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Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na + channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other β-subunit genes (Scn2b - Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na + current peak amplitude in myocytes (i.e., native I Na ) were compared with its inhibitory potency on recombinant Na V 1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native I Na and recombinant Na V 1.6 expressed in HEK293 cells (i.e., recombinant I Na ). The current decay of native I Na was similar to the recombinant Na V 1.6 current co-expressed with β 1 -subunits. The current-voltage (I-V) relationships of native I Na were similar to those of recombinant Na V 1.6 currents co-expressed with β 1 -subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native I Na (K i  = 510 nM), recombinant I Na (K i  = 112 nM), and recombinant I Na co-expressed with β 1 -subunits (K i  = 92 nM). The half-maximal (V half ) activation and inactivation of native I Na values were similar to those observed in recombinant I Na co-expressed with β 1 -subunits. These results suggest that β 1 -subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native I Na but also recombinant I Na and recombinant I Na co-expressed with β 1 -subunits in a concentration-dependent manner.

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.)

Patch-clamp experiments were performed to investigate the molecular properties of resurgent-like currents in single smooth muscle cells dispersed from mouse vas deferens, utilizing both Na(V)1.6-null mice (Na(V)1.6(-/-)), lacking the expression of the Scn8a Na(+) channel gene, and their wild-type littermates (Na(V)1.6(+/+)). Na(V)1.6 immunoreactivity was clearly visible in dispersed smooth muscle cells obtained from Na(V)1.6(+/+), but not Na(V)1.6(-/-), vas deferens. Following a depolarization to +30 mV from a holding potential of -70 mV (to produce maximal inactivation of the Na(+) current), repolarization to voltages between -60 and +20 mV elicited a tetrodotoxin (TTX)-sensitive inward current in Na(V)1.6(+/+), but not Na(V)1.6(-/-), vas deferens myocytes. The resurgent-like current in Na(V)1.6(+/+) vas deferens myocytes peaked at approximately -20 mV in the current-voltage relationship. The peak amplitude of the resurgent-like current remained at a constant level when the membrane potential was repolarized to -20 mV following the application of depolarizing rectangular pulses to more positive potentials than +20 mV. 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX), a selective Na(V)1.6 blocking toxin, purified from a crude mixture of TTX analogues by LC-FLD techniques, reversibly suppressed the resurgent-like currents. β-Pompilidotoxin, a voltage-gated Na(+) channel activator, evoked sustained resurgent-like currents in Na(V)1.6(+/+) but not Na(V)1.6(-/-) murine vas deferens myocytes. These results strongly indicate that, primarily, resurgent-like currents are generated as a result of Na(V)1.6 channel activity.

Patch-clamp experiments were performed to investigate the behavior of voltage-activated inward currents in vas deferens myocytes from Na(V)1.6-null mice (Na(V)1.6(-/-)) lacking the expression of the Na(+) channel gene, Scn8a, and their wild-type littermates (Na(V)1.6(+/+)). Immunohistochemistry confirmed expression of Na(V)1.6 in the muscle of Na(V)1.6(+/+), but not Na(V)1.6(-/-), vas deferens. PCR analysis revealed that the only beta(1)-subunit gene expressed in Na(V)1.6(+/+) vas deferens was Scn1b. In Na(V)1.6(+/+) myocytes, the threshold for membrane currents evoked by 20 msec voltage ramps (-100 mV to 60 mV) was -38.5 +/- 4.6 mV and this was shifted to a more positive potential (-31.2 +/- 4.9 mV) by tetrodotoxin (TTX). In Na(V)1.6(-/-) myocytes, the threshold was -30.4 +/- 3.4 mV and there was no TTX-sensitive current. The Na(+) current (I(Na)) in Na(V)1.6(+/+) myocytes had a bell-shaped current-voltage relationship that peaked at approximately -10 mV. Increasing the duration of the voltage ramps beyond 20 msec reduced the peak amplitude of I(Na). I(Na) displayed both fast (tau approximately 10 msec) and slow (tau approximately 1 sec) recovery from inactivation, the magnitude of the slow component increasing with the duration of the conditioning pulse (5-40 msec). During repetitive activation (5-40 msec pulses), I(Na) declined at stimulation frequencies > 0.5 Hz and at 10 Hz

