Single passive leg movement assessment of vascular function: contribution of nitric oxide

Passive leg movement (PLM), a novel assessment of vascular function, has been simplified to a single PLM (sPLM), thereby increasing the clinical utility of this technique. However, the role of nitric oxide (NO) in mediating the robust sPLM hemodynamic responses is unknown. This study revealed that sPLM induces a hyperemic and vasodilatory response that is predominantly NO-mediated and, as such, appears to be a promising simple, in vivo, clinical assessment of NO-mediated vascular function and, therefore, NO bioavailability.

Abstract

Broxterman RM, Trinity JD, Gifford JR, Kwon OS, Kithas AC, Hydren JR, Nelson AD, Morgan DE, Jessop JE, Bledsoe AD, Richardson RS. Single passive leg movement assessment of vascular function: contribution of nitric oxide. J Appl Physiol 123: 1468–1476, 2017. First published August 31, 2017; doi:10.1152/japplphysiol.00533.2017.—The assessment of passive leg movement (PLM)-induced leg blood flow (LBF) and vascular conductance (LVC) is a novel approach to assess vascular function that has recently been simplified to only a single PLM (sPLM), thereby increasing the clinical utility of this technique. As the physiological mechanisms mediating the robust increase in LBF and LVC with sPLM are unknown, we tested the hypothesis that nitric oxide (NO) is a major contributor to the sPLM-induced LBF and LVC response. In nine healthy men, sPLM was performed with and without NO synthase inhibition by intra-arterial infusion of N^G-monomethyl-l-arginine (l-NMMA). Doppler ultrasound and femoral arterial pressure were used to determine LBF and LVC, which were characterized by the peak change (ΔLBF_peak and ΔLVC_peak) and area under the curve (LBF_AUC and LVC_AUC). l-NMMA significantly attenuated ΔLBF_peak [492 ± 153 (l-NMMA) vs. 719 ± 238 (control) ml/min], LBF_AUC [57 ± 34 (l NMMA) vs. 147 ± 63 (control) ml], ΔLVC_peak [4.7 ± 1.1 (l-NMMA) vs. 8.0 ± 3.0 (control) ml·min⁻¹·mmHg⁻¹], and LVC_AUC [0.5 ± 0.3 (l-NMMA) vs. 1.6 ± 0.9 (control) ml/mmHg]. The magnitude of the NO contribution to LBF and LVC was significantly correlated with the magnitude of the control responses (r = 0.94 for ΔLBF_peak, r = 0.85 for LBF_AUC, r = 0.94 for ΔLVC_peak, and r = 0.95 for LVC_AUC). These data establish that the sPLM-induced hyperemic and vasodilatory response is predominantly (~65%) NO-mediated. As such, sPLM appears to be a promising, simple, in vivo assessment of NO-mediated vascular function and NO bioavailability.

NEW & NOTEWORTHY Passive leg movement (PLM), a novel assessment of vascular function, has been simplified to a single PLM (sPLM), thereby increasing the clinical utility of this technique. However, the role of nitric oxide (NO) in mediating the robust sPLM hemodynamic responses is unknown. This study revealed that sPLM induces a hyperemic and vasodilatory response that is predominantly NO-mediated and, as such, appears to be a promising simple, in vivo, clinical assessment of NO-mediated vascular function and, therefore, NO bioavailability.

the bioavailability of nitric oxide (NO), a molecule with important cardioprotective effects, is mechanistically linked to the endothelial dysfunction that is commonplace in aging and disease (3, 9, 13, 16, 17, 25, 28, 33, 35, 39, 44, 45, 52). As such, robust techniques for the assessment of NO-mediated vascular function are paramount for the early detection and mechanistic assessment of vascular dysfunction. Historically, the most pervasive assessment of NO-mediated vascular function has been flow-mediated dilation (FMD) in response to postischemic cuff occlusion-induced hyperemia (8). However, enthusiasm for this technique has been dampened by the complex methodologies and mounting evidence that FMD may not be NO-mediated (37, 38, 49, 57). This has stimulated the development of new methodologies for the assessment of NO-mediated vascular function.

