Dataset Information

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells.

ABSTRACT:

Aim

To investigate RBC-NOS dependent NO signaling during in vivo RBC aging in health and disease.

Method

RBC from fifteen healthy volunteers (HC) and four patients with type 2 diabetes mellitus (DM) were separated in seven subpopulations by Percoll density gradient centrifugation.

Results

The proportion of old RBC was significantly higher in DM compared to HC. In both groups, in vivo aging was marked by changes in RBC shape and decreased cell volume. RBC nitrite, as marker for NO, was higher in DM and increased in both HC and DM during aging. RBC deformability was lower in DM and significantly decreased in old compared to young RBC in both HC and DM. RBC-NOS Serine1177 phosphorylation, indicating enzyme activation, increased during aging in both HC and DM. Arginase I activity remained unchanged during aging in HC. In DM, arginase I activity was significantly higher in young RBC compared to HC but decreased during aging. In HC, concentration of L-arginine, the substrate of RBC-NOS and arginase I, significantly dropped from young to old RBC. In DM, L-arginine concentration was significantly higher in young RBC compared to HC and significantly decreased during aging. In blood from healthy subjects, RBC-NOS activation was additionally inhibited by N5-(1-iminoethyl)-L-Ornithine dihydrochloride which decreased RBC nitrite, and impaired RBC deformability of all but the oldest RBC subpopulation.

Conclusion

This study first-time showed highest RBC-NOS activation and NO production in old RBC, possibly to counteract the negative impact of cell shrinkage on RBC deformability. This was even more pronounced in DM. It is further suggested that highly produced NO only insufficiently affects cell function of old RBC maybe because of isolated RBC-NOS in old RBC thus decreasing NO bioavailability. Thus, increasing NO availability may improve RBC function and may extend cell life span in old RBC.

SUBMITTER: Bizjak DA

PROVIDER: S-EPMC4406474 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells.

Bizjak Daniel Alexander DA Brinkmann Christian C Bloch Wilhelm W Grau Marijke M

PloS one 20150422 4

<h4>Aim</h4>To investigate RBC-NOS dependent NO signaling during in vivo RBC aging in health and disease.<h4>Method</h4>RBC from fifteen healthy volunteers (HC) and four patients with type 2 diabetes mellitus (DM) were separated in seven subpopulations by Percoll density gradient centrifugation.<h4>Results</h4>The proportion of old RBC was significantly higher in DM compared to HC. In both groups, in vivo aging was marked by changes in RBC shape and decreased cell volume. RBC nitrite, as marker ...[more]

PMID: 25902315

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Project description:ObjectiveWe determined, for packed red blood cells (PRBC) and fresh frozen plasma, the maximum content, and ability to release the endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and monomethylarginine (LNMMA).BackgroundADMA and LNMMA are near equipotent NOS inhibitors forming blood's total NOS inhibitory content. The balance between removal from, and addition to plasma determines their free concentrations. Removal from plasma is by well-characterized specific hydrolases while formation is restricted to posttranslational protein methylation. When released into plasma they can readily enter endothelial cells and inhibit NOS. Fresh rat and human whole blood contain substantial protein incorporated ADMA however; the maximum content of ADMA and LNMMA in PRBC and fresh frozen plasma has not been determined.MethodsWe measured total (free and protein incorporated) ADMA and LNMMA content in PRBCs and fresh frozen plasma, as well as their incubation induced release, using HPLC with fluorescence detection. We tested the hypothesis that PRBC and fresh frozen plasma contain substantial inhibitory methylarginines that can be released chemically by complete in vitro acid hydrolysis or physiologically at 37°C by enzymatic blood proteolysis.ResultsIn vitro strong-acid-hydrolysis revealed a large PRBC reservoir of ADMA (54.5 ± 9.7 µM) and LNMMA (58.9 ± 28.9 ?M) that persisted over 42-d at 6° or -80°C. In vitro 5h incubation at 37°C nearly doubled free ADMA and LNMMNA concentration from PRBCs while no change was detected in fresh frozen plasma.ConclusionThe compelling physiological ramifications are that regardless of storage age, 1) PRBCs can rapidly release pathologically relevant quantities of ADMA and LNMMA when incubated and 2) PRBCs have a protein-incorporated inhibitory methylarginines reservoir 100 times that of normal free inhibitory methylarginines in blood and thus could represent a clinically relevant and proximate risk for iatrogenic NOS inhibition upon transfusion.

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

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells.

Aim

Method

Results

Conclusion

Publications

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells.

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