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. 2005 Jun 15;565(Pt 3):815-25.
doi: 10.1113/jphysiol.2005.086447. Epub 2005 Apr 7.

The nitric oxide donor sodium nitroprusside stimulates the Na+-K+ pump in isolated rabbit cardiac myocytes

Maged William  1 , Jimmy Vien, Elisha Hamilton, Alvaro Garcia, Henning Bundgaard, Ronald J Clarke, Helge H Rasmussen
Affiliations

The nitric oxide donor sodium nitroprusside stimulates the Na+-K+ pump in isolated rabbit cardiac myocytes

Maged William et al. J Physiol. .

Abstract

Nitric oxide (NO) affects the membrane Na(+)-K(+) pump in a tissue-dependent manner. Stimulation of intrinsic pump activity, stimulation secondary to NO-induced Na(+) influx into cells or inhibition has been reported. We used the whole-cell patch clamp technique to measure electrogenic Na(+)-K(+) pump current (I(p)) in rabbit ventricular myocytes. Myocytes were voltage clamped with wide-tipped patch pipettes to achieve optimal perfusion of the intracellular compartment, and I(p) was identified as the shift in holding current induced by 100 microm ouabain. The NO donor sodium nitroprusside (SNP) in concentrations of 1, 10, 50 or 100 microm induced a significant increase in I(p) when the intracellular compartment was perfused with pipette solutions containing 10 mm Na(+), a concentration near physiological levels. SNP had no effect when the pump was near-maximally activated by 80 mm Na(+) in pipette solutions. Stimulation persisted in the absence of extracellular Na(+), indicating its independence of transmembrane Na(+) influx. The SNP-induced pump stimulation was abolished by inhibition of soluble guanylyl cyclase (sGC) with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, by inhibition of protein kinase G (PKG) with KT-5823 or by inhibition of protein phosphatase with okadaic acid. Inclusion of the non-hydrolysable cGMP analogue 8pCPT-cGMP, activated recombinant PKG or the sGC-activator YC-1 in patch pipette filling solutions reproduced the SNP-induced pump stimulation. Pump stimulation induced by YC-1 was dependent on the Na(+) concentration but not the K(+) concentration in pipette filling solutions, suggesting an altered sensitivity of the Na(+)-K(+) pump to intracellular Na(+).

