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In order to identify functional sulphonylurea receptor (SUR.x) subunits of native ATP-sensitive K + channels (K ATP channels) in mouse portal vein, the effects of ZD0947, a SUR.x modulator, were investigated on spontaneous portal vein contractions, macroscopic membrane currents and unitary currents recorded (using patch-clamp techniques) in freshly dispersed mouse portal vein myocytes. Spontaneous contractions in mouse portal vein were reversibly reduced by ZD0947 in a concentration-dependent manner (K i =293nM). The relaxation elicited by 3µM ZD0947 was antagonized by the additional application of glibenclamide (300nM), but not gliclazide (100-300nM). In the conventional whole-cell configuration, 100µM ZD0947 elicited inward glibenclamide-sensitive currents at a holding potential of -60mV that demonstrated selectivity for K + (i.e. K ATP currents). The peak amplitude of the membrane current elicited by 30µM or 100µM ZD0947 was smaller than that elicited by 100µM pinacidil at -60mV. In the cell-attached mode, 100µM ZD0947 activated glibenclamide-sensitive K + channels with a conductance (35 pS) similar to that of recombinant Kir6.1/SUR2B channels that were expressed in HEK293 cells and activated by 100µM ZD0947. These results demonstrate that ZD0947 caused a significant vascular relaxation through the activation of K ATP channels and that SUR2B may be the major functional subunit of SUR.x in mouse portal vein K ATP channels, based on its pharmacological selectivity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The effects of ZD0947, a novel ATP-sensitive K + channel (K ATP channel) opener, on the activity of reconstituted K ATP channels were investigated using cell-attached recordings. K ATP channels were studied in HEK 293 cells by co-expression of inwardly rectifying-6 family K + channel subunits (Kir6.x: Kir6.1 and Kir6.2) with 3 different types of sulphonylurea receptors (SUR.x: SUR1, SUR2A and SUR2B). ZD0947 (100µM) activated SUR2B/Kir6.2 channels in a concentration-dependent manner, but caused only weak activation of SUR1/Kir6.2 channels and SUR2A/Kir6.2 channels expressed in HEK 293 cells. ZD0947 reversibly suppressed diazoxide-elicited SUR1/Kir6.2 channels activity and pinacidil-elicited SUR2A/Kir6.2 channel activity. However, ZD0947 did not affect SUR2B/Kir6.2 channels fully activated by 100µM pinacidil. ZD0947 had little inhibitory effects on the activity of Kir6.2ΔC26 channels (a truncated isoform of Kir6.2) or its mutant channels (i.e. Kir6.2ΔC26C166A) expressed in HEK 293 cells. ZD0947 also elicited activity in SUR2B/Kir6.1 channels expressed in HEK 293 cells, in a concentration-dependent manner. Therefore, ZD0947 is a relatively effective activator of smooth muscle-type K ATP channels (SUR2B/Kir6.1 and SUR2B/Kir6.2) but is a partial antagonist of pancreatic-type K ATP channels (i.e. SUR1/Kir6.2) and cardiac-type K ATP channels (i.e. SUR2A/Kir6.2). These results suggest that a pharmacological agent can possess either agonist or antagonist actions on the activity of K ATP channels, depending on the subtype of SUR.x., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Several combinations of inwardly rectifying K(+) channel 6.x family pore-forming (KIR6.x) subunits associated with sulphonylurea receptor (SUR.x) subunits have been detected among ATP-sensitive K(+) (KATP) channels. It remains to be established which of these is expressed in native vascular smooth muscle., Methods: Pharmacological and electrophysiological properties of KATP channels in mouse portal vein were investigated using tension measurements and patch-clamp techniques. Molecular biological analyses were also performed to investigate the structural properties of these channels., Results: Spontaneous contractions in mouse portal vein were reversibly reduced by pinacidil and MCC-134, and the pinacidil-induced relaxation was antagonized by glibenclamide and U-37883A. In cell-attached mode, pinacidil activated glibenclamide-sensitive K(+) channels with a conductance (35 pS) similar to that of KIR6.1. RT-PCR analysis revealed the expression of KIR6.1, KIR6.2 and SUR2B transcripts. Using real-time PCR methods, the quantitative expression of KIR6.1 was much greater than that of KIR6.2. Immunohistochemical studies indicated the presence of KIR6.1 and SUR2B proteins in the smooth muscle layers of mouse portal vein and in single smooth muscle cells dispersed from mouse portal vein., Conclusions: The results indicate that native KATP channels in mouse portal vein are likely to be composed of a heterocomplex of KIR6.1 and SUR2B subunits., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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 Ca(2+) 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 alpha,beta methylene ATP (alpha,beta-MeATP) (1 microM) to inhibit responses to NA and ATP respectively. Exogenous ACh (10 microM) elicited Ca(2+) transients, an effect blocked by the muscarinic receptor antagonist, cyclopentolate (1 microM). Ca(2+) transients were evoked by electrical stimulation of intrinsic nerves in the presence of the cholinesterase inhibitor neostigmine (10 microM). Stimulation produced a marked increase in the frequency and number of Ca(2+) transients. Cyclopentolate reduced the frequency of occurrence of spontaneous and evoked events to control levels. The alpha(2)-adrenoceptor antagonist yohimbine (300 nM) did not affect the spontaneous Ca(2+) 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 alpha(2)-adrenoceptors. Tetrodotoxin (TTX, 300 nM) abolished the nerve-evoked Ca(2+) events, with no effect on Ca(2+) transients elicited by exogenous ACh. In conclusion, the presence of spontaneous and evoked cholinergic Ca(2+) transients in smooth muscle cells of the mouse isolated vas deferens has been revealed. These events are mediated by ACh acting at M(3) muscarinic receptors. This action stands in marked contrast to the lack of effect of neuronally-released NA on smooth muscle Ca(2+) dynamics in this tissue.

