Nitric oxide (NO) derived from endothelial cell NO synthase (eNOS) is a critical regulator of vascular function. Red blood cells (RBCs) also express eNOS but it remains controversial whether this enzyme is functional and whether any NO export from RBCs can escape scavenging by hemoglobin. Arginase, an enzyme that reciprocally regulates NO production by competitively metabolizing the eNOS substrate arginine to ornithine and urea, is also expressed in RBCs. Arginase inhibitors reduce myocardial infarct size in vivo due to a shift in the metabolism of arginine from arginase to NOS. However, it is unknown whether arginase controls NO formation in RBCs.
Aim: To test the hypothesis that arginase in RBCs is an important regulator of RBC eNOS and NO production that mediates protection against myocardial ischemia-reperfusion injury. Methods: Rat and mouse hearts were isolated, buffer perfused and subjected to global ischemia and reperfusion. Left ventricular pressures were recorded. Vehicle and the arginase inhibitor N-hydroxy-nor-arginine (nor-NOHA 1 mM) were diluted in buffer, whole blood, RBCs+plasma or plasma and injected at the start of ischemia. The involvement of NOS was evaluated using pharmacological blockade (L-NAME 0.1 mM) or eNOS-/- or wild type mice. Arginase level and activity in RBCs and nitrite/nitrate export were also determined.
Results: Rodent and human RBCs contain functional arginase 1. Inhibition of arginase caused a 4- to 5-fold increase (P<0.05) in export of nitrite/nitrate from RBCs of wild type but not eNOS-/- mice. Inhibition of arginase significantly improved post-ischemic functional recovery in rat hearts if administered in whole blood or with RBCs in plasma (Fig. 1A). By contrast, arginase inhibition did not improve post-ischemic recovery when administered with buffer or plasma alone (Fig. 1B). The protective effect of arginase inhibition was lost in the presence of a NOS inhibitor and when given with blood from eNOS-/- mice (Fig. 1C).
Figure 1. Effect of vehicle and the arginase inhibitor nor-NOHA on recovery of left ventricular develop pressure (LVDP) in rat (panel A and B) and mouse (panel C) hearts during reperfusion following global ischemia. Vehicle and nor-NOHA were administered to rat hearts in red blood cells (RBC) + plasma (panel A) or plasma only (panel B) at the onset of ischemia. In panel C blood from eNOS-/- mice were given with vehicle or nor-NOHA to wild type mouse hearts. Recovery of LVDP during reperfusion is expressd in percentage of baseline; n=6–8.
Conclusion: The present study demonstrates a novel role of arginase 1 in control of eNOS function in RBCs. Inhibition of arginase unravels an important functional effect of RBC-derived NO that mediates protection against myocardial ischemia-reperfusion injury. These observations provide evidence for an important regulatory role of RBCs in control of NO bioactivity with pathophysiological consequences in ischemia-reperfusion.
