ATP and acetylcholine interact to modulate vascular tone and α₁-adrenergic vasoconstriction in humans

Abstract

The vascular endothelium senses and integrates numerous inputs to regulate vascular tone. Recent evidence reveals complex signal processing within the endothelium, yet little is known about how endothelium-dependent stimuli interact to regulate blood flow. We tested the hypothesis that combined stimulation of the endothelium with adenosine triphosphate (ATP) and acetylcholine (ACh) elicits greater vasodilation and attenuates α₁-adrenergic vasoconstriction compared with combination of ATP or ACh with the endothelium-independent dilator sodium nitroprusside (SNP). We assessed forearm vascular conductance (FVC) in young adults (6 women, 7 men) during local intra-arterial infusion of ATP, ACh, or SNP alone and in the following combinations: ATP + ACh, SNP + ACh, and ATP + SNP, wherein the second dilator was coinfused after attaining steady state with the first dilator. By design, each dilator evoked a similar response when infused separately (ΔFVC, ATP: 48 ± 4; ACh: 57 ± 6; SNP: 53 ± 6 mL·min⁻¹·100 mmHg⁻¹; P ≥ 0.62). Combined infusion of the endothelium-dependent dilators evoked greater vasodilation than combination of either dilator with SNP (ΔFVC from first dilator, ATP + ACh: 45 ± 9 vs. SNP + ACh: 18 ± 7 and ATP + SNP: 26 ± 4 mL·min⁻¹·100 mmHg⁻¹, P < 0.05). Phenylephrine was subsequently infused to evaluate α₁-adrenergic vasoconstriction. Phenylephrine elicited less vasoconstriction during infusion of ATP or ACh versus SNP (ΔFVC, −25 ± 3 and −29 ± 4 vs. −48 ± 3%; P < 0.05). The vasoconstrictor response to phenylephrine was further diminished during combined infusion of ATP + ACh (−13 ± 3%; P < 0.05 vs. ATP or ACh alone) and was less than that observed when either dilator was combined with SNP (SNP + ACh: −26 ± 3%; ATP + SNP: −31 ± 4%; both P < 0.05 vs. ATP + ACh). We conclude that endothelium-dependent agonists interact to elicit vasodilation and limit α₁-adrenergic vasoconstriction in humans.

NEW & NOTEWORTHY The results of this study highlight the vascular endothelium as a critical site for integration of vasomotor signals in humans. To our knowledge, this is the first study to demonstrate that combined stimulation of the endothelium with ATP and ACh results in enhanced vasodilation compared with combination of either ATP or ACh with an endothelium-independent dilator. Furthermore, we show that ATP and ACh interact to modulate α₁-adrenergic vasoconstriction in human skeletal muscle in vivo.

INTRODUCTION

Vascular tone is regulated in a highly specific manner that relies on integration of numerous vasoactive molecules and signaling mechanisms. The vascular endothelium constantly monitors physiological cues and processes multiple chemical and mechanical stimuli arising from the bloodstream and surrounding tissues to regulate vessel diameter. Precisely how the endothelium integrates simultaneous sensory inputs to govern an appropriate response is not clear; however, it appears to involve complex interactions and communication between neighboring endothelial cells (ECs) and underlying vascular smooth muscle cells (VSMCs) (1, 2). Investigating how diverse stimuli interact to regulate vascular tone has important implications for understanding blood flow regulation and may inform future strategies to improve tissue oxygen delivery. Interactions between stimuli are particularly relevant during physiological stressors such as hypoxia or exercise, which induce concomitant changes in vasodilatory molecules, sympathetic nerve activity, and mechanical forces acting on the vasculature.

Activation of the endothelium by physiological stimuli is largely mediated by fluctuations in cytosolic Ca²⁺ (3). Vasodilatory molecules such as adenosine triphosphate (ATP) and acetylcholine (ACh) stimulate an increase in cytosolic Ca²⁺ when they bind to their respective purinergic (P_2Y) and muscarinic (M₃) receptors on the endothelium (4, 5). Likewise, shear forces exerted by blood flowing along the endothelium initiate cytosolic Ca²⁺ signals (6, 7). In addition to stimulating the production and release of vasodilatory autacoids such as nitric oxide and prostaglandins, elevated Ca²⁺ within ECs stimulates Ca²⁺-activated K⁺ (K_Ca) channels (8). The resulting efflux of K⁺ results in endothelium-derived hyperpolarization, which is conducted to adjacent ECs and spreads to underlying VSMCs to promote relaxation (9).

