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J Appl Physiol 97: 1268-1274, 2004. First published May 28, 2004; doi:10.1152/japplphysiol.00242.2004
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Age-related alterations in NOS and oxidative stress in mesenteric arteries from male and female rats

Jennifer C. Sullivan,1,2 Elizabeth Dabbs Loomis,1 Matthew Collins,1 John D. Imig,1,3 Edward W. Inscho,3 and Jennifer S. Pollock1,2

1Vascular Biology Center, and Departments of 2Pharmacology and Toxicology and 3Physiology, Medical College of Georgia, Augusta, Georgia 30912

Submitted 5 March 2004 ; accepted in final form 21 May 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Epidemiological evidence suggests that advancing age affects the cardiovascular system of men and women differently. The purpose of this study was to determine whether the effects of aging on nitric oxide synthase (NOS), oxidative stress, and vascular function are different in males and females. Mesenteric arteries from young (3 mo) and old (24 mo) male and female Fischer 344/Brown Norway rats were studied. Western blot analysis and NOS activity were performed on the homogenized mesenteric arterial bed separated into cytosolic and membrane-associated fractions. Plasma 8-isoprostane measurements assessed oxidative stress. Vascular reactivity was determined by using a wire myograph in the absence and presence of a NOS inhibitor, N{omega}-nitro-L-arginine, to examine endothelial function and basal and stimulated nitric oxide release. In additional arteries, reactivity was performed in the presence of polyethylene glycol-SOD to assess the impact of superoxide on vascular function. Among females, aging was associated with a decline in membrane-associated NOS activity and membrane-associated NOS III protein expression. Advancing age in males was associated with increased cytosolic NOS III protein expression. Among both males and females, advancing age resulted in increased oxidative stress. Vascular function was maintained with age in arteries from both males and females, and there was no difference in either basal or stimulated nitric oxide release with age. Despite sex-specific effects of advancing age on the NOS system and increases in markers of oxidative stress, vascular function is maintained in mesenteric arteries from aged Fischer 344/Brown Norway rats. These data suggest that age-related alterations in the resistance vasculature are complex and likely involve multiple compensating vasoactive pathways.

aging; nitric oxide; oxygen radicals; vascular reactivity

THE VASCULAR ENDOTHELIUM IS a dynamic structure that plays a central role in maintaining cardiovascular health, in part via the release of vasoactive substances such as nitric oxide (NO) (11). NO is a potent vasodilator whose formation is catalyzed by the enzyme endothelial NO synthase (NOS III). Several risk factors for the development of cardiovascular disease, including advancing age (1, 2, 10, 36, 37, 39) and the male sex (20, 22), are associated with dysfunction of the vascular endothelium. While the mechanism that mediates this decline in endothelial function is not established, a decrease in endothelium-derived NO has been suggested. A decrease in bioavailable NO associated with aging or the male sex may be attributed to 1) a decrease in NOS III activity and/or expression (7–9), 2) a decrease in NOS III substrate or cofactor availability (28), or 3) an increase in the degradation of NO (2).

Early studies characterizing NOS III in cultured endothelial cells reported that a majority of NOS III is located in the membrane fraction of cells, with only a small fraction localized in the cytosol (13, 27). Furthermore, in vitro studies suggest that subcellular localization of NOS III regulates enzymatic activity (30, 31). Subcellular localization of NOS III with aging and sex has not been examined. Our laboratory (34) has previously published that alterations in compartmentalization of NOS III may contribute to endothelial dysfunction in hypertension, supporting the hypothesis that appropriate subcellular localization of NOS III is important in the regulation of enzyme activity. Therefore, alterations in NOS III compartmentalization may also contribute to endothelial dysfunction with age and sex.

Superoxide (O2–·) is a free radical that rapidly scavenges NO, thereby decreasing NO bioavailability (14). Oxidative stress is defined as an imbalance between the production of reactive oxygen species and the ability of antioxidant systems to neutralize them. Increased oxidative stress has been shown to contribute to the development of endothelial dysfunction in many forms of cardiovascular disease (16). There are numerous reports of increased levels of O2–· production with advancing age in epidemiological as well as experimental studies (10, 15, 29, 36, 39). There is also evidence that oxidative stress is enhanced in males compared with females (3, 18, 20).

