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Examines the role of delayed rectifier K+ current (IK) in rabbit left atrium. Application of the whole cell voltage-clamp technique to isolated single myocytes; Contribution of IK with other K+ currents; Cell isolation; Electrical measurements; Identification of IK and sensitivity to E-4031.

Transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable nonselective cation channel expressed in neuronal and nonneuronal cells and plays an important role in acute and inflammatory pain. Here, we show that an NADPH oxidase (NOX) inhibitor, diphenyleneiodonium (DPI), functions as a TRPA1 activator in human embryonic kidney cells expressing human TRPA1 (HEK-TRPA1) and in human fibroblast-like synoviocytes. Application of DPI at 0.03-10 μM induced a Ca(2+) response in HEK-TRPA1 cells in a concentration-dependent manner. The Ca(2+) response was effectively blocked by a selective TRPA1 antagonist, HC-030031 (HC). In contrast, DPI had no effect on HEK cells expressing TRPV1-V4 or TRPM8. Four other NOX inhibitors, apocynin (APO), VAS2870 (VAS), plumbagin, and 2-acetylphenothiazine, also induced a Ca(2+) response in HEK-TRPA1 cells, which was inhibited by pretreatment with HC. In the presence of 5 mM glutathione, the Ca(2+) response to DPI was effectively reduced. Moreover, mutation of cysteine 621 in TRPA1 substantially inhibited the DPI-induced Ca(2+) response, while it did not inhibit the APO- and VAS-induced responses. The channel activity was induced by DPI in excised membrane patches with both outside-out and inside-out configurations. Internal application of neomycin significantly inhibited the DPI-induced inward currents. In inflammatory synoviocytes with TRPA1, DPI evoked a Ca(2+) response that was sensitive to HC. In mice, intraplantar injection of DPI caused a pain-related response which was inhibited by preadministration with HC. Taken together, our findings demonstrate that DPI and other NOX inhibitors activate human TRPA1 without mediating NOX., (Copyright © 2014 the American Physiological Society.)

Gold compounds, which were widely used to treat rheumatoid arthritis, have been recently used as experimental agents for tumor treatment. Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1) is a Ca(2+)-permeable ion channel that senses acute and inflammatory pain signals. Electrophilic compounds such as mustard oil and cinnamaldehyde activate TRPA1 by interacting with TRPA1 cysteine residues. Here we investigate the effects of the gold compound auranofin (AUR) on TRPA1 channels. Intracellular Ca(2+) and whole cell patch-clamp recordings were performed on human embryonic kidney cells transiently expressed with TRPA1, TRP melastatin 8 (TRPM8), and vanilloid type TRP (TRPV1-4) channels. AUR stimulated TRPA1 in a concentration-dependent manner with a half-maximum potency of around 1.0 μM. The AUR-induced response was effectively blocked by HC030031, a TRPA1 antagonist. On the other hand, AUR failed to activate TRPM8 and TRPV1-4 channels, which are highly expressed in sensory neurons as nociceptors. The stimulatory effect on TRPA1 channels depended on the C414, C421, C621, and C633 cysteine residues and not on the inhibition of thioredoxin reductase by AUR. Moreover, AUR effectively activated TRPA1 channels expressed in human differentiated neuroblastoma cell lines. The study shows that AUR is a potent stimulator of TRPA1 channels.

In articular cartilage inflammation, histamine release from mast cells is a key event. It can enhance cytokine production and matrix synthesis and also promote cell proliferation by stimulating chondrocytes. In this study, the functional impact of Ca(2+)-activated K(+) (K(Ca)) channels in the regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in chondrocytes in response to histamine was examined using OUMS-27 cells, as a model of chondrocytes derived from human chondrosarcoma. Application of histamine induced a significant [Ca(2+)](i) rise and also membrane hyperpolarization, and both effects were mediated by the stimulation of H(1) receptors. The histamine-induced membrane hyperpolarization was attenuated to approximately 50% by large-conductance K(Ca) (BK) channel blockers, and further reduced by intermediate (IK) and small conductance K(Ca) (SK) channel blockers. The tonic component of histamine-induced [Ca(2+)](i) rise strongly depended on the presence of extracellular Ca(2+) ([Ca(2+)](o)) and was markedly reduced by La(3+) or Gd(3+) but not by nifedipine. It was significantly attenuated by BK channel blockers, and further blocked by the cocktail of BK, IK, and SK channel blockers. The K(Ca) blocker cocktail also significantly reduced the store-operated Ca(2+) entry (SOCE), which was induced by Ca(2+) addition after store-depletion by thapsigargin in [Ca(2+)](o) free solution. Our results demonstrate that the histamine-induced membrane hyperpolarization in chondrocytes due to K(Ca) channel activation contributes to sustained Ca(2+) entry mainly through SOCE channels in OUMS-27 cells. Thus, K(Ca) channels appear to play an important role in the positive feedback mechanism of [Ca(2+)](i) regulation in chondrocytes in the presence of articular cartilage inflammation.