Ca(2+) channel antagonists are useful to reduce the abnormal motility in patients with irritable bowel syndrome. We have therefore examined the effects of a newly synthesized antagonist AJG049, on voltage-dependent L-type Ca(2+) channels in gastric antrum. Intracellular recordings were made from sheets of the circular muscle layer of guinea-pig gastric antrum, with simultaneous measurement of spontaneous contraction activity, and the effects of AJG049 were studied. The effects of AJG049 on voltage-dependent Ba(2+) currents (I(Ba)) and the basal membrane currents at -70 mV in dispersed smooth muscle cells were also investigated by the use of conventional whole-cell patch-clamp techniques. Although AJG049 (100 nM) enhanced the peak amplitude of spontaneous contractions, high concentrations (>or=10 microM) had inhibitory effects. In whole-cell configuration, AJG049 (10 nM) reversibly enhanced the peak amplitude of I(Ba) in a voltage-dependent manner whilst high concentrations (>or=100 nM) suppressed the peak amplitude in a concentration- and voltage-dependent manner. AJG049 (300 nM) caused little shift in the activation curve at a holding potential of -70 mV although the voltage dependence of the steady-state inactivation was shifted to more negative potentials by 5 mV in its presence. AJG049 caused a delay of the recovery from the inactivated state of I(Ba). Furthermore, AJG049 reduced the amplitude of the basal membrane currents at -70 mV in a concentration-dependent manner. These results suggest that AJG049 possesses a dual action on voltage-dependent Ca(2+) channels in circular layer of guinea-pig antrum.

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 U-37883A, a vascular ATP-sensitive K(+) channel (K(ATP) channel) blocker, on membrane currents were investigated in pig urethral myocytes by use of patch-clamp techniques (conventional whole-cell recordings, nystatin-perforated patches and cell-attached configuration). Tension measurement was also performed to study the effects of U-37883A on the levcromakalim-induced urethral relaxation and the urethral resting tone in the absence and presence of Bay K 8644. Although cumulative application of U-37883A produced a concentration-dependent inhibitory effect on the levcromakalim-induced urethral relaxation, U-37883A did not abolish the relaxation. In nystatin-perforated patch recording, K(ATP) currents activated by levcromakalim were inhibited by U-37883A in a concentration-dependent manner (K(i), 4.7 microM). Approximately 10% of the K(ATP) currents still remained even in the presence of 300 microM U-37883A. In cell-attached mode, extracellular application of U-37883A (100 microM) irreversibly inhibited the activity of the levcromakalim-induced K(ATP) channels. In whole-cell configuration, U-37883A suppressed the peak amplitude of voltage-dependent Ba(2+) currents in a concentration- and voltage-dependent manner, and at 30 microM, shifted the steady-state inactivation curve of the Ba(2+) currents to the left at -90 mV. These results demonstrate that U-37883A reduces not only the activities of K(ATP) channels but also voltage-dependent Ca(2+) channels. Therefore, it is not appropriate to define U-37883A as solely a vascular K(ATP) channel blocker.

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.

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.

To investigate the possible mechanisms involved in the stable and long-lasting levcromakalim-induced relaxation of the resting urethral tone, we have performed mechanical and voltage-clamp experiments using intact tissue and isolated cells from pig urethra, respectively. At negative membrane potentials, levcromakalim induced time- and voltage-independent membrane currents in whole-cell configurations. In cell-attached patches, levcromakalim not only increased the open-state probability (the NP(0) value) of the glibenclamide-sensitive 43 pS K+ channel (K(GS)) in a concentration-dependent manner, but also activated K(GS) with a time- and voltage-independence. During long burst-like channel activity, neither the mean open lifetime nor the mean closed time of K(GS) exhibited voltage-dependency between -100 and - 40 mV. It is concluded that levcromakalim causes a stable and potent relaxation of pig urethra through opening of K(GS) which possesses time- and voltage-independent activating mechanisms.

For the diagnosis of allergic rhinitis we measured the specific IgE and the total serum IgE in the blood. Although we had measured them by the conventional Phadebas RAST and the Pharmacia IgE RIA before, we now adopted the new Pharmacia CAP System (CAP RAST FEIA and CAP IgE FEIA). To compare these methods, we measured the specific IgE to the cedar pollen and total serum IgE in the same blood of 224 hospital staff at the regular physical examination. As a result the new Pharmacia CAP System proved to be simpler, safer, faster, easier, more specific, and more sensitive than the Phadebas RAST and the Pharmacia IgE RIA. And also these methods correlated well with each other.