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Figures

Figure 1
Figure 1. Sodium dependence of ouabain-induced shifts in holding currents
Patch pipette filling solutions and superfusates included 10 and 150 mm Na+ for experiments shown in A, 0 and 150 mm for B and 10 and 0 mm for experiments shown in C. The traces show holding currents before and after exposure to ouabain (Oua). The membrane capacitance, Cm (in pF) is indicated to facilitate comparisons.
Figure 2
Figure 2. Effect of SNP on Na+–K+ pump current
The Na+ concentration in patch pipette filling solutions was 10 mm in all experiments. Mean (± s.e.m.) Ip values recorded in a superfusate that included 150 mm Na+ (A) or was Na+ free (B) are shown. The extracellular Na+ concentration ([Na+]o) is indicated in the figure. The number of experiments is indicated in parentheses. *P < 0.05.
Figure 3
Figure 3. Dependence of Na+–K+ pump current on the concentration of SNP
The Na+ concentration in patch pipette filling solutions was 10 mm. The superfusate contained 150 mm Na+ and 5.6 mm K+. Superoxide dismutase (SOD) was included in the patch pipette solution as indicated. Mean (± s.e.m.) Ip values at different concentrations of SNP are indicated. The numbers of experiments are indicated in parenthesis. Asterisk indicates a statistically significant SNP-induced increase in Ip.
Figure 4
Figure 4. Intracellular messenger pathways linking SNP to Na+–K+ pump stimulation
Myocytes were superfused with control solutions or solutions containing SNP. Patch pipettes contained control solutions or solutions containing inhibitors of soluble guanylyl cyclase (ODQ), protein kinase G (KT-5823) or protein phosphatase (okadaic acid, OA). Numbers in parentheses indicate the number of myocytes studied with each set of conditions. * Significant difference between mean (± s.e.m.) Ip values of myocytes exposed or not exposed to SNP; # significant difference between Ip of myocytes exposed to SNP and patch clamped using control pipette filling solutions and solutions containing ODQ, KT-5823 or OA. Results of experiments using an SNP-free superfusate and ODQ, KT-5823 or OA in pipette solutions are also shown.
Figure 5
Figure 5. Effect of 8-pCPT-cGMP, TEA and recombinant PKG on Na+–K+ pump current
Myocytes were exposed to control superfusates, and the compounds indicated in the figure were included in patch pipette solutions. Numbers in parentheses indicate the number of myocytes for each set of experimental conditions. Asterisk indicates a significant difference (P < 0.05) between Ip values of myocytes patch clamped using control patch pipette filling solutions and solutions containing 8-pCPT-cGMP, TEA. The protein kinase A inhibitor H-89 did not prevent the activation of Ip induced by the highest concentration of 8-pCPT-cGMP,TEA. # Significant difference (P < 0.05) between Ip values of myocytes patch clamped using filling solutions containing 20 μm 8-pCPT-cGMP, TEA with or without recombinant bovine protein kinase G (PKG).
Figure 6
Figure 6. Effect of activation of soluble guanylyl cyclase with YC-1 on Na+–K+ pump current
Inclusion of 1 μm YC-1 in pipette solutions is indicated in the figure. A shows shifts in holding current induced by ouabain (Oua) recorded using Na+ at a concentration ([Na+]pip) of 10 mm Na+ in patch pipette solutions. The Na+ concentration in superfusates ([Na+]o) was 150 mm (upper traces) or 0 mm (lower traces). B summarizes mean (± s.e.m.) Ip values recorded in Na+-containing and Na+-free superfusates. C shows ouabain-induced shifts in holding current recorded using 80 mm Na+ in patch pipette solutions, recorded in Na+-containing superfusates. D summarizes mean Ip values recorded using 80 mm Na+ in pipette solutions. Cm indicates membrane capacitance in pF. *P < 0.05.
Figure 7
Figure 7. Intracellular messenger pathways linking YC-1 to Na+–K+ pump stimulation
Patch pipettes contained control solutions or solutions that contained YC-1 and inhibitors of soluble guanylyl cyclase (ODQ), protein kinase G (KT-5823) or protein phosphatase (okadaic acid, OA) as indicated. The Na+ concentrations in pipette solutions and superfusates were 10 and 150 mm, respectively. Numbers in parentheses indicate the number of myocytes studied with each set of conditions. * Significant difference (*P < 0.05) between mean (± s.e.m.) Ip values of myocytes perfused or not perfused with pipette solutions containing YC-1; # significant difference (*P < 0.05) between mean Ip values of myocytes perfused with pipette solutions containing YC-1 alone and solutions containing both YC-1 and ODQ, KT-5823 or OA. Results of control experiments using ODQ, KT-5823 or OA alone are shown in Fig. 4.
Figure 8
Figure 8. Dependence of Na+–K+ pump stimulation induced by YC-1 on intracellular K+
Ip was measured in myocytes voltage clamped using control pipette filling solutions (^) or solutions containing YC-1 (•). The pipette filling solutions contained Na+ at a concentration of 10 mm and K+ at a concentration ([K+]pip) ranging from 0 to 140 mm. The numbers of experiments for each set of conditions are indicated in parentheses. Mean (± s.e.m.) Ip values for myocytes perfused with pipette solutions containing YC-1 were significantly (*P < 0.05) higher than for myocytes perfused with control solutions at all levels of [K]pip.
Figure 9
Figure 9. Dependence of Na+–K+ pump stimulation induced by YC-1 on intracellular Na+
Ip was measured in myocytes voltage clamped using control pipette filling solutions (▵) or solutions containing YC-1 (•). The pipette filling solutions contained Na+ at concentrations ranging from 0 to 80 mm. They were nominally K+ free. The numbers of experiments for each set of conditions are indicated in parentheses. Mean (± s.e.m.) Ip values for myocytes perfused with pipette solutions containing YC-1 were significantly higher than for myocytes perfused with control solutions when the pipette Na+ concentration was at rate-limiting levels.
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