The activation of a vanilloid type 4 transient receptor potential channel (TRPV4) has an obligatory role in regulation of intracellular Ca(2+) (Ca(2+)(i)) in several types of cells including vascular and sensory organs. In this study, we provide evidence that TRPV4 is a functional regulator of Ca(2+)(i) in human synoviocytes. Although significant expression of TRPV4 in synoviocytes from patients with (RA) and without (CTR) rheumatoid arthritis was detected at mRNA and protein level, those in the human fibroblast-like synoviocyte line MH7A were rather lower. Consistently, the selective TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alphaPDD) effectively elevated Ca(2+)(i) in the RA and CTR cells, which was abolished by the removal of external Ca(2+). Moreover, the elevation was inhibited by ruthenium red, a blocker of TRPVs. In MH7A cells transfected with human TRPV4 (MH7A-V4), 4alphaPDD elevated the Ca(2+)(i) in a similar manner to those in the RA and CTR cells. Electrophysiological analysis also revealed that 4alphaPDD activated nonselective cationic currents in RA cells. Application of 227 mosM solution to the RA and MH7A-V4 cells elevated their Ca(2+)(i), but this does not occur when it was applied to MH7A cells. Treatment of RA but not MH7A cells with 4alphaPDD for 24 h reduced their production of IL-8. These results suggest that an environmental sensor, TRPV4, is a novel regulator of intracellular Ca(2+) in human synoviocytes.

Changes in K(+) conductances and their contribution to membrane depolarization in the setting of an acidic pH environment have been studied in myocytes from aortic smooth muscle cells of spontaneously hypertensive rats (SHR) compared with those from Wistar-Kyoto (WKY) rats. The resting membrane potential (RMP) of aortic smooth muscle at extracellular pH (pH(o)) of 7.4 was significantly more depolarized in SHR than in WKY rats. Acidification to pH(o) 6.5 made this difference in RMP between SHR and WKY rats more significant by further depolarizing the SHR myocytes. Large-conductance Ca(2+)-activated K(+) (BK) currents, which were markedly suppressed by acidification, were larger in aortic myocytes of SHR than in those of WKY rats. In contrast, acid-sensitive, non-BK currents were smaller in SHR. Western blot analyses showed that expression of BK-alpha- and -beta(1) subunits in SHR aortas was upregulated and comparable with those in WKY rats, respectively. Additional electrophysiological and molecular studies showed that pH- and halothane-sensitive two-pore domain weakly inward rectifying K(+) channel (TWIK)-like acid-sensitive K(+) (TASK) channel subtypes were functionally expressed in aortas, and TASK1 expression was significantly higher in WKY than in SHR. Although the background current through TASK channels at normal pH(o) (7.4) was small and may not contribute significantly to the regulation of RMP, TASK channel activation by halothane or alkalization (pH(o) 8.0) induced significant hyperpolarization in WKY but not in SHR. In conclusion, the larger depolarization and subsequent abnormal contractions after acidification in aortic myocytes in the setting of SHR hypertension are mainly attributable to the larger contribution of BK current to the total membrane conductance than in WKY aortas.

