Passive leg movement and nitric oxide-mediated vascular function: the impact of age

Abstract

In young healthy men, passive leg movement (PLM) elicits a robust nitric oxide (NO)-dependent increase in leg blood flow (LBF), thus providing a novel approach to assess NO-mediated vascular function. While the magnitude of the LBF response to PLM is markedly reduced with age, the role of NO in this attenuated response in the elderly is unknown. Therefore, this study sought to determine the contribution of NO in the PLM-induced LBF with age. Fourteen male subjects (7 young, 24 ± 1 yr; and 7 old, 75 ± 3 yr) underwent PLM with and without NO synthase (NOS) inhibition achieved by intra-arterial infusion of N^G-monomethyl-l-arginine (l-NMMA). LBF was determined second-by-second by Doppler ultrasound, and central hemodynamics were measured by finger photoplethysmography. NOS inhibition blunted the PLM-induced peak increase in LBF in the young (control: 668 ± 106; l-NMMA: 431 ± 95 Δml/min; P = 0.03) but had no effect in the old (control: 266 ± 98; l-NMMA: 251 ± 92 Δml/min; P = 0.59). Likewise, the magnitude of the reduction in the overall (i.e., area under the curve) PLM-induced LBF response to NOS inhibition was less in the old (LBF: −31 ± 18 ml) than the young (LBF: −129 ± 21 ml; P < 0.01). These findings suggest that the age-associated reduction in PLM-induced LBF in the elderly is primarily due to a reduced contribution to vasodilation from NO and therefore support the use of PLM as a novel approach to assess NO-mediated vascular function across the lifespan.

passive leg movement (PLM) evokes a robust leg blood flow (LBF) response (34, 56, 61), the magnitude of which is markedly reduced by aging (26, 35). Importantly, aging is also characterized by impaired NO-mediated vascular function (20, 45, 51), suggesting that the magnitude of the PLM-induced LBF response may be directly associated with impaired vascular function. Interestingly, in young healthy men, utilizing NO synthase (NOS) blockade, it has been documented that nearly 80% of the PLM-induced LBF response is NO dependent (55). This high reliance on NO coupled with a relatively simple methodology supports the use of the PLM paradigm as a novel approach for the in vivo assessment of NO bioavailability and endothelial function. However, the contribution of NO to PLM-induced LBF in populations other than the healthy young, most notably the elderly, is not yet known, thus limiting the implications of this assessment across the lifespan.

The assessment of endothelial function is of significance in both clinical and research settings as endothelial dysfunction precedes overt cardiovascular disease including hypertension, atherosclerosis, and coronary artery disease (1, 9, 17, 53). In the research arena, endothelial function is commonly assessed by flow-mediated dilation (FMD) following circulatory occlusion (10). However, recent studies have questioned the NO-dependent nature of this technique (42–44, 57, 63). Given the recognized vasoprotective and cardioprotective properties of NO, the growing uncertainty regarding the role of NO in the traditional FMD technique, and its failure to be embraced clinically, there is reason to search for a novel approach to assess NO-dependent endothelial function.

Therefore, this investigation sought to determine the mechanism responsible for the recognized impairment in PLM-induced LBF with age (26, 35) by assessing the role of NO in the peripheral hemodynamic response to this stimulus. Specifically, we hypothesized that due to the age-associated reduction in NO bioavailability, aging would be associated with a limited contribution of NO to the PLM-induced increase in LBF. Documentation of the capacity for PLM to discern changes in NO bioavailability across the lifespan will help to solidify the practical usefulness of PLM as a novel and clinically relevant approach to assess NO-mediated endothelial function.

METHODS

Subjects

Seven young healthy men (24 ± 1 yr of age) and seven older healthy men (75 ± 3 yr of age) volunteered to participate in this research study. Subjects were normally active, not taking any prescription medications, and free from overt cardiovascular disease. Protocol approval and written informed consent were obtained according to the University of Utah and Veterans Affairs Salt Lake City Institutional Review Boards, in accordance with the principles outlined in the Declaration of Helsinki. All data collection took place at the Utah Vascular Research Laboratory located in the Salt Lake City Veteran Affairs Medical Center's Geriatric Research, Education, and Clinical Center.