Passive leg movement (PLM) has emerged as a robust assessment of vascular function. In healthy humans, following the onset of passive movement, there is a transient, yet robust, increase in limb blood flow (LBF) and limb vascular conductance (LVC) (29, 46, 55). This hyperemic and vasodilatory response is likely initiated by the mechanical deformation of blood vessels and local release of vasodilators (10, 12, 21, 26, 34, 48) that set in motion a cascade of events that transiently sustain this hyperemic and vasodilatory response, such as microvascular flow-mediated dilation and increases in heart rate (HR) and cardiac output (CO) (21, 29, 34, 46, 48). PLM is an easy to administer technique that relies on the relatively simple and robust measurement of LBF with no change in conduit artery diameter, rather than on small changes in vessel diameter. As such, PLM assesses microvascular function, rather than conduit vessel function, which may be more clinically relevant. PLM has been demonstrated to be a robust assessment of NO-mediated vascular function, where ~70% of the hyperemic and vasodilatory response, as measured by area under the curve (AUC), is NO-mediated (21, 34, 48). As such, PLM has been utilized to unveil alterations in NO-mediated vascular function and NO bioavailability with aging (20–22, 30, 47), physical activity (19), and disease (53, 54). Single PLM (sPLM), a recently developed PLM variant (51), has been documented to be a valid assessment of vascular function that is sensitive to differences in vascular function between patients with heart failure and age-matched controls (54). sPLM retains the features of PLM, while further simplifying the methodology by requiring only a single movement. Thus sPLM has the potential to further enhance the clinical and research utility of passive movement as an assessment of vascular function. It remains imperative, however, to assess the contribution of NO to the sPLM hyperemic and vasodilatory response.

Therefore, the purpose of this study was to assess the role of NO in the sPLM-induced hyperemic and vasodilatory response. Specifically, we utilized NO synthase (NOS) inhibition by an intra-arterial infusion of N^G-monomethyl-l-arginine (l-NMMA) to quantify the contribution of NO to the sPLM-induced hemodynamic and vasodilatory response. We hypothesized that inhibition of NOS would significantly attenuate the LBF and LVC responses induced by sPLM, documenting that sPLM is also an assessment of NO-mediated vascular function and NO bioavailability.

METHODS

Subjects

Nine healthy, recreationally active men (age 27 ± 5 yr, stature 179 ± 6 cm, body mass 79 ± 10 kg) volunteered to participate in the study and provided written informed consent before testing. All subjects were free from overt cardiovascular or metabolic disease, as determined from medical health history evaluations. The experimental procedures were approved by the Institutional Review Boards of the University of Utah and the Salt Lake City Veterans Affairs Medical Center and were conducted in accordance with the Declaration of Helsinki.

Experimental Design

Subjects arrived at the laboratory having abstained from food and caffeine on the day of testing and from vigorous activity for 24 h. In the experimental leg, the common femoral artery was catheterized using the Seldinger technique under sterile conditions. After catheterization, the subjects were allowed to rest for ~30 min before commencement of the experiments. The experimental protocols were conducted in an upright-seated posture and consisted of passive knee flexion-extension through a ~90 degree range of motion at 1 Hz for 60 s for PLM or 1 s for sPLM. For sPLM, the experimental leg was maintained fully extended after the single movement. An occlusion cuff (Hokanson, Bellevue, WA) placed just distal to the knee was inflated (≥250 mmHg) throughout the l-NMMA infusion and passive movement protocols to localize the drug effects to the thigh. After any hemodynamic alterations from the cuffing subsided (<5 min), resting baseline hemodynamic measurements were recorded for 60 s with the experimental leg supported at a 180 degree knee joint angle. Passive movement hemodynamic measurements were recorded for 60 s following the onset of passive knee flexion-extension. Subjects were instructed to remain relaxed during the protocols, and the contralateral leg was supported at a 180 degree knee joint angle and remained motionless during both protocols. To avoid the startle reflex and active resistance to the passive movement, subjects were informed that the movement would take place in ~1 min, but they were not informed of the exact timing of the movement to minimize the chance of an anticipatory response. A member of the research team moved the leg and verified the absence of muscle contraction. The passive movement protocols were each performed with and without l-NMMA infusion, and the subjects were allowed ≥20 min of rest between protocols. The testing order for PLM and sPLM was counterbalanced, and the drug infusion tests were performed last because of the potential lasting effects of l-NMMA.

l-NMMA Infusion

l-NMMA (Bachem, Bubendorf, Switzerland) was diluted in normal saline to a concentration 5 mg/ml from 250 mg of lyophilized powder. Anthropometrically determined thigh volume (27) was utilized to calculate drug dosing, and l-NMMA was infused at a dose of 0.24 mg·dl⁻¹·min⁻¹ for 5 min. This dosing was based on previous dose-response curves that demonstrated a plateau in the reduction of resting arm blood flow at this dose (56).

