Dinenno, Frank A.

Introduction Adenosine triphosphate (ATP) is a vasodilating metabolite that plays a role in exercise hyperemia. In humans, exogenous infusion of ATP elevates skeletal muscle blood flow values to that observed during moderate‐intensity exercise. Recently ATP has been shown to evoke prolonged vasodilation over 150 minutes without tachyphylaxis. However, the steady‐state hyperemic responses to superimposed forearm exercise remain unaltered. The kinetics of blood flow during the initiation of exercise remains not well understood. Therefore we evaluated the effects of prolonged exogenous infusion of ATP on exercise hyperemia during the transition between rest and initiation of exercise. Methods While supine, eight healthy young participants (4M/4F; 25±1 years) performed 5 minutes of rhythmic dynamic handgrip exercise bouts (20% of maximum), first during saline (control), then at minute 5 and minute 150 during a continuous ATP infusion into the brachial artery. Each duty cycle consisted of 1 second contraction and 2 second relaxation. Mean blood velocity (MBV; cm/s), forearm vascular conductance (FVC; ml/minutes/100 mmHg), and shear rate (8*MBV/diameter; seconds −1 ) were calculated. The kinetics at the onset of exercise was assessed by calculating the slope of the FVC responses for the first 20 duty cycles after the start of contractions. Data are presented as means ± SEM. Results During saline control FVC was 3.9±0.3 at rest and with exercise rose to 22.7±0.84 and 27.8±1.2 ml/minute/100mmHg at 30 seconds and 1 minute, respectively (P<0.05 for both). Infusion of ATP increased FVC (3.9±0.3 vs 43.1±2.0 ml/minutes/100 mmHg, P<0.05) at five minutes and to 61.0±1.1 ml/minutes/100 mmHg at 150 minutes (P<0.05) with no systemic hemodynamic changes. Prolonged infusion of ATP did not alter the kinetics at the onset of contractions over 20 duty cycles of exercise hyperemic responses compared to control (0.71±0.1 control vs 1.01±0.1 ml/minutes/100mmHg/cycle at 5 minutes and vs 0.65±0.1 at 150 minutes, P>0.05 for all). Shear rate significantly increased compared to control (92±5 vs 751±57 seconds −1 , P<0.05) but was not different across exercise responses during ATP infusion (751±57 at 5 minutes vs 549±46 seconds −1 at 150 minutes, P>0.05). Conclusions Our findings demonstrate that despite the ability of ATP to elevate blood flow to that observed during exercise, the blood flow kinetics for the initiation of dynamic exercise hyperemic response are unaffected and remain unaltered after 150 minutes of prolonged ATP infusion. Further investigation in needed to understand the role of ATP in regulating the blood flow kinetics during dynamic exercise in humans. Support or Funding Information This research was supported by National Institutes of Health Research Grants HL‐119337 and by CTSA UL1 TR000135. The Caywood Professorship via the Mayo Foundation also supported this study.