Experimental Protocol

Before the experimental day all subjects reported to the laboratory for a familiarization trial. During this session, PLM and Doppler ultrasound imaging of the femoral artery were performed to both familiarize the subjects and ensure that acceptable images could be obtained with and without movement. Upon arrival at the laboratory on the experimental day, body mass and height were recorded and the right femoral artery was catheterized (18-gauge central venous catheter; Arrow International, Reading, PA) using the Seldinger technique. Following a 30-min rest period, subjects were positioned in the supine position. Due to the lasting effects of N^G-monomethyl-l-arginine (l-NMMA), the control trials were always performed before l-NMMA infusion. Trials were separated by at least 30 min to ensure blood flow and central hemodynamics returned to baseline values. Before the commencement of PLM, stable baseline central and peripheral hemodynamic measures were documented. An automated occlusion cuff (Hokansen, Bellevue, WA) was placed just below the knee of the passively moved leg and inflated to 250 mmHg during both control and l-NMMA trials to ensure that the infusate did not perfuse the lower portion of the limb. Single leg PLM was performed by a member of the research team who moved the leg through a 90° range of motion at 1 Hz. The starting position of the leg was the full extension at the knee (i.e., 180°), and therefore, the first movement served to passively flex the knee from 180 to 90°. Real-time knee joint angle feedback was provided to the researcher by digital display. Before the start and throughout the protocol, subjects were encouraged to remain passive and resist the urge to assist with leg movement. To avoid the startle reflex and active resistance to the PLM, subjects were made aware that PLM would take place in ∼1 min, but to minimize the chance of an anticipatory response, they were not informed of exactly when the movement would begin. When commenced, PLM was performed for 2 min at 1 Hz. We previously reported no discernable difference in the time course and magnitude of the PLM-induced LBF response when performed on separate days (coefficient of variation = 5%), indicating the peripheral hemodynamic response to PLM is reproducible (55).

l-NMMA infusion.

Thigh volume was determined anthropometrically (31) and then used for the calculation of drug dosing. l-NMMA (Bachem, Bubendorf, Switzerland) was diluted from 250 mg lypholyzed powder in normal saline to a concentration of 5 mg/ml. l-NMMA was infused at a priming dose of 0.48 mg/dl thigh volume for 5 min before PLM. During the minute before PLM, l-NMMA was infused at a maintenance dose of 0.24 mg/dl thigh volume and this infusion rate was maintained for the duration of PLM. During control trials, normal saline was infused intra-arterially at the same rate and duration as l-NMMA.

Measurements

Femoral blood flow.

Measurements of femoral arterial blood velocity and vessel diameter were performed in the passively moved and the nonmoved legs distal to the inguinal ligament and proximal to the deep and superficial femoral bifurcation with Logic 7 and Logic e Doppler ultrasound systems (General Electric Medical Systems, Milwaukee, WI). The ultrasound systems were equipped with a linear transducer operating at an imaging frequency of 10 MHz. Vessel diameter was determined at a perpendicular angle along the central axis of the scanned area. Blood velocity was measured using the same transducer with a frequency of 5 MHz. All blood velocity measurements were obtained with the probe appropriately positioned to maintain an insonation angle of 60° or less. The sample volume was maximized according to vessel size and was centered within the vessel. Arterial diameter was measured, and mean velocity (V_mean; angle corrected and intensity-weighted area under the curve) was automatically calculated (Logic 7 and Logic e). with the use of femoral arterial diameter and V_mean, LBF was calculated as LBF = V_mean·π (vessel diameter/2)²·60, where LBF is in milliliters per minute. Leg vascular conductance (LVC) was calculated as LVC = LBF/mean arterial pressure (MAP).

Central hemodynamic variables.

