Nitric oxide (NO)-induced activation of large conductance Ca 2+ -dependent K + channels (BK Ca ) in smooth muscle cells isolated from the rat mesenteric artery

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 [Ca 2+ ] i . The channels had a linear slope conductance of 212 (cid:43) 5 pS ( n =12) in symmetrical (140 m M ) 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 m M ) applied to the intracellular face of the channel produced a voltage-dependent block and external tetraethylammonium (TEA; at 1 m M ) 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 (cid:109) M ) produced an increase in BK Ca activity. In inside-out patches, BK Ca activity was also enhanced in some patches with 100 or 200 (cid:109) M 3-morpholino- sydnonimine (SIN-1) (4/11) and 100 (cid:109) M 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 (cid:109) M SIN-1 failed to increase BK Ca activity (in all 4 patches tested), while at a higher (500 (cid:109) M ) concentration SIN-1 had a direct blocking e(cid:128)ect on the channels ( n =3). NO applied directly to inside-out patches increased ( P (cid:53) 0.05) BK Ca activity in two patches. 5 In the majority of cells (6 out of 7), application of NO (45 or 67 (cid:109) M ) evoked an increase in the amplitude of whole-cell currents in perforated patches. This action was not a(cid:128)ected 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 (cid:109) M ). With SIN-1, the increase in current was blocked with the BK Ca channel blocker, iberiotoxin (50 n M ). 6 With conventional whole-cell voltage clamp, the increase in the outward K + current evoked with SIN-1 (50– 300 (cid:109) M ) showed considerable variability. Either no e(cid:128)ect was obtained (11 out of 18 cells), or in the remaining cells, an average increase in current amplitude of 38.7 (cid:43) 10.2% was recorded at 40 mV. 7 In cell-attached patches, large conductance voltage-dependent K + channels were stimulated by SIN-1 (100 (cid:109) M ) 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.