Impairment of flow-induced vasodilation in coronary resistance arterioles may contribute to the decline in coronary vasodilatory reserve that occurs with advancing age. This study investigated the effects of age on flow-induced signaling and activation of nitric oxide (NO)-mediated vasodilation in coronary resistance arterioles. Coronary arterioles were isolated from young (∼6 mo) and old (∼24 mo) male Fischer-344 rats to assess vasodilation to flow, vascular endothelial growth factor (VEGF), and ACh. Flow- and VEGF-induced vasodilation of coronary arterioles was impaired with age (P ≤ 0.05); however, ACh-induced vasodilation was preserved with age. NG-nitro-l-arginine methyl ester (l-NAME) (1 × 10−5 M) eliminated vasodilation to flow, VEGF, and ACh, indicating dependence of these responses on NO. SU-1498, an inhibitor of vascular endothelial growth factor receptor 2 (VEGFR, also known as Flk-1), abolished age-related differences in flow-induced vasodilation. Flow-stimulated phosphorylation of Flk-1 in coronary arterioles from young but not old rats and Flk-1 protein was reduced in coronary arterioles from old rats compared with those from young rats. Flow stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) in coronary arterioles from both young and old rats. VEGF induced phosphorylation of both protein kinase B (Akt) and eNOS in coronary arterioles. VEGF-induced phosphorylation of Akt, but not eNOS, was significantly reduced in arterioles from old rats compared with arterioles from young rats. Wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinase, eliminated age-related differences in both flow- and VEGF-induced vasodilation. These results indicate that impairment of Flk-1/PI3-kinase signaling contributes to the reduction of flow-induced vasodilation in coronary arterioles with advancing age.
- phosphatidylinositol 3-kinase
- endothelial nitric oxide synthase
- vascular endothelial growth factor
- phosphorylated endothelial nitric oxide synthase
- phophorylated protein kinase B
the risk for heart failure increases substantially with advancing age and may be linked to decrements in coronary flow reserve that occur in senescent animals and humans (7, 17). Reductions in both maximal and submaximal coronary blood flow responses to pharmacological interventions have been reported in aged animals (17, 40) and humans (11). These age-related alterations in coronary vasomotor reactivity may contribute to the age-induced decline in coronary vascular reserve and increased risk for heart failure.
The endothelium functions as an important modulator of coronary vasomotor tone (26, 34, 35). Aging-associated impairment of endothelium-dependent vasodilation has been reported in both conduit arteries and resistance arterioles of peripheral vascular beds (15, 20, 21, 37, 38); however, less is known regarding the effects of age on endothelial function in the coronary vasculature. In coronary conduit arteries, relaxation responses to ACh are maintained with age (16, 39), whereas in resistance arteries from middle-aged rats dilation to ACh is impaired (6), indicating that age-associated adaptations of the endothelium are heterogeneous within the coronary vasculature. The effects of age on cellular signaling mechanisms that regulate endothelial function in the coronary resistance arterioles remain to be determined.
Increases in intraluminal flow produce profound changes in the caliber of coronary resistance arterioles (6, 26); thus, flow-induced dilation is a critical regulator of coronary vascular resistance. In coronary arterioles, nitric oxide (NO) signaling through activation of endothelial nitric oxide synthase (eNOS) is critical to flow-induced vasodilation (25, 30). The mechanisms of flow-induced mechanotransduction and eNOS activation are not completely understood; however, recent evidence indicates that flow-induced phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2, also known as Flk-1) results in phophorylation of phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt) and eNOS activation (18, 19). Therefore, the purpose of this study was to determine whether age impairs flow-induced, NO-mediated vasodilation of coronary resistance arterioles through a reduction of Flk-1/PI3-kinase/Akt signaling in coronary resistance arterioles.
Young (6 mo; n = 75) and old (24 mo; n = 74) male Fischer-344 rats were obtained from Harlan (Indianapolis, IN). All procedures were approved by the Institutional Animal Care and Use Committee at West Virginia University and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC, Revised 1996). Rats were housed individually at 23°C and were maintained on a 12:12-h light-dark cycle. All rats were fed standard rat chow and water ad libitum.
