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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

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

¹Department of Cardiology, Royal North Shore Hospital, Sydney, Australia; and ²Department of Medicine, University of Sydney, Sydney, Australia

Submitted 8 June 2007 ; accepted in final form 30 November 2007

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 (I_p). Mean I_p 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 1H-[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 I_p with 100 µmol/l paraquat included in patch pipette solutions. This decreased I_p 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.

myocyte; nitric oxide; superoxide; oxidative stress

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