Background and Purpose: The effects of veratridine, an alkaloid found in Liliaceae plants, on tetrodotoxin (TTX)-sensitive voltage-gated Na(+) channels were investigated in mouse vas deferens., Experimental Approach: Effects of veratridine on TTX-sensitive Na(+) currents (I(Na)) in vas deferens myocytes dispersed from BALB/c mice, homozygous mice with a null allele of Na(V)1.6 (Na(V)1.6(-/-)) and wild-type mice (Na(V)1.6(+/+)) were studied using patch-clamp techniques. Tension measurements were also performed to compare the effects of veratridine on phasic contractions in intact tissues., Key Results: In whole-cell configuration, veratridine had a concentration-dependent dual action on the peak amplitude of I(Na): I(Na) was enhanced by veratridine (1-10 microM), while higher concentrations (> or =30 microM) inhibited I(Na). Additionally, two membrane current components were evoked by veratridine, namely a sustained inward current during the duration of the depolarizing rectangular pulse and a tail current at the repolarization. Although veratridine caused little shift of the voltage dependence of the steady-state inactivation curve and the activation curve for I(Na), veratridine enhanced a non-inactivating component of I(Na). Veratridine caused no detectable contractions in vas deferens from Na(V)1.6(-/-) mice, although in tissues from Na(V)1.6(+/+) mice, veratridine (> or =3 microM) induced TTX-sensitive contractions. Similarly, no detectable inward currents were evoked by veratridine in Na(V)1.6(-/-) vas deferens myocytes, while veratridine elicited both the sustained and tail currents in cells taken from Na(V)1.6(+/+) mice., Conclusions and Implications: These results suggest that veratridine possesses a dual action on I(Na) and that the veratridine-induced activation of contraction is induced by the activation of Na(V)1.6 channels.

Kurtoxin is described as a selective inhibitor of Ca(V)3.1. Using patch-clamp techniques, the modulatory effects of kurtoxin on tetrodotoxin-sensitive voltage-gated Na(+) currents (I(Na)) recorded from mouse vas deferens myocytes were investigated. Kurtoxin increased the peak amplitude of I(Na) between -40 and -30 mV, whilst inhibited the peak amplitude at more positive potentials than -10 mV, thereby demonstrating a dual action on the peak amplitude of I(Na). The time to reach the peak amplitude of I(Na) became significantly longer in the presence of kurtoxin in comparison with that of the controls. Kurtoxin also slowed the deactivation of I(Na) at more positive membrane potentials than -30 mV. Kurtoxin enhanced the total amount of electrical charge of I(Na) in a concentration-dependent manner. These results suggest that kurtoxin is a modulator of Na(V)1.6 in native freshly dispersed smooth muscle cells from mouse vas deferens as well as its action on Ca(V)3.1.

The inwardly rectifying properties and molecular basis of ATP-sensitive K(+) channels (K(ATP) channels) have now been established for several cell types. However, these aspects of nonvascular smooth muscle K(ATP) channels still remain to be defined. In this study, we investigated the molecular basis of the pore of K(ATP) channels of pig urethral smooth muscle cells through a comparative study of the inwardly rectifying properties, conductance, and regulation by PKC of native and homo- and heteroconcatemeric recombinant Kir6.x channels coexpressed with sulfonylurea receptor subunit SUR2B in human embryonic kidney (HEK) 293 cells by the patch-clamp technique (conventional whole-cell and cell-attached modes). In conventional whole-cell clamp recordings, levcromakalim (> or = 1 microM) caused a concentration-dependent increase in current that demonstrated strong inward rectification at positive membrane potentials. In cell-attached mode, the unitary amplitude of levcromakalim-induced native and recombinant heteroconcatemeric Kir6.1-Kir6.2 K(ATP) channels also showed strong inward rectification at positive membrane potentials. Phorbol 12,13-dibutyrate, but not the inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate, enhanced the activity of native and heteroconcatemeric K(ATP) channels at -50 mV. The conductance of the native channels at approximately 43 pS was consistent with that of heteroconcatemeric channels with a pore-forming subunit composition of (Kir6.1)(3)-(Kir6.2). RT-PCR analysis revealed the expression of Kir6.1 and Kir6.2 transcripts in pig urethral myocytes. Our findings provide the first evidence that the predominant K(ATP) channel expressed in pig urethral smooth muscle possesses a unique, heteromeric pore structure that differs from the homomeric Kir6.1 channels of vascular myocytes and is responsible for the differences in inward rectification, conductance, and PKC regulation exhibited by the channels in these smooth muscle cell types.

