Nitric oxide (NO)-induced activation of large conductance Ca²⁺-dependent K⁺ channels (BK_Ca) in smooth muscle cells isolated from the rat mesenteric artery

Abstract

1. To assess the action of nitric oxide (NO) and NO-donors on K⁺ current evoked either by voltage ramps or steps, patch clamp recordings were made from smooth muscle cells freshly isolated from secondary and tertiary branches of the rat mesenteric artery.

2. Inside-out patches contained channels, the open probability of which increased with [Ca²⁺]_i. The channels had a linear slope conductance of 212±5 pS (n=12) in symmetrical (140 mM) K⁺ solutions which reversed in direction at 4.4 mV. In addition, the channels showed K⁺ selectivity, in that the reversal potential shifted in a manner similar to that predicted by the Nernst potential for K⁺. Barium (1 mM) applied to the intracellular face of the channel produced a voltage-dependent block and external tetraethylammonium (TEA; at 1 mM) caused a large reduction in the unitary current amplitude. Taken together, these observations indicate that the channel most closely resembled BK_Ca.

3. In five out of six inside-out patches, NO (45 or 67 μM) produced an increase in BK_Ca activity. In inside-out patches, BK_Ca activity was also enhanced in some patches with 100 or 200 μM 3-morpholino-sydnonimine (SIN-1) (4/11) and 100 μM sodium nitroprusside (SNP) (3/8). The variability in channel opening with the NO donors may reflect variability in the release of NO from these compounds.

4. In inside-out patches, 100 μM SIN-1 failed to increase BK_Ca activity (in all 4 patches tested), while at a higher (500 μM) concentration SIN-1 had a direct blocking effect on the channels (n=3). NO applied directly to inside-out patches increased (P<0.05) BK_Ca activity in two patches.

5. In the majority of cells (6 out of 7), application of NO (45 or 67 μM) evoked an increase in the amplitude of whole-cell currents in perforated patches. This action was not affected by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). An increase in whole-cell current was also evoked with either of the NO donors, SIN-1 or SNP (each at 100 μM). With SIN-1, the increase in current was blocked with the BK_Ca channel blocker, iberiotoxin (50 nM).

6. With conventional whole-cell voltage clamp, the increase in the outward K⁺ current evoked with SIN-1 (50–300 μM) showed considerable variability. Either no effect was obtained (11 out of 18 cells), or in the remaining cells, an average increase in current amplitude of 38.7±10.2% was recorded at 40 mV.

7. In cell-attached patches, large conductance voltage-dependent K⁺ channels were stimulated by SIN-1 (100 μM) applied to the cell (n=5 patches).

8. These data indicate that NO and its donors can directly stimulate BK_Ca activity in cells isolated from the rat mesenteric artery. The ability of NO directly to open BK_Ca channels could play an important functional role in NO-induced relaxation of the vascular smooth muscle cells in this small resistance artery.

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