Recent evidence suggests that activation of the endothelium enhances its sensitivity to further stimulation. Using freshly isolated arteries from rats, Lee et al. (10) recently determined that muscarinic and purinergic receptors are heterogeneously distributed among ECs such that clusters of ECs detect each agonist and communicate the information with neighboring cells to coordinate an appropriate response. When a combination of ATP and ACh is applied to the endothelium, a synergistic rise in intracellular Ca²⁺ occurs with distinct Ca²⁺ signals that are not observed when either dilator is applied in isolation. This suggests that ATP and ACh influence endothelial signaling in response to each other, yet it is unclear how the augmented Ca²⁺ response impacts vasodilation, as diameter responses were not assessed. A similar interaction has been observed in the cutaneous microcirculation, where administration of ATP enhances subsequent cholinergic vasodilation (11). However, it is unclear whether this reflects a nonspecific effect of baseline vasodilation, as the response was not assessed during administration of a control dilator. ATP activates K_IR channels (12, 13), which have been shown to amplify electrical signaling in ECs in response to cholinergic vasodilation (14); thus, circulating ATP could act on the endothelium to influence its sensitivity to other stimuli. Indeed, we recently demonstrated in humans that vasodilation to ACh is selectively amplified in contracting skeletal muscle (15) when circulating ATP is elevated (16, 17). Collectively, these observations highlight the ability of the endothelium to process complex interactions between multiple signals to regulate vascular tone.

In addition to sensing circulating vasodilators, the endothelium receives input from and communicates with VSMCs to regulate vascular tone. This is particularly important during physiological stressors such as exercise, when elevated sympathetic nerve activity and norepinephrine release stimulate α-adrenergic receptors on VSMCs to elicit vasoconstriction even within the vasculature of contracting skeletal muscle. Although muscle sympathetic nerve activity is increased during exercise, α-adrenergic vasoconstriction is attenuated within resistance vessels of contracting muscle, which facilitates redistribution of blood flow to active muscle (18–20). This phenomenon of “functional sympatholysis” is essential to ensure adequate delivery of oxygen to the working muscle despite elevated sympathetic outflow (21, 22). The mechanisms that attenuate postjunctional α-adrenergic signaling in the microcirculation of exercising muscle are unclear, though accumulating evidence suggests that the endothelium may play a central role in limiting sympathetic vasoconstriction (23). In rodents, endothelium-dependent hyperpolarization counteracts sympathetic vasoconstriction (24), and in humans, we have shown that activating the endothelium with either ATP or ACh enhances functional sympatholysis (25, 26). In contrast, vasodilators that bypass the endothelium and act directly on vascular smooth muscle, such as sodium nitroprusside (SNP), do not enhance functional sympatholysis (25). Thus, the endothelium appears to play a key role in limiting sympathetic vasoconstriction, yet it is unknown whether endothelium-dependent stimuli interact to modulate α-adrenergic vasoconstriction.

Therefore, in the present study, we aimed to determine how the endothelium-dependent agonists ATP and ACh interact to regulate vascular tone in humans. To distinguish the role of the endothelium from general effects of hyperemia, we used the endothelium-independent dilator SNP as a comparison to control for nonspecific vasodilation. We hypothesized that the combined actions of ATP and ACh would elicit greater vasodilation and limit α₁-adrenergic vasoconstriction compared with the combined action of either endothelium-dependent dilator with SNP.

METHODS

Participants and Ethical Approval

All experimental protocols were approved by the Institutional Review Board at Colorado State University (Protocol No. 14-5392H) and performed in accordance with the Declaration of Helsinki, except for registration in a database. After providing written, informed consent, 13 healthy volunteers (6 women, 7 men; 24 ± 0.7 yr old, 26 ± 2% body fat; means ± SE) completed the study. Participants were free from cardiovascular and metabolic diseases and were not taking medication aside from oral contraceptives (3 participants).

