, 14.5±0.4 ng/100 g body wt; P<0.05). The
percent MAP decrease calculated from the respective basal level was
also significantly less in aging than in young rats (Figure 2B).
The endothelium-independent agent SNP produced a
dose-related decrease in arterial pressure in aging and
young rats. However, no significant differences in the hypotensive
response to SNP were found between aging and young rats (Figure 1C)
(threshold dose: young rats, 0.6±0.1 µg/kg body wt; aging rats,
0.9±0.7 µg/kg body wt; EC50: young rats,
35.1±0.5 µg/kg body wt; aging rats, 36.7±0.6 µg/kg body wt;
P=NS). Furthermore, no differences in the percent MAP
decrease by SNP were found between young and aging rats (Figure 2C). As
occurred with ACh, SNP concentrations up to 250 µg/kg body wt
slightly increased the heart rate (percent increase: young rats,
20±7%; aging rats, 17±4%; P<0.05 with respect to each
corresponding value) (Figure 3B).
Effects of the L-Arginine Antagonist and
Ang II on MAP and Their Interaction
As shown in Figure 4, aging rats
demonstrated a higher sensitivity to the arterial
pressure–increasing response produced by inhibiting NO generation with
L-NwNLA.
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The dose-response curve for Ang II was shifted to the right in aging
with respect to young rats (Figure 5A).
The approximate threshold dose and EC50 for the
Ang II–induced increase in MAP were higher in aging rats (threshold
dose, 1.2±0.2 ng/100 g body wt; EC50, 4.8±0.8
ng/100 g body wt) than in young rats (threshold dose, 0.2±0.05 ng/100
g body wt; EC50, 2.0±0.4 ng/100 g body wt).
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The administration of a subpressor dose of L-NwNLA (0.65 mg/kg
body wt) significantly shifted the dose-response curve of Ang II to the
left in both young and aging rats (Figure 5B). However, such an effect
was more marked in the aging animals (Figure 5B). In fact, in the
presence of L-NwNLA, the response to Ang II was more intense in aging
than in young rats (Figure 5B). It is important to note that the
response to Ang II in the presence of the L-arginine
antagonist was determined with respect to the new resting
level of blood pressure achieved with L-NwNLA for each group of rats
(basal MAP in rats after administration of 0.65 mg/kg body wt L-NwNLA:
young rats, 105±4 mm Hg; aging rats, 138±6 mm Hg).
Plasma NOx Concentration, cGMP Content, and NOS Isoform Expression
in the Aortic Vascular Wall
The concentration of NOx in plasma samples was significantly
higher in aging than in young rats (Figure 6). NOx plasma levels were normalized in
young and aging rats to the corresponding value of plasma
creatinine (plasma creatinine: young rats,
0.53±0.02 mg/dL; aging rats, 0.57±0.04 mg/dL; P=NS). The
cGMP content in aortic segments of aging rats was also significantly
increased with respect to that found in aortic segments of young rats
(Figure 6).
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As depicted in Figures 7A and 7B, aortic
segments isolated from young rats showed a marked expression of eNOS
protein that was found to be further increased in the aging rats.
Immunolocalization of eNOS protein demonstrated that in both young and
aging rats this enzyme was expressed only on the luminal side (Figure 8A and 8B).
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On the other hand, the expression of the iNOS protein was almost absent
in the aortic segments of young rats (Figure 9A and 9B). However, a marked expression
of the iNOS protein was observed in the segments of aging rats (Figure 9A and 9B). The histological examination also revealed
that although the vessels from young rats were negative for iNOS
protein, the aging vessels showed a marked iNOS protein expression in
the cells of the luminal surface and in the internal and external media
(Figure 10A and 10B). No changes in the
expression of ß-actin protein were observed between young and aging
rats (Figures 7A and 9A).
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The autopsies of young and aging rats revealed that all the animals were free of infections and tumors.
