Reduced deformability contributes to impaired deoxygenation-induced ATP release from red blood cells of older adult humans

Abstract

Key points: Red blood cells (RBCs) release ATP in response to deoxygenation, which can increase blood flow to help match oxygen supply with tissue metabolic demand. This release of ATP is impaired in RBCs from older adults, but the underlying mechanisms are unknown. In this study, improving RBC deformability in older adults restored deoxygenation-induced ATP release, whereas decreasing RBC deformability in young adults reduced ATP release to the level of that of older adults. In contrast, treating RBCs with a phosphodiesterase 3 inhibitor did not affect ATP release in either age group, possibly due to intact intracellular signalling downstream of deoxygenation as indicated by preserved cAMP and ATP release responses to pharmacological G_i protein activation in RBCs from older adults. These findings are the first to demonstrate that the age-related decrease in RBC deformability is a primary mechanism of impaired deoxygenation-induced ATP release, which may have implications for treating impaired vascular control with advancing age.

Abstract: In response to haemoglobin deoxygenation, red blood cells (RBCs) release ATP, which binds to endothelial purinergic receptors and stimulates vasodilatation. This ATP release is impaired in RBCs from older vs. young adults, but the underlying mechanisms are unknown. Using isolated RBCs from young (24 ± 1 years) and older (65 ± 2 years) adults, we tested the hypothesis that age-related changes in RBC deformability (Study 1) and cAMP signalling (Study 2) contribute to the impairment. RBC ATP release during normoxia ( $P_{O_2}$ ∼112 mmHg) and hypoxia ( $P_{O_2}$ ∼20 mmHg) was quantified with the luciferin-luciferase technique following RBC incubation with Y-27632 (Rho-kinase inhibitor to increase deformability), diamide (cell-stiffening agent), cilostazol (phosphodiesterase 3 inhibitor), or vehicle control. The mean change in RBC ATP release from normoxia to hypoxia in control conditions was significantly impaired in older vs. young (∼50% vs. ∼120%; P < 0.05). RBC deformability was also lower in older vs. young as indicated by a higher RBC transit time (RCTT) measured by blood filtrometry (RCTT: 8.541 ± 0.050 vs. 8.234 ± 0.098 a.u., respectively; P < 0.05). Y-27632 improved RBC deformability (RCTT: 8.228 ± 0.083) and ATP release (111.7 ± 17.2%) in older and diamide decreased RBC deformability (RCTT: 8.955 ± 0.114) and ATP release (67.4 ± 11.8%) in young (P < 0.05), abolishing the age group differences (P > 0.05). Cilostazol did not change ATP release in either age group (P > 0.05), and RBC cAMP and ATP release to pharmacological G_i protein activation was similar in both groups (P > 0.05). We conclude that decreased RBC deformability is a primary contributor to age-related impairments in RBC ATP release, which may have implications for impaired vascular control with advancing age.

Keywords: ATP release; deoxygenation; hypoxia; red blood cells.

Figure 1.. Effect of donor age, Y-27632, and diamide on red blood cell deformability

RBCs from older adults were less deformable than RBCs from young adults in control (saline) conditions, as indicated by a greater RCTT. Incubation of RBCs with 1 μM Y-27632 restored deformability in older adults, whereas 500 μM diamide decreased RBC deformability similarly in young and older adults. *P < 0.05 vs. saline (within age); †P < 0.05 vs. young (within condition)

Figure 2.. Effect of donor age and Y-27632 on red blood cell ATP release in normoxia and hypoxia

A: extracellular ATP (i.e., ATP release) from RBCs of older adults was significantly lower compared to young adults during hypoxia with saline (control) but not Y-27632, as Y-27632 significantly increased extracellular ATP during hypoxia from RBCs of older adults. B: the mean percent increase in extracellular ATP from normoxia to hypoxia was impaired from RBCs of older adults in the saline condition. Incubation with Y-27632 normalized this response from RBCs of older adults relative to the young saline control. *P < 0.05 vs. saline (within age); †P < 0.05 vs. young (within condition); ‡P < 0.05 vs. normoxia (within condition)

Figure 3.. Effect of donor age and diamide on red blood cell ATP release in normoxia and hypoxia

A: extracellular ATP (i.e., ATP release) from RBCs of older adults was significantly lower compared to young adults during hypoxia with saline (control) but not diamide, as diamide significantly decreased extracellular ATP during hypoxia from RBCs of both age groups. B: the mean percent increase in extracellular ATP from normoxia to hypoxia was impaired from RBCs of older adults in the saline condition. Incubation with diamide significantly decreased ATP release from RBCs of young adults such that it was no longer different from older adults. *P < 0.05 vs. saline (within age); †P < 0.05 vs. young (within condition); ‡P < 0.05 vs. normoxia (within condition)

Figure 4.. Effect of donor age and cilostazol on red blood cell ATP release in normoxia and hypoxia

A: extracellular ATP (i.e., ATP release) from RBCs of older adults trended towards being lower compared to young adults during hypoxia with DMF (control; P = 0.07) and was significantly lower with cilostazol. Cilostazol had no effect on extracellular ATP from RBCs of young or older adults during normoxia or hypoxia. B: the mean percent increase in extracellular ATP from normoxia to hypoxia was impaired from RBCs of older adults in both the DMF (control) and cilostazol conditions. †P < 0.05 vs. young (within condition); ‡P < 0.05 vs. normoxia (within condition)

Figure 5.. Effect of donor age on RBC responses to Mas 7

A: the G_i activator Mas 7 increased intracellular cAMP similarly from RBCs of young and older adults. B: extracellular ATP (i.e. ATP release) from RBCs of young and older adults was unaffected by incubation with saline (vehicle and time control) and increased significantly following incubation with Mas 7. ◊P < 0.05 vs. zero; *P < 0.05 vs. saline

Figure 6.. Effect of donor age and pharmacology on red blood cell intracellular ATP in normoxia and hypoxia

RBC intracellular ATP increased significantly during hypoxia in all age groups and drug conditions. A: intracellular ATP was significantly lower in older (n = 10) compared to young (n = 9) adults during hypoxia with Y-27632, although the mean change from normoxia to hypoxia was unaffected by age. B: diamide significantly decreased intracellular ATP vs. saline during normoxia and hypoxia in young (n = 11) and older (n = 10) adults. C: intracellular ATP in young (n = 10) and older (n = 12) adults was unaffected by age group or drug condition. *P < 0.05 vs. control (within age); ‡P < 0.05 vs. normoxia (within condition)

Figure 7.. Experimental targets and working hypothesis for the mechanism of impaired deoxygenation-induced from RBCs of older adults

Pharmacological manipulations in Study 1 targeted the RBC cytoskeleton and in Study 2 targeted cAMP-related components of the proposed signalling cascade for deoxygenation-induced ATP release (adapted from Ellsworth & Sprague, 2012). Our findings indicate that reductions in RBC deformability with advancing donor age may limit activation of the signalling cascade downstream of haemoglobin desaturation, ultimately leading to impaired deoxygenation-induced RBC ATP release in older adults.