EFFECTS OF THE BKCA CHANNEL ACTIVATOR, NS1619, ON RAT CEREBRAL-ARTERYSMOOTH-MUSCLE

1 We have investigated the actions of NS1619, a putative activator of large conductance calcium-activated potassium channels (BKCa) by use o f the patch-clamp technique on smooth muscle cells enzymatically isola ted from the rat basilar artery. 2 Using whole cell current-clamp to m easure membrane potential, addition of 30 mu M NS1619 produced cellula r hyperpolarization, moving the membrane potential towards the calcula ted equilibrium potential for potassium. This hyperpolarization was ra pidly reversed by IbTX (100 nM), a selective inhibitor of BKCa. 3 In w hole 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 Thi s outward current was unaffected by application of either glibenclamid e (5 mu M), an inhibitor of ATP-sensitive potassium channels, or apami n (100 nM), an inhibitor of small-conductance calcium-activated potass ium 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 NS161 9 (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 unitar y 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 contain ed in excised inside-out and outside-out membrane patches. In both con figurations NS1619 (10-30 mu M) rapidly increased the open probability of a large conductance calcium-dependent channel. The activation prod uced by NS1619 was calcium-dependent and inhibited by external IbTX (1 00 nM). The unitary current amplitude was unaffected by NS1619. 8 By u se of conventional whole cell recording methods and conditions that su ppressed 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 N S1619, recovering to 60-90% of control values within 2 min. 9 Ba2+ cur rents, measured by conventional whole cell recording, were activated b y 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 appro aching unity. This inhibition was reversible, with the currents recove ring to 65-100% of control values after washout of NS1619 for 2 min. 1 0 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 hyperpolari zation of basilar artery myocytes through direct activation of BKCa an d also directly inhibits Ca2+ currents and voltage-activated K+ channe ls. We discuss the implications of these results for its vasorelaxant actions.