Dataset Information

Shear Stress Ameliorates Superoxide Impairment to Erythrocyte Deformability With Concurrent Nitric Oxide Synthase Activation.

ABSTRACT: The cellular deformability of red blood cells (RBC) is exceptional among mammalian cells and facilitates nutrient delivery throughout the microcirculation; however, this physical property is negatively impacted by oxidative stress. It remains unresolved whether the molecular determinants of cellular deformability - which in the contemporary model of RBC are increasingly recognized - are sensitive to free radicals. Moreover, given cellular deformability has recently been demonstrated to increase following exposure to specific doses of mechanical stimulation, the potential for using shear "conditioning" as a novel method to reverse free-radical induced impairment of cell mechanics is of interest. We thus designed a series of experiments that explored the effects of intracellular superoxide (O₂ ^-) generation on the deformability of RBC and also activation of pivotal molecular pathways known to regulate cell mechanics - i.e., PI3K/Akt kinase and RBC nitric oxide synthase (NOS). In addition, RBC exposed to O₂ ^- were conditioned with specific shear stresses, prior to evaluation of cellular deformability and activation of PI3K/Akt kinase and RBC-NOS. Intracellular generation of O₂ ^- decreased phosphorylation of RBC-NOS at its primary activation site (Ser¹¹⁷⁷) (p < 0.001), while phosphorylation of Akt kinase at its active residue (Ser⁴⁷³) was also diminished (p < 0.001). Inactivation of these enzymes following O₂ ^- exposure occurred in tandem with decreased RBC deformability. Shear conditioning significantly improved cellular deformability, even in RBC previously exposed to O₂ ^-. The improvement in cellular deformability may have been the result of enhanced molecular signaling, given RBC-NOS phosphorylation in RBC exposed to O₂ ^- was restored following shear conditioning. Impaired RBC deformability induced by intracellular O₂ ^- may be due, in part, to impaired activation of PI3K/Akt, and downstream signaling with RBC-NOS. These findings may shed light on improved circulatory health with targeted promotion of blood flow (e.g., exercise training), and may prove fruitful in future development of blood-contacting devices.

SUBMITTER: Kuck L

PROVIDER: S-EPMC6370721 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Shear Stress Ameliorates Superoxide Impairment to Erythrocyte Deformability With Concurrent Nitric Oxide Synthase Activation.

Kuck Lennart L Grau Marijke M Bloch Wilhelm W Simmonds Michael J MJ

Frontiers in physiology 20190205

The cellular deformability of red blood cells (RBC) is exceptional among mammalian cells and facilitates nutrient delivery throughout the microcirculation; however, this physical property is negatively impacted by oxidative stress. It remains unresolved whether the molecular determinants of cellular deformability - which in the contemporary model of RBC are increasingly recognized - are sensitive to free radicals. Moreover, given cellular deformability has recently been demonstrated to increase ...[more]

PMID: 30804795

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Project description:OBJECTIVE:Impaired endothelial cell (EC) autophagy compromises shear stress-induced nitric oxide (NO) generation. We determined the responsible mechanism. APPROACH AND RESULTS:On autophagy compromise in bovine aortic ECs exposed to shear stress, a decrease in glucose uptake and EC glycolysis attenuated ATP production. We hypothesized that decreased glycolysis-dependent purinergic signaling via P2Y1 (P2Y purinoceptor 1) receptors, secondary to impaired autophagy in ECs, prevents shear-induced phosphorylation of eNOS (endothelial nitric oxide synthase) at its positive regulatory site S1117 (p-eNOSS1177) and NO generation. Maneuvers that restore glucose transport and glycolysis (eg, overexpression of GLUT1 [glucose transporter 1]) or purinergic signaling (eg, addition of exogenous ADP) rescue shear-induced p-eNOSS1177 and NO production in ECs with impaired autophagy. Conversely, inhibiting glucose transport via GLUT1 small interfering RNA, blocking purinergic signaling via ectonucleotidase-mediated ATP/ADP degradation (eg, apyrase), or inhibiting P2Y1 receptors using pharmacological (eg, MRS2179 [2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetrasodium salt]) or genetic (eg, P2Y1-receptor small interfering RNA) procedures inhibit shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Supporting a central role for PKC?T505 (protein kinase C delta T505) in relaying the autophagy-dependent purinergic-mediated signal to eNOS, we find that (1) shear stress-induced activating phosphorylation of PKC?T505 is negated by inhibiting autophagy, (2) shear-induced p-eNOSS1177 and NO generation are restored in autophagy-impaired ECs via pharmacological (eg, bryostatin) or genetic (eg, constitutively active PKC?) activation of PKC?T505, and (3) pharmacological (eg, rottlerin) and genetic (eg, PKC? small interfering RNA) PKC? inhibition prevents shear-induced p-eNOSS1177 and NO generation in ECs with intact autophagy. Key nodes of dysregulation in this pathway on autophagy compromise were revealed in human arterial ECs. CONCLUSIONS:Targeted reactivation of purinergic signaling and PKC? has strategic potential to restore compromised NO generation in pathologies associated with suppressed EC autophagy.

Dataset Information

Shear Stress Ameliorates Superoxide Impairment to Erythrocyte Deformability With Concurrent Nitric Oxide Synthase Activation.

Publications

Shear Stress Ameliorates Superoxide Impairment to Erythrocyte Deformability With Concurrent Nitric Oxide Synthase Activation.

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