The relative contributions of Ca(2+)-induced Ca(2+) release (CICR) versus Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs) to excitation-contraction coupling has not been defined in most smooth muscle cells (SMCs). The present study was undertaken to address this issue in mouse urinary bladder (UB) smooth muscle cells (UBSMCs). Confocal Ca(2+) images were obtained under voltage- or current-clamp conditions. When UBSMCs were activated by a 30-ms depolarization to 0 mV, intracellular Ca(2+) concentration ([Ca(2+)](i)) increased in several small, discrete areas just beneath the cell membrane. These Ca(2+) "hot spots" then spread slowly through the myoplasm as Ca(2+) waves, which continued even after repolarization. Shorter depolarizations (5 ms) elicited only a few Ca(2+) sparks, which declined quickly. The number of Ca(2+) sparks, or hot spots, was closely related to the depolarization duration in the range of approximately 5-20 ms. There was an apparent threshold depolarization duration of approximately 10 ms within which to induce enough Ca(2+) transients to spread globally and then induce a contraction. Application of 100 microM ryanodine to the pipette solution did not change the resting [Ca(2+)](i) or the VDCC current, but it did abolish Ca(2+) hot spots elicited by depolarization. Application of 3 microM xestospongin C reduced ACh-induced Ca(2+) release but did not affect depolarization-induced Ca(2+) events. The addition of 100 microM ryanodine to tissue segments markedly reduced the amplitude of contractions triggered by direct electrical stimulation. In conclusion, global [Ca(2+)](i) rise triggered by a single action potential is not due mainly to Ca(2+) influx through VDCCs but is attributable to the subsequent two-step CICR.

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca2+-activated K+ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the alpha-subunit of the BK channel (BK-alpha), both alpha- and beta1-subunits (BK-alphabeta1), or both alpha- and beta4-subunits (BK-alphabeta4) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-alphabeta1 within a similar concentration range. Because methAEA could potentiate BK-alpha, BK-alphabeta1, and BK-alphabeta4 with similar efficacy, the beta-subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK-alpha and HEK-BK-alphabeta1 did not change intracellular Ca2+ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB2 agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed.

The molecular components of the large-conductance Ca(2+)-activated K(+) channels that are functionally expressed in mitochondria (mitoK(Ca)) in cardiac myocytes have not been identified. Our experimental results show that the transcript corresponding to the large-conductance Ca(2+)-activated K(+) channel beta1-subunit (BK-beta1) is substantially expressed in mammalian heart. A yeast two-hybrid assay showed the BK-beta1 protein can interact with a mitochondrial protein, cytochrome c oxidase subunit I (Cco1). Results from immunocytochemical experiments also demonstrated that BK-beta1 interacted with Cco1 and colocalized in rat cardiac mitochondria. Furthermore, 17beta-estradiol, which enhances the activity of the BK channel alpha-subunit only in the presence of the beta1-subunit, significantly increased flavoprotein oxidation in rat ventricle myocytes and decreased the rate of cell death under simulated ischemia. Single-channel recordings from mitochondrial inner membrane indicated that the activity of mitoK(Ca), which had a conductance of approximately 270 pS, was enhanced by 17beta-estradiol and blocked by paxilline. In combination, the present study revealed a new mechanism for the cardioprotective effects of 17beta-estradiol, which include the activation of mitoK(Ca) via the interaction with BK-beta1. BK-beta1 may be an important molecular component that functionally couples with both Cco1 and mitoK(Ca) pore-forming alpha-subunit.

Contribution of K(+) channels derived from the expression of ERG, KCNQ, and KCNE subtypes, which are responsible for rapidly and slowly activating delayed rectifier K(+) currents (I(Kr) and I(Ks), respectively) in cardiac myocytes, to membrane currents was examined in stomach circular smooth muscle cells (SMCs). The region-qualified multicell RT-PCR showed that ERG1/KCNE2 transcripts were expressed in rat stomach fundus and antrum SMCs and that KCNQ1/KCNE1 transcripts were expressed in antrum but not fundus. Western blotting and immunocytochemical analyses indicate that ERG1 proteins were substantially expressed in both regions, whereas KCNE1 proteins were faintly expressed in antrum and not in fundus. Both I(Kr)- and I(Ks)-like currents susceptible to E-4031 and indapamide, respectively, were identified in circular SMCs of antrum but only I(Kr)-like current was identified in fundus. It is strongly suggested that I(Kr)- and I(Ks)-like currents functionally identified in rat stomach SMCs are attributable to the expression of ERG1/KCNE2 and KCNQ1/KCNE1, respectively. The membrane depolarization by 1 microM E-4031 indicates the contribution of K(+) channels encoded by ERG1/KCNE2 to the resting membrane potential in stomach SMCs.