Epidemiological evidence suggests that aging affects the cardiovascular system of men and women differently. Women reportedly lag behind men in the development of coronary heart disease, atherosclerotic lesions, and endothelial dysfunction by approximately a decade (5, 6, 19). The purpose of this study was to determine whether the effect of aging on the NOS system, oxidative stress, and endothelial function is different between males and females. Most aging studies focus on responses in males, whereas the majority of sex studies examine only young subjects. In addition, most of these studies have examined responses in large, conduit arteries, not small resistance-sized arteries, which are more involved in the regulation of blood pressure. We hypothesize that advancing age will be associated with decreased NOS activity and NOS III protein expression and increased oxidative stress, contributing to a decrease in endothelial function. Furthermore, we hypothesize that these alterations will be more pronounced in arteries from males compared with arteries from females. This study provides a novel assessment of the interaction of age and sex on the vascular NOS system and endothelial function in resistance arteries.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   Young (3–4 mo; YM) and old male (24–25 mo; OM) and female (YF and OF, respectively) Fisher 344/Brown Norway (F344/BN) rats were used in this study. The rats are maintained by the National Institute on Aging (NIA) specifically for aging studies and were barrier raised; thus the changes seen with age are due to the passage of time and not disease due to environmental exposure. At 24 mo of age, the survival rate for both male and female rats is ~50%. The investigation conforms with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication No. 85–23, revised 1996). All animal protocols were performed in accordance with guidelines and approved by the Medical College of Georgia Committee for Animal Use in Research and Education. Rats were anesthetized by using pentobarbital sodium (50 mg/kg ip), the abdomen was opened, and blood was collected from the abdominal aorta followed by a thoracotomy.

Measurement of NOS activity by conversion of [3H]arginine to [3H]citrulline.   The mesenteric arterial bed (arteries from 100–500 µm) was isolated, homogenized, and separated into cytosolic and membrane-associated fractions, as previously described (34). The entire bed was used to allow for sufficient protein levels to perform both NOS activity assays, as well as Western blot analysis. Protein concentrations were determined by standard Bradford assay (Bio-Rad, Hercules, CA) with the use of BSA as the standard. Aliquots of cytosolic and membrane-associated fractions were incubated with [3H]arginine (10 µmol/l final arginine, 71 Ci/mmol) in the presence of excess cofactors, as previously described (4), in a final volume of 50 µl for 30 min at room temperature. The remainder of the assay was performed as previously described (4). NOS activity was normalized to milligrams of protein (pmol·30 min–1·mg protein–1) (n = 11 for YF, n = 9 for OF, YM, and OM).

Western blotting.   Western blotting was performed, as previously described (4). Thirty-five micrograms of protein were loaded per lane. The primary antibody was a monoclonal anti-NOS III (1:500, Transduction Laboratories, Franklin Lakes, NJ), and the secondary antibody was horseradish peroxidase-conjugated goat anti-mouse antibody (1:2,000; Amersham, Piscataway, NJ). The primary antibodies were stripped by using ReBlot Plus Mild Antibody Stripping Solution (Chemicon International, Temecula, CA), and equal protein loading was verified by {beta}-actin (1:5,000, Sigma, St. Louis, MO). Specific bands were detected with enhanced chemiluminescence (SuperSignal Chemiluminescent Substrate, Pierce, Rockford, IL), and densitometry was performed by using a digital imaging system (Alpha Innotech, Staffordshire, UK). N = 7 for each group; each rat sample was only used once for Western blot analysis.