Instrumentation

Participants arrived at the laboratory at 7:00 AM following an overnight fast and lay supine with the nondominant arm abducted 90° resting on a table slightly above heart level throughout the study visit. After local anesthesia (1% lidocaine), a 20-g, 7-cm catheter was inserted in the brachial artery using sterile technique. The catheter was continuously flushed with heparinized saline and connected to a three-way port for infusion of vasoactive drugs. Mean arterial pressure (MAP) was determined using a pressure transducer connected to the catheter, and heart rate (HR) was assessed with a three-lead electrocardiogram (Cardiocap 5). Studies were conducted in a temperature-controlled room with a fan directed at the experimental arm to minimize skin blood flow (27).

Forearm Blood Flow

Forearm blood flow was determined using Doppler ultrasound as previously described by our laboratory (25, 28). Brachial artery blood velocity and diameter were measured proximal to the catheter using a 12-MHz linear array ultrasound probe (Vivid7, General Electric, Milwaukee, WI). Velocity was measured with a probe insonation angle <60° at a frequency of 5 MHz, and a spectral analyzer (Multigon 500 M, Multigon Industries, Mount Vernon, NY) was used to determine mean blood velocity (MBV) as the weighted mean of the spectrum of Doppler shift frequencies. Brachial artery diameter was measured at end-diastole at 30-s intervals throughout each trial from images recorded on a DVD. Forearm blood flow (FBF) was calculated as FBF = MBV × π × (diameter ÷ 2)² and expressed in mL/min, and forearm vascular conductance (FVC) was calculated as FVC = FBF ÷ MAP × 100 and expressed in mL/min/100 mmHg (25).

Endothelium-Dependent and -Independent Vasodilation

All vasoactive drugs were infused through the arterial catheter at low inflow rates (∼2 mL/min) to elicit local effects within the experimental forearm without affecting central hemodynamics. Doses were adjusted according to each participant’s forearm volume (FAV) determined by dual X-ray absorptiometry (29, 30).

Adenosine triphosphate (ATP; A7699, Sigma Aldrich, St. Louis, MO) and acetylcholine (ACh; Miochol-E, Novartis, Basel, Switzerland) were used to stimulate endothelium-dependent vasodilation, and sodium nitroprusside (SNP; Hospira, Lake Forest, IL) was used to evoke vascular smooth muscle relaxation that occurs independently of the endothelium. Each drug was infused at a low dose with the intention of elevating FBF to ∼3 times baseline FBF (15). The doses were adjusted to match the average, steady-state response to each dilator as best as possible (25, 29). ATP was infused at 1.28 μg/dL FAV/min in all participants, and the doses of ACh and SNP were adjusted to elicit similar steady-state vasodilation (ACh, 1.48 ± 0.01, range: 1.36–1.50 μg/dL FAV/min; SNP, 0.28 ± 0.02, range: 0.18–0.35 μg/dL FAV/min). The same dose of each dilator was used for the single-dilator and combination trials (described under Experimental Protocol below) within each participant.

Sympathetic α₁-Adrenergic Vasoconstriction

To assess responsiveness to α-adrenergic receptor stimulation, the α₁-agonist phenylephrine (PE) was infused via the arterial catheter at 0.125 μg/dL FAV/min. The rate of PE was adjusted according to FBF to carefully control the vasoconstrictor stimulus, as described previously (25, 31). Briefly, the infusion rate of PE was adjusted to account for the extent of forearm hyperemia, which was assessed as the ratio of FBF during dilator infusion compared with baseline. For example, a threefold increase in FBF upon dilator infusion would correspond to a threefold increase in the PE infusion rate in an effort to normalize the arterial concentration of PE across trials.

Experimental Protocol

Figure 1 illustrates the general experimental protocol (A) and timeline for each trial (B). Vasodilation and α₁-adrenergic vasoconstrictor responsiveness were assessed during infusion of endothelium-dependent (ATP and ACh) and endothelium-independent (SNP) dilators alone and in combination. Initially, the response to each dilator was evaluated separately in randomized order; then, the following combinations were tested in randomized order: 1) ATP + ACh, 2) SNP + ACh, and 3) ATP + SNP.

Figure 1.

Experimental protocol. A: following brachial artery catheterization, vasodilation and α₁-adrenergic vasoconstrictor responsiveness were assessed during infusion of endothelium-dependent (ATP, ACh) and endothelium-independent (SNP) dilators alone and in combination in trials separated by ≥10 min. B: when each dilator was infused separately, baseline values were recorded, then the dilator was infused and steady-state (SS) measurements were obtained at 4 min. PE was infused at the end of each trial, and the steady-state vasoconstrictor response was measured at 2 min of PE infusion (SS_PE). In the dilator combination trials, the first dilator was infused for 4 min to achieve steady-state vasodilation (SS₁), then the second dilator was coinfused and steady-state (SS₂) measurements were obtained after 4 min of combined infusion. PE was infused for 2 min at the end of the trial. ACh, acetylcholine, ATP, adenosine triphosphate; PE, phenylephrine; SNP, sodium nitroprusside.