Heart rate (HR), stroke volume (SV), and cardiac output (CO) were determined with a finometer (Finapres Medical Systems, Amsterdam, The Netherlands). SV was calculated from beat-by-beat pressure waveforms assessed by photoplethysmography using the Modelflow method (Beatscope version 1.1; Finapres Medical Systems), which, in combination with HR, has been documented to accurately estimate CO during a variety of experimental protocols (3, 18, 19, 50, 58). Intravascular systolic and diastolic arterial pressures were measured with an in-line pressure transducer (Baxter, Deerfield, IL) placed at the level of the catheter. MAP was calculated as diastolic + 1/3(systolic − diastolic).

Data acquisition.

Throughout each protocol, HR, SV, CO, MAP, and ECG signals underwent analog-to-digital conversion and were simultaneously acquired (200 Hz) using a data acquisition system (AcqKnowledge; Biopac Systems, Goleta, CA).

Data and Statistical Analysis

The data acquisition software (AcqKnowledge; Biopac Systems) allowed second-by-second analyses of HR, SV, CO, and MAP. The second-by-second blood velocities were analyzed on the ultrasound systems (GE Logic 7 Logic e) for the first 60 s of movement, and 12-s averages were assessed from 60 to 120 s of PLM. All analyses were performed using a 5-s moving average. Baseline was analyzed using the 60 s before initiation of PLM. A 2 × 2 repeated-measures ANOVA was used to determine significant differences in the absolute change from baseline to peak for HR, SV, CO, MAP, LBF, LVC, and area under the curve (AUC) for LBF, LVC, and MAP. Cumulative AUC was calculated as the summed second-by-second response during the first 60 s of PLM after normalizing for baseline differences. Post hoc comparisons were made using Fischer's least significant difference when appropriate. Subject characteristics and blood parameters were compared by independent sample t-tests.

The rate of increase in the hyperemic response from baseline to peak was determined a posteriori by linear regression using an iterative process minimizing the sum of squared residuals between the fitted function and the observed values. To avoid the inclusion of nonessential points in the regression, the data were initially fit up to the peak of the hyperemic response and then decreased iteratively by 1 s. The best fitting window was then established using the following three indexes: 1) the flat profile of the residual plot being no longer sustained as determined by visual inspection, 2) the demonstration of a local “threshold” in the χ²-squared values, and 3) a significant correlation (range: r = 0.89 ± 0.05 to 0.98 ± 0.01) between the actual data and the predicted values as determined by linear regression (46). A 2 × 2 repeated-measures ANOVA was used to test differences in the rate of increase of the hyperemic response and post hoc analysis was performed with Fischer's least significant difference when appropriate. Significance was set at an α-level of 0.05, and data are presented as means ± SE.

RESULTS

Young and old subjects were well-matched for stature, thigh volume, MAP, and blood characteristics (Table 1).

Table 1.

Subject stature and blood characteristics

	Young	Old
Age, yr	24 ± 1	75 ± 3*
Height, cm	177 ± 3	177 ± 3
Weight, kg	75 ± 4	78 ± 5
Body mass index, kg/m2	24 ± 1	25 ± 2
Thigh volume, dl	74 ± 4	63 ± 4
Mean arterial pressure, mmHg	95 ± 3	98 ± 4
Glucose, mg/dl	68 ± 4	75 ± 4
Cholesterol, mg/dl	172 ± 14	178 ± 13
Triglycerides, mg/dl	89 ± 15	102 ± 17
HDL, mg/dl	49 ± 3	50 ± 4
LDL, mg/dl	107 ± 12	117 ± 9

Values are means ± SE.

^*P < 0.05, significant difference between young and old.

Baseline Effects of l-NMMA

Baseline hemodynamics with and without l-NMMA are presented in Table 2. Baseline LBF was ∼30% lower in the old compared with the young; however, this difference did not reach statistical significance (P = 0.19). NOS inhibition, achieved by the intra-arterial infusion of l-NMMA, decreased baseline LBF in the young (P < 0.01) and the old (P = 0.02); however, the magnitude of this reduction was not different between groups (P = 0.21). l-NMMA reduced baseline HR (P = 0.03) and CO (P = 0.024) in the young but had no effect in the old. SV and MAP were not different between groups and were not altered by l-NMMA (Table 2).

