Opposing effects of coupled and uncoupled NOS activity on the Na+-K+ pump in cardiac myocytes.
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 O(2)(.-), 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 micromol/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 micromol/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 O(2)(.-)-sensitive dye dihydroethidium compared with control (n = 9; P < 0.05). To examine the functional effects of uncoupling, we measured I(p) with 100 micromol/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 O(2)(.-)-mediated pump inhibition.
Department of Cardiology, Royal North Shore Hospital, Sydney, Australia.
C N
CN
White
E J
EJ
Hamilton
A
A
Garcia
D
D
Wang
K K M
KK
Chia
G A
GA
Figtree
H H
HH
Rasmussen
D000818:Animals; D001120:Arginine / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004996:Ethidium / Q000031:analogs & derivatives; D019793:Fluorescein; D005456:Fluorescent Dyes; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018613:Microscopy, Confocal; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000187:drug effects / Q000378:metabolism*; D010269:Paraquat / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology
C414689:4,5-diaminofluorescein diacetate; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D014475:Uncoupling Agents; C067883:dihydroethidium; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D006162:Guanylate Cyclase; D000254:Sodium-Potassium-Exchanging ATPase; D004996:Ethidium; D010269:Paraquat; D019793:Fluorescein