Vasoreactivity.   A third-order mesenteric artery was isolated, as previously described (33), and placed in the chamber of a wire myograph (Danish Myo Technology) containing warmed (37°C), gassed (95% O2-5% CO2) physiological salt solution (composition in mM: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, and 11.1 dextrose). Passive tension was set to 4 mN, and alterations in tension were monitored and recorded by using the appropriate software (Myodaq, Danish Myo Technology). Vessels were equilibrated for 30 min before the viability of the vessel was determined by contracting the vessel with 1 µmol/l phenylephrine (PE), followed by 10 µmol/l ACh. Drugs were rinsed out, the vessels were reequilibrated for 30 min, and a cumulative concentration-response curve was performed.

Cumulative concentration-response curves were performed to the vasoconstrictors PE (1 nmol/l to 31.6 µmol/l, n = 12–17) in the absence or presence of the nonselective NOS inhibitor N{omega}-nitro-L-arginine (L-NNA, 100 µmol/l) or SOD-polyethylene glycol (PEG) (200 U/ml) and KCl (4.7–100 mmol/l, n = 14–19). This concentration of PEG-SOD was shown in independent studies to decrease the production of O2–· from xanthine/xanthine oxidase (data not shown). Cumulative concentration-response curves were also performed to the endothelium-dependent vasodilators ACh (1 nmol/l to 31.6 µmol/l, n = 13–17) and A-23187 (100 pmol/l to 10 µmol/l, n = 4–8) and the endothelium-independent vasodilator sodium nitroprusside (SNP; 100 pmol/l and 31.6 µmol/l, n = 5–9) in the presence of L-NNA (100 µmol/l). ACh concentration-response curves were performed in the absence or presence of L-NNA (100 µmol/l). A-23187 concentration-response curves were performed in the absence or presence of PEG-SOD (200 U/ml). Arteries were preconstricted with PE to 80% maximum constriction, determined when testing artery viability [precontracted force (in mN) for ACh curves: YF 12.2 ± 0.87, OF 12.0 ± 0.89, YM 12.5 ± 0.68, OM 12.5 ± 0.80]. Each agonist concentration was added only after the vessel had reached a plateau from the previous dose, and one curve was obtained from each vessel.

Plasma 8-isoprostane.   Blood was collected on ice over 7.5% EDTA with the use of a syringe and spun at 1,500 rpm at 4°C for 15 min, and plasma was collected, treated with butylated hydroxytoluene (0.005%), and stored in plastic tubes at –80°C. 8-Isoprostane concentrations were determined according to the kit manufacturer's instructions.

Materials.   Acetylcholine, SNP, PE, L-NNA, PEG-SOD, and physiological salt solution reagents were purchased from Sigma. [3H]arginine was obtained from Amersham (Arlington Heights, IL).

Data analysis.   All data are expressed as means ± SE. Concentration-response curves were analyzed by using nonlinear regression of sigmoidal dose-response curves (GraphPad Prism), which calculated the EC50, maximum response, and slope. The negative log EC50 values (pD2) were calculated, and control responses between groups were made by using a two-way ANOVA, followed by a Student- Newman-Keuls test (STATISTICA for Windows 4.0; StatSoft, Tulsa, OK). To analyze the effects of L-NNA, within-group comparisons were made by using a t-test for independent samples, and between-group comparisons were made by calculating the shift in the pD2 followed by a t-test (STATISTICA). All other comparisons were made by using a two-way ANOVA (STATISTICA) followed by a Student-Newman-Keuls test. For all comparisons, P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   Advancing age was associated with a significant increase in body weight. YF were significantly smaller than the other three rat groups, whereas OM were significantly larger [body weight (in g): YF 193 ± 4.3; OF 298 ± 3.5; YM 296 ± 10.2; OM 585 ± 18.1].

NOS activity and NOS III expression in mesenteric arteries.   NOS activity was calculated by measuring the conversion of [3H]arginine to [3H]citrulline and defined as conversion that was inhibited by L-NNA. NOS activity was expressed as picomoles of NOS activity normalized to milligrams of protein and is shown in Fig. 1A. Membrane-associated NOS activity declined with age among females but was maintained with age in males. In addition, there was a sex difference in NOS activity, with YF having greater cytosolic and membrane-associated NOS activity compared with YM.



