Effects of Nitric Oxide on Voltage-Gated K+ Currents in Human Cardiac Fibroblasts through the Protein Kinase G and Protein Kinase A Pathways but Not through S-Nitrosylation

Figures

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  Figure 1. Identification of two types of voltage-gated K+ (KV) channels in human cardiac fibroblasts by reverse transcription polymerase chain reaction (RT-PCR). (A) Strong gene expression was shown for KV1.1, KV1.2 KV1.5, and KV3.1 for delayed rectifier K+ (IK) channels and (B) KV3.3 and KV3.4 for transient outward K+ (Ito) channels. Weak expression was observed for KV1.6, KV1.7 and KV2.1 (for IK) and KV1.4, KV4.1, KV4.2, and KV4.4 (for Ito). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a positive control.

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  Figure 2. Effect of 4-aminopyridine (4-AP) on KV currents in HCFs. (A) The representative rapid activated and slow inactivated, IK recorded in a single HCF in control conditions and after application of 4-AP (1 or 10 mM). The current-voltage (I-V) relationship of steady-state currents with IK changed in the presence of 4-AP, and the concentration-response bar graphs for the 4-AP effect on IK are shown. (B) Typical rapid-activated and inactivated Ito in control and after 4-AP (1 or 10 mM) addition. The I-V relationship of peak currents changed by the presence of 4-AP on Ito and concentration-response bar graphs are shown. * p < 0.05, ** p < 0.01 versus the control.

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  Figure 3. Effect of tetraethylammonium chloride (TEA) on KV currents in HCFs. (A) The representative IK recorded in a single HCF in control conditions and after application of TEA (1 or 10 mM) are shown. The I-V relationship of the steady-state current change of IK by TEA and bar graphs for concentration change are also shown. (B) The typical Ito was not changed by TEA. The I-V relationship of the peak current change for the TEA of Ito and bar graph for concentration response are shown. * p < 0.05 versus the control.

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  Figure 4. Effect of nitric oxide (NO) on two subtypes of KV currents. Raw data and the I-V relationship of steady-state currents changed by the presence of SNAP on (A) delayed rectifier K+ current (IK) and (B) transient outward K+ current (Ito). (C) The concentration-response curve of S-nitroso-N-acetylpenicillamine (SNAP) on IK. The continuous line represents the fit to the Hill equation. The normalized currents (percent inhibition) were calculated from the IK in the absence of SNAP and plotted against various concentrations of SNAP. * p < 0.05 versus the control.

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  Figure 5. Effects of the PKG pathway on the stimulating effect of NO on IK. Representative currents showing the effect of SNAP on IK after pretreatment with (A) 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or (B) KT5823. (C) Effect of 8-Br-cyclic GMP on IK. (D) Bar graph summarizing current density changes for the effects of SNAP on IK after pre-incubation with ODQ or KT5823, and the effect of 8-Br-cyclic GMP on IK current. * p < 0.05 versus the control.

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  Figure 6. Effects of the protein kinase A (PKA) pathway on the stimulating effect of NO on IK current. Representative currents showing the effect of SNAP on IK after pretreatment with (A) KT5720 or (B) SQ22536. Effect of (C) forskolin or (D) 8-Br-cyclic AMP on IK. (E) Bar graph summarizing current density changes for the effects of SNAP on IK after pre-incubation with KT5720 or SQ22536, and the effect of forskolin or 8-Br-cAMP on IK. * p < 0.05, ** p < 0.01 versus the control.

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  Figure 7. Influence of S-nitrosylation on the stimulating effect of NO on IK. (A) Representative currents showing the effect of SNAP on IK after pretreatment with N-ethylmaleimide (NEM). (B) Effect of DL-dithiothreitol (DTT) on SNAP-stimulating IK. (C) Bar graph summarizing current density changes for SNAP effects on IK after pretreatment with NEM and DTT. * p < 0.05 versus the control.