Single-dilator trials.

In each trial, baseline hemodynamics were assessed during 2 min of saline infusion, then the dilator was infused and steady-state hemodynamics were assessed at 4 min of dilator infusion. PE was infused for 2 min at the end of each trial, and hemodynamics were reassessed during the final 30 s to determine the steady-state vasoconstrictor response. The dose of PE was adjusted according to FBF to match the concentration of PE in the blood across trials, as described previously by our laboratory (25, 29, 32).

Combination trials.

Baseline hemodynamics were recorded during 2 min of saline infusion before each trial, after which the first dilator was infused for 4 min to obtain steady-state values. The second dilator was then coinfused, and the steady-state response was assessed at 4 min of combined dilator infusion (see Fig. 1B). PE was infused for 2 min at the end of each trial, and hemodynamics were reassessed during the final 30 s to calculate PE-mediated vasoconstriction (see Fig. 1B).

High-SNP trial.

Because we hypothesized that vasodilation would be augmented during combination of two endothelium-dependent dilators (ATP + ACh) compared with combination of either dilator with SNP (ATP + SNP and SNP + ACh), an additional trial was performed in which a high dose of SNP (2.22 ± 1.5 μg/dL FAV/min) was used to elicit vasodilation comparable with that observed in the ATP + ACh trial. The purpose of this trial was to serve as a “high-flow” control condition to better compare vasoconstrictor responsiveness to PE (25, 31).

Data Acquisition and Analysis

Data were collected at 250 Hz and stored on a computer for analysis with signal processing software (WinDaq; DATAQ Instruments, Akron, OH). Baseline hemodynamics reflect 30-s average values immediately preceding the start of dilator infusions. Steady-state hemodynamics reflect 30-s average values obtained between minutes 3 and 4 of each dilator infusion. Vasodilatory responses were expressed as the absolute change in FVC from baseline (single-dilator trials) or the first dilator (combination trials). Hemodynamics during PE reflect 30-s average values at the end of 2 min of PE infusion. The vasoconstrictor response to PE was calculated as the percent change in FVC from steady-state vasodilator infusion (before PE) to the end of PE infusion: (FVC_PE – FVC_Pre−PE) ÷ FVC_Pre−PE × 100 (Fig. 1B) (25, 27, 29). We chose to evaluate the percent reduction in FVC because this is the most appropriate way to compare vasoconstrictor responses across conditions where vascular tone differs (25, 33), as FVC was greater before PE in the ATP + ACh and high-SNP trials compared with other combinations.

Statistics

All values are presented as means ± SE. Baseline hemodynamics before each trial were compared using one-way repeated-measures ANOVA. Steady-state responses to each dilator were compared using one-way repeated-measures ANOVA, and hemodynamics before and after PE were compared using two-way repeated-measures ANOVA (drug × condition: pre-PE vs. post-PE). α₁-adrenergic vasoconstrictor responses were compared with one-way repeated-measures ANOVA. Tukey’s post hoc testing was performed when appropriate based on significant main effects or interactions between factors. All comparisons were performed using R statistical software, and significance was evaluated as P < 0.05. FBF and FVC values for one participant were consistently ≥2 standard deviations below the mean; therefore, we excluded data from this participant from analysis of vasodilatory parameters. Despite low overall FVC values corresponding with a small FAV (572 mL), vasoconstrictor responses to PE were within the normal range (i.e., not statistical outliers) in this participant; thus, we included her data in the analysis for α₁-adrenergic vasoconstriction.

RESULTS

Vasodilation

Resting hemodynamics were similar before the start of all trials (Fig. 2, A and B). As intended, each dilator evoked similar forearm vasodilation when infused individually (Fig. 2, A and C). In the combination trials, steady-state hemodynamics were similar during administration of the first dilator (Fig. 2B). When the second dilator was added, combination of the two endothelium-dependent dilators (ATP + ACh) resulted in greater forearm vasodilation than combination of either dilator with SNP (Fig. 2, B and D).