Ca2+-Dependent and Ca2+-Independent
NOS Activity
We analyzed the conversion of
[14C]L-arginine into
[14C]L-citrulline by
homogenates of aortic segments obtained from young and
aging rats in the presence (eNOS activity) or in the absence (iNOS
activity) of Ca2+. As shown in Figure 11, total NOS activity was greater in
aging than in young rats. In order to prove that the conversion back to
L-arginine did not result in a different
L-citrulline accumulation, further experiments were
performed by analyzing the conversion of
[14C]L-citrulline into
[14C]L-arginine in the aortic
extracts. The conversion of
[14C]L-citrulline back into
[14C]L-arginine was not
significantly different between young and aging rats (1.6±0.7% for
young rats and 2.6±0.6% for aging rats with respect to the total
[14C]L-arginine added in the assay;
n=3 different aortic segments, P=NS).
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The Ca2+-dependent NOS activity was found
decreased in the homogenates of aging aortic segments with
respect to that observed in young rats (Figure 11). In young rats,
there was no detection of the Ca2+-independent
NOS activity. However, a marked Ca2+-independent
iNOS activity was detected in the aortic extracts of the aging animals
(Figure 11).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present results provide new evidence about the function of the NO-dependent vasorelaxing mechanisms in aging rats. The first functional disturbance found in the aging animals was a reduction of the hypotensive effect of ACh, an NO-dependent vasorelaxing agent. However, blood vessels from aging rats showed a marked ability to produce NO through the presence of the iNOS isoform.
The possibility that in the aging animals the decreased response to ACh involved a muscarinic receptor impairment was discarded, since aging rats also showed a decreased pressure-lowering response to bradykinin.
Basal MAP levels were significantly higher in aging than in young rats. Therefore, this difference could contribute to the higher threshold and EC50 doses for ACh and bradykinin observed in aging rats. However, this hypothesis could be discarded, since the hypotensive response induced by either ACh or bradykinin calculated as percent MAP decrease remained decreased in aging with respect to young rats.
The question then raised was whether the altered ACh and bradykinin effects in the aging animals were due to a disturbance in guanylate cyclase activity. Our results demonstrated a preserved pressure-lowering response to SNP in aging rats, discarding this possibility.
In accordance with the present results, Gerhard et al13 have recently demonstrated that endothelium-dependent vasodilation is progressively impaired with increasing age, whereas age does not change the endothelium-independent vasodilation.
Aging rats showed an enhanced sensitivity response to the L-arginine analogue L-NwNLA. Furthermore, the evidence showing that inhibition of the NO-dependent vasodilating response normalized the pressor effect of Ang II in the aging animals suggests that NO generated by the vessels of these animals is critical in the regulation of the vasoconstricting effect of Ang II.
One potential mechanism for the reduced endothelium-dependent hypotensive response in aging could include intimal thickness or structural changes in the blood vessel wall that oppose NO action. In this regard, the loss of elastin fibers and replacement of the elastin for collagen have been demonstrated.25 26 This mechanism was discarded by the preserved response to SNP in aging rats.
NOx plasma concentration was higher in aging than in young rats, which could reflect a higher NO generation by the vascular wall of these animals. In addition, the intracellular second messenger of most of the NO effects, cGMP, was increased in the aging vessels. These findings suggest that the vascular wall of aging rats has an increased NO-forming mechanism.
Apparently, the present data could have paradoxical interpretations related to the coexistence in the aging rats of high plasma NO levels with an impaired NO-dependent vasorelaxing response. To clarify this issue, we determined the expression of the NOS isoforms in the vessels. The Western-blot experiments demonstrated that in the aortas from aging rats, the expression of both eNOS and iNOS isoforms was significantly increased and that although the eNOS isoform appeared to be expressed in the luminal side, the iNOS protein was distributed in both the lumen and media.
An increased remodeling processes associated with aging could be occurring. Therefore, the increased expression of eNOS and iNOS in the vessel wall of aging rats could be attributed to a different amounts of protein within their vascular walls, which could be discarded, since the expression of the ß-actin protein was not different in the vascular walls of young and aging animals. Moreover, the appearance of iNOS expression in the aging rats was not related to infections or tumors.