Measurements

Peripheral hemodynamics.

Common femoral artery blood velocity and diameter were measured in the passively moved leg distal to the inguinal ligament and proximal to the bifurcation of the superficial and deep femoral arteries using an ultrasound system (Logiq e9, General Electric Medical Systems, Milwaukee, WI) that was equipped with a linear array transducer probe operating at an imaging frequency of 9 MHz and a pulse wave frequency of 5.0 MHz. Based on real-time Duplex visualization of the ultrasound image and the pulse wave spectra, a sustained insonation angle of ≤60 degrees was ensured, and the sample volume was centered within the vessel and maximized according to vessel size.

Central hemodynamics.

Arterial pressure was measured continuously from within the common femoral artery with a pressure transducer (Transpac IV, Hospira, Lake Forest, IL) placed at the level of the catheter. HR, stroke volume (SV), and CO were measured with a Finometer (Finapres Medical Systems, Amsterdam, The Netherlands). This device calculates SV from beat-by-beat pressure waveforms assessed by photoplethysmography using the Modelflow method (Beatscope, Finapres Medical Systems), which, in combination with HR, has been documented to accurately estimate CO during various experimental protocols (4, 14, 15, 42, 50).

Data Acquisition and Analysis

Arterial pressure, HR, SV, and CO were obtained at 200 Hz via a data acquisition system (AcqKnowledge, Biopac Systems, Goleta, CA). Mean arterial pressure (MAP) was calculated as the mean of the femoral arterial catheter pressure recording. Common femoral artery diameter (D) was determined at a perpendicular angle along the central axis of the scanned area. LBF was calculated using D and intensity-weighted mean blood velocity (V_mean)

$$\text{LBF}={\text{V}}_{\text{mean}}\text{π}{\left(\frac{\text{D}}{\text{2}}\right)}^{\text{2}}\times \text{60}$$

LVC was then calculated as

$$\text{LVC}=\frac{\text{LBF}}{\text{MAP}}$$

Baseline values for all variables were determined as the average over the 60-s period before the onset of the passive movement. For each passive movement, all variables were analyzed second-by-second and smoothed using a 3-s rolling average. ΔLBF_peak and ΔLVC_peak were calculated as the peak minus the baseline. LBF_AUC and LVC_AUC were calculated, after normalization for baseline, as the summed response over 45 s for the sPLM and 60 s for the PLM. The initial rates of change in LBF and LBF were calculated over the first 3 s for sPLM and 7 s for PLM.

Statistical Analysis

Baseline and passive movement-induced hemodynamic measurements in control and l-NMMA conditions were compared within the sPLM and PLM protocols using Student’s paired t-tests or nonparametric Wilcoxon tests, where appropriate. The central hemodynamic responses in control and l-NMMA conditions were compared within the sPLM and PLM protocols using a two-way (time × condition) ANOVA with repeated measures. Within the sPLM and PLM protocols, the relationships between the control value and the l-NMMA-induced change for ΔLBF_peak, ΔLVC_peak, LBF_AUC, and LVC_AUC were assessed with Pearson’s product-moment correlation coefficients. Control ΔLBF_peak and ΔLVC_peak were compared between sPLM and PLM using Student’s paired t-tests. The relationships between the l-NMMA-induced changes in LBF_AUC and LVC_AUC between sPLM and PLM were assessed with Pearson’s product-moment correlation coefficients. Statistical significance was accepted at P ≤ 0.05. Values are means ± SD, except in Figs. 1, 2, and 4, were SE is used for clarity.

Fig. 1.

Peripheral hemodynamic responses to passive leg movement (PLM) and single PLM (sPLM) performed with and without intra-arterial infusion of N^G-monomethyl-l-arginine (l-NMMA): leg blood flow (LBF) and vascular conductance (LVC). Insets: area under the curve (AUC), calculated as the summed response after normalization for baseline. Values are means ± SE. †Significantly different from control.