Rats were anesthetized (isoflurane 5%-O2 balance) and killed by removal of the heart. Hearts were placed in cold (4°C) physiological saline solution (PSS) containing 145.0 mM NaCl, 4.7 mM KCl, 2.0 mM CaCl2, 1.17 mM MgSO4, 1.2 mM NaH2PO4, 5.0 mM glucose, 2.0 mM pyruvate, 0.02 mM EDTA, 3.0 mM 3-(N-morpholino)propanesulfonic acid buffer, and 1 g/100 ml BSA, pH 7.4. Resistance arterioles (<150 μm) were isolated from the left anterior descending coronary artery distribution as previously described (32). Arterioles were cannulated on pipettes matched (within 1%) for size and resistance in a Lucite chamber that contained PSS equilibrated with room air. Intraluminal pressure is ∼40–50 mmHg in coronary arterioles <150 μm (5), and mean arterial pressure and cardiac output are similar between young and old Fischer-344 male rats (9); therefore, mean intraluminal pressure was maintained at 60 cmH2O in arterioles from both young and old rats throughout the course of experiments. Arterioles unable to hold pressure due to leaks were discarded. Those without leaks were warmed to 37°C and allowed to develop spontaneous tone. Only arterioles that developed and maintained >20% baseline tone were used to assess vasodilatory responses.
Evaluation of vasodilator responses to intraluminal flow.
Arterioles were exposed to graded increases in intraluminal flow by adjusting the height of the fluid reservoirs in equal but opposite directions, thereby creating a pressure difference across the arterioles without altering intraluminal pressure within the arterioles (26). Diameter measurements were determined in response to pressure differences of 2, 4, 10, 20, 40, and 60 cmH2O, corresponding to physiologically relevant flow rates from 5 to 60 nl/s (29). In a subset of vessels of similar diameter to those used to assess functional dilation to flow, red blood cell velocity (Vrbc) was measured at the same pressure differences. The pipettes used in these experiments were of similar inner tip diameter and resistance as those used in subsequent flow experiments. Volumetric flow (Q) was then calculated from mean Vrbc and inner diameter (D) of these vessels according to the equation (26):
Corresponding shear stress was then calculated from volumetric flow (Q) according to the following equation: where η is viscosity (0.8 cp), Q is volumetric flow rate (calculated as described above), and r is vessel radius.
Responses to vascular endothelial growth factor and ACh.
To assess the effects of age on receptor-dependent vasodilation that occurs through diverse intracellular signaling pathways within the endothelium, concentration-responses to vascular endothelial growth factor (VEGF) and ACh were determined at intraluminal pressure of 60 cmH2O, in the absence of flow. Intraluminal diameter was recorded after cumulative additions of VEGF [1 × 10−15 to 1 × 10−10 M (4.2 × 10−8 to 4.2 × 10−3 mg/ml), 5-min stages] or ACh (1 × 10−9 to 1 × 10−4 M; 3-min stages).
Responses to diethylamineNONOate.
To evaluate vascular smooth muscle responsiveness to exogenous NO, a concentration-response to diethylamineNONOate (Dea-NONOate) (1 × 10−9 to 1 × 10−4 M; 2-min stages) was determined.
Blockade of nitric oxide synthase and cyclooxygenase.
To assess the role of eNOS in flow-, ACh-, and VEGF-induced vasodilation, diameter responses were reevaluated in the presence of NG-nitro-l-arginine methyl ester (l-NAME; 1 × 10−5 M; 30-min incubation). To determine the contribution of the cyclooxygenase (COX) pathway to flow-induced vasodilation, a flow-diameter curve was constructed in the presence of indomethacin (1 × 10−5 M; 30-min incubation). In some experiments, responses to flow and ACh were repeated in the same arteriole used to evaluate control responses. In some experiments, the effects of ACh and flow were performed in vessels without prior performance of a complete control response. The order of interventions was randomized except that flow was always evaluated before any pharmacological evaluation was performed. In vessels where a complete control response was not performed before intervention with an inhibitor, the responsiveness of the endothelium was verified by assessment of dilation to a brief exposure to flow (5 nl/s). Vehicle controls were performed to verify that repeated exposure to flow and ACh did not alter the vasodilatory responses of the arterioles. Vasodilation to VEGF was not repeatable in a single arteriole; therefore, the assessments of VEGF-induced vasodilation under control conditions and in the presence of l-NAME were always performed in separate arterioles. Vessels that did not respond to flow under control conditions were discarded. The effects of inhibitors on baseline tone were evaluated by comparing steady-state tone before and after incubation with the inhibitor (l-NAME or indomethacin).