Table 2.

Baseline hemodynamics with and without l-NMMA

	Young		Old
	Control	l-NMMA	Control	l-NMMA
Leg blood flow, ml/min	310 ± 27	161 ± 16†	217 ± 62	121 ± 50†
Leg vascular conductance, ml·min−1·mmHg−1	3.3 ± 0.3	1.7 ± 0.1†	2.3 ± 0.6	1.3 ± 0.5†
Heart rate, beats/min	54 ± 2	50 ± 2†	59 ± 4	61 ± 6
Stroke volume, ml/beat	98 ± 6	96 ± 8	93 ± 19	79 ± 15
Cardiac output, l/min	5.5 ± 0.4	4.7 ± 0.3†	5.2 ± 1.0	4.1 ± 0.8
Mean arterial pressure, mmHg	95 ± 3	96 ± 3	98 ± 4	98 ± 5

Values are means ± SE.

^†P < 0.05, significant difference between control and N^G-monomethyl-l-arginine (l-NMMA).

Peripheral Hemodynamics During PLM

In both groups and trials, LBF increased immediately at the onset of PLM (Fig. 1A). To take into account the l-NMMA-induced reduction in baseline LBF, the peak change in LBF was calculated (Fig. 1B). The peak change in LBF during control PLM was 2.5-fold greater in the young (668 ± 106 ml/min) than the old (266 ± 98 ml/min; P = 0.016). NOS inhibition evoked a significant reduction in peak LBF in the young (Δ −237 ± 83 ml/min; P = 0.03) but not the old (Δ −15 ± 26 ml/min; P = 0.59). Similarly, l-NMMA reduced the overall LBF response (as measured by LBF AUC) in the young (−129 ± 21 ml; P < 0.01) but not the old (−31 ± 18 ml, P = 0.13; Fig. 1C). PLM-induced changes in LVC (second-by-second data not illustrated), an indicator of leg vasodilation, mirrored the changes in LBF, with a threefold greater peak change in LVC in the young (7.7 ± 1.1 ml·min⁻¹·mmHg⁻¹) compared with the old (2.5 ± 0.9 ml·min⁻¹·mmHg⁻¹; P < 0.01). Similar to LBF, NOS inhibition significantly attenuated the peak change in LVC in young (Δ −3.3 ± 1.0 ml·min⁻¹·mmHg⁻¹; P = 0.02) but not the old (Δ −0.2 ± 0.4 ml·min⁻¹·mmHg⁻¹; P = 0.62). The overall vasodilatory response to PLM (as measured by LVC AUC) was reduced with age (P < 0.05) and by l-NMMA in both the young (P < 0.01) and the old (P < 0.05; Fig. 2A); however, the magnitude of the l-NMMA-induced reduction in LVC AUC was far greater in the young (P = 0.02; Fig. 2B).

Fig. 1.

Passive limb movement (PLM)-induced hyperemia in young and old with and without intra-arterial N^G-monomethyl-l-arginine (l-NMMA) infusion. A: absolute leg blood flow (LBF; ml/min). B: change in LBF, normalized for the resting reduction in LBF due to l-NMMA. C: LBF area under the curve (AUC) calculated as the summed second-by-second response during the first 60 s of movement. Values are means ± SE. To increase clarity of the data presented, only the first 60 s of PLM are displayed. One minute of baseline data was collected before PLM occurred. *P < 0.05, significant difference between control and l-NMMA.

Fig. 2.

PLM-induced vasodilatory response in young and old with and without intra-arterial l-NMMA infusion. A: leg vascular conductance (LVC) AUC calculated as the summed second-by-second response during the first 60 s of movement. B: l-NMMA-induced reduction in LVC AUC. Values are means ± SE. *P < 0.05, significant difference between control and l-NMMA. †P < 0.05, significant difference between young and old. #P < 0.05, significant reduction due to l-NMMA.

