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Background and Purpose: ATP-sensitive K(+)(K(ATP)) channels, which are composed of K(IR)6.x associated with sulphonylurea receptor (SUR) subunits, have been detected in native smooth muscle cells, but it is currently not known which of these is expressed in mouse vas deferens myocytes., Experimental Approach: Pharmacological and electrophysiological properties of K(ATP) channels in mouse vas deferens myocytes were investigated using patch clamp techniques. Molecular biological analyses were performed to examine the properties of these K(ATP) channels., Key Results: During conventional whole-cell recording, pinacidil elicited an inward current that was suppressed by glibenclamide, a sulfonylurea agent, and by U-37883A, a selective K(IR)6.1 blocker. When 0.3 mM ATP was added to the pipette solution, the peak amplitude of the pinacidil-induced current was much smaller than that recorded in its absence. When 3 mM UDP, GDP or ADP was included in the pipette solution, an inward current was elicited after establishment of the conventional whole-cell configuration, with potency order being UDP > GDP > ADP. These nucleoside diphosphate-induced inward currents were suppressed by glibenclamide. MCC-134, a SUR modulator, induced glibenclamide-sensitive K(ATP) currents that were similar to those induced by 100 μM pinacidil. In the cell-attached configuration, pinacidil activated channels with a conductance similar to that of K(IR)6.1. Reverse transcription PCR analysis revealed the expression of K(IR)6.1 and SUR2B transcripts and immunohistochemical studies indicated the presence of K(IR)6.1 and SUR2B proteins in the myocytes., Conclusions and Implications: Our results indicate that native K(ATP) channels in mouse vas deferens myocytes are a heterocomplex of K(IR)6.1 channels and SUR2B subunits., (© 2013 The British Pharmacological Society.)

Potassium channels that are inhibited by intracellular ATP (ATP(i)) were first identified in ventricular myocytes, and are referred to as ATP-sensitive K+ channels (i.e. K(ATP) channels). Subsequently, K+ channels with similar characteristics have been demonstrated in many other tissues (pancreatic beta-cells, skeletal muscle, central neurones, smooth muscle). Approximately one decade ago, K(ATP) channels were cloned and were found to be composed of at least two subunits: an inwardly rectifying K+ channel six family (Kir6.x) that forms the ion conducting pore and a modulatory sulphonylurea receptor (SUR) that accounts for several pharmacological properties. Various types of native K(ATP) channels have been identified in a number of visceral and vascular smooth muscles in single-channel recordings. However, little attention has been paid to the molecular properties of the subunits in K(ATP) channels and it is important to determine the relative expression of K(ATP) channel components which give rise to native K(ATP) channels in smooth muscle. The aim of this review is to briefly discuss the current knowledge available for K(ATP) channels with the main interest in the molecular basis of native K(ATP) channels, and to discuss their possible linkage with physiological functions in smooth muscle.

We have investigated the effects of MCC-134 (1-[4-(1H-imidazol-1-yl)benzoyl]-N-methyl-cyclobutanecarbothioamide) on membrane currents and ATP-sensitive K(+) channel (K(ATP) channel) opener-induced currents in pig urethra by use of patch-clamp techniques (conventional whole-cell configuration and nystatin perforated patch recordings). Tension measurement was also performed to study the effects of MCC-134 on the resting tone of pig urethral strips. MCC-134 reduced the resting tone of pig urethra in a concentration-dependent manner (EC(50)=6 microM). The MCC-134 (30 microM)-induced relaxation was suppressed by glibenclamide. In voltage-clamp experiments, MCC-134 produced a concentration-dependent inward K(+) current which was suppressed by application of glibenclamide at a holding potential of -50 mV (symmetrical 140 mM K(+) conditions). Application of MCC-134 enhanced diazoxide-induced inward currents and inhibited pinacidil-induced inward currents in a concentration-dependent manner at -50 mV. These results suggest that MCC-134 induces glibenclamide-sensitive K(ATP) currents in pig urethra.

