Effects of levcromakalim and nucleoside diphosphates on glibenclamide‐sensitive K⁺ channels in pig urethral myocytes

Abstract

1 E ects 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 con®guration, nystatin perforated patch, cell-attached con®guration and inside-out patches). 2 Levcromakalim produced a concentration-dependent outward current at a holding potential of 750 mV. The peak current amplitude showed little variation when measured by either conventional whole-cell or nystatin perforated patch con®gurations. 3 In conventional whole-cell con®guration, the levcromakalim (100 mM)-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 mM)-induced outward membrane current recorded by the conventional whole-cell con®guration was greatly reduced by inclusion of 5 mM EDTA in the pipette. The much smaller but signi®cant 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 mM 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 con®guration was used. 6 In the cell-attached mode in symmetrical 140 mM K + conditions, glibenclamide inhibited the 100 mM levcromakalim-activated 43 pS K + channel in a concentration-dependent manner, showing an inhibitory dissociation constant (K i ) 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 Mg 2+ 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 Mg 2+ present (either UDP in 5 mM EGTA or UDP in 5 mM EDTA with Mg 2+ ). 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 glibenclamidesensitive K + channel in the presence of 100 mM 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 levcromaka...

Characterization of outward K+currents in isolated smooth muscle cells from sheep urethra

“…The only other single studies that have been carried out were in the pig, in which an ATP-sensitive K ϩ current has been found (25)(26)(27). There is evidence that this current may contribute to the resting membrane conductance (27). However, because this current was neither voltage nor Ca 2ϩ sensitive, it is unlikely to play a part in determining the shape of the slow wave, which in any case has not been demonstrated in the pig (26).…”

“…However, because so far there have been no electrophysiological studies of the human urethra, the validity of different animal models remains to be established. The only other single studies that have been carried out were in the pig, in which an ATP-sensitive K ϩ current has been found (25)(26)(27). There is evidence that this current may contribute to the resting membrane conductance (27).…”

“…Both currents contributed to the generation of spontaneous slow waves in isolated cells, similar in configuration to those found in whole tissue recordings (10,13,14,17). However, with the exception of one group that has described an ATPsensitive K ϩ channel in the pig (25)(26)(27), so far there has been no attempt to study the K ϩ currents present in the urethra. This is an important omission, because the shape of the action potential in other smooth muscles is modified by both voltage-dependent and Ca 2ϩ -dependent K ϩ currents that can act to attenuate the spike and shorten the plateau and, therefore, reduce contraction amplitude (21,29,33).…”

Levcromakalim and MgGDP Activate Small Conductance ATP-Sensitive K+ Channels of K+ Channel Pore 6.1/Sulfonylurea Receptor 2A in Pig Detrusor Smooth Muscle Cells: Uncoupling of cAMP Signal Pathways

“…In accordance with previous studies, it has been shown that K ATP channels in pig urethra are activated by cromakalim and intracellular NDPs and inhibited by glibenclamide (Teramoto et al, 1997). On the other hand, K ATP channel properties in pig detrusor smooth muscle cells are poorly understood.…”

“…Effect of Intracellular MgGDP and cAMP on the Levcromakalim-Induced Outward Current. In a wide variety of preparations, intracellular NDPs such as GDP, UDP, IDP, and ADP have been reported to augment K ATP channel activity (Kajioka et al, 1991;Beech et al, 1993;Kamouchi and Kitamura, 1994;Teramoto et al, 1997). In addition, protein kinase (PK)A and/or PKC have been reported to activate a levcromakalim-sensitive K ϩ channel, presumably K ATP (Standen et al, 1998).…”