Erythrocytes may synthesize their own nitric oxide

Abstract

The ability of hemoglobin and myoglobin to bind nitric oxide (NO) produced by other cells and circulating vasodilators is well known. To characterize erythrocytes (RBCs), we used NADPH diaphorase labeling, as well as antibodies to the three known types of NO synthase (NOS 1, 2, and 3), and to a molecule usually associated with NOS, calmodulin. We show that the NADPH-diaphorase label labels myenteric neurons, endothelial cells, and the blood cells trapped in the lumen of the blood vessels running through the intestinal wall. The myenteric neurons are also positive for neuronal NOS (NOS1), calmodulin, and neuropeptide Y, indicating that they are NO-producing neurons. Endothelial cells are positive for NOS3 (a constitutive form of NOS), while macrophages and lymphocytes are positive for NOS2 (an inducible form of NOS). All are positive for calmodulin. Surprisingly, the RBCs are positive for NOS2 and 3, as well as calmodulin. Thus the RBCs possess all the cellular machinery to synthesize their own NO. We suggest that erythrocytes would synthesize and use NO to modulate their own physiology.

Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

“…Two earlier studies suggested that RBCs also contain iNOS. 20,25 We are unable to confirm these reports as we failed to isolate any iNOS from crude human RBC lysates of healthy donors using specific anti-NOS2 antibodies for immunoprecipitation.Although a L-NNA/L-NAME-inhibitable NOS activity was demonstrated by measuring NO metabolites or citrulline in the supernatant of intact erythrocytes 2,15,16,19 and RBC membranes,16,23 2 reports suggested RBCs harbor an inactive NOS. 20,22 It was important to confirm, therefore, that the protein identified as eNOS in our study was catalytically active, L-arginine:citrulline conversion was dependent on the presence of Ca 2ϩ /calmodulin, and activity was inhibited by L-NAME.…”

“…RBCs contain both a constitutive Ca 2ϩ -dependent NOS containing epitopes of an eNOS 6,15,16,25 and an active XOR. 6 Thus, enzymatic NO synthesis in RBCs might occur either via NOS-catalyzed L-arginine to citrulline conversion 15,16,18 or by XOR-mediated nitrite reduction.…”

“…6,15,16,20,[23][24][25] However, positive staining with anti-iNOS antibodies has also been reported, 20,25 and others suggested that RBCs might express a novel NOS isoform. 23,25 Thus, doubts about the functional significance of the NOS-like activity in RBCs remain, and little is known about NOS isoform identity and disease relevance.We therefore sought to definitively identify and characterize the activity of human red cell NOS using an advanced multilevel analytical approach. Using HPLC, LC-MS/MS, flow cytometry, laser scanning microscopy, and enzymatic assay together with functional studies we here demonstrate that human RBCs contain an active eNOS that gives rise to constitutive intracellular NO formation under normoxic conditions.…”

“…on May 10, 2018. by guest www.bloodjournal.org From protein with eNOS epitopes. Two groups hypothesized that RBCs express a novel NOS isoform, 23,25 but no sequence confirmation analysis was provided.The main technical hurdle in the biochemical characterization of proteins in RBCs is the presence of overwhelmingly high level of hemoglobin, which represents a formidable obstacle to purification, activity determination, immunostaining, and colorimetric/ fluorimetric assay. In this study, we chose to isolate NOS from hemolysates under native conditions to permit combined structural and functional analysis.…”

Red blood cell K+ could be a marker of K+ changes in other cells involved in blood pressure regulation

“…33 Red blood cells, although lacking a nucleus, are useful models for membrane transport studies 34,35 and share membrane properties related to VSMC and the endothelium. RBC, which are equipped with IK Ca and inducible nitric oxide synthase, 36,37 respond to K Ca activators producing vasodilatation 7 or to 8Br-cGMP (Delgado et al, 2002, unpublished data) in a manner similar to the VSMC with the activation of K Ca and the efflux of K + . 8 Consequently, K + changes in RBC could be a marker for K + changes in other cells such as VSMC and the endothelium.…”