Racine, Matthew L.

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

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

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

We have recently demonstrated that vasodilation to the endothelium‐dependent agonist acetylcholine is amplified in contracting compared with quiescent skeletal muscle, highlighting the importance of the vascular endothelium in integrating vasomotor signals to regulate vascular tone in humans. In the present study, we tested the hypothesis that sensitivity to the endothelium‐dependent vasodilator ATP is augmented during exercise and systemic hypoxia, whereas sensitivity to the nitric oxide donor and endothelium‐independent dilator sodium nitroprusside (SNP) is not affected by these physiological conditions. In 12 young adults (7 M, 5 F), we measured forearm blood flow (FBF; Doppler ultrasound) and calculated changes in vascular conductance (FVC: quotient of FBF and mean arterial pressure) in response to local, intra‐arterial infusion of ATP or SNP during 1) control resting conditions (CON), 2) dynamic handgrip exercise (EX, 20 contractions per minute at 10% of maximal voluntary contraction), or 3) systemic hypoxia (HYP, 80% O 2 saturation). Three doses of each vasodilator were infused via a brachial artery catheter during each condition (ATP: 1.25, 2.50, and 5.00 μg·dl forearm volume −1 ·min −1 ; SNP: 0.25, 0.50, and 1.00 μg·dl forearm volume −1 ·min −1 ). The order of drugs and conditions was randomized. Within each trial, infusions began after 3 min of rest, steady‐state exercise, or hypoxia, and each dose was infused for 2 min while the exercise or hypoxic stimulus was sustained. Compared to control conditions, vasodilatory sensitivity to the high dose of ATP was enhanced by 53 ± 18% during mild handgrip exercise (Δ FVC, CON: 120 ± 16 vs. EX: 176 ± 28 ml·min −1 ·100 mmHg −1 ; P < 0.05), whereas vasodilatory sensitivity was not affected at lower doses of ATP ( P = NS; Dose × Condition interaction P < 0.05). In contrast, exercise had no effect on vascular sensitivity to any dose of SNP ( P = NS). Similarly, during systemic hypoxia, vasodilation to ATP was enhanced by 18 ± 10% at the medium dose (Δ FVC, CON: 100 ± 15 vs. HYP: 125 ± 24 ml·min −1 ·100 mmHg −1 ; P < 0.05) and 28 ± 11% at the high dose (Δ FVC, CON: 120 ± 16 vs. HYP: 158 ± 27 ml·min −1 ·100 mmHg −1 ; P < 0.05; Dose × Condition interaction P < 0.05), whereas vasodilation to SNP was not different from control conditions at any dose ( P = NS). These findings provide evidence that muscle contractions and hypoxia augment ATP‐mediated, endothelium‐dependent vasodilation in human skeletal muscle. Moreover, given that intravascular ATP increases under these physiological conditions, the results indicate that small changes in plasma ATP may result in augmented vasodilation and contribute to blood flow and oxygen delivery in humans. Support or Funding Information HL119337 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .