Abstract
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 microM 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 microM) activated an outward current, which reversed following washout of NS1619. 4. This outward current was unaffected by application of either glibenclamide (5 microM), 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 microM) 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 microM) directly activated single channels contained in excised inside-out and outside-out membrane patches. In both configurations NS1619 (10-30 microM) 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 microM) 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 microM) inhibited the evoked current in a concentration-dependent manner, yielding an IC50 value of 7 microM 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 microM) induced concentration-dependent relaxation of basilar artery segments contracted with histamine/5-HT (IC50 = 12.5 +/- 2.0 microM; 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.
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