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  		Fig. 1. Nitric oxide synthase (NOS) activity and NOS III protein expression. A: NOS activity is normalized to milligrams of protein. Solid lines indicate differences in sex, whereas dashed lines indicate differences with aging. Values are means ± SE; n = 11 for young female (YF); n = 9 for old female (OF), young male (YM), and old male (OM) rats. *Significant difference in activity, P < 0.05. B: representative Western blot and densitometry; n = 7. The lanes are the cytosolic (C) and membrane-associated (P) fractions from YF, OF, YM, and OM. Thirty-five micrograms of protein were loaded per lane. Values are means ± SE. {dagger}Significant difference from YF, P < 0.05. *Significant difference from all groups, P < 0.05.

 
Western blot analysis was performed to examine NOS III distribution in cytosolic and membrane-associated subcellular fractions (Fig. 1B). As was seen with NOS activity, advancing age in females was associated with a decrease in membrane-associated NOS III expression, whereas membrane-associated NOS III expression was not altered with age in males. Cytosolic NOS III expression was significantly less in YM compared with the other three rat groups.

Oxidative stress.   Isoprostanes are generated by free radical-induced peroxidation of arachidonic acid, as such enhanced isoprostane levels suggest an increase in oxidative stress. Therefore, plasma 8-isoprostane was measured as an indicator of total body oxidative stress. There was a significant increase in 8-isoprostane concentrations with age in both males [values in pg/ml: YM (n = 12), 11.6 ± 0.7; OM (n = 13), 28.8 ± 6.7; P > 0.05] and females [YF (n = 12), 11.9 ± 0.7; OF (n = 11), 21.1 ± 1.4; P > 0.05]. There were no differences in 8-isoprostane concentrations with sex in either young or aged animals.

Vasoconstriction.   The biochemical studies of the NOS system suggest that there is a sex difference in the effect of advancing age on NOS. Therefore, vascular reactivity studies were performed to assess the functional impact of altered biochemistry on basal and stimulated NO release. Cumulative concentration-response curves to PE mesenteric arteries from YM, OM, YF, and OF rats are shown in Fig. 2. There were no significant differences in either sensitivity or maximum percent increase in force generation to PE with either sex or age (Fig. 2A, see Table 1). Incubation with L-NNA, used to assess basal NO release, significantly increased sensitivity to PE in all four groups; however, there were no differences in maximum increases in force (Table 1). The increase in sensitivity to PE in the presence of L-NNA was comparable among the four groups, suggesting that basal NO release was similar.



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  		Fig. 2. Phenylephrine (PE) cumulative concentration-response curves in mesenteric arteries from females (A) and males (B) in the absence and presence of N{omega}-nitro-L-arginine (L-NNA). Values are means ± SE. Nos. in parentheses are n values. Neither age nor sex alters sensitivity or maximum response to PE. L-NNA significantly increase sensitivity to PE in all groups, P < 0.05 for all comparisons of L-NNA vs. control.

 

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  		Table 1. Sensitivity and maximum increases in force generation to PE

 
The biochemical data suggest that, with advancing age, there is an overall increase in oxidative stress. To assess the physiological impact on vascular function, cumulative concentration-response curves were performed to PE in the presence of PEG-SOD. Incubation with PEG-SOD did not alter either the sensitivity or maximum increases in force (Table 1).

Cumulative concentration-response curves were also performed to KCl (Fig. 3). OM were significantly more sensitive to KCl compared with YM and YF. In addition, arteries from OM generated significantly more force at maximal concentrations of KCl compared with the other three rat groups (see Table 1).



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  		Fig. 3. KCl cumulative concentration-response curves in mesenteric arteries. Values are means ± SE. Nos. in parentheses are n values. Advancing age among males is associated with an increase in sensitivity and maximum increase in force to KCl.