Effects of norepinephrine (NE) on voltage-dependent Ca channel current (ICa) were examined applying whole cell patch-clamp technique to single smooth muscle cells freshly isolated from vas deferens of the guinea pig. K currents and contraction of the cell were abolished by Cs and EGTA in the pipette solution, respectively. The peak ICa and Ba current (IBa) elicited by depolarization from -60 mV in a solution containing 2.2 mM Ca or Ba were reduced by 10-60% in voltage- and dose-dependent manners by the application of NE or phenylephrine. This effect was greatly attenuated in the presence of prazosin. The decrease in IBa was always smaller than that in ICa at any potential. Even after simultaneous application of 5 mM caffeine and 10 microM NE to the cells in a Ba-containing solution, the second challenge with NE again reduced IBa in a similar manner. The decrease in IBa by 10 microM NE could not be explained well by a small shift (-5 mV) of the voltage dependence of the steady-state inactivation. The effect of NE on IBa was irreversibly enhanced by 0.1 mM guanosine 5'-O-(3-thiotriphosphate) and almost abolished by 1 mM guanosine 5'-O-(2-thiodiphosphate) added to the pipette solution but appeared not to be affected by the treatment with pertussis toxin. It can be concluded that, under these experimental conditions, the activation of alpha 1-adrenoceptor in vas deferens smooth muscle cells reduces Ca channel activity possibly via a mechanism involving GTP-binding protein in addition to Ca-mediated Ca channel inactivation mechanism.

This study examines the effects of acid, acid secretagogues, and pepstatin on gastric luminal concentrations of xenopsin-like immunoreactivity (XPLI) by in situ luminal perfusion of the stomach in anesthetized rats. During perfusion with saline over a 2-h period, the concentration of XPLI fell in parallel with acid output. Levels of XPLI fell more rapidly when the saline contained 20 micrograms/ml pepstatin A and when phosphate-buffered saline (pH 7.0) was used. These treatments did not, however, alter acid output. After a basal condition was established at 90 min, intravenous injection of carbachol, pentagastrin, or histamine stimulated acid and pepsin secretion and also led to an increase in XPLI concentration, which was pepstatin sensitive. Acid itself was also a stimulus for pepsin and XPLI output, which were correlated at various levels of acidity. Although pepstatin blocked the effect of acid on XPLI output, it did not lead to an accumulation of xenopsin (XP) precursor in the luminal fluid. However, the decrement in acid-stimulated XPLI output seen in the presence of pepstatin was matched by an increment in XP precursor associated with the mucosal surface. During high-pressure liquid chromatography, approximately 70% of the acid-generated XPLI eluted at the position of mammalian XP. These data support the notion that, during heightened acid output, XP is secreted by a pepsin-dependent process or generated by the action of pepsin on XP precursor present on the mucosal surface of the rat stomach.

An attempt was made to obtain electrophysiological evidence for continuous influx of Ca ion through voltage-dependent Ca channel (VDCC) in smooth muscle during long depolarization, for example in high K solution. Noninactivated Ca current [ICa(ni)] remaining after the accomplishment of voltage-dependent inactivation by prolonged depolarization for approximately 1 min was detected by three means under whole cell voltage clamp in several types of smooth muscle cells. The measurement of ICa(ni) was performed by micropuff application of Cd2+ or Ca2+ in the presence or absence of 5 mM extracellular Ca, respectively, or jump of extracellular Ca concentration [( Ca]o). The current-voltage relationship of ICa(ni) evaluated by these means had a peak at approximately -10 mV. The peak amplitude ranged from 5 to 25 pA, depending on whether the cells were isolated from guinea pig urinary bladder, ureter, vas deferens, taenia caecum, or rabbit portal vein. The ICa(ni) may be large enough to explain sustained contraction in high K solution, at least in these smooth muscle tissues. A window current simulated from the steady-state activation and inactivation curves and the maximum conductance of Ca current (ICa) in these cells suggests a theoretical basis for the observed ICa(ni).