Inhibition of PI3-kinase.
Wortmannin [5 × 10−7 M; 30-min incubation (2)] was used to determine the effects of PI3-kinase inhibition on flow-, VEGF-, and ACh-induced vasodilation in coronary arterioles. Responses to VEGF were not assessed in the same vessels evaluated under control conditions. In all vessels, the viability of the endothelium was established before wortmannin incubation by demonstration of dilation of the vessels to flow (2 min at 5 nl/s). The effect of wortmannin on baseline tone was evaluated by comparing steady-state tone before and after 30 min of incubation. To ensure that wortmannin inhibition did not alter responsiveness of the vascular smooth muscle to NO, vasodilation to DEA-NONOate (1 × 10−4 M) was assessed in the absence and presence of wortmannin (5 × 10−7 M).
To determine whether age altered signaling through Flk-1, a flow-diameter curve was assessed in the presence of SU-1498 [1 × 10−5 M; 30-min incubation (18, 19)]. Dilation of the arterioles to flow (2 min at 5 nl/s) was verified before incubation with SU-1498. The effect of SU-1498 on baseline tone was evaluated by comparing steady-state tone before and after 30 min incubation.
At the conclusion of each experiment, the vessels were washed with Ca2+-free PSS every 15 min for 1 h to obtain maximal passive diameter at 60 cmH2O.
Flow and VEGF stimulation of arterioles.
Flow stimulation of coronary arterioles was performed by cannulating and subjecting the arterioles to a flow of 13 nl/s for 5 min at 37°C. Control vessels were cannulated, warmed to 37°C, and maintained under no-flow conditions for 5 min. In VEGF experiments, arterioles were stimulated at 37°C with either VEGF (1 × 10−10 M for 5 min), wortmannin (5 × 10−7 M for 30 min), or wortmannin (5 × 10−7 M for 30 min) followed by VEGF (1 × 10−10 M for 5 min). At the end of the stimulation period, arterioles were removed from the pipettes and immediately solubilized in lysis buffer.
Coronary arterioles were snap-frozen and stored at −80°C. Arterioles were solubilized in 20 μl lysis buffer, and protein content was assessed by NanoOrange assay (Molecular Probes). Protein (10 μg) was electrophoresed on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Following blocking (5% nonfat dry milk), membranes were incubated overnight at 4°C with primary antibodies to eNOS (1:1,000; BD Transduction Laboratories), Akt (1:1,000; Cell Signaling Technology), Flk-1 (1:750; Santa Cruz Biotechnology), phosphorylated (p)-Akt (1:500; Cell Signaling Technology), p-eNOS (Cell Signaling Technology; 1:500), p-Flk-1 (1:1,000; Sigma), or β-actin (1:1,500; Cell Signaling Technology). After being washed, membranes were incubated with the appropriate horseradish peroxidase-conjugated species-specific anti-IgG (1 h). Peroxidase activity was detected by enhanced chemiluminescence (Super Signal West Femto; Pierce). Densitometric analysis of immunoblot films was performed using NIH ImageJ 1.38 × Analysis Software (National Institutes of Health, Bethesda, MD). Normalization for loading differences was accomplished using ratios of the densitometry signals for Flk-1, eNOS, AKT, p-Flk-1, p-eNOS, or p-AKT vs. β-actin.
Solutions and drugs.