The biological and molecular properties of tetrodotoxin (TTX)-sensitive voltage-gated Na(+) currents (I(Na)) in murine vas deferens myocytes were investigated using patch-clamp techniques and molecular biological analyses. In whole-cell configuration, a fast, transient inward current was evoked in the presence of Cd(2+), and was abolished by TTX (K(d) = 11.2 nM), mibefradil (K(d) = 3.3 microM), and external replacement of Na(+) with monovalent cations (TEA(+), Tris(+), and NMDG(+)). The fast transient inward current was enhanced by veratridine, an activator of voltage-gated Na(+) channels, suggesting that the fast transient inward current was a TTX-sensitive I(Na). The values for half-maximal (V(half)) inactivation and activation of I(Na) were -46.3 mV and -26.0 mV, respectively. RT-PCR analysis revealed the expression of Scn1a, 2a, and 8a transcripts. The Scn8a transcript and the alpha-subunit protein of Na(V)1.6 were detected in smooth muscle layers. Using Na(V)1.6-null mice (Na(V)1.6(-/-)) lacking the expression of the Na(+) channel gene, Scn8a, I(Na) were not detected in dispersed smooth muscle cells from the vas deferens, while TTX-sensitive I(Na) were recorded in their wild-type (Na(V)1.6(+/+)) littermates. This study demonstrates that the molecular identity of the voltage-gated Na(+) channels responsible for the TTX-sensitive I(Na) in murine vas deferens myocytes is primarily Na(V)1.6.

The effects of ZD0947, a novel urinary bladder selective ATP-sensitive potassium channel (K(ATP) channel) opener, on carbachol-induced contractions of isolated guinea pig urinary bladder strips were investigated to compare its ability to relax norepinephrine-induced contraction of the aorta. Electrophysiological techniques were also utilized to compare the effects of ZD0947 on membrane currents between guinea pig detrusor and aortic myocytes. ZD0947 caused a significant reduction of the carbachol-induced contractile activity, demonstrating a biphasic relaxation (the first and second components). Although glibenclamide antagonized the effects of two components for the ZD0947-induced relaxation, gliclazide, a selective sulphonylurea receptor 1 (SUR1) antagonist, reduced the effects of the first component but not the second component of the ZD0947-induced relaxation. ZD0947 also reduced the norepinephrine-induced contraction of the aorta. ZD0947 reduced electrical excitability of detrusor smooth muscles, inhibiting spike discharges and also hyperpolarizing the membrane as measured with microelectrodes. In conventional whole-cell configuration, ZD0947 caused a glibenclamide-sensitive K(+) current (i.e., K(ATP) current) at a holding potential of -60 mV in guinea pig detrusor and aortic myocytes. The current density of ZD0947-induced K(ATP) currents in guinea pig detrusor myocytes was significantly larger than that in aortic smooth muscle cells. These results show that ZD0947 caused a significant relaxation through the activation of K(ATP) channels in detrusor muscle.