Skeletal muscle blood flow during exercise is typically reduced in older compared with young adults, which may be mediated in part by age‐related reductions in local vasodilator molecules such as nitric oxide (NO) and ATP. Despite this, muscle blood flow does increase substantially to contracting muscle of older adults. We questioned whether reductions in muscle blood flow with age, particularly at the onset of exercise, leads to greater accumulation of local metabolites that could play a more prominent role in contraction‐induced vasodilation compared with young adults. Thus, in the present study we tested the hypothesis that the contribution of adenosine (ADO) to contraction‐induced vasodilation is greater in older vs. young adults. In young (25 ± 2 years; n=8) and older (65 ± 2 years; n=8) adults, forearm vascular conductance (FVC) responses to a single 20% maximum voluntary contraction (MVC) handgrip contraction and to 5 minutes of 10% MVC rhythmic handgrip exercise were measured using Doppler ultrasound of the brachial artery before and after brachial artery infusion of the ADO receptor blocker aminophylline (APH). In order to determine the effect of APH infusion that was due specifically to the blockade of ADO receptors as opposed to the elevated baseline FVC that often occurs as an off‐target effect of APH, FVC responses to contraction were also measured after sodium nitroprusside (SNP) infusion to match the APH‐induced increase in baseline FVC. In response to a single contraction, both total FVC area under the curve (AUC) and the absolute change in FVC from baseline to peak were lower in older vs. young adults (−61.2% and −56.1%, respectively; p < 0.05) under control conditions. There was no significant effect of APH on total FVC AUC in young (−5.4 ± 7.8%) or older (16.6 ± 12.4%) adults vs. control, whereas the absolute change in FVC from baseline to peak was significantly lower in young adults (−22.5 ± 7.7%; p < 0.05), but unaffected in older adults (4.1 ± 7.8%) vs. control. During rhythmic handgrip exercise, FVC was significantly lower in older adults in the first 60 sec of exercise (~45–50%) as well as during steady‐state (~25%; 110.0 ± 14.9 vs. 149.5 ±12.6 mL/min/100mmHg; both p < 0.05) under control conditions. APH had no effect on the FVC response throughout the first 3 min of exercise onset in young (range: 0.8–6.2%) or older (range: −3.9 to 10.5%) adults vs. control (p > 0.05), and did not alter the absolute change in FVC from baseline to steady‐state exercise in either group (p > 0.05). SNP had no effect on these FVC responses during single contractions or rhythmic handgrip exercise in either age group (p > 0.05). We conclude that that ADO contributes to the peak vasodilatory response to a single contraction in young but not older adults, and that advancing age is not associated with a greater reliance on ADO for local vasodilation during skeletal muscle contractions. Support or Funding Information HL095573, HL119337

Introduction Adenosine triphosphate (ATP) is postulated to be a major player in the local control of skeletal muscle vascular tone during exercise. Exercise during hypoxic conditions significantly augments skeletal muscle blood flow and elevates venous plasma levels of ATP compared to normoxic conditions in young healthy adults. This suggests that the elevated levels of ATP during hypoxic exercise contribute to the augmented vasodilation. Nitric oxide synthase (NOS) inhibition has also been shown to blunt the effects of hypoxia on exercise vasodilation. Therefore, the aim of the current study was to evaluate the effect of NOS inhibition and hypoxia on vasodilator responses and plasma ATP levels during exercise in healthy young adults. Methods 10 young volunteers (age = 28 ± 1 years, 6 men/4 women) performed four minutes of forearm handgrip exercise at 20% of their maximal voluntary contraction (MVC). The forearm handgrip exercise was performed under normoxia and hypoxia (SpO2 = ~80%) with and without NOS inhibitor, LNMMA administered via a brachial artery catheter. Beat to beat blood pressure and heart rate were recorded. Forearm blood flow was measured using Doppler ultrasound and normalized for arterial pressure to determine forearm vascular conductance (FVC). Venous blood was obtained from a retrograde intravenous catheter at rest and during the last minute of each exercise bout. Plasma ATP was measured using the luciferin‐luciferase technique. Results During saline and normoxia FVC was 43.4 ± 8.4 at rest and 307.8 ± 22.6 ml/min/100mmHg during exercise. With hypoxia FVC increased to 59.35 ± 10.5 at rest and 370.9 ± 33.3 ml/min/100mmHg during exercise (both p<0.05 vs. normoxia). During LNMMA and normoxia FVC did not change significantly compared to saline 33.9 ± 4.4 at rest and 283.5 ± 23.2 ml/min/100mmHg during exercise. With hypoxia and LNMMA FVC was 35.04 ± 4.1 at rest (p>0.05 vs. normoxia) and 326.0 ± 23.9 ml/min/100mmHg during exercise (p<0.05 vs. normoxia). When changes from baseline are evaluated, hypoxia augmented the vasodilator