In the vessels, shear stress and hemodynamic
forces have been found to increase eNOS expression by the
endothelium27 28 because of the
presence of a shear stress–responsive element in the promotor region
of the eNOS gene.29 Therefore, the increased MAP
found in the aging rats could be stimulating eNOS protein expression by
increasing shear stress on the vascular wall. On the other hand, iNOS
isoform expression has been shown to be stimulated in response to
cytokines.11 Recent studies have also
shown that the production of cytokines, eg, TNF-, is
increased in the arterial wall of aging
rats.17
We then determined the activity of eNOS and iNOS isoforms in the aortic vessels of young and aging rats, demonstrating that although in aging rats the expression of the eNOS isoform was markedly enhanced, the eNOS activity was reduced, a finding that was accompanied by the appearance of iNOS activity. Moreover, the conversion of L-citrulline back to L-arginine was not different between young and aging rats, discarding the hypothesis that the differences found in NO production between both group of rats could be due to this fact.
Several reports have demonstrated that the vasodilator responses to endothelial receptor-dependent agonists, which act through the eNOS activity,1 3 are substantially decreased after endotoxemia, a pathological situation with increased iNOS activity in the vessel wall.30 31 Minor et al32 also demonstrated in atherosclerotic rabbits an impaired endothelium-dependent vasorelaxation that was accompanied by an increased NO production by the blood vessels of these animals. Similar to our finding, an iNOS and eNOS protein up-expression has been demonstrated in chronic hypoxia, whereas the NO-dependent vasodilator response to ACh was found to be impaired.33
The present study did not allow us to identify the mechanism responsible for the eNOS inactivation. Different experimental works have shown that eNOS activity is markedly inhibited by NO itself, whereas iNOS activity is highly resistant to inhibition by NO.34 35 In this regard, in vivo experiments in rabbits demonstrated that a constant infusion of exogenous NO caused antagonism of the endothelium-dependent vasodilatation elicited by ACh or bradykinin.36 Therefore, the NO released by the iNOS activity could be involved in the diminished eNOS activity found in aging rats. However, further experiments are needed to elucidate this hypothesis. It is also important to note that the NOS activity assay was performed under conditions that maximize substrate and cofactors that may not be available in in vivo conditions and therefore could limit the interpretation of the NOS activity assays.
In summary, at sites of endothelial damage, the locally released cytokines could potentially stimulate iNOS expression in the vascular wall. The NO released by the iNOS activity could decrease the eNOS activity, thus favoring the impaired endothelium-dependent vasorelaxation. On the other hand, the iNOS activity has an important role in the maintenance of vascular tone with increasing age.
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Acknowledgments |
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This study was supported by a grant from SAF 97/0022 of Plan Nacional de Comision Interministerial de Ciencia y Tecnologia. Dr Montón is a postdoctoral fellow of Laboratorios Bayer SA and the Nephrology, Hypertension, and Cardiovascular Research Laboratory. Drs Sánchez de Miguel, García-Durán, de Frutos, Rodríguez-Feo, Guerra, and González-Fernández are fellows of Fundación Conchita Rábago de Jiménez Díaz. The authors thank María Begoña Ibarra for secretarial assistance and Liselotte Gulliksen for English corrections.
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Footnotes |
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Correspondence to Antonio López-Farré, PhD, Nephrology, Hypertension, and Cardiovascular Research Laboratory, Fundación Jiménez Díaz, Avenida Reyes Católicos 2, Madrid 28040, Spain.
Received September 22, 1997; accepted April 9, 1998.