The anticholinergic propiverine (1-methyl-4-piperidyl diphenylpropoxyacetate), which is used for the treatment of overactive bladder syndrome, has functionally active metabolites [M-1 (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide) and M-2 (1-methyl-4-piperidyl benzilate N-oxide)], but the site of actions of these metabolites is uncertain. Propiverine is rapidly absorbed after oral administration and is extensively biotransformed in the liver, giving rise to several active metabolites (M-1 and M-2). This study determines the effect of M-1 and M-2 on voltage-dependent nifedipine-sensitive inward Ca(2+) currents (I(Ca)) using patch-clamp techniques and fluorescent Ca(2+) imaging [after electrical field stimulation (EFS) and acetylcholine (ACh)] in the murine urinary bladder. In conventional whole-cell recording, propiverine and M-1 but not M-2 inhibited the peak amplitude of I(Ca) in a concentration-dependent manner at a holding potential of -60 mV (propiverine, K(i) = 10 microM; M-1, K(i) = 118 microM). M-1 shifted the steady-state inactivation curve of I(Ca) to the left at -90 mV by 7 mV. Carbachol (CCh) reversibly inhibited I(Ca). This inhibition probably occurred through muscarinic type 3 receptors, coupling with G-proteins, because nanomolar concentrations of 4-diphenylacetoxy-N-methyl-piperidine greatly reduced this inhibition, whereas pirenzepine or 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) at concentrations up to 1 microM was almost ineffective. In the presence of M-2, the CCh-induced inhibition of I(Ca) was blocked. In fluorescent Ca(2+) imaging, M-2 inhibited EFS-induced and ACh-induced Ca(2+) transients. These results suggest that M-1 acts, at least in part, as a Ca(2+) channel antagonist (as it inhibited I(Ca)), whereas M-2 has more direct antimuscarinic actions.

Background and Purpose: ATP-sensitive K+ channels (K(ATP) channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)-induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated., Experimental Approach: Tension measurements and patch-clamp techniques were utilized to study the effects of ZD0947 in segments of human urinary bladder. Immunohistochemistry was also performed to detect the expression of the sulphonylurea receptor 1 (SUR1) and the SUR2B antigens in human detrusor muscle., Key Results: ZD0947 (> or = 0.1 microM) caused a concentration-dependent relaxation of the CCh-induced contraction of human detrusor, which was reversed by glibenclamide. The rank order of the potency to relax the CCh-induced contraction was pinacidil > ZD0947 > diazoxide. In conventional whole-cell configuration, ZD0947 (> or = 1 microM) caused a concentration-dependent inward K+ current which was suppressed by glibenclamide at -60 mV. When 1 mM ATP was included in the pipette solution, application of pinacidil or ZD0947 caused no inward K+ current at -60 mV. Gliclazide (< or =1 microM), a selective SUR1 blocker, inhibited the ZD0947-induced currents (Ki = 4.0 microM) and the diazoxide-induced currents (high-affinity site, Ki1 = 42.4 nM; low-affinity site, Ki2 = 84.5 microM) at -60 mV. Immunohistochemical studies indicated the presence of SUR1 and SUR2B proteins, which are constituents of KATP channels, in the bundles of human detrusor smooth muscle., Conclusions and Implications: These results suggest that ZD0947 caused a glibenclamide-sensitive detrusor relaxation through activation of glibenclamide-sensitive KATP channels in human urinary bladder.

We have investigated whether tyrosine kinases modify the activity of voltage-dependent Ba(2+) currents (I(Ba)) recorded from guinea-pig gastric myocytes by use of patch-clamp techniques. All experiments were carried on single smooth muscle cells, dispersed from the circular layer of the guinea-pig gastric antrum. Genistein ( > or = 10 microM), a specific tyrosine kinase inhibitor, reduced the peak amplitude of I(Ba) in a voltage- and concentration-dependent manner. Daidzein ( > or = 30 microM), an inactive analog of genistein, also inhibited I(Ba) in a concentration-dependent manner. Similarly, other types of tyrosine kinase inhibitors (lavendustin A and tyrphostin 23) suppressed the peak amplitude of I(Ba) in a concentration-dependent manner. These results indicate that tyrosine kinases may be essential to regulate Ca(2+) mobilization through voltage-dependent Ca(2+) channels in gastric myocytes.