Figure 2.

Vasodilatory responses are greater during combined infusion of endothelium-dependent agonists. Each dilator evoked similar vasodilation when administered separately (A and C). Combination of the endothelium-dependent dilators ATP + ACh resulted in greater vasodilation than combination of either dilator with SNP (B and D). Data are presented as means ± SE for n = 12 participants (5 women, 7 men) and were analyzed using one-way ANOVA. ACh, acetylcholine; ATP, adenosine triphosphate; SNP, sodium nitroprusside. *P < 0.05.

Sympathetic α₁-Adrenergic Vasoconstriction

The vasoconstrictor response to PE during the individual dilator trials is presented in Fig. 3, A and C. Steady-state FVC was similar during infusion of each dilator before administration of PE (Fig. 3A). PE significantly reduced FVC in each dilator condition; however, the vasoconstrictor response was less during ATP and ACh compared with SNP (Fig. 3, A and C).

Figure 3.

α₁-Adrenergic vasoconstriction is attenuated during combined infusion of endothelium-dependent agonists. When administered separately, ATP and ACh limited vasoconstriction to PE (α₁-agonist) compared with SNP (A and C). Combination of the endothelium-dependent dilators limited vasoconstriction to PE compared with other combinations or high SNP (B and D). α₁-Adrenergic vasoconstrictor responsiveness was further reduced during combination of ATP + ACh (D) than when either dilator was administered alone (C). Data are presented as means ± SE for n = 13 participants (6 women, 7 men) and were analyzed using one-way and two-way ANOVA. ACh, acetylcholine; ATP, adenosine triphosphate; PE, phenylephrine; SNP, sodium nitroprusside. *P < 0.05 vs. all other single dilators or combinations at the same time point; †P < 0.05 vs. high SNP.

The vasoconstrictor response to PE during the combination dilator trials is presented in Fig. 3, B and D. Administration of PE reduced FVC in each dilator condition; however, the vasoconstrictor response was less during combined infusion of the endothelium-dependent dilators ATP + ACh compared with SNP + ACh, ATP + SNP, and high SNP (Fig. 3, B and D). Interestingly, combined infusion of ATP + ACh further limited PE-mediated vasoconstriction compared with either endothelium-dependent dilator alone (Fig. 3, C and D; P = 0.02 vs. ATP alone; P < 0.01 vs. ACh alone) or in combination with SNP (Fig. 3, B and D).

DISCUSSION

The present study was designed on the premise that the vascular endothelium simultaneously processes numerous stimuli and integrates information in a complex manner to regulate vasomotor tone. We tested the hypothesis that stimulating the endothelium with a combination of ATP and ACh would 1) elicit greater vasodilation and 2) limit α₁-adrenergic vasoconstriction compared with the combined action of either dilator with SNP, which bypasses the endothelium to act directly on vascular smooth muscle. To our knowledge, this is the first study to demonstrate that endothelium-dependent agonists combine to enhance vasodilation and attenuate α₁-adrenergic vasoconstriction compared with combination of either agonist with an endothelium-independent vasodilator. These findings highlight the unique capacity of the endothelium to synthesize inputs from multiple vasoactive stimuli and regulate vascular tone in human skeletal muscle.

The interactions between vasoactive stimuli reported in this study have important implications for blood flow regulation. Along with previous studies, our results support the notion that stimulating the endothelium alters the response to subsequent vasodilatory signals (10, 15, 34). This may be particularly important during stressors such as exercise or hypoxia when a number of stimuli act in concert to regulate skeletal muscle blood flow (35, 36). Moreover, the observation that endothelium-dependent agonists combine to limit α₁-adrenergic vasoconstriction provides insight into the mechanisms of functional sympatholysis. The microcirculation within contracting skeletal muscle is exposed to a number of endothelium-dependent stimuli that may combine to attenuate sympathetic vasoconstriction in a manner similar to that observed in the present study (25, 37, 38). How interactions between endogenous vasoactive stimuli regulate vascular tone under physiological conditions is a topic for future investigations.