 
Vasodilation.   Endothelium-dependent vasodilation was also assessed in mesenteric arteries from YM, OM, YF, and OF rats. Cumulative concentration-response curves were performed to ACh in the absence and presence of L-NNA, to assess stimulated NO release (Fig. 4). There were no significant differences in either sensitivity or maximum relaxation to ACh with either sex or age (see Table 1). Incubation with L-NNA significantly decreased both sensitivity and maximum relaxation to ACh to a similar degree in all four groups, suggesting that stimulated NO release was comparable (see Table 1, Fig. 4). To verify that endothelium-dependent vasodilator responses were maintained with age, cumulative concentration-response curves were also performed to calcium ionophore A-23187. Similar to our findings with ACh, there were no significant differences in either sensitivity or maximum relaxation with age; however, YF were significantly more sensitive to A-23187 compared with YM (see Table 1). Incubation with PEG-SOD did not alter either the sensitivity or the maximum response to A-23187 in any of the rat groups (see Table 1).



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  		Fig. 4. Acetylcholine cumulative concentration-response curves in mesenteric arteries from females (A) and males (B) in the absence and presence of L-NNA. Values are means ± SE. Nos. in parentheses are n values. Arteries are preconstricted to 80% maximal contraction using PE. Neither age nor sex alters sensitivity or maximum relaxation to acetylcholine. L-NNA significantly decreased sensitivity and maximum relaxation to acetylcholine in all groups, P < 0.05 for all comparisons of L-NNA vs. control.

 
Endothelium-independent vasodilation was assessed by performing cumulative concentration-response curves to SNP in the presence of L-NNA (Fig. 5). There were no significant differences in either sensitivity or maximum relaxation to SNP with either sex or age (see Table 1 for values).



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  		Fig. 5. Sodium nitroprusside (SNP) cumulative concentration-response curves in mesenteric arteries treated with 100 µmol/l L-NNA. Values are means ± SE. Nos. in parentheses are n values. Neither age nor sex alters sensitivity or maximum relaxation to sodium nitroprusside.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Males tend to develop cardiovascular disorders at an earlier age compared with females, and age- and sex-related alterations in cardiovascular function have been suggested to be related to NOS. This study examined the interaction of age and sex on the NOS system in small mesenteric arteries. We found a sex-specific effect of advancing age on NOS activity and expression, supporting the postulate that sex influences the effect of aging on the vasculature. In addition, aging in both males and females was found to be associated with increases in oxidative stress. Paradoxically, despite alterations in NOS biochemistry and increased oxidative stress, vascular function of isolated arteries was maintained with age. These data suggest that age-related alterations in the resistance vasculature are complex and likely involve multiple compensating vasoactive pathways.

There is a notable lack of consensus as to the effects of aging on arterial function. A majority of aging studies have been performed with the use of large-conduit arteries, not resistance-sized arteries as are used in the present study. Age has been shown to either increase (7, 9, 39) or decrease (7, 8) NOS activity and cGMP content in isolated aorta. Plasma nitrite (NOx) levels have been shown to be either unchanged (9), decreased (10, 17, 35), or increased (7) with age in male rats, and NOS III mRNA expression is decreased with age in aorta and coronary arteries from male rats (2, 8, 10). Urinary NOx levels decrease with age in male rats as well as NOS activity and NOS III protein expression in the renal cortex and medulla (12). The studies that have examined the effect of age on NOS III protein expression have found protein expression to be either unchanged (9) or increased (37) in both the aorta (9, 37) and the mesentery (26, 23, 35); however, none of these studies determined subcellular localization.

Early studies characterizing NOS III in cultured endothelial cells reported that a majority of NOS III is located in the membrane fraction of cells (13, 27). Experiments in which human embryonal kidney 293 cells were transfected with a mutated form of NOS III that does not associate with the membrane reported decreased NOx following stimulation with Ca2+ ionophore, suggesting that cytosolic localization of NOS III is associated with decreased NO production in vitro (30, 31). Examination of subcellular localization of NOS III protein in conduit arteries agrees with in vitro studies: NOS III is localized in the membrane-associated fraction (39). Our laboratory has previously published that, in mesenteric arteries from healthy male rats, a majority of NOS III is in the membrane fraction (34). In the present study, we also describe the subcellular localization of NOS III in arteries from females.