Fig. 2.

Absolute peak changes in peripheral hemodynamic responses to passive leg movement (PLM) and single PLM (sPLM) performed with and without intra-arterial infusion of N^G-monomethyl-l-arginine (l-NMMA): peak changes in leg blood flow (ΔLBF_peak) and vascular conductance (ΔLVC_peak). Values are means ± SE. †Significantly different from control.

Fig. 4.

Central hemodynamic responses to passive leg movement (PLM) and single PLM (sPLM) performed with and without intra-arterial infusion of N^G-monomethyl-l-arginine (l-NMMA). MAP, mean arterial pressure; CO, cardiac output; SV, stroke volume; HR, heart rate. Values are means ± SE.

RESULTS

Peripheral and Central Hemodynamics During sPLM

Baseline sPLM peripheral and central hemodynamics are presented in Table 1, and the passive movement-induced peripheral hemodynamic responses are presented in Fig. 1. Baseline LBF and LVC were significantly reduced with l-NMMA (Table 1). l-NMMA significantly attenuated the sPLM LBF_AUC [57 ± 34 (l-NMMA) vs. 147 ± 63 (control) ml], ΔLBF_peak [492 ± 153 (l-NMMA) vs. 719 ± 238 (control) ml/min], LVC_AUC [0.5 ± 0.3 (l-NMMA) vs. 1.6 ± 0.9 (control) ml/mmHg], and ΔLVC_peak [4.7 ± 1.1 (l-NMMA) vs. 8.0 ± 3.0 (control) ml·min⁻¹·mmHg⁻¹] (Figs. 1 and 2). The control response was significantly related to the magnitude of the l-NMMA-induced change for the sPLM ΔLBF_peak (r = 0.94), LBF_AUC (r = 0.85), ΔLVC_peak (r = 0.94), and LVC_AUC (r = 0.95) (Fig. 3). l-NMMA significantly altered the initial rate of change in sPLM LBF [66 ± 79 (l-NMMA) vs. 129 ± 74 (control) ml·min⁻¹·s⁻¹] and LVC [0.6 ± 0.7 (l-NMMA) vs. 1.3 ± 0.8 (control) ml·min⁻¹·mmHg⁻¹·s⁻¹] (Fig. 1). The CO, HR, and SV responses changed significantly from baseline during sPLM in the l-NMMA and control conditions, but there were no significant differences in these responses between conditions (Fig. 4). The MAP response during sPLM was significantly higher in the l-NMMA than control condition, but MAP did not change significantly from baseline in either condition (Fig. 4).

Table 1.

Baseline peripheral and central hemodynamics before PLM and sPLM protocols with and without intra-arterial infusion of l-NMMA

	sPLM		PLM
	Control	l-NMMA	Control	l-NMMA
LBF, ml/min	379 ± 277	269 ± 101†	368 ± 201	253 ± 88†
LVC, ml·min−1·mHg−1	3.7 ± 2.7	2.6 ± 1.0†	3.6 ± 1.9	2.4 ± 0.9†
MAP, mmHg	103 ± 11	104 ± 11	104 ± 11	106 ± 12
CO, l/min	5.9 ± 1.0	5.8 ± 1.1	6.0 ± 1.5	5.9 ± 1.4
SV, ml/beat	94 ± 25	98 ± 24	100 ± 34	100 ± 24
HR, beats/min	63 ± 13	63 ± 13	64 ± 13	62 ± 12

Values are means ± SD. PLM, passive leg movement; sPLM, single PLM; l-NMMA, N^G-monomethyl-l-arginine; LBF, leg blood flow; LVC, leg vascular conductance; MAP, mean arterial pressure; CO, cardiac output; SV, stroke volume; HR, heart rate.

^†Significantly different from control within the same protocol.

Fig. 3.

Relationship between peripheral hemodynamic responses and the contribution of nitric oxide (NO) during passive leg movement (PLM) and single PLM (sPLM) performed with and without intra-arterial infusion of N^G-monomethyl-l-arginine (l-NMMA): leg vascular conductance (LVC) peak changes (ΔLVC_peak) and area under the curve (LVC_AUC).