Albumin was purchased from USB Chemicals (Cleveland, OH). All other chemicals were purchased from Sigma Chemical (St. Louis, MO).
Data are expressed as means ± SE. Spontaneous tone was calculated by the following equation: where DM is the maximal diameter recorded at 60 cmH2O and DT is the steady-state baseline diameter recorded at the same pressure. The vasodilator responses to flow, VEGF, ACh, and Dea-NONOate are expressed using the following equation: where DS is the arteriolar diameter and DB is the diameter recorded immediately before initiation of the flow- or concentration-diameter curves.
Flow-diameter and concentration-diameter curves were evaluated by repeated-measures ANOVA to detect differences within and between factors. Pairwise comparisons were made by post hoc analysis (Bonferroni) when a significant main effect was found. t-Tests were used for comparisons of animal and vessel characteristics, and sensitivity (IC50) to ACh and VEGF. Significance was set at P ≤ 0.05.
Animal and vessel characteristics.
Body weight, heart weight, and heart weight-to-body weight ratio all increased with age (Table 1). Neither maximal diameter nor tone achieved before any intervention differed between arterioles from young and old rats (Table 1). Treatment with l-NAME increased tone to a similar degree in arterioles from both young and old rats (Table 1). Indomethacin, wortmannin, and SU-1498 treatments did not alter tone in arterioles from either young or old rats.
Vasodilator responses to intraluminal flow, VEGF, and ACh.
Flow- and VEGF-induced vasodilatory responses were impaired in arterioles from old rats (Figs. 1A and 2A). In contrast to the age-induced impairment of vasodilation to flow and VEGF, maximal ACh-induced dilation of coronary arterioles was similar in young and old rats (Fig. 3A). Sensitivity to ACh (IC50) was slightly reduced in arterioles from old rats (young = 9.69 × 10−7 M; old = 9.25 × 10−8 M), but maximal vasodilation was unaltered by age. Treatment with l-NAME abolished flow-, VEGF-, and ACh-induced vasodilation, demonstrating the critical role of NO in mediating these responses (Figs. 1A, 2A, and 3A). Flow-induced vasodilation in coronary arterioles was not affected by treatment with indomethacin, a COX inhibitor (Fig. 1C), indicating that NO, rather than prostanoid compounds, is the primary mediator of flow-induced vasodilation.
Vasodilation to shear stress.
The shear stress to which the vascular endothelium is exposed depends on vessel radius; therefore, we calculated shear stress in coronary arterioles from young and old rats at each level of volumetric flow. Spontaneous tone, and thus, initial radii were similar in coronary arterioles from young and old rats, resulting in similar levels of shear stress in both groups at the onset of intraluminal flow (Fig. 4). Upon exposure to flow, arterioles from young animals displayed greater dilation than arterioles from aged animals. Thus the slope of the relationship between shear stress and dilation was significantly greater in coronary arterioles from young rats when compared with coronary arterioles from old rats (Fig. 4).
To inhibit PI3-kinase, and subsequent phosphorylation of Akt and eNOS, vasodilator responses to flow, VEGF, and ACh were assessed in vessels treated with wortmannin. Incubation with wortmannin reduced vasodilation to flow in coronary arterioles from young rats (Fig. 1B), and abolished vasodilation to VEGF in arterioles from both young and old rats (Fig. 2B). Importantly, the age-related differences in flow- and VEGF-induced vasodilation were eliminated in the presence of wortmannin (Figs. 1B and 2B), suggesting that the loss of these vasodilatory responses is linked to impaired PI3-kinase signaling. In contrast, vasodilation to ACh, although dependent on NO in arterioles from both young and old rats, was reduced by wortmannin only in arterioles from young rats (Fig. 3B). Wortmannin had no effect on ACh-induced vasodilation in arterioles from old rats (Fig. 3B). These findings indicate that ACh-induced dilation is maintained in coronary arterioles despite a loss of wortmannin-sensitive dilation.