Vasodilation

Recent ex vivo evidence revealed a synergistic effect of ATP and ACh receptor activation on Ca²⁺ signaling within the endothelium (10), yet it was previously unknown whether interactions between these two endothelium-dependent dilators occur in vivo and affect vascular tone in human skeletal muscle. Therefore, we determined the interaction between ATP and ACh in regulating vasomotor tone when infused intra-arterially. The results demonstrate that combination of ATP + ACh causes vasodilation that is augmented compared with combination of either dilator with SNP (Fig. 2, B and D). These findings suggest that ATP and ACh interact to influence the magnitude of vasodilation, which adds to the growing body of evidence that activating the endothelium alters the response to subsequent vasodilatory stimuli.

Sympathetic α₁-Adrenergic Vasoconstriction

Accumulating evidence suggests that the endothelium merges inputs from vasodilatory stimuli and the sympathetic nervous system to regulate vascular tone (2). Myoendothelial communication has important implications for blood flow distribution during exercise, when elevated sympathetic nerve activity limits blood flow to inactive tissues to preserve arterial pressure. Despite increased sympathetic outflow, α-adrenergic vasoconstriction is reduced within the vasculature of contracting skeletal muscle (18), which facilitates redistribution of blood flow to metabolically active tissue. Although the mechanisms underlying functional sympatholysis are unknown, hyperpolarization arising from the endothelium limits sympathetic vasoconstriction in rodents (24). Infusion of ATP, which evokes endothelium-dependent hyperpolarization (39, 40), reduces the vasoconstrictor response to α-adrenergic agonists in humans (29). Furthermore, we recently demonstrated that stimulating the endothelium with low doses of ATP or ACh enhances sympatholysis in contracting muscle (25, 26), which indicates that stimulation of the endothelium attenuates α₁-adrenergic vasoconstriction in humans.

Therefore, in the present study, we sought to determine whether endothelium-dependent agonists interact to limit α₁-adrenergic vasoconstriction in resting skeletal muscle. When each dilator was administered separately, the α₁-adrenergic receptor agonist PE elicited less vasoconstriction during infusion of either ATP or ACh compared with SNP (Fig. 3C). Remarkably, combined infusion of ATP + ACh further limited α₁-mediated vasoconstriction compared with infusion of either dilator alone (Fig. 3, C and D) or combined infusion of either dilator with SNP (Fig. 3D). Moreover, when a high dose of SNP was infused to match vasodilation to that observed in the ATP + ACh trial, an even greater response to PE was observed such that threefold more vasoconstriction occurred in the high-SNP trial compared with the ATP + ACh trial. These findings indicate that combined activation of the endothelium with ATP and ACh enhances its capacity to regulate vascular tone through specific interactions with postjunctional α₁-adrenergic signaling, which further highlights the endothelium as a critical site of integration controlling vasomotor tone in humans.

Potential Cellular Signaling Mechanisms

The cellular signaling mechanisms by which ATP and ACh interact to elicit vasodilation and limit α₁-adrenergic vasoconstriction are unclear. Many chemical and mechanical stimuli that act on the endothelium are transduced as localized cytosolic Ca²⁺ signals, which give rise to endothelium-dependent hyperpolarization and stimulate production of vasodilatory autacoids (3). The results of this study indicate an interaction between ATP and ACh, both of which bind to G_q/11-coupled protein receptors on the endothelium. Activation of these receptors mobilizes Ca²⁺ from both intracellular stores and influx across the cell membrane, and the resulting rise in cytosolic Ca²⁺ propagates to neighboring ECs (41). Interestingly, ATP and ACh initiate distinctive Ca²⁺ oscillations that are influenced by the presence of the other agonist such that combined activation of ATP and ACh receptors generates a new, synergistic Ca²⁺ signal (10). Augmented Ca²⁺ signaling in response to combination of ATP + ACh coupled with the dense localization of K_Ca at myoendothelial projections (4) points toward involvement of K_Ca-mediated electrical signaling in the ability of the endothelium to modulate sympathetic vasoconstriction (23, 24). The findings from the present study are consistent with previous observations from our laboratory that strongly implicate endothelium-dependent hyperpolarization as a key signaling event in modulating α₁-mediated vasoconstriction in humans (13, 25–27).