Peripheral and Central Hemodynamics During PLM

Baseline peripheral and central hemodynamics for PLM are presented in Table 1, and the peripheral hemodynamic responses to the passive movement are presented in Fig. 1. l-NMMA significantly reduced baseline LBF and LVC (Table 1). For PLM, l-NMMA significantly attenuated LBF_AUC [172 ± 128 (l-NMMA) vs. 490 ± 221 (control) ml], ΔLBF_peak [777 ± 247 (l-NMMA) vs. 1,207 ± 382 (control) ml/min], LVC_AUC [1.7 ± 1.4 (l-NMMA) vs. 4.6 ± 2.1 (control) ml/mmHg], and ΔLVC_peak [7.8 ± 3.1 (l-NMMA) vs. 12.1 ± 4.6 (control) ml·min⁻¹·mmHg⁻¹] (Figs. 1 and 2). ΔLBF_peak and ΔLVC_peak were significantly greater for PLM than sPLM. The magnitude of the l-NMMA-induced change was significantly correlated with the control values during PLM for ΔLBF_peak (r = 0.78), LBF_AUC (r = 0.92), ΔLVC_peak (r = 0.74), and LBF_AUC (r = 0.82) (Fig. 3). The PLM initial rate of change was significantly attenuated for LBF [87 ± 32 (l-NMMA) vs. 140 ± 50 (control) ml·min⁻¹·s⁻¹] and LVC [0.9 ± 0.3 (l-NMMA) vs. 1.4 ± 0.6 (control) ml·min⁻¹·mmHg⁻¹·s⁻¹] (Fig. 1). The CO, HR, SV, and MAP responses during PLM changed significantly from baseline in l-NMMA and control conditions, but there were no significant differences in these responses between conditions (Fig. 4).

DISCUSSION

This study sought to determine the contribution of NO to the sPLM-induced hyperemic and vasodilatory response by utilizing an intra-arterial infusion of l-NMMA to inhibit NOS. Consistent with our hypothesis, l-NMMA significantly attenuated the sPLM LBF and LVC responses by ~65% from control. Further evidence that the magnitudes of the sPLM hemodynamic responses are strongly related to NO bioavailability was demonstrated by the direct relationships between the magnitudes of the control responses and the magnitudes of the l-NMMA-induced changes. Collectively, the current data establish that the robust hyperemic and vasodilatory response induced by sPLM is predominantly NO-mediated. The uncomplicated methodology and NO dependency of sPLM make this a promising, simple, in vivo assessment of NO-mediated vascular function and NO bioavailability.

Contribution of NO to sPLM Hemodynamic Responses

Accruing evidence indicates that sPLM is able to assess vascular function with a simple, streamlined methodology (51, 54). Indeed, with a single movement, vascular function could be characterized (51) and differences between patients with heart failure and aged-matched controls could be distinguished (54). However, the vital question regarding the extent to which sPLM assessed NO-mediated vascular function remained unknown. This study demonstrated that inhibition of NOS with l-NMMA greatly attenuated the magnitude and duration of the sPLM-induced LBF and LVC responses. Quantitatively, ~65% of the sPLM-induced hyperemic and vasodilatory responses were documented to be NO-mediated (Fig. 1). Further evidence that the magnitudes of the sPLM hemodynamic responses are strongly related to NO bioavailability was demonstrated by the direct relationships between the magnitudes of the control responses and the magnitudes of the l-NMMA-induced changes (Fig. 3). The initial rates of change in sPLM LBF and LVC responses were significantly decreased by l-NMMA, demonstrating an NO-dependent hyperemia and vasodilation at the onset of sPLM (Fig. 1). Interestingly, transient attenuated peaks in LBF and LVC were induced by sPLM, despite the inhibition of NOS. These findings reveal that the remaining peaks in LBF and LVC are likely mediated by mechanisms other than NO, which is consistent with previous reports that NO, prostaglandins, and neural input are not requisite for immediate vasodilation (5, 6, 18, 40, 41). Such mechanisms as mechanical deformation-induced changes in ion channels and release of non-endothelial-derived vasodilators may be mediating these early sPLM peripheral hemodynamic responses (10–12, 23, 26, 31, 32). Overall, this study establishes that sPLM induces a peripheral hemodynamic response that is predominantly NO-mediated, achieved by a simple, streamlined methodology.