Electorophysiological and pharmacological properties of the levcromakalim-induced inward ATP-sensitive K+ currents (K(ATP) currents) in pig proximal urethra were investigated by use of two different whole-cell patch-clamp techniques, namely conventional whole-cell and nystatin-perforated patch recordings. In conventional whole-cell configuration, the levcromakalim (100 microM)-induced K(ATP) current decayed by about 30% in 8 min at a holding potential of -50 mV. In contrast, with the nystatin-perforated patch, 96% of the levcromakalim-induced K(ATP) current still remained even after 8 min application of levcromakalim. The peak amplitude of the levcromakalim-induced inward K(ATP) currents in nystatin-perforated patch was approximately half of those observed in conventional whole-cell configuration. When cytosolic extract of pig urethra was included in the pipette solution, approximately 90% of the levcromakalim (100 microM)-induced K(ATP) current remained at 8 min, even after the establishment of conventional whole-cell configuration. In conventional whole-cell configuration, glibenclamide suppressed the levcromakalim-induced K(ATP) currents in a concentration-dependent manner (Ki=175 nM). Inclusion of 1 mM uridine 5'-diphosphate (UDP) in the pipette solution shifted the glibenclamide-sensitivity (Ki=640 nM) to the right in comparison with that in the absence of UDP (i.e., control). In contrast, using nystatin-perforated patch, glibenclamide inhibited the levcromakalim-induced K(ATP) currents with two affinity sites (high-affinity site, Ki1=10 nM; low-affinity site, Ki2=9 microM). The concentration response curves regarding the inhibitory effects of K(ATP) channel pore blockers (Ba2+ and flecainide) on the levcromakalim-induced K(ATP) currents in conventional whole-cell recording nearly overlapped with those in nystatin-perforated patch recording. These results indicate that the glibenclamide-sensitivity of pig urethral K(ATP) channels in nystatin-perforated patch recording was significantly different from that in a conventional whole-cell configuration, and that the glibenclamide-sensitivity may be modified by some cytosolic factor(s).

The effects of flavoxate hydrochloride (Bladderon, piperidinoethyl-3-methylflavone-8-carboxylate; hereafter referred as flavoxate) on voltage-dependent nifedipine-sensitive inward Ba(2+) currents in human detrusor myocytes were investigated using a conventional whole-cell patch-clamp. Tension measurement was also performed to study the effects of flavoxate on K(+)-induced contraction in human urinary bladder. Flavoxate caused a concentration-dependent reduction of the K(+)-induced contraction of human urinary bladder. In human detrusor myocytes, flavoxate inhibited the peak amplitude of voltage-dependent nifedipine-sensitive inward Ba(2+) currents in a voltage- and concentration-dependent manner (K(i) = 10 microM), and shifted the steady-state inactivation curve of Ba(2+) currents to the left at a holding potential of -90 mV. Immunohistochemical studies indicated the presence of the alpha(1C) subunit protein, which is a constituent of human L-type Ca(2+) channels (Ca(V)1.2), in the bundles of human detrusor smooth muscle. These results suggest that flavoxate caused muscle relaxation through the inhibition of L-type Ca(2+) channels in human detrusor.

We have investigated whether the activation of cAMP- and cGMP-dependent pathways modifies the properties of voltage-dependent Ba(2+) currents (I(Ba)) recorded from guinea-pig gastric myocytes using patch-clamp techniques. All experiments were carried on single smooth muscle cells, dispersed from the circular layer of the guinea-pig gastric antrum. Both dibutyryl cAMP (db-cAMP, 0.1-1 mM), a membrane-permeable ester of cAMP, and isoproterenol, a selective beta-stimulant, inhibited I(Ba) in a concentration-dependent manner. Forskolin, but not dideoxy-forskolin, an inactive isomer of forskolin, inhibited the peak amplitude of I(Ba). In the presence of either Rp-cAMP or the PKA (cAMP-dependent protein kinase) inhibitor peptide 5-24 (PKA-IP), neither forskolin nor db-cAMP inhibited I(Ba). After establishing a conventional whole-cell recording, the peak amplitude of I(Ba) gradually decreased when the catalytic subunit of PKA was included in the pipette. The further application of Rp-cAMP reversibly enhanced I(Ba). Sodium nitroprusside (0.1-1 mM) and 8-Br-cGMP (0.1-1 mM) also inhibited I(Ba) in a concentration-dependent manner. The inhibitory effects of forskolin or db-cAMP on I(Ba) were not significantly changed by pretreatment with a cGMP-dependent protein kinase (PKG) inhibitor. Similarly, the inhibitory actions of 8-Br-cGMP on I(Ba) were not modified by PKA-IP. The membrane-permeable cyclic nucleotides db-cAMP and 8-Br-cGMP caused little shift of the voltage dependence of the steady-state inactivation and reactivation curves. Neither of the membrane-permeable cyclic nucleotides db-cAMP or 8-Br-cGMP had additive inhibitory effects on I(Ba). These results indicate that two distinct cyclic nucleotide-dependent pathways are present in the guinea-pig gastric antrum, and that both inhibited I(Ba) in an independent manner.