The rate of the initial (first 7–10 s) increase in the LBF response to PLM in the control trial was nearly three times faster in the young (74 ± 11 ml·min⁻¹·s) than the old (26 ± 6 ml·min⁻¹·s; P = 0.004). l-NMMA, however, had no impact on the rate of the LBF response in either group (young: 65 ± 15 ml·min⁻¹·s, P = 0.63; old: 35 ± 12 ml·min⁻¹·s, P = 0.53).

LBF in the nonmoved leg exhibited a slight increase following the onset of PLM and then fluctuated around the baseline for the remaining portion of PLM in the young and old in the control trial and the old l-NMMA trial (Fig. 3). In contrast, during the l-NMMA trial in the young, LBF in the nonmoved leg exhibited a clear transient increase during the first minute of PLM (Fig. 3).

Fig. 3.

Change in LBF in the nonmoved limb during PLM in young and old with and without intra-arterial l-NMMA infusion. Significant main effect of interaction for the young (trial × time). One minute of baseline data was collected before PLM. To increase clarity of the data presented, only the first 60 s of PLM are displayed. Values are means ± SE.

Central Hemodynamics During PLM

PLM evoked significant increase in HR, SV, and CO in both groups and across all trials (Table 3). Peak changes in HR and CO were not different between groups or conditions. The change in SV was greater in the young during the control trial compared with the l-NMMA trial while there was no effect of l-NMMA on SV in the old. Due to the dynamic changes in MAP during PLM (Fig. 4A), the overall MAP response, as determined by AUC during the first minute of PLM, was compared between groups and trials. Young subjects exhibited an overall decrease in MAP during the control trial, whereas a net increase was evident with l-NMMA infusion (Fig. 4B). In contrast, MAP in the old increased during PLM, and when expressed as AUC, the overall MAP response also revealed a net increase that was similar between the control and l-NMMA trials (Fig. 4D).

Table 3.

Peak changes in central hemodynamics during passive limb movement

	Young		Old
	Control	l-NMMA	Control	l-NMMA
Heart rate, beats/min	10 ± 3	10 ± 3	7 ± 2	7 ± 3
Stroke volume, ml/beat	7 ± 2	3 ± 1†	10 ± 5	6 ± 2
Cardiac output, l/min	0.9 ± 0.2	1.1 ± 0.3	1.4 ± 0.6	0.7 ± 0.4

Values are means ± SE.

^†P < 0.05, significant difference between control and l-NMMA.

Fig. 4.

PLM-induced changes in mean arterial pressure in young and old with and without intra-arterial l-NMMA infusion. Absolute mean arterial pressure (MAP; mmHg; A) and MAP AUC (B) calculated as the summed second-by-second response during the first 60 s of movement. Values are means ± SE. *P < 0.05, significant difference between control and l-NMMA.

DISCUSSION

Previously, we (55) and others (37) have reported that NO plays an essential role in the magnitude and duration of the PLM-induced increase in LBF, suggesting that such a paradigm may provide a novel assessment of NO-mediated vascular function and cardiovascular disease risk. The current study is the first to directly examine the contribution of NO to PLM-induced LBF with age. The PLM-induced increase in peak LBF and the contribution of NO to this response were severely attenuated with age, as NOS inhibition diminished the LBF response in the young but had no effect in the old. Moreover, the overall LBF response (i.e., AUC) was significantly blunted following NOS inhibition in the young but not the old. These findings reveal that aging reduces the role of NO in PLM-induced LBF and provide further support for PLM as a novel and clinically relevant approach to assess NO-mediated vascular function across the lifespan.