To inhibit Flk-1 phosphorylation, vasodilation to flow and VEGF was assessed in arterioles treated with SU-1498. Incubation with SU-1498 decreased flow-induced dilation in coronary arterioles and abolished age-related differences in the responses to flow (Fig. 1D). To confirm the specificity of SU-1498, dilation to VEGF (1 × 10−10 M) was assessed in arterioles treated with SU-1498. Dilation to VEGF was completely abolished after pretreatment with SU-1498 (young: 0.70 ± 0.85, old: 0.09 ± 0.87 % relaxation), indicating that phosphorylation of Flk-1 is imperative for dilation to VEGF to occur (data not shown).
Vasodilator responses to Dea-NONOate.
To determine whether the age-related impairment of vasodilation in coronary arterioles was due to a decrease in smooth muscle responsiveness to NO, vasodilation to Dea-NONOate was measured. Dea-NONOate elicited similar vasodilatory responses in coronary arterioles from young (maximal relaxation = 90 ± 4%, n = 6) and old (maximal relaxation = 84 ± 5%, n = 6) rats (Fig. 5). Pretreatment with wortmannin did not alter maximal vasodilation to Dea-NONOate in young (85 ± 6%) or old (95 ± 1%) rats (Fig. 5, inset).
Akt, eNOS, and Flk-1 protein levels.
Akt and eNOS protein levels tended to be lower in coronary arterioles from old rats compared with arterioles from young rats (P = 0.10 and P = 0.09 for eNOS and Akt, respectively; Fig. 6, A and B). Flk-1 protein was reduced by 20% in coronary arterioles from old rats (P < 0.05) (Fig. 6C).
VEGF-induced phosphorylation of eNOS and Akt.
To demonstrate that VEGF stimulates phosphorylation of eNOS through a PI3-kinase-dependent pathway, levels of p-eNOS and p-Akt were determined in coronary arterioles stimulated with VEGF in the presence and absence of wortmannin. Basal levels of p-eNOS did not differ between arterioles from young and old rats. VEGF increased phosphorylation of eNOS significantly, and to a similar degree, in coronary arterioles from both young and old rats (Fig. 7A). Wortmannin inhibited the VEGF-induced increase in coronary arterioles from both young and old rats, reducing p-eNOS to basal, unstimulated levels (Fig. 7A). Wortmannin alone had no effect on phosphorylated eNOS levels (Fig. 7A). Basal p-Akt was lower in arterioles from young rats compared with arterioles from old rats (Fig. 7B). In contrast, VEGF-stimulated p-Akt was greater in arterioles from young rats compared with arterioles from old rats (p-Akt increased 700% above basal in young and 75% above basal in old, Fig. 7B, inset). p-Akt protein was not visible in coronary arterioles from young and old rats after wortmannin treatment alone or in combination with VEGF-stimulation (Fig. 7B).
Flow-stimulated phosphorylation of Flk-1 and eNOS.
Flow has been shown to activate Flk-1 in a ligand-independent manner, resulting in activation of PI3-kinase/Akt and phosphorylation of eNOS (18). To demonstrate that flow induces phosphorylation of Flk-1 in coronary arterioles, p-Flk-1 protein was assessed in arterioles exposed to 13 nl/s flow for 5 min. Exposure to flow increased phosphorylation of Flk-1 significantly in coronary arterioles from young rats, but phosphorylation of Flk-1 did not increase with flow exposure in coronary arterioles from old rats (Fig. 8A). Flow stimulation also increased phosphorylation of eNOS, but the flow-induced increase in p-eNOS was similar in arterioles from young and old rats (Fig. 8B).
The primary finding of this investigation is that impairment of Flk-1-mediated PI3-kinase/Akt signaling contributes to the age-induced reduction of flow-dependent, NO-mediated vasodilation in coronary arterioles from male Fischer-344 rats. This finding confirms earlier observations of reduced NO-mediated vasodilation in coronary resistance arteries of middle-aged rats (6) and provides novel insight into the effects of aging on cellular signaling mechanisms that activate eNOS in coronary arterioles. Our current findings indicate that age-related decrements in several components of the Flk-1 PI3-kinase/Akt signaling pathway contribute to the loss of flow- and VEGF-induced vasodilation that occurs with age in coronary arterioles. In contrast, ACh-induced signaling through G protein-coupled receptors is maintained with age, suggesting that Flk-1-mediated signaling is a critical target in age-induced coronary endothelial dysfunction.