responses exercise to exercise during both saline and LNMMA. ATP was higher during 20% MVC exercise with saline during hypoxia as compared to normoxia (65.6 ± 12.1 vs. 108.1 ± 23.2 versus nmol/L, p=0.058). It was also higher during 20% MVC exercise with LNMMA (81.7 ± 9.5 vs. 106.1 ± 14.7 nmol/L, p=0.055). When changes from baseline are evaluated, hypoxia augmented venous ATP concentration with exercise during both saline and LNMMA. Conclusions Our results suggest that increased endogenous ATP release during hypoxic exercise continued to evoke vasodilation during NOS inhibition. A likely source of this ATP is from red blood cells and this interpretation is consistent with findings demonstrating that NOS inhibition has limited effects on ATP mediated dilation in the human forearm. Support or Funding Information This research was supported by National Institutes of Health Research Grants HL‐119337, CTSA UL1 TR000135 and the Caywood Professorship via the Mayo Foundation

Red blood cells (RBCs) release ATP in direct proportion to the degree of hemoglobin (Hb) deoxygenation, which binds to purinergic receptors on the endothelium and stimulates vasodilation. Accordingly, RBCs act as both a ‘sensor’ for oxygen demand and an ‘effector’ for increasing oxygen delivery to facilitate the matching of tissue oxygen supply and demand. We have recently demonstrated that Hb deoxygenation‐induced ATP release is impaired in RBCs from healthy older vs. young adults, but the underlying mechanisms are not fully understood. Cyclic AMP (cAMP) has been shown to be an important component of the signaling cascade for RBC ATP release, and decreased cAMP signaling contributes to impaired ATP release from RBCs of humans with type 2 diabetes. As such, treatment with cilostazol to inhibit phosphodiesterase 3 (PDE3) hydrolysis of cAMP improves Hb deoxygenation‐induced ATP release from RBCs of these patients. Whether similar alterations in cAMP signaling contribute to impaired RBC ATP release in healthy older adults is unclear. Thus, in the present study we tested the hypothesis that PDE3 inhibition would restore Hb deoxygenation‐induced ATP release from RBCs of healthy older adults. Isolated RBC ATP release in response to normoxic (PO 2 ~111 mmHg) and hypoxic (PO 2 ~26 mmHg) stimuli was quantified in RBCs from young (25 ± 1 years; n = 4) and older (65 ± 3 years; n = 6) adults using the luciferin‐luciferase technique following RBC incubation with either dimethylformamide (DMF; vehicle control) or 100 μM cilostazol (PDE3 inhibitor). The relative change in ATP release from normoxia to hypoxia in control conditions was impaired in RBCs from older (56.1 ± 11.0%) vs. young (129.1 ± 16.1%) adults (p < 0.05). Cilostazol resulted in a slight, but non‐significant improvement in RBC ATP release in older adults (74.2 ± 15.0%; p = 0.32 vs. control), whereas there was no effect on RBC ATP release in young adults (123.4 ± 37.5%; p = 0.84 vs. control). RBC intracellular ATP was not different between age groups or treatments (p > 0.05). These preliminary data suggest that greater cAMP hydrolysis via PDE3 in response to Hb deoxygenation is not a primary contributor to impaired RBC ATP release in older adults. Support or Funding Information HL119337, F31HL126377

Adenosine triphosphate (ATP) is postulated to be a major exercise related vasodilating metabolite. In humans, ATP infusion mimics exercise hyperemia. However, it remains unknown whether ATP can evoke the prolonged vasodilation seen during exercise. Therefore we tested two hypotheses during a three‐hour intra‐arterial infusion of a high dose of ATP (20 μgrams ·100 mL forearm vol −1 ·min −1 ): (1) skeletal muscle blood flow would wane over time (tachyphalaxis); (2) exercise hyperemic responses during ATP administration would be blunted compared to baseline. Nine healthy young participants (25 ± 1 years) performed seven rhythmic handgrip exercise bouts (20% of maximum), two during saline (control) and five during 180 minutes of continuous ATP infusion. Forearm vascular conductance (FVC; ml min −1 (100 mmHg) −1 ) and percent (%) change from control were calculated. Five minutes of ATP infusion resulted in a 710% increase in FVC (4.8±0.3 vs 35.0±1.9 ml min −1 *100 mmHg −1 , P<0.05). FVC did not wane over time with values of 35.0±1.9 and 36±2.6 ml min −1 (100 mmHg) −1 (P>0.05) seen prior to the exercise bouts at 5 vs 150 minutes respectively. With superimposed exercise FVC increased from 35.0±1.9 to 49.6±2.2 ml min −1 *100 mmHg −1 at five minutes and 36.0±2.6 to 54.5±1.1 at 150 min (P<0.05). Our findings demonstrate (1) ATP vasodilation is sustained over time without tachyphalaxis; (2) vasodilator responses to exercise during high dose ATP infusion remain intact. This latter finding questions the extent to which ATP is obligatory for exercise hyperemia. Support or Funding Information This research was supported by National Institutes of Health Research Grants HL‐119337 and by CTSA UL1 TR000135. The Caywood Professorship via the Mayo Foundation also supported this research.

