title: Effects of levcromakalim and nucleoside diphosphates on glibenclamide-sensitive K+ channels in pig urethral myocytes.
abstract: 1. Effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in pig proximal urethra were investigated by use of patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, cell-attached configuration and inside-out patches). 2. Levcromakalim produced a concentration-dependent outward current at a holding potential of -50 mV. The peak current amplitude showed little variation when measured by either conventional whole-cell or nystatin perforated patch configurations. 3. In conventional whole-cell configuration, the levcromakalim (100 microM)-induced outward current decayed by about 90% in 18 min. In contrast, with the nystatin perforated patch, approximately 86% of the levcromakalim-induced outward current still remained after 18 min. 4. The peak amplitude of the levcromakalim (100 microM)-induced outward membrane current recorded by the conventional whole-cell configuration was greatly reduced by inclusion of 5 mM EDTA in the pipette. The much smaller but significant outward membrane current remaining was abolished by glibenclamide. 5. In conventional whole-cell recordings, inclusion of an NDP in the pipette solution induced a small outward current which slowly reached a maximal amplitude (in 2 to 10 min) and was suppressed by glibenclamide. Addition of 100 microM levcromakalim after the NDP-induced current had peaked activated a further outward current which was larger than that recorded in the absence of NDPs. Approximately 50% of this current still remained at 18 min, even when conventional whole-cell configuration was used. 6. In the cell-attached mode in symmetrical 140 mM K+ conditions, glibenclamide inhibited the 100 microM levcromakalim-activated 43 pS K+ channel in a concentration-dependent manner, showing an inhibitory dissociation constant (Ki) of approximately 520 nM. 7. In inside-out patches in which the glibenclamide-sensitive K+ channel had run down after exposure to levcromakalim, both uridine 5'-diphosphate (UDP) and MgATP were capable of reactivating the channel. Further application of Mg2+ to the UDP-reactivated K+ channels enhanced the channel activity reversibly. 8. In inside-out patches UDP was capable of activating the glibenclamide-sensitive K+ channel without levcromakalim, providing that there was free Mg2+ present (either UDP in 5 mM EGTA or UDP in 5 mM EDTA with Mg2+). Additional application of levcromakalim caused a further reversible activation of channel opening. 9. In the presence of levcromakalim, application of adenosine 5'-triphosphate (ATP) to the inner surface of the membrane patch inhibited UDP-reactivated channel opening in a concentration-dependent manner. 10. Addition of an untreated cytosolic extract of pig proximal urethra reactivated the glibenclamide-sensitive K+ channel in the presence of 100 microM levcromakalim in inside-out patches. 11. These results demonstrate the presence in the pig proximal urethra of a glibenclamide-sensitive K+ channel that is blocked by intracellular ATP and can be activated by levcromakalim. Intracellular UDP can reactivate the channel after rundown. Additionally, intracellular Mg2+ may play an important role in regulating the channel activity.
authors.affiliation: University Department of Pharmacology, Oxford.
authors.forename: N
authors.initials: N
authors.lastname: Teramoto
authors.forename: G
authors.initials: G
authors.lastname: McMurray
authors.forename: A F
authors.initials: AF
authors.lastname: Brading
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology*; D019806:Cromakalim; D005260:Female; D005905:Glyburide / Q000494:pharmacology*; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015221:Potassium Channels / Q000187:drug effects*; D011758:Pyrroles / Q000494:pharmacology*; D013552:Swine; D014521:Urethra / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014530:Uridine Diphosphate / Q000494:pharmacology*
chemical_list: D001578:Benzopyrans; D015221:Potassium Channels; D011758:Pyrroles; D019806:Cromakalim; D014530:Uridine Diphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium; D005905:Glyburide