Shear stress is a potent physiological stimulus for release of NO and is dependent on an intact endothelium (22). Flow-induced vasodilation is a key mediator of local vascular control in the coronary circulation (34, 35) and is critically dependent on endothelium-dependent release of NO, as evidenced by reports demonstrating that nitric oxide synthase inhibition eliminates flow-induced vasodilation in coronary arterioles of several species (25, 30, 41). Recent studies have indicated that VEGFR2, also known as Flk-1 (4), is rapidly tyrosine phosphorylated by flow (18, 24) and VEGF (14), leading to PI3-kinase-Akt-eNOS activation. Our results show that flow- and VEGF-induced vasodilatory responses of coronary arterioles are dramatically reduced by inhibition of Flk-1 with SU-1498. Importantly, our current results show that exposure to flow increases phosphorylation of Flk-1 in coronary arterioles from young rats, but not in coronary arterioles from old rats. Thus our results indicate that flow-induced vasodilation is impaired in coronary arterioles of senescent rats (24 mo) and extend previous findings in vessels from cardiac (6) and skeletal (29, 42) muscle by demonstrating that deficits in Flk-1/PI3-kinase/Akt signaling contribute to age-related reductions of NO-mediated vasodilation in coronary arterioles. Our current work also indicates that the decrease in Flk-1 activation that occurs with age is accompanied by a decrease in Flk-1 protein levels.
Both fluid shear stress and VEGF stimulate tyrosine phosphorylation of Flk-1, which thereafter phosphorylates eNOS through PI3-kinase-dependent activation of the protein kinase Akt (10, 13, 18, 28). In this study, we found that wortmannin, a PI3-kinase inhibitor, abolished the responses to flow and VEGF in coronary arterioles from both young and old rats; however, the magnitude of this inhibition was greater in arterioles from young rats compared with those from old rats. These findings indicate that the contribution of PI3-kinase/Akt signaling to NO-mediated vasodilation declines with age, resulting in reduced vasodilator responses to flow and VEGF in arterioles from old rats. Vasodilation to ACh was significantly decreased by wortmannin in coronary arterioles from young but not old rats, supporting the conclusion that the contribution of the PI3-kinase/Akt pathway to NO-mediated vasodilation wanes with age. The finding that wortmannin had no effect on ACh-induced vasodilation in coronary arterioles from old rats demonstrates that wortmannin inhibition of NO-mediated vasodilation was specifically due to blockade of PI3-kinase/Akt signaling. Additionally, vasodilation to DEA-NONOate was not affected by treatment with wortmannin, further validating that wortmannin does not alter NO-mediated dilation of vascular smooth muscle in a nonspecific manner.
To test the possibility that age-related impairments of Flk-1/PI3-kinase/Akt signaling contribute to a loss of NO-mediated vasodilation, we evaluated flow- and VEGF-stimulated phosphorylation of eNOS in coronary arterioles from young and old rats. Both flow and VEGF increased phosphorylation of eNOS; however, stimulation of p-eNOS did not differ between arterioles from young and old rats. Thus, we did not find that age altered phosphorylation of eNOS despite the decline in flow-induced phosphorylation of Flk-1 (Fig. 8A) and VEGF-induced phosphorylation of Akt (Fig. 7B) that occurred in coronary arterioles with age. Previous reports investigating the effects of aging on PI3-kinase signaling pathways vary with regard to directional changes in both Akt and eNOS. For example, p-eNOS has been shown to decrease in the vasculature of aged mice (3), despite a lack of decrease in p-Akt. Sun et al. (36) reported a decrease in p-eNOS in mesenteric arteries of aged rats, but the specific signaling mechanisms involved in this impairment were not investigated. Additionally, in endothelial cells harvested from young and old rats, p-Akt and p-eNOS declined with advancing age, but the decrease in p-Akt was accompanied by an increase in total Akt protein (33). Recent findings from several laboratories now indicate that several phosphorylation sites regulate the activity of eNOS (1, 10, 27, 44). Our current data indicate that phosphorylation of eNOS at Ser1177/1179 is not altered with age in coronary arterioles despite evidence of reduced Flk-1/PI3-kinase/Akt signaling. Further investigation will be needed to determine whether the age-related reduction of Flk-1 signaling results in impaired eNOS function due to adverse alterations of other regulatory sites on the eNOS enzyme. Our findings also do not rule out the possibility that lesions in other signaling pathways contribute to the loss of NO-mediated dilation that occurs with age in coronary arterioles. For example, Flk-1 activates mitogen-activated protein kinase (MAPK) (8), and, although regulation of eNOS function by MAPK has not been reported, the possibility exists that other signaling pathways involving kinases other than Akt (e.g., MAPK, AMP-activated kinase, or protein kinase A) regulate eNOS function. We specifically evaluated phosphorylation of eNOS at Ser1177/1179 because this is the regulatory site on eNOS reported to undergo phosphorylation in response to flow stimulation and activation of PI3-kinase/Akt signaling (18).
