Opposing effects of coupled and uncoupled NOS activity on the Na+-K+ pump in cardiac myocytes

Abstract

Pharmacological delivery of nitric oxide (NO) stimulates the cardiac Na+-K+ pump. However, effects of NO synthesized by NO synthase (NOS) often differ from the effects of NO delivered pharmacologically. In addition, NOS can become “uncoupled” and preferentially synthesize O2·−, which often has opposing effects to NO. We tested the hypothesis that NOS-synthesized NO stimulates Na+-K+ pump activity, and uncoupling of NOS inhibits it. To image NO, we loaded isolated rabbit cardiac myocytes with 4,5-diaminofluorescein-2 diacetate (DAF-2 DA) and measured fluorescence with confocal microscopy. l-Arginine (l-Arg; 500 μmol/l) increased DAF-2 DA fluorescence by 51% compared with control ( n = 8; P < 0.05). We used the whole cell patch-clamp technique to measure electrogenic Na+-K+ pump current ( Ip). Mean Ip of 0.35 ± 0.03 pA/pF ( n = 44) was increased to 0.48 ± 0.03 pA/pF ( n = 7, P < 0.05) by 10 μmol/l l-Arg in pipette solutions. This increase was abolished by NOS inhibition with radicicol or by NO-activated guanylyl cyclase inhibition with 1 H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. We next examined the effect of uncoupling NOS using paraquat. Paraquat (1 mmol/l) induced a 51% increase in the fluorescence intensity of O2·−-sensitive dye dihydroethidium compared with control ( n = 9; P < 0.05). To examine the functional effects of uncoupling, we measured Ip with 100 μmol/l paraquat included in patch pipette solutions. This decreased Ip to 0.28 ± 0.03 pA/pF ( n = 12; P < 0.001). The paraquat-induced pump inhibition was abolished by superoxide dismutase (in pipette solutions). We conclude that NOS-mediated NO synthesis stimulates the Na+-K+ pump, whereas uncoupling of NOS causes O2·−-mediated pump inhibition.