An original impetus for the development of sPLM was the minimization of any central hemodynamic responses that could potentially confound the assessment of vascular function. Indeed, it has been suggested that the central hemodynamic responses evoked by passive movement may contribute to the increase in blood flow (29, 36, 46, 55). Previously, in healthy young individuals, sPLM did not evoke significant central hemodynamic responses (51). However, in the current study, there were significant, albeit relatively minor, changes in SV, HR, and CO following sPLM (Fig. 4). This discrepancy is likely the result of the nearly twofold greater hemodynamic responses in the current study than in the previous study (51). Importantly, this previous study confirmed that the central hemodynamic responses evoked by sPLM do not contribute to the peripheral hemodynamic responses, as expected from Ohm’s law. Rather, the peripheral hemodynamic responses appear to be dependent on pressure and resistance changes within the leg vasculature (51). In the current study the central and peripheral hemodynamic responses were dissociated by l-NMMA, as LBF and LVC responses were greatly attenuated with no or minimal changes in central hemodynamic variables. These data collectively suggest that the central hemodynamic responses do not dictate the peripheral hemodynamic responses during sPLM.

Contribution of NO to PLM Hemodynamic Responses

The PLM-induced hyperemic and vasodilatory response was previously demonstrated to be predominantly (~70%) NO-mediated (21, 34, 48). This was confirmed in the current study, as l-NMMA reduced the PLM response by ~70% from control. Beyond the important corroboration of previous findings, the current study serves as an important experimental control by confirming that the subjects were sensitive to alterations in NO bioavailability with a previously established methodology. The current findings also confirm that a component of the transient peaks in LBF and LVC induced by PLM are NO-independent, as a portion of this response was still induced in the presence of l-NMMA. However, the initial rates of increase in LBF and LVC were significantly attenuated with NOS inhibition (Fig. 1). It has been documented that these initial rates of increase were not influenced by NOS inhibition when PLM was conducted in the supine posture (21, 48). However, when PLM was performed in the upright posture, these initial rates of change were blunted with l-NMMA (21). Additionally, the comparison of the magnitude of control responses with the l-NMMA-induced changes revealed that the magnitude of the PLM peripheral hemodynamic responses was strongly related to NO bioavailability (Fig. 3). Overall, the current findings regarding the PLM-induced hyperemic and vasodilatory response provide an important experimental control, while corroborating that the PLM-induced LBF and LVC responses are predominantly NO-mediated.

There are robust central hemodynamic responses evoked by PLM (21, 29, 36, 48, 51, 54, 55). Consistent with previous reports (21, 48), l-NMMA did not significantly alter the central hemodynamic responses evoked by PLM (Fig. 4). The large reduction in the PLM-induced LBF and LVC responses with l-NMMA, despite no central hemodynamic effect, further supports the concept that the central responses play a minimal role in the peripheral hemodynamic responses. Interestingly, the magnitude of the hemodynamic responses in the current study are much larger than previously reported (21, 48, 51). It appears that the greater magnitudes of the central hemodynamic responses evoked by PLM were likely due to differences in joint and muscle mechanoreceptors (1, 2, 24, 46), and not the baroreflex, as the change in MAP (~5 mmHg) was similar to that in previous studies. Thus the current findings further suggest that the primary drive to increase the central hemodynamic variables is afferent feedback, rather than the baroreflex (46, 51).

Insight from Comparison of sPLM and PLM Hemodynamic Responses

Interestingly, the same general effect of l-NMMA was apparent in sPLM and PLM protocols in this study. Initially, l-NMMA decreased resting blood flow similarly by ~30% for both protocols, which is consistent with previous reports of the contribution of NO to resting blood flow (21, 43, 48, 56). Therefore, from this similar starting point, the effect of l-NMMA on the sPLM and PLM responses was strikingly similar, ~65–70% attenuation for both protocols (Figs. 1 and 2). The magnitude of the NO contribution to the sPLM and PLM responses was correlated, suggesting that the hyperemic and vasodilatory responses are predominantly NO-mediated and, as such, have a common underlying mechanism. Intriguingly, the mechanistic role of NO in the sPLM and PLM hyperemic and vasodilatory responses reported in the current study is similar to that reported for a single muscle contraction in young, healthy subjects (7). Therefore, it is possible that greater insight regarding the relationship between vascular function and vasodilation in active muscle may be gained by considering both types of protocols. However, until future studies investigate a mechanistic link between these protocols, combined inferences from these protocols should be made cautiously because of the potential mechanistic differences responsible for vasodilation in these two scenarios. Previously, the peak peripheral hemodynamic responses (i.e., ΔLBF_peak and ΔLVC_peak) were, somewhat unexpectedly, not significantly different between sPLM and PLM (51). In the current study, we noticed unanticipated potential differences in the peak responses between the two protocols, which were indeed statistically different. Importantly, NO was also documented to contribute ~65% to the sPLM peripheral hemodynamic responses, despite the smaller magnitude of these responses. Furthermore, the initial rates of change in LBF and LVC for both sPLM and PLM were NO-dependent (Fig. 1). The control sPLM and PLM peripheral hemodynamic responses were directly related to the contribution of NO, as assessed by the change induced by NOS blockade (Fig. 3). These relationships provide further evidence that the hyperemic and vasodilatory responses to sPLM and PLM are reliant on and, therefore, indicative of NO-mediated vascular function.