In the current study, l-NAME eliminated vasodilation to flow, VEGF, and ACh in coronary arterioles from both young and old rats, indicating the dependence of these responses on NO. Although vasodilator responses to flow and VEGF were reduced with age, ACh-induced dilation was preserved. These findings are in contrast to reports of reduced ACh-induced vasodilation in skeletal muscle and septal resistance arteries from old and middle-aged rats (6, 29, 43). ACh binding to muscarinic receptors stimulates Ca2+/calmodulin-dependent activation of eNOS through a pathway that can be modulated by, but does not require, Akt-dependent phosphorylation of eNOS (12, 31). Thus maintenance of signaling through G proteins and Ca2+-dependent mechanisms may function to preserve ACh-stimulated eNOS activity with advancing age. Our results suggest that impairment of Flk-1 PI3-kinase/Akt signaling is a primary factor contributing to the old age-related reduction of NO-mediated dilation in coronary arterioles and are consistent with the notion that G protein-coupled activation of eNOS that occurs independently of PI3-kinase signaling is maintained or increased with age.
The NO donor DEA-NONOate elicited similar responses from coronary arterioles in young and old rats (Fig. 5), indicating that the responsiveness of smooth muscle to NO is unchanged with advancing age, a finding consistent with reports from a number of vascular beds (6, 23, 29, 36, 42). Furthermore, spontaneous tone development and the level of tone induced by l-NAME were similar in arterioles from young and old rats, suggesting that basal NO production and vascular smooth muscle tone are not modified by age in coronary arterioles. The vasodilator responses reported in this study are assessed under in vitro conditions in which hemoglobin and other endogenous scavengers of NO are absent from the perfusate medium. Therefore, it is possible that the vasodilator responses reported here could be mitigated by the presence of red blood cells in vivo. Collectively, our data suggest that mechanisms within smooth muscle that regulate NO-mediated dilation are not altered with age in coronary arterioles. It is unlikely that extracellular scavengers of NO regulate the activity of intracellular signaling molecules such as PI3-kinase; thus, the age-induced impairment of NO-mediated dilation in coronary arterioles appears to be confined to endothelial pathways that signal through receptor tyrosine kinases.
In conclusion, advancing age impairs flow- and VEGF-induced vasodilation in coronary arterioles through reductions in Flk-1 activation and PI3-kinase/Akt signaling. NO-mediated vasodilation that occurs independently of Flk-1/PI3-kinase/Akt signaling is maintained with age in coronary arterioles, contributing to the preservation of ACh-induced vasodilation. These results suggest that Flk-1/PI3-kinase/Akt signaling mechanisms could be targeted for therapeutic interventions designed to improve coronary endothelial function in old age.
This research was funded by grants from the American Heart Association (J. Muller-Delp), the American College of Sports Medicine (R. Shipley), and the National Institute of Health (R01-HL-077224; J. Muller-Delp).
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.
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