Advancing age among males was associated with increased cytosolic NOS III protein expression, but a corresponding increase in cytosolic NOS activity was not apparent. Our laboratory has previously shown increased cytosolic expression of NOS III in mesenteric arteries from salt-dependent hypertensive rats, which are known to have impaired vascular function (34). These data may be interpreted in two ways: 1) cytosolic NOS III is increased to compensate for age-associated alterations in vascular function or 2) increased cytosolic localization of NOS III does not translate into increased NO and is thereby indicative of dysfunction. In YM, there was more membrane-associated NOS III; however, there was also a substantial amount of cytosolic NOS III expression. This is not seen in Sprague-Dawley rats (34) and may represent an important strain-specific trait in F344/BN rats. Additional studies will be required to further resolve the physiological significance of cytosolic NOS III expression in F344/BN rats and the relevance of increases with age.

The majority of studies examining the effects of aging on the cardiovascular system have focused on male animals. Uniquely, this study simultaneously establishes the effects of advancing age on the NOS system in the vasculature of males and females. Unexpectedly, a majority of NOS III in mesenteric arteries from YF and OF rats was localized to the cytosolic fraction. The presence of cytosolic NOS III in otherwise healthy animals is surprising. Accordingly, alterations in activity or translocation of NOS III between cytosol and membrane compartments may represent a physiologically important issue in age-related alterations in vascular function. Among females, membrane-associated NOS III protein expression decreased with age, with a corresponding decline in NOS activity. Urinary NOx levels have been shown to decrease with age in female rats while NOS activity and NOS III protein expression are maintained in the renal cortex and medulla (12).

Oxidative stress contributes to the development of endothelial dysfunction in many forms of cardiovascular disease. The production of O2–· is increased with advancing age (10, 15, 27, 36, 39) and in the male sex (3, 18, 21). In the present study, we found that a measure of whole body oxidative stress (plasma 8-isoprostanes) increased with advancing age similarly in both male and female rats. This observation may suggest that age-related increases in oxidative stress occur uniformly in males and females. Alternatively, the direct influences of oxidative stress may need to be examined in an organ-specific manner. For example, differences in free radical production between males and females may be subtle and localized to specific tissues. If this is true, then determination of whole body oxidative stress may only be suitable to establish that aging results in increased oxidative stress. More specific measures of local superoxide production will be needed to identify sources, mechanisms, and gender specificity of enhanced oxidative stress.

To assess functional consequences of alterations in the NOS system and increases in oxidative stress, we examined vascular function in isolated mesenteric arteries. Surprisingly, there were no differences in endothelium-dependent vasodilation to either ACh or A-23187 with age. This is in contrast to many epidemiological and experimental studies that report an age-specific decline in endothelial function (1, 2, 10, 23, 36, 37, 39). Only one other study has examined vascular reactivity in aged F344/BN rats. Van der Loo et al. (39) reported a decline in ACh-induced relaxation in aortas of F344/BN animals that were 32–35 mo of age, suggesting that, if we had aged the animals for additional time, a decrease in endothelial function may have become apparent. In aging studies using F344 rats, previous studies using mesenteric arteries from male and female rats found that YF were more sensitive to PE and electrical field stimulation-induced vasoconstriction compared with OF (33) and that endothelium-dependent electrical field stimulation-induced vasodilation was blunted with age in males but maintained in females (32). Whereas both F344 and F344/BN rats are colonies maintained at the NIA, F344 rats have more health problems and tumors with age, suggesting that F344 rats may be more susceptible to insult and altered vascular function. Endothelium-dependent vasodilation has also been reported to be blunted in soleus feed arteries with age, but maintained in gastrocnemius feed arteries from aged male F344 rats, suggesting that, even within the same strain of rat, aging may have contrary effects on different vascular beds (25, 40). In agreement with our findings, Barton et al. (2) reported that ACh relaxation in femoral arteries is maintained with age in female Ro-Ro Wistar rats.