PLM as a Novel Method for the Assessment of NO-Mediated Vascular Function

The assessment of NO-mediated vascular function is important from both clinical and research perspectives as reductions in NO bioavailability and endothelial function precede overt cardiovascular disease (1, 17, 25, 53). Indeed, the prognostic and diagnostic importance of accurately and reliably assessing NO-mediated vascular function has led to the development and sustained interest in the occlusion-based FMD (10). This traditional FMD technique, as first described by Celermajer et al. (10), has been widely used as a noninvasive approach to determine endothelial derived NO bioavailability. However, recent evidence has challenged the usefulness of FMD in determining NO-dependent vascular function (42–44, 57, 63). This uncertainty and the failure of FMD to become adopted clinically prompted interest in determining whether an alternative method, PLM, can serve as a functional bioassay for endothelium-derived NO bioavailability and assess NO-dependent vascular function in humans.

This study provides novel evidence that aging greatly diminishes the contribution of NO to PLM-induced hyperemia. Confirming our previous findings (55), NOS inhibition reduced the peak change and overall LBF in the young, while having no effect in the old (Fig. 1). Interestingly, the significant reduction in overall LVC (Fig. 2A) and not LBF (Fig. 1C) in the older adults is likely due to the slight elevation in MAP during PLM in the NOS-inhibited trial in the old. Thus normalizing the LBF response for this minor elevation in MAP revealed that the overall vasodilatory response to PLM in the old is at least partially NO mediated, albeit at a level much less than that reported in the young (Fig. 2B). Overall, these findings imply that the attenuated PLM-induced increases in LBF and vasodilation associated with aging are largely due to a reduced contribution of NO.

Reductions in vascular function with advancing age are well established. Indeed, both intra-arterial infusion of acetylcholine and occlusion-based FMDs have predominantly revealed impaired endothelial function with aging (5, 11, 20, 24, 38, 39). Compared with these conventional methods of assessing NO-dependent endothelial function, PLM may be a superior alternative. First, unlike the intra-arterial acetylcholine infusion approach, PLM is noninvasive, significantly reducing the risks associated with the technique. Mortensen et al. (37) recently reported a strong correlation between acetylcholine infusion and PLM induced-LBF, thus providing additional support in favor of the NO dependency of the PLM-induced hyperemic response. Second, acute tissue ischemia, which is required to provide the shear stimulus for vasodilation in the traditional FMD, is uncomfortable, and may be contraindicated in some individuals, is not a component of PLM. Third, compared with cuff-occlusion FMD, PLM does not require the precise sequential measurement of conduit artery diameter as the common femoral artery does not dilate during passive movement or exercise, greatly reducing the technical requirements in terms of both sonography and analysis (29). Finally, our recent observations, along with the current study, reveal that the PLM-induced increase in LBF is primarily NO dependent (55), a contention that has recently been challenged with traditional FMD (43, 63). Establishing PLM as a biomarker of NO-dependent vascular function will undoubtedly require additional investigation and validation; however, recent investigations by our group indicate that in addition to aging (26, 35), conditions commonly associated with vascular dysfunction including spinal cord injury (60) and heart transplantation (30) exhibit reduced vasodilation during PLM. Taken together, these findings suggest that PLM is capable of determining impairments in NO-dependent vascular function in clinically relevant patient groups.

In terms of the mechanisms responsible for the reduced NO-mediated LBF response to PLM with age, upstream factors, including a reduction in eNOS protein expression, have been suggested. However, Donato et al. (22) reported no difference in endothelial cell eNOS protein expression between young and old men, despite impaired endothelial function, suggesting that the enzymatic machinery for NO production is present but may simply not be functioning optimally. Alternatively, downstream factors such as elevated oxidative stress and the concomitant reduction in NO bioavailability by the inactivation of NO to peroxynitrite (8, 13, 27) may add to the diminished role of NO with age. In agreement with this hypothesis our group recently reported that acute antioxidant treatment resulted in improved blood antioxidant status, reduced oxidative stress, and improved vascular function in older healthy individuals (62). Additionally, intra-arterial infusion of ascorbic acid markedly improved endothelium-dependent vasodilation during prolonged handgrip exercise (16, 32) and during coinfusion of acetylcholine (52), suggesting a causative relationship between oxidative stress and impaired endothelium-dependent vascular function with age. Therefore, further research is warranted to determine if the impaired age-associated hyperemic response to PLM can be augmented by either acute or chronic antioxidant supplementation.