The effects of mefenamic acid and Bay K 8644 on voltage-dependent nifedipine-sensitive inward Ba2+ currents in pig urethral myocytes were investigated by use of conventional whole-cell configuration patch clamp. Mefenamic acid increased the peak amplitude of voltage-dependent nifedipine-sensitive inward Ba2+ current without shifting the position of the current-voltage relationship. Mefenamic acid (300 microM) caused little shift in the activation curve although the voltage dependence of the steady-state inactivation was shifted to more positive potentials by 11 mV in the presence of mefenamic acid. Bay K 8644 (> or = 100 nM) enhanced voltage-dependent nifedipine-sensitive inward Ba2+ currents in a concentration- and voltage-dependent manner, shifting the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction. Bay K 8644 (1 microM) significantly shifted the voltage dependence of the activation curve to more negative potentials by approximately 9 mV although Bay K 8644 caused little shift in the steady-state inactivation curve. These results indicate that mefenamic acid increased voltage-dependent nifedipine-sensitive inward Ba2+ currents through the activation of L-type Ca2+ channels with different kinetics from those of Bay K 8644 in pig urethral myocytes.

Kinetic studies of U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl-hydrochloride), a vascular ATP-sensitive K+ channel (KATP channel) blocker, were performed on pig urethral myocytes to investigate inhibitory effects on large-conductance intracellular Ca2+ -sensitive K+ channels (i.e., BKCa channels; 225 pS K+ channels) by use of single-channel recordings (outside-out and inside-out configuration). BKCa channels in pig urethral smooth muscles showed extracellular iberiotoxin (300 nM) sensitivity and voltage dependency. The alpha subunit of BKCa channel proteins was detected in the membrane fraction by use of Western blot technique. Application of U-37883A (> or =10 microM) reduced the activity of BKCa channels in a concentration-dependent manner, not only by decreasing mean openlife time but also by prolonging the mean closed time. These results shows that U-37883A affects channels other than the vascular KATP channel, and demonstrates how it inhibits the activities of BKCa channels in urethral smooth muscles.

The inhibitory effects of glibenclamide on the levcromakalim-induced ATP-sensitive K+ (K(ATP)) channels were investigated with cell-attached configuration in pig proximal urethra. Application of 10 microM glibenclamide reversibly inhibited the activity of the 100 microM levcromakalim-induced K(ATP) channel, decreasing not only the channel open probability but also the amplitude of unitary current. The inhibitory concentration-response curve of the glibenclamide-induced sublevel conductance of K(ATP) channel was shifted to the right (IC50 = 4.7 microM), compared with the levcromakalim-induced K(ATP) channel (full conductance, IC50 = 0.5 microM). Glibenclamide is the first reported sulphonylurea to selectively block K(ATP) channel, not only by decreasing the channel activity but also by reducing the unitary amplitude in smooth muscle.

The effects of mefenamic acid on both membrane potential and K+ currents in pig urethral myocytes were investigated using patch-clamp techniques (conventional whole-cell, cell-attached, outside-out and inside-out configuration). In the current-clamp mode, mefenamic acid caused a concentration-dependent hyperpolarization, which was inhibited by preapplication of 1 microm glibenclamide. In the voltage-clamp mode, mefenamic acid induced an outward current that was blocked by glibenclamide even in the presence of iberiotoxin (IbTX, 300 nm) at -50 mV. ATP-sensitive K+ channels (KATP 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. In outside-out recording, external application of mefenamic acid activated intracellular Ca2+-activated IbTX-sensitive large-conductance K+ channels (BKCa channels). Mefenamic acid (or=100 microm) increased sustained outward currents, diminishing the activity of STOCs. Over the whole voltage range, mefenamic acid caused opposite effects on the membrane currents in the absence and presence of 5 microm glibenclamide. In the presence of 10 mm 4-aminopyridine (4-AP), mefenamic acid only increased the outward currents. These results indicate that mefenamic acid increases the channel activities of two distinct types of K+ channels (i.e. BKCa channels and KATP channels) and decreased 4-AP-sensitive K+ channels in pig urethral myocytes.