(1) We have investigated the possible roles of sulphonylurea receptor (SUR) type 1 and 2B in the activity of pig urethral ATP-sensitive K(+) channels (K(ATP) channels) by use of patch-clamp and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. (2) In voltage-clamp experiments, not only diazoxide, a SUR1 and weak SUR2B activator, but also pinacidil, a selective SUR2 activator, caused an inward current at a holding potential of -50 mV in symmetrical 140 mM K(+) conditions. (3) Gliclazide (

It is a common sequelae of bladder outlet obstruction caused by benign prostatic hyperplasia in adult males and gives rise to significant bladder dysfunction such as frequency and urgency of micturition. The unstable detrusor contractions may lead to urge incontinence. Since it has been reported that experimentally-induced bladder instability can be abolished by ATP-sensitive K+ channel (KATP channel) openers, various types of detrusor-selective KATP channels have been newly synthesized, targeting KATP channels in urinary bladder. Thus, the significant differences in molecular and pharmacological properties of KATP channels between urinary bladder and urethra hold out some hope for the development of tissue-selective KATP channel openers for urge urinary incontinence, and detrusor-selective KATP channel openers should be screened against urethral as well as vascular smooth muscle. In functional expression experiments, pharmacological and electrophysiological studies have reported that SUR1/Kir6.2 represents the pancreatic beta-cell KATP channel and that SUR2A/Kir6.2 is thought to represent the cardiac KATP channel, whereas SUR2B/Kir6.1 represents the smooth muscle-type KATP channel. In general, the smooth muscle type-KATP channel is (i) of a relatively small conductance (about 20 pS under quasi-physiological conditions, approximately 40 pS in symmetrical 140 mM K+ conditions), (ii) intracellular Ca(2+)-insensitive, (iii) inhibited by intracellular ATP, (iv) abolished by glibenclamide at a submicromolar concentration, and (v) reactivated by intracellular nucleoside diphosphates (NDPs). There has been no report concerning the properties of KATP channels in human detrusor by use of single-channel recordings. We would like to introduce our recent evidence of novel synthesized detrusor-selective KATP channel openers and properties of KATP channels in the lower urinary tract.

1. The effects of ZD6169, a novel K(+) channel opener, on both membrane and unitary currents in pig urethra were investigated using patch-clamp techniques. Its effect was also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K(+) channel (Kir6.2) subunits (Kir6.2C36) which form ATP-sensitive K(+) channels (K(ATP) channels). 2. In current-clamp mode, ZD6169 (< or = 10 microM) induced a concentration-dependent membrane hyperpolarization. Higher concentrations (> or = 30 microM) caused a transient membrane hyperpolarization, followed by a gradual membrane depolarization. On removal of ZD6169, an after hyperpolarization was observed. 3. In conventional voltage-clamp configuration, at -50 mV in symmetrical 140 mM K(+) conditions, ZD6169 (100 microM) caused a transient inward current which gradually decayed. Removal of ZD6169 evoked a much larger amplitude K(+) current with a similar time course. 4. ZD6169 produced an inward glibenclamide-sensitive K(+) current, demonstrating a bell-shaped concentration-response relationship. 5. In cell-attached configuration in symmetrical 140 mM K(+) conditions, ZD6169 (< or = 30 microM) activated an K(ATP) channel which was reversibly suppressed by application of glibenclamide. In contrast, ZD6169 (100 microM) inhibited the activity of the levcromakalim-induced K(ATP) channels. 6. ZD6169 (100 microM) had no significant effect on the channel activity of Kir6.2C36 in inside-out configuration, although cibenzoline greatly suppressed the channel activity. 7. These results demonstrate that ZD6169 possesses a dual effect on the activity of the K(ATP) channel; activating at low concentration and inhibiting at higher concentration.

The relaxing effects of the adenosine 5'-triphosphate (ATP)-sensitive K+ channel openers (K(ATP) openers; diazoxide, minoxidil, pinacidil, (+/-)-cromakalim, (+)-cromakalim and (-)-cromakalim) were investigated on the resting tone of pig proximal urethra. In addition, patch clamp techniques were utilized for recording cromakalim-induced ionic currents in cells dispersed from the same urethral region. The (-)-cromakalim-induced relaxation of urethral muscle strips was stable, reversible and reproducible. The rank order of potency regarding of K(ATP) openers in lowering the resting urethral tone was (-)-cromakalim>pinacidil>diazoxide>minoxidil. K(ATP) opener-induced urethral relaxation was suppressed by subsequent application of glibenclamide (1 microM). (+)-Cromakalim (< or =10 microM) did not relax the urethra nor antagonize the (-)-cromakalim-induced urethral relaxation. However, at higher concentrations, (+)-cromakalim (> or =30 microM) caused a small but significant urethral relaxation. In accordance with these observations, the relaxation induced by 5 microM (-)-cromakalim was identical to that induced by 10 microM (+/-)-cromakalim, as expected from a theoretical half potency for (+/-)-cromakalim. In whole-cell recording, (-)-cromakalim and (+) cromakalim (100 microM) activated a glibenclamide-sensitive outward current which was due to the activation of the glibenclamide-sensitive 43 pS K+ channel (K(GS)-43 pS). The potency of (+)-cromakalim to activate K(GS)-43 pS was much weaker than that of (-)-cromakalim. These results indicate that the ability of K(ATP) openers to relax pig urethral smooth muscle can be accounted for by activation of glibenclamide-sensitive K+ channels.

