Effects of the BKCa channel activator, NS1619, on rat cerebral artery smooth muscle

1 We have investigated the actions of NS1619, a putative activator of large conductance calcium-activated potassium channels (BKCa) by use of the patch-clamp technique on smooth muscle cells enzymatically isolated from the rat basilar artery. 2 Using whole cell current-clamp to measure membrane potential, addition of 30 mu M NS1619 produced cellular hyperpolarization, moving the membrane potential towards the calculated equilibrium potential for potassium. This hyperpolarization was rapidly reversed by IbTX (100 nM), a selective inhibitor of BKCa. 3 In whole cell recordings made from cells voltage-clamped at 0 mV using the perforated-patch technique, addition of NS1619 (10-30 mu M) activated an outward current, which reversed following washout of NS1619. 4 This outward current was unaffected by application of either glibenclamide (5 mu M), an inhibitor of ATP-sensitive potassium channels, or apamin (100 nM), an inhibitor of small-conductance calcium-activated potassium channels. However, this current was almost completely abolished by iberiotoxin (IbTX; 50-100nM). 5 Depolarizing voltage steps activated small outward currents from cells held at -15 mV. Application of NS1619 (10-30 mu M) increased the size of these currents, producing a shift to the left of the current-voltage (I-V) relationship. These currents were largely inhibited by IbTX (100 nM). 6 Measurements of the unitary amplitude of the single channels activated by NS1619 which could be resolved in whole cell recordings yielded a value of 5.6+/-0.14 pA at 0 mV. 7 NS1619 (10-30 mu M) directly activated single channels contained in excised inside-out and outside-out membrane patches. In both configurations NS1619 (10-30 mu M) rapidly increased the open probability of a large conductance calcium-dependent channel. The activation produced by NS1619 was calcium-dependent and inhibited by external IbTX (100 nM). The unitary current amplitude was unaffected by NS1619. 8 By use of conventional whole cell recording methods and conditions that suppressed BKCa, openings, outward potassium currents were activated by depolarizing potentials positive to -35 mV from a holding potential of -65 mV. NS1619 (10-30 mu M) inhibited this current in a concentration-dependent manner. This inhibition was reversed following washout of NS1619, recovering to 60-90% of control values within 2 min. 9 Ba2+ currents, measured by conventional whole cell recording, were activated by depolarizing voltage steps from negative holding potentials. NS1619 (1-30 mu M) inhibited the evoked current in a concentration-dependent manner, yielding an IC50 value of 7 mu M with a Hill coefficient approaching unity. This inhibition was reversible, with the currents recovering to 65-100% of control values after washout of NS1619 for 2 min. 10 NS1619 (0.3-100 mu M) induced concentration-dependent relaxation of basilar artery segments contracted with histamine/5-HT (IC50=12.5+/-2.0 mu M; n=4). This relaxation curve was shifted to the right, but not abolished, when the tissue was treated with a blocker of BKCa channels (IbTX; 100nM). Additionally, NS1619 produced concentration-dependent relaxation of basilar artery contracted with a depolarizing, isotonic salt solution containing 80 mM K+. 11 Thus NS1619 produces hyperpolarization of basilar artery myocytes through direct activation of BKCa and also directly inhibits Ca2+ currents and voltage-activated K+ channels. We discuss the implications of these results for its vasorelaxant actions.