Expression of Constitutive and Inducible Nitric Oxide Synthases in the Vascular Wall of Young and Aging Rats
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-Nω-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.
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
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-α (TNF-α) 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-α 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.
Materials and Methods
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.
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.
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-Nω-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.
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.
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 β-actin by Western blot using a β-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 β-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
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).
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.
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⇓).
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.
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).
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⇓).
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⇓).
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 β-actin protein were observed between young and aging rats (Figures 7A⇑ and 9A⇓).
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).
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⇑).
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 β-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-α, 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.
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.
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.
- © 1998 American Heart Association, Inc.
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