Insight into the Initial PLM-Induced Hyperemic Response

Based on our previous investigation (55), and confirmed by the current findings, the LBF response to PLM in young healthy individuals involves two distinct phases: an initial rapid NO-independent component, in which LBF increases, followed by an NO-dependent component that consists of a continued rise to peak LBF that gradually decays toward baseline. Interestingly, the current study reveals that despite the initial onset of LBF apparently not being NO-dependent (Fig. 1B), aging is associated with a threefold slower rate of LBF increase with the onset of PLM (i.e., within the first 7–10 s; Fig. 1B). This suggests that additional, non-NO-mediated, mechanisms associated with vasodilation are impaired with age. It has been suggested that the immediate or rapid vascular response to muscle contraction, or changes in muscle length with PLM, occur following the first muscle “contraction,” without a measurable delay and without a contribution from NO (47, 48). The current finding of an age-associated reduction in PLM-induced LBF hyperemia agrees with previous reports (Kirby et al. 32; Crecelius et al. 16) utilizing a single brief muscle contraction (handgrip exercise), which also implied that endothelial function, per se, does not contribute to the age-associated reduction in blood flow following a single contraction. Additionally, endothelium denuded isolated rat feed arteries display a reduced, but not abolished, immediate vasodilation in response to mechanical compression (12), providing evidence that an intact endothelium is not essential for the initial response.

Although the exact mechanisms responsible for the initial LBF response to PLM are not known, the current findings and those of others indicate that local endothelium-dependent vasodilators (NO and prostaglandins) and neural mechanisms do not appear to be essential for the initial vasodilatory response (6, 7, 23, 47, 49). A smooth muscle-related mechanism may involve potassium (K⁺) flux resulting in smooth muscle hyperpolarization, as the inhibition of inwardly rectifying K⁺ channels has been demonstrated to result in a reduction in the immediate vasodilator response in animals (2, 28, 36) and humans (14, 15). Mechanosensitive ion channels present on the endothelium may respond to stretch- and shear-induced changes during PLM leading to vasodilation. Additionally, myogenic vasodilation may play a role as we have previously reported that an increase in perfusion pressure, evoked by an upright posture, augmented the initial onset of LBF at the start of PLM (26, 56).

Contribution of Central Hemodynamics to PLM-Induced Hyperemia

At the onset of passive movement, group III mechanosensitive skeletal muscle afferent fibers are stimulated eliciting a reflex-mediated cardio-acceleration and subsequently a central hemodynamic response (30, 34, 40, 54, 61). This central hemodynamic response (HR, SV, and CO) to PLM-induced hyperemia was confirmed by the current study; however, differences due to age and NOS inhibition were unremarkable (Fig. 4). Central factors may contribute to the differences in PLM-induced hyperemia; however, the contribution of the central hemodynamic response (< 30%) (54) is much less than that associated with peripheral factors (∼ 80%), indicating that the differences between individuals or groups are largely peripherally mediated.

Interestingly, the overall MAP response to PLM was affected by both NOS inhibition and age (Fig. 4). Notably, in the young subjects PLM resulted in a transient reduction in MAP corresponding temporally with the peak increase in LBF. Theoretically, this reduction in MAP should act to decrease LBF; however, PLM-induced vasodilation appears to outstrip the capacity of the CO increase to raise pressure. During the infusion of l-NMMA in the young, MAP exhibited a net increase due to the expected reduction in vasodilation or increased vascular tone. Importantly, this peripherally induced reduction in vascular conductance due to intra-arterial infusion of l-NMMA had a profound impact on systemic blood flow distribution as the control leg (nonmoved and noninfused leg) of the young subjects experienced a significant increase in LBF during PLM (Fig. 3). This increase in LBF in the nonmoved leg of the young is likely the direct hemodynamic consequence of eliminating the previously available vascular sink (the passively moved limb) for the increase in CO during PLM. Interestingly, the accompanying increase in MAP in the young was modest, likely due to the accommodation of the increased CO by the nonmoved leg, presumably by non-NOS-mediated mechanisms due to the systemic effects of circulating l-NMMA. In both control and NOS-inhibited trials, the old subjects displayed marked increases in MAP during PLM, providing further evidence of a reduced vasodilatory capacity with age. Again, perhaps, non-NOS mediated, because of the anticipated effect of circulating l-NMMA. Interestingly, in stark contrast to the young, blood flow distribution between the infused and noninfused legs of the old was not affected by the l-NMMA as the overall contribution of NO to PLM-induced LBF and vasodilation was greatly reduced in the old.