High doses of adenosine (ADO) infused over a prolonged period (3 hours) produces marked vasodilation. However, this vasodilation wanes over time in an isolated canine skeletal muscle preparation. To our knowledge the waning of vasodilation with high doses of ADO has not been shown in humans. In addition, ADO plays a controversial role in exercise hyperemia. Therefore, our primary hypothesis was that in humans during a continuous three‐hour intra‐arterial infusion of a high dose of ADO (23 μgrams ·100 mL forearm vol −1 ·min −1 ) skeletal muscle blood flow would wane over time. Our secondary hypothesis was exercise hyperemic responses during ADO administration would be similar compared to control. Nine healthy young participants (29 ± 2 years) performed rhythmic handgrip (HG) exercise bouts (20% of maximum) during saline and 180 minutes of continuous ADO infusion. Forearm vascular conductance (FVC; ml min −1 (100 mmHg) −1 ) was calculated from measured forearm blood flow and mean blood pressure. FVC during minute 5 versus minute 175 during ADO infusion were 24.22 ± 3.2 versus 23.24 ± 1.95 ml min −1 (100 mmHg) −1 respectively (p>0.05). FVC during exercise increased significantly from baseline (4.78 ± 0.63 to 31.13 ± 4.17 ml min −1 *100 mmHg −1 ) during saline infusion and from (24.22 ± 3.23 to 56.33 ± 4.82 ml min −1 *100 mmHg −1 ) at five minutes and 21.99 ± 1.25 to 59.16 ± 5.21 seen at 150 minutes during ADO infusion. There was no significant difference in the delta FVC (exercise FVC – baseline FVC) between saline exercise bout versus ADO exercise bout (26.34 ± 3.75 versus 32.11 ± 5.55 ml min −1 *100 mmHg −1 ). Our findings show that contrary to the animal model, ADO vasodilatation does not wane in humans. Also, the vasodilator responses to exercise are similar during saline and prolonged ADO infusion compared to control condition. These findings suggest a minimal role of ADO in exercise hyperemia. Support or Funding Information Supported by National Institutes of Health Research Grants HL‐119337 (to M.J. Joyner and F.A. Dinenno) and by CTSA UL1 TR000135. The Caywood Professorship (M.J. Joyner) via the Mayo Foundation also supported this research.