The broad variability among studies related to the effects of aging on vascular reactivity can be attributed to many possible explanations. Across the field of aging research, there is no standard definition of what constitutes an aged model. In the last 10 yr, the term "aged rat" has been used to refer to animals >20 mo of age, whereas earlier studies included rats 15–18 mo of age. Vascular reactivity is also studied in a wide variety of large- and small-caliber arteries that exhibit broad variation in vasoconstrictor and vasodilatory characteristics. In the present study, we focused on small-caliber resistance arteries, as these are the vessels that participate in blood pressure regulation and the regulation of organ perfusion. Reports in the literature suggest, however, that, even within the same strain of rat, and in similar-sized arteries, there are variations in vascular responses between different vascular beds. Finally, aging studies have been conducted with the use of animal models from many sources and genetic backgrounds. In our studies, we have focused on the F344/BN rat strain, as provided by the NIA. These animals are barrier raised for the specific purpose of standardizing the animal composition used for aging research. These animals are very robust and appear healthy, even at advanced ages, making them well suited to aging studies.

It may seem paradoxical for vascular function to be maintained in the face of the biochemical alterations in the vasculature with age. It is not unprecedented, however, for vascular function and biochemistry to differ. Matz et al. (23) reported a decline in endothelial function in both the aorta and the superior mesenteric artery, despite increased NOS III protein expression, due to enhanced production of vasoconstrictor prostaglandins. Therefore, multiple vasoactive pathways modulate vascular function, and the effects of advancing age on each of these vasoactive pathways are complex. Our data suggest that F344/BN rats can compensate for alterations in NOS III protein expression and increased oxidative stress with age to maintain vascular function. This could involve alterations in the regulation of NOS III activity, which is carefully controlled by substrate availability, protein-to-protein interactions, cofactor association, and phosphorylation and dephosphorylation. With age, NOS III may become hyperphosphorylated or protein interactions may be enhanced, resulting in increased NOS activity and NO production, despite alterations in protein expression and localization. Additionally, when viewed together, the biochemical and functional data may support the hypothesis that cytosolic NOS III expression is conferring protection in the aged animals to maintain vascular function. Future studies will examine the physiological relevance of cytosolic NOS III expression. Alternatively, the effectiveness of endogenous antioxidant systems may be enhanced, thus conferring increased capability of the aged animal to respond to increases in oxidative stress and prevent significant oxidative damage. In support of this concept, Van der Loo et al. (38) have reported that aged male rats exhibit elevated levels of the endogenous antioxidant, vitamin E. Finally, in the mesenteric vasculature, ACh-induced relaxation is mediated by the stimulated release of NO, vasodilator prostaglandins (PGI2), and endothelium-derived hyperpolarizing factor. In response to alterations in the NOS system, there may also be compensatory increases in PGI2/endothelium-derived hyperpolarizing factor release or a decrease in vasoconstrictor prostaglandin release to maintain endothelial function (23, 24).

In summary, we found that advancing age alters vascular function in a sex-specific manner. In particular, there are sex differences in the effects of aging on the NOS system. Advancing age in females is associated with a decrease in membrane-associated NOS activity and NOS III protein expression, whereas, among males, cytosolic NOS III protein expression increased. Despite alteration in the NOS system with age, endothelial function was maintained. Together, our data support the hypothesis that age-related alterations in the vasculature are complex and not solely dependent on alterations in the NOS pathway.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by National Institutes of Health Grants HL-60653 (to J. S. Pollock) and AG-18187 (to E. W. Inscho), and grants from the American Physiological Society (to J. C. Sullivan and M. Collins), and the American Heart Association (to E. D. Loomis). J. S. Pollock and J. D. Imig are Established Investigators of the American Heart Association.


   ACKNOWLEDGMENTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors acknowledge the excellent technical assistance of Janet L. Hobbs and Jacqueline B. Musall.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. C. Sullivan, Medical College of Georgia, Vascular Biology Center, 1459 Laney-Walker Blvd., Augusta, GA 30912 (E-mail: jsullivan{at}mail.mcg.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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