Experimental Considerations

The primary finding of this investigation is the novel determination that aging is associated with an attenuated contribution of NO to the PLM-induced increase in LBF. However, several experimental limitations should be recognized. First, the overall sample size (n = 14) is relatively small and may have contributed to our inability to detect age-associated differences in resting LBF. Several investigations (4, 21, 33, 41) report a significant 20–30% reduction in resting LBF, which is in agreement with the nonsignificant magnitude of reduction reported here (30%). Second, despite a numerically lower thigh volume in the old, there was not a detectable significant difference between groups, which, if actually altered by age, could influence the PLM-induced hyperemia (59). However, even after normalization for thigh volume the peak change in LBF during PLM remained attenuated in the older men compared with their young counterparts (young: 90 Δml·min⁻¹·l thigh volume⁻¹; old: 48 Δml·min⁻¹·l thigh volume⁻¹; P = 0.035), indicating that the smaller thigh volume was not the driving factor for age-related reduction in LBF during PLM. Third, unlike the subjects selected for this study, the proportion of older men (∼75 yr of age) in good health and not taking prescription medications in the general population is quite low (<20% based on recent statistics from the Centers for Disease Control). However, our careful matching of young and old subjects was required to determine the true impact of aging, independent of other confounding factors, on the contribution of NO to LBF during PLM across the lifespan. Finally, pharmacological inhibition of NOS with l-NMMA revealed a critical role of NO bioavailability in the PLM-induced hyperemic response in the young that was absent in the old; however, the contribution of factors upstream to NO release, for example, NOS phosphorylation and expression, remains to be elucidated. Indeed, future investigations are warranted to determine if directly augmenting NO bioavailability, with an NO donor, improves the LBF response to PLM in those with vascular dysfunction.

Conclusions

In young healthy men PLM evokes a robust increase in LBF, the magnitude of which is largely NO mediated. In contrast, the LBF response to PLM in the old is severely attenuated and the contribution of NO to this response in the old is markedly reduced as NOS inhibition failed to impact the peak and the overall hyperemic response in the old. Taken together, these findings indicate that PLM provides a novel noninvasive and relatively simple model capable of assessing NO-dependent changes in vascular function across the lifespan.

GRANTS

This study was supported by Veteran Affairs Rehabilitation Research and Development Grant 1IK2RX001215 CDA2 (to J. D. Trinity) and Merit Grants E1697R and E6910R (to R. S. Richardson); American Heart Association Grant 14SDG1850039 (to J. D. Trinity); and National Heart, Lung, Blood Institute Grant PO1-HL-091830 (to R. S. Richardson).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: J.D.T., H.J.G., and R.S.R. conception and design of research; J.D.T., H.J.G., G.L., M.J.R., S.J.I., D.E.M., B.S.G., and A.D.B. performed experiments; J.D.T. and H.J.G. analyzed data; J.D.T., H.J.G., G.L., M.J.R., S.J.I., and R.S.R. interpreted results of experiments; J.D.T. and H.J.G. prepared figures; J.D.T. drafted manuscript; J.D.T., H.J.G., G.L., M.J.R., S.J.I., and R.S.R. edited and revised manuscript; J.D.T., H.J.G., G.L., M.J.R., S.J.I., D.E.M., B.S.G., A.D.B., and R.S.R. approved final version of manuscript.