During muscle contractions, an increase in interstitial potassium (K + ) and subsequent activation of inwardly‐rectifying K + (K IR ) channels is thought to be a functional vasodilatory signal contributing to exercise hyperemia. Indeed, our laboratory has recently demonstrated that inhibition of K IR channels blunts exercise hyperemia ~30% in humans. In theory, the vasodilatory response to changes in interstitial K + during muscle contraction could be minimized by artificially elevating the concentration of K + perfusing the vasculature prior to the onset of muscle contractions, thus limiting further recruitment of this pathway and blunting the dilatory response to exercise. We tested the hypothesis that infusion of potassium chloride (KCl) to elevate extracellular potassium concentrations and induce hyperpolarization prior to the onset of muscle contractions would reduce the contribution of this mechanism to the dilatory response to exercise. In young adults (n = 10), we measured forearm blood flow (FBF; Doppler ultrasound) and calculated changes in vascular conductance (FVC; FBF/intra‐arterial blood pressure) during 4 minutes of rhythmic handgrip exercise at 10% of maximal voluntary contraction during 1) control conditions (CTRL), 2) local, intra‐arterial infusion of KCl to elevate the concentration of K + prior to the initiation of exercise, and 3) intra‐arterial infusion of sodium nitroprusside (SNP) as a high flow control to match the K + ‐induced vasodilation observed at rest. As intended, infusion of KCl or SNP elevated resting FVC similarly prior to the onset of exercise (CTRL: 40 ± 7 vs. KCl: 84 ± 13 and SNP: 85 ± 14 ml·min −1 ·100 mmHg −1 ; both P < 0.01 vs. CTRL). Infusion of KCl diminished the subsequent vasodilatory response to handgrip exercise by ~31% when compared to control conditions (ΔFVC: KCl: 92 ± 14 vs. CTRL: 133 ± 16 ml·min −1 ·100 mmHg −1 ; P < 0.01), whereas elevation of resting FVC with SNP prior to contractions did not change the vasodilatory response to exercise (ΔFVC, SNP: 117 ± 15 vs . CTRL: 133 ± 16 ml·min −1 ·100 mmHg −1 ; P = NS). These findings indicate that K + ‐mediated hyperpolarization prior to the onset of muscle contractions blunts the normal vasodilatory response to exercise, which supports a role for elevated K + signaling in the hyperemic response to exercise in humans. Support or Funding Information HL119337, HL095573

Exercise‐induced rapid onset vasodilation produces a temporary rise in skeletal muscle blood flow in humans. The underlying mechanisms for this rapid hyperemia and regulation of muscle blood flow with single contraction are still not well understood. We tested the hypothesis that during a prolonged high dose intrarterial infusion of adenosine triphosphate (ATP; 20 μgrams ·100 mL forearm vol −1 ·min −1 ), the rapid onset vasodilatory response post‐single exercise contraction would be reduced compared to that of adenosine (ADO; 23 μgrams ·100 mL forearm vol −1 ·min −1 ). 14 healthy young participants (30 ± 1 years) performed a forearm single contraction exercise (20% of maximum) at minute 75 of either ATP (N=8) or ADO (N=6) continuous infusion. Artery diameter and blood velocity were measured using Doppler ultrasound. Forearm vascular conductance (FVC; ml·min −1 ·100 mmHg −1 ), time to peak, and area under curve were calculated. The mean resting FVC was higher during ATP (307.15 ± 50.56) and ADO (353.23 ± 43.98) infusion compared to saline infusion (45.17 ± 5.61 and 47.14 ± 5.57) (p<0.05). Compared to ADO, ATP infusion had a lower delta FVC from post‐ to pre‐contraction (174.06 ± 30.75 vs. 276.53 ± 24.90, p<0.05). Area under the curve and time of peak FVC did not significantly differ between drugs. These results suggest a prolonged ATP infusion may have blunted the rapid vasodilatory response more‐so than ADO. Thus, our findings indicate that ATP may be necessary but not obligatory in the rapid onset vasodilation response post‐single muscle contraction in humans. Support or Funding Information Supported by National Institutes of Health Research Grants HL‐119337 (to M.J. Joyner and F.A. Dinenno) and by CTSA UL1 TR000135. The Caywood Professorship (M.J. Joyner) via the Mayo Foundation also supported this research.