1 The effects of mefenamic acid on both membrane potential and K[sup+] currents in pig urethral myocytes were investigated using patch-clamp techniques (conventional whole-cell, cell-attached, outside-out and inside-out configuration). 2 In the current-clamp mode, mefenamic acid caused a concentration-dependent hyperpolarization, which was inhibited by preapplication of 1 μM glibenclamide. In the voltage-clamp mode. mefenamic acid induced an outward current that was blocked by glibenclamide even in the presence of iberiotoxin (IbTX, 300nM) at -50mV. 3 ATP-sensitive K[sup+] channels (K[subATP] channels) could be activated in the same patch by mefenamic acid and levcromakalim, with the same unitary amplitude and the similar opening gating at -50 mV in cell-attached configuration. 4 In outside-out recording, external application of mefenamic acid activated intracellular Ca[sup2+]-activated 1bTX-sensitive large-conductance K[sup+] channels (BK[subca] channels). 5 Mefenamic acid (≤ 30 μM) activated spontaneous transient outward currents (STOCs). In contrast, mefenamic acid ( ≥ 100 μM) increased sustained outward currents, diminishing the activity of STOCs. 6 Over the whole voltage range, mefenamic acid caused opposite effects on the membrane currents in the absence and presence of 5 μM glibenclamide. In the presence of l0mM 4-aminopyridine (4-A.P). mefenamic acid only increased the outward currents. 7 These results indicate that mefenamic acid increases the channel activities of two distinct types of K[sup+] channels (i.e. BK[subCa] channels and K[subATP] channels) and decreased 4-AP-sensitive K[sup+] channels in pig urethral myocytes. [ABSTRACT FROM AUTHOR]

Abstract: It is acknowledged that neurotransmission in the mouse vas deferens is predominantly mediated by ATP and noradrenaline (NA) released from sympathetic nerves while cholinergic transmission in the rodent vas deferens is often overlooked despite early literature. Recently we have characterized a cholinergic component of neurogenic contraction of mouse isolated vas deferens. In the present paper, by confocal imaging of Ca2+ dynamics we detected acetylcholine (ACh) action at muscarinic cholinergic neuroeffector junctions at high-resolution. Experiments were carried out in the presence of prazosin (100 nM) and α,β methylene ATP (α,β-MeATP) (1 μM) to inhibit responses to NA and ATP respectively. Exogenous ACh (10 μM) elicited Ca2+ transients, an effect blocked by the muscarinic receptor antagonist, cyclopentolate (1 μM). Ca2+ transients were evoked by electrical stimulation of intrinsic nerves in the presence of the cholinesterase inhibitor neostigmine (10 μM). Stimulation produced a marked increase in the frequency and number of Ca2+ transients. Cyclopentolate reduced the frequency of occurrence of spontaneous and evoked events to control levels. The α2-adrenoceptor antagonist yohimbine (300 nM) did not affect the spontaneous Ca2+ transients, but increased the frequency of occurrence of evoked transients, an effect inhibited by cyclopentolate. The postjunctional effects of neuronally-released ACh are limited by the action of cholinesterase. Release of ACh appears to be tonically inhibited by NA released from sympathetic nerve terminals through action at prejunctional α2-adrenoceptors. Tetrodotoxin (TTX, 300 nM) abolished the nerve-evoked Ca2+ events, with no effect on Ca2+ transients elicited by exogenous ACh. In conclusion, the presence of spontaneous and evoked cholinergic Ca2+ transients in smooth muscle cells of the mouse isolated vas deferens has been revealed. These events are mediated by ACh acting at M3 muscarinic receptors. This action stands in marked contrast to the lack of effect of neuronally-released NA on smooth muscle Ca2+ dynamics in this tissue. [Copyright &y& Elsevier]