Experimental Considerations

This study has a relatively small sample size of nine subjects; however, this sample size was likely sufficient, given the robust significant differences accompanied by high statistical power and a large effect size. In addition, because of the invasive nature of the experimental protocol, there are some ethical concerns in terms of increasing the number of subjects when the findings are clear. In this study the contribution of NO to the sPLM-induced hyperemic and vasodilatory response was investigated in young, healthy men. It remains to be determined if the predominant role of NO in the sPLM-induced hyperemic and vasodilatory response holds true for women. Furthermore, future investigations should assess the sPLM-induced hyperemic and vasodilatory response in conditions of altered NO bioavailability, such as age and disease. Because of the likely long-lasting effects of l-NMMA, the control trials were always performed in advance of the l-NMMA infusion trials. Thus the potential for an ordering effect cannot be ruled out. However, because of the magnitude and clarity of the results, this is unlikely to have had an impact on the current findings.

Conclusion

The current study utilized an intra-arterial infusion of the NOS inhibitor l-NMMA to quantify the contribution of NO to the hyperemic and vasodilatory response induced by sPLM. This approach documented that the robust hyperemic and vasodilatory response induced by sPLM is predominantly (~65%) NO-mediated. It was also revealed that the sPLM peripheral hemodynamic responses were directly related to NO bioavailability, with greater responses having a greater NO contribution. Collectively, the uncomplicated methodology and NO dependency of sPLM make this a simple, streamlined, in vivo assessment of NO-mediated vascular function and NO bioavailability.

GRANTS

This study was supported by National Heart, Lung, and Blood Institute Grant HL-091830; Veterans Affairs Rehabilitation Research and Development Merit Awards E6910-R and E1697-R, Spire Award E1433-P, Senior Research Career Scientist Award E9275-L, and Career Development Award IK2RX001215; and American Heart Association Grant 14SDG18850039.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.M.B., J.D.T., J.R.G., O.S.K., A.C.K., J.R.H., A.D.N., D.E.M., J.E.J., A.D.B., and R.S.R. conceived and designed research; R.M.B., J.D.T., J.R.G., O.S.K., A.C.K., J.R.H., A.D.N., D.E.M., J.E.J., A.D.B., and R.S.R. performed experiments; R.M.B., J.D.T., J.R.G., O.S.K., A.C.K., J.R.H., A.D.N., D.E.M., J.E.J., A.D.B., and R.S.R. analyzed data; R.M.B., J.D.T., J.R.G., O.S.K., A.C.K., J.R.H., A.D.N., D.E.M., J.E.J., A.D.B., and R.S.R. interpreted results of experiments; R.M.B., J.D.T., J.R.G., O.S.K., A.C.K., J.R.H., A.D.N., D.E.M., J.E.J., A.D.B., and R.S.R. prepared figures; R.M.B., J.D.T., J.R.G., O.S.K., A.C.K., J.R.H., A.D.N., D.E.M., J.E.J., A.D.B., and R.S.R. drafted manuscript; R.M.B., J.D.T., J.R.G., O.S.K., A.C.K., J.R.H., A.D.N., D.E.M., J.E.J., A.D.B., and R.S.R. edited and revised manuscript; R.M.B., J.D.T., J.R.G., O.S.K., A.C.K., J.R.H., A.D.N., D.E.M., J.E.J., A.D.B., and R.S.R. approved final version of manuscript.