Dual modulation of swelling-activated chloride current by NO and NO donors in rabbit portal vein myocytes

Abstract

1. The effects of authentic NO and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) on swelling-activated chloride currents (Iswell) were investigated in freshly dispersed rabbit portal vein smooth muscle cells. Iswell was recorded with the perforated patch configuration of the whole-cell patch clamp technique. 2. In approximately 50 % of cells NO and SNAP inhibited the amplitude of Iswell by about 45 % in a voltage-independent manner. Iswell was also inhibited by an inhibitor of NO-sensitive guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and by KT5823, an inhibitor of cGMP-dependent protein kinase. 3. In other cells both NO and SNAP enhanced Iswell by about 40 % in a voltage-independent manner. A similar increase was produced by application of the cell-permeable cGMP analogue 8-bromo-guanosine 3', 5'-cyclic monophosphate (8-Br-cGMP). However, 8-Br-cGMP had no effect on current amplitude in cells pre-treated with KT5823. In contrast 8-Br-cGMP increased the amplitude of Iswell in cells which had been pre-treated with ODQ. 4. SNAP also modulated Iswell recorded in the conventional whole-cell configuration with internal solutions containing 10 mM EGTA to rule out any contribution from Ca2+-activated Cl- currents. 5. These data suggest that the amplitude of Iswell can be enhanced by NO via a cGMP-dependent phosphorylation and inhibited by NO in a cGMP-independent manner.

Figure 1

Inhibitory effect of NO on I_swell A, representative trace showing a control cell where I_swell developed following exposure of a single portal vein smooth muscle cell to hypotonic solution. Note that the current is sustained throughout the application of hypotonic solution. B, the inhibitory effect of NO on I_swell. The concentration of NO was about 1 μm (see Methods) and in A and B large deflections represent the ramp protocol from a holding potential of −50 mV. C, the mean time dependence of the NO-induced inhibition shown at −50 mV (○) and +100 mV (•). Currents were normalised to peak I_swell prior to application of NO. Each point is the mean ± s.e.m. of 5 cells.

Figure 2

Inhibitory effect of SNAP on I_swell A, the mean time dependence of the SNAP-induced inhibition of I_swell shown at −50 mV (○) and +100 mV (•). Each point is the mean ±s.e.m. of 15 cells. B, comparison of the maximum SNAP- and NO-induced inhibition of I_swell recorded at −50 mV. Currents were normalised to peak I_swell prior to application of SNAP or NO.

Figure 3

Cells in which SNAP and NO increased I_swell A, typical cell showing the increase of I_swell by 10 μm SNAP. B, time dependence of the potentiating effect of SNAP (○) and NO (•) on I_swell recorded at −50 mV. Each point is the mean ±s.e.m. of 9 cells for SNAP and 6 cells for NO.

Figure 4

Effect of 8-Br-cGMP on I_swell A, record illustrating the excitatory effect of 100 μm 8-Br-cGMP on I_swell. B, time dependence of the 100 μm 8-Br-cGMP-induced increase shown at −50 mV (○) and +100 mV (•). Each time point is the mean ±s.e.m. of 6 cells. C, comparison of the potentiating effect of SNAP, NO and 8-Br-cGMP on I_swell recorded at −50 mV. Currents were normalised to peak I_swell prior to application of SNAP, NO or 8-Br-cGMP and the number of cells is shown in parentheses.

Figure 5

Effect of ODQ and KT5823 on I_swell A, the inhibitory effect of 10 μm ODQ on I_swell. B, the inhibitory effect of 1 μm KT5823 on I_swell. C, comparison of ODQ- and KT5823-induced inhibition of I_swell recorded at −50 mV. Currents were normalised to peak I_swell prior to application of ODQ or KT5823.

Figure 6

Effect of KT5823 on the modulation of I_swell by 8-Br-cGMP and effect of ODQ on the action of SNAP A, representative trace showing the inhibitory effect of 1 μm KT5823. Application of 8-Br-cGMP had no effect on I_swell in the continued presence of KT5823. B, representative trace showing the combined inhibitory effect of 10 μm ODQ and 10 μm SNAP.