The theory that red blood cells (RBCs) generate and release nitric oxide (NO)-like bioactivity has gained considerable interest. However, it remains unclear whether it can be produced by endothelial NO synthase (eNOS), which is present in RBCs, and whether NO can escape scavenging by hemoglobin. The aim of this study was to test the hypothesis that arginase reciprocally controls NO formation in RBCs by competition with eNOS for their common substrate arginine and that RBC-derived NO is functionally active following arginase blockade. We show that rodent and human RBCs contain functional arginase 1 and that pharmacological inhibition of arginase increases export of eNOS-derived nitrogen oxides from RBCs under basal conditions. The functional importance was tested in an ex vivo model of myocardial ischemia-reperfusion injury. Inhibitors of arginase significantly improved postischemic functional recovery in rat hearts if administered in whole blood or with RBCs in plasma. By contrast, arginase inhibition did not improve postischemic recovery when administered with buffer solution or plasma alone. The protective effect of arginase inhibition was lost in the presence of a NOS inhibitor. Moreover, hearts from eNOS^−/− mice were protected when the arginase inhibitor was given with blood from wild-type donors. In contrast, when hearts from wild-type mice were given blood from eNOS^−/− mice, the arginase inhibitor failed to protect against ischemia-reperfusion. These results strongly support the notion that RBCs contain functional eNOS and release NO-like bioactivity. This process is under tight control by arginase 1 and is of functional importance during ischemia-reperfusion.

Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O₂C) were studied in awake mice lacking one or both of the adenosine A₁or A_2Areceptors (A₁R or A_2AR, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A₁R knockout (A₁RKO) mice but lower in A_2ARKO mice and intermediate in A₁-A_2AR double KO mice. A single dose of an unselective β-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A₁Rs had little effect on Temp, whereas deletion of A_2ARs increased it in females and decreased it in males. A₁-A_2ARKO mice had lower Temp than WT mice. LA was unaltered in A₁RKO mice and lower in A_2ARKO and A₁-A_2ARKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A_2AR. Caffeine ingestion also increased LA in an A_2AR-dependent manner in male mice. Caffeine ingestion significantly increased O₂C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A₁R or A_2AR blockade. Selective A_2Bantagonism had little or no effect. Thus A₁R and A_2AR influence HR, Temp, LA, and O₂C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A_2AR plays an important role in the modulation of O₂C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A₁R and A_2AR.

This study tested the hypothesis that red blood cell (RBC) arginase represents a potential therapeutic target in ischemia-reperfusion in type 2 diabetes. Post-ischemic cardiac recovery was impaired in hearts from db/db mice compared with wild-type hearts. RBCs from mice and patients with type 2 diabetes attenuated post-ischemic cardiac recovery of nondiabetic hearts. This impaired cardiac recovery was reversed by inhibition of RBCs arginase or nitric oxide synthase. The results suggest that RBCs from type 2 diabetics impair cardiac tolerance to ischemia-reperfusion via a pathway involving arginase activity and nitric oxide synthase-dependent oxidative stress.

Caffeine is believed to exert its stimulant effects by blocking A_2Aand A₁adenosine receptors (A_2AR and A₁R). Although a genetic knockout of A_2AR eliminates effects of caffeine, the phenotype of the knockout animal does not resemble that of caffeine treatment. In this study we explored the possibility that a mere reduction of the number of A₁Rs and A_2ARs, achieved by deleting one of the two copies of the A₁R and A_2AR genes, would mimic some aspects of long-term caffeine ingestion. The A₁R and A_2AR double heterozygous (A₁R-A_2AR dHz) mice indeed had approximately one-half the number of A₁R and A_2AR, and there were little compensatory changes in A_2Bor A₃adenosine receptor (A_2BR or A₃R) expression. The ability of a stable adenosine analog to activate receptors was shifted to the right by caffeine and in A₁R-A_2AR dHz tissue. Caffeine (0.3 g/l in drinking water for 7–10 days) and A₁R-A_2AR dHz genotype increased locomotor activity (LA) and decreased heart rate without significantly influencing body temperature. The acute stimulatory effect of a single injection of caffeine was reduced in A₁R-A_2AR dHz mice and in mice treated long term with oral caffeine. Thus at least some aspects of long-term caffeine use can be mimicked by genetic manipulation of the A₁R and A_2AR.

Red blood cells (RBCs) induce endothelial dysfunction in type 2 diabetes (T2D), but the mechanism by which RBCs communicate with the endothelium is unknown. This study tested the hypothesis that extracellular vesicles (EVs) secreted by RBCs act as mediators of endothelial dysfunction in T2D. Despite a lower production of EVs derived from RBCs of T2D patients (T2D RBC-EVs), their uptake by endothelial cells was greater than that of EVs derived from RBCs of healthy individuals (H RBC-EVs). T2D RBC-EVs impaired endothelium-dependent relaxation, and this effect was attenuated following inhibition of arginase in EVs. Inhibition of vascular arginase or oxidative stress also attenuated endothelial dysfunction induced by T2D RBC-EVs. Arginase-1 was detected in RBC-derived EVs, and arginase-1 and oxidative stress were increased in endothelial cells following coincubation with T2D RBC-EVs. T2D RBC-EVs also increased arginase-1 protein in endothelial cells following mRNA silencing and in the endothelium of aortas from endothelial cell arginase-1–knockout mice. It is concluded that T2D-RBCs induce endothelial dysfunction through increased uptake of EVs that transfer arginase-1 from RBCs to the endothelium to induce oxidative stress and endothelial dysfunction. These results shed important light on the mechanism underlying endothelial dysfunction mediated by RBCs in T2D.

Red blood cells (RBCs) mediate cardioprotection via nitric oxide–like bioactivity, but the signaling and the identity of any mediator released by the RBCs remains unknown. We investigated whether RBCs exposed to hypoxia release a cardioprotective mediator and explored the nature of this mediator. Perfusion of isolated hearts subjected to ischemia-reperfusion with extracellular supernatant from mouse RBCs exposed to hypoxia resulted in improved postischemic cardiac function and reduced infarct size. Hypoxia increased extracellular export of cyclic guanosine monophosphate (cGMP) from mouse RBCs, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on RBC-soluble guanylate cyclase and cGMP transport and was sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice to increase nitric oxide bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. In a placebo-controlled clinical trial, a clear cardioprotective, soluble guanylate cyclase–dependent effect was induced by RBCs collected from patients randomized to 5 weeks nitrate-rich diet. It is concluded that RBCs generate and export cGMP as a response to hypoxia, mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention, suggesting preventive and therapeutic opportunities in ischemic heart disease.