The vasoactive properties of exogenous adenosine triphosphate (ATP) are unique because it has the dual ability to induce robust vasodilation as well as blunt sympathetic vasoconstriction, similar to what is observed in contracting human skeletal muscle. ATP‐mediated vasodilation and exercise hyperemia are dependent, in part, on activation of inwardly rectifying potassium (K IR ) channels and subsequent smooth muscle cell hyperpolarization, with a modest reliance on the production of nitric oxide (NO) and prostaglandins (PGs). However, recent studies from our laboratory indicate that these pathways are not obligatory for contracting skeletal muscle to blunt α 1 ‐adrenergic vasoconstriction. Given the specific role of K IR channels in ATP‐mediated vasodilation, the primary purpose of this study was to determine whether the ability of ATP to blunt α 1 ‐adrenergic vasoconstriction is dependent upon K IR channel activity in humans. A secondary purpose was to determine whether the ability of ATP to blunt α 1 ‐adrenergic vasoconstriction is dependent upon activation of K IR channels and Na + /K + ‐ATPase, as well as the synthesis of NO and PGs. In a total of 13 young subjects we measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to local intra‐arterial infusion of phenylephrine (PE; α 1 ‐agonist) during infusion of ATP or a control vasodilator(s) (Protocol 1 (n=7): adenosine (ADO), and sodium nitroprusside (SNP); Protocol 2 (n=6): ADO). Vasomotor responses were assessed before and after blockade of signaling pathways that contribute to ATP‐mediated vasodilation (Protocol 1: K IR channels (barium chloride); Protocol 2: combined blockade of K IR channels, Na + /K + ‐ATPase (ouabain), NO (L‐NMMA), PGs (Ketorolac)). Protocol 1: ATP significantly attenuated PE‐mediated vasoconstriction relative to both SNP and ADO (Δ FVC: ATP: −16 ± 2; ADO: −38 ± 6; SNP: −59 ± 6%; P<0.05 vs ADO and SNP). Blockade of K IR channels significantly attenuated ATP mediated vasodilation ~40% (P<0.05 vs. control), but had no effect on the ability of ATP to blunt PE‐ mediated vasoconstriction (Δ FVC: −6 ± 5%; P>0.05 vs. control). Protocol 2: Despite combined blockade of K IR , Na + /K + ‐ATPase, NO, and PGs resulting in profound attenuation of ATP‐mediated vasodilation (−~60%; P<0.01 vs. control), ATP was still able to blunt PE‐mediated vasoconstriction (Δ FVC: −4 ± 14%; P=0.50 vs. control). Together these studies highlight that while NO, PGs, and vascular smooth muscle hyperpolarization (via K IR channel activation) are contributors to the normal vasodilatory response to exercise and exogenous ATP, these pathways are not obligatory for exercise or ATP to blunt α 1 ‐adrenergic vasoconstriction in humans. Support or Funding Information Support: HL119337, HL095573

Substantial evidence indicates that red blood cells (RBCs) can act as a ‘sensor’ for oxygen demand in addition to their traditional role as an oxygen ‘carrier’, in that they are able to stimulate vasodilation and increase oxygen delivery to the tissue through the release of ATP in direct proportion to the degree of hemoglobin (Hb) deoxygenation. However, RBCs from older adults have an impaired ability to release ATP in response to Hb deoxygenation compared to RBCs from young adults, yet the underlying cause of this remains unknown. RBC membrane deformability has been shown to decrease with advancing donor age, thus we hypothesized that increasing membrane deformability (via the Rho‐kinase inhibitor Y‐27632) would restore ATP release from RBCs of older adults, while decreasing membrane deformability (via the cell‐stiffening agent diamide) would impair ATP release from RBCs of young adults. Isolated RBC ATP release in response to normoxic (PO 2 ~110 mmHg) and hypoxic (PO 2 ~20 mmHg) stimuli was quantified in RBCs from young (23 ± 3 years; n = 11 for Y‐27632 and diamide) and older (68 ± 7 years; n = 11 and 6 for Y‐27632 and diamide, respectively) adults using the luciferin‐luciferase technique following RBC incubation with either saline (control), Y‐27632, or diamide. The relative change in ATP release from normoxia to hypoxia in saline control conditions was impaired in RBCs from older (41.5 ± 12.1% and 63.5 ± 18.5%) vs. young (110.4 ±12.7% and 146.0 ± 29.3%) adults (P < 0.05; control trials for Y‐27632 and diamide, respectively). Y‐27632 improved RBC ATP release in older adults (98.8 ± 19.8%) such that it was not different from the young control (P > 0.05), whereas diamide decreased RBC ATP release in young adults (57.4 ± 8.4%) such that it was not different from the older control (P > 0.05). Preliminary data from our laboratory indicate that these drugs affected RBC membrane deformability as expected based on other reports of the effects of Y‐27632 and diamide on RBC membrane deformability. We conclude that decreased membrane deformability contributes to the impaired ability of RBCs from older adults to release ATP in response to Hb deoxygenation, and that this may have implications for impaired vascular control with advancing age. Support or Funding Information HL095573, HL119337, F31HL126377