Implications for Blood Flow Regulation

Interactions involving ATP and ACh may have physiological relevance for controlling peripheral vascular tone. Endothelial cells and erythrocytes release ATP in response to deoxygenation and mechanical forces such as shear stress and deformation (42–44); thus, ATP serves as a vasodilatory signal in areas with low tissue PO₂ during physiological stimuli such as hypoxia and muscle contractions (37) Elevations in ATP may also serve to enhance the endothelium’s sensitivity to further stimuli. In support of this idea, ATP potentiates flow-mediated vasodilation in isolated rat mesenteric arteries; indeed, the presence of ATP is required for sustained dilation in response to shear (34). Although it is not clear whether endogenous ACh plays a physiological role in blood flow regulation in humans (45), studies of cultured endothelial cells and isolated arteries suggest that endothelial cells synthesize and release ACh in response to shear stress to facilitate flow-mediated vasodilation (7, 43, 46). Interestingly, we have observed that ACh-mediated vasodilation is amplified within contracting skeletal muscle (15), and both ATP and ACh augment functional sympatholysis in humans (25, 26). Further experiments will be required to determine whether such interactions occur to influence vascular tone in humans under normal physiological conditions.

Limitations

The present investigation was limited to three vasodilatory substances and one vasoconstrictor. Moreover, vasodilators were always infused in the same order during the combination trials. It is therefore unclear whether the interactions we observed between ATP and ACh also occur with other endothelium-dependent vasodilators or whether the same interactions would occur if the order of dilators was reversed. Similarly, it is unclear whether combined activation of the endothelium also affects constriction mediated via α₂-adrenergic receptors or other vasoconstrictors such as angiotensin II or endothelin-1. Therefore, our interpretation is limited to interactions between the specific dilators and specific vasoconstrictor used in the present study.

In the combination trials, although the vasodilatory response to the second dilator was clearly greatest when two endothelium-dependent agonists were combined (ATP + ACh), the net response to ACh (Fig. 2D) was comparable with infusion of ACh alone (Fig. 2B). In contrast, the second dilator in the SNP + ACh and ATP + SNP trials evoked only half as much vasodilation (∼20–30 mL/min/100 mmHg; Fig. 2D) as was observed when each drug was infused separately (∼50–60 mmHg; Fig. 2C). Although the reasons for this are unclear, several potential explanations exist. First, it is possible that inhibitory interactions occurred between endothelium-dependent and -independent stimuli (47, 48). Second, overperfusion of resting tissue relative to its metabolic demand may have caused other mechanisms to restrain vasodilation (49, 50). Third, the elevation in blood flow generated by the first dilator may have diluted the concentration of the second dilator in the arterial circulation, rendering it less effective in evoking vasodilation. Interestingly, the latter two possibilities would have been present during all combination trials, which again highlights the unique interaction between the two endothelium-dependent dilators ATP and ACh.

The present study was not designed to determine the impact of sex and/or circulating hormone levels on the interactions between endothelium-dependent vasodilators in the regulation of vascular tone, thus we did not control for menstrual cycle. Future studies will be needed to address these specific issues.

Perspectives

Many patient populations at elevated risk for cardiovascular disease display both endothelial dysfunction and elevated sympathetic nervous system activity. Thus, understanding the interactions between vasodilator and vasoconstrictor signaling may lead to strategies to improve tissue blood flow and oxygen delivery in at-risk populations (51). We have observed impaired ATP release from erythrocytes of older adults, and circulating ATP is lower in older adults during exercise and systemic hypoxia (17). Therefore, therapeutic approaches to enhance circulating ATP (52) could potentially improve vasodilatory sensitivity to ACh, which declines with age (53, 54), while also improving the capacity to overcome exaggerated sympathetic activation.

Conclusions

The present study was designed to investigate potential interactions between endothelium-dependent vasodilators in regulating skeletal muscle vascular tone in humans. To our knowledge, these findings are the first to demonstrate that the endothelium-dependent agonists ATP and ACh combine to augment vasodilation compared with that observed when either agonist is paired with the endothelium-independent dilator SNP, which suggests that activation of the endothelium modulates its response to further stimuli. Moreover, combination of ATP and ACh significantly attenuates α₁-adrenergic vasoconstriction in resting skeletal muscle, which may have implications for functional sympatholysis and blood flow regulation in contracting muscle. Collectively, these observations highlight the endothelium as a key site for integrating multiple vasoactive inputs to regulate tissue blood flow. Yet how such interactions occur to regulate vascular tone in humans under physiological conditions remains to be determined.

GRANTS

This research was supported by the National Institutes of Health Awards HL119337 (to F.A.D.) and HL142240 (to J.D.T. and F.A.D.).

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

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