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S. J. Zieman, G. Gerstenblith, E. G. Lakatta, G. O. Rosas, K. Vandegaer, K. M. Ricker, and J. M. Hare Upregulation of the Nitric Oxide-cGMP Pathway in Aged Myocardium : Physiological Response to l-Arginine Circ. Res., January 19, 2001; 88(1): 97 - 102. [Abstract] [Full Text] [PDF]
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M. M. ARRIERO, J. A. RODRÍGUEZ-FEO, A. CELDRÁN, L. S. D. MIGUEL, F. GONZÁLEZ-FERNÁNDEZ, J. FORTES, A. REYERO, O. FRIEYRO, J. C. D. L. PINTA, A. FRANCO, et al. Expression of Endothelial Nitric Oxide Synthase in Human Peritoneal Tissue: Regulation by Escherichia Coli Lipopolysaccharide J. Am. Soc. Nephrol., October 1, 2000; 11(10): 1848 - 1856. [Abstract] [Full Text]
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K. Asai, R. K. Kudej, Y.-T. Shen, G.-P. Yang, G. Takagi, A. B. Kudej, Y.-J. Geng, N. Sato, J. B. Nazareno, D. E. Vatner, et al. Peripheral Vascular Endothelial Dysfunction and Apoptosis in Old Monkeys Arterioscler Thromb Vasc Biol, June 1, 2000; 20(6): 1493 - 1499. [Abstract] [Full Text] [PDF]
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K. G. Stewart, Y. Zhang, and S. T. Davidge Aging Increases PGHS-2-Dependent Vasoconstriction in Rat Mesenteric Arteries Hypertension, June 1, 2000; 35(6): 1242 - 1247. [Abstract] [Full Text] [PDF]
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A. Orlandi, M. Marcellini, and L. G. Spagnoli Aging Influences Development and Progression of Early Aortic Atherosclerotic Lesions in Cholesterol-Fed Rabbits Arterioscler Thromb Vasc Biol, April 1, 2000; 20(4): 1123 - 1136. [Abstract] [Full Text] [PDF]
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M. E. Safar, J. Blacher, J. J. Mourad, and G. M. London Stiffness of Carotid Artery Wall Material and Blood Pressure in Humans : Application to Antihypertensive Therapy and Stroke Prevention Stroke, March 1, 2000; 31(3): 782 - 790. [Abstract] [Full Text] [PDF]
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Z.-q. Yan, A. Sirsjo, M.-L. Bochaton-Piallat, G. Gabbiani, and G. K. Hansson Augmented Expression of Inducible NO Synthase in Vascular Smooth Muscle Cells During Aging Is Associated With Enhanced NF-{kappa}B Activation Arterioscler Thromb Vasc Biol, December 1, 1999; 19(12): 2854 - 2862. [Abstract] [Full Text] [PDF]
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A. Koller and A. Huang Development of Nitric Oxide and Prostaglandin Mediation of Shear Stress-Induced Arteriolar Dilation With Aging and Hypertension Hypertension, November 1, 1999; 34(5): 1073 - 1079. [Abstract] [Full Text] [PDF]
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T. de Frutos, L. S. de Miguel, M. Garcia-Duran, F. Gonzalez-Fernandez, J. A. Rodriguez-Feo, M. Monton, J. Guerra, J. Farre, S. Casado, and A. Lopez-Farre NO from smooth muscle cells decreases NOS expression in endothelial cells: role of TNF-alpha Am J Physiol Heart Circ Physiol, October 1, 1999; 277(4): H1317 - H1325. [Abstract] [Full Text] [PDF]
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H. Ruetten, U. Zabel, W. Linz, and H. H. H. W. Schmidt Downregulation of Soluble Guanylyl Cyclase in Young and Aging Spontaneously Hypertensive Rats Circ. Res., September 17, 1999; 85(6): 534 - 541. [Abstract] [Full Text] [PDF]
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F. Gonzalez-Fernandez, A. Lopez-Farre, J. A. Rodriguez-Feo, J. Farre, J. Guerra, J. Fortes, I. Millas, M. Garcia-Duran, L. Rico, P. Mata, et al. Expression of Inducible Nitric Oxide Synthase After Endothelial Denudation of the Rat Carotid Artery : Role of Platelets Circ. Res., November 30, 1998; 83(11): 1080 - 1087. [Abstract] [Full Text] [PDF]
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H. Matsushita, E. Chang, A. J. Glassford, J. P. Cooke, C.-P. Chiu, and P. S. Tsao eNOS Activity Is Reduced in Senescent Human Endothelial Cells: Preservation by hTERT Immortalization Circ. Res., October 26, 2001; 89(9): 793 - 798. [Abstract] [Full Text] [PDF]
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M. M. Arriero, J. C. de la Pinta, M. Escribano, A. Celdran, L. Munoz-Alameda, J. Garcia-Canete, A. M. Jimenez, S. Casado, J. Farre, and A. Lopez-Farre Aspirin Prevents Escherichia coli Lipopolysaccharide- and Staphylococcus aureus-Induced Downregulation of Endothelial Nitric Oxide Synthase Expression in Guinea Pig Pericardial Tissue Circ. Res., April 5, 2002; 90(6): 719 - 727. [Abstract] [Full Text] [PDF]
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