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Takefumi Mizutani,
Masahiro Morise, Yasushi Ito,
Yoshitaka Hibino,
Tadakatsu Matsuno,
Satoru Ito,
Naozumi Hashimoto,
Mitsuo Sato,
Masashi Kondo,
Kazuyoshi Imaizumi,
Yoshinori Hasegawa
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ABSTRACT: The present study investigated physiological effects of inhaled corticosteroids, which are used widely to treat asthma. Application of fluticasone propionate (FP, 100 µM) induced sustained increases in short-circuit current (I(SC)) in human airway Calu-3 epithelial cells. The FP-induced I(SC) was prevented by the presence of H89 (10 µM, a protein kinase A inhibitor) and SQ22536 (100 µM, an adenylate cyclase inhibitor). The FP-induced responses were composed of bumetanide (a Na(+)-K(+)-2Cl(-) cotransporter inhibitor)- and 4,4'-dinitrostilbene-2,2'-disulfonic acid (an inhibitor of HCO(3)(-)-dependent anion transporters)-sensitive components, both of which reflect basolateral anion transport. Further, FP augmented apical membrane Cl(-) current (I(Cl)), reflecting cystic fibrosis transmembrane conductance regulator (CFTR)-mediated conductance, in the nystatin-permeabilized monolayer. In I(SC) and I(Cl) responses, FP failed to enhance the responses to forskolin (10 µM, an adenylate cyclase activator). Nevertheless, we found that FP synergistically increased cytosolic cAMP levels in combination with forskolin. All these effects of FP were reproduced by the use of budesonide. Collectively, inhaled corticosteroids such as FP and budesonide stimulate CFTR-mediated anion transport through adenylate-cyclase-mediated mechanisms in a non-genomic fashion, thus sharing elements of a common pathway with forskolin. Notwithstanding, the corticosteroids cooperate with forskolin for synergistic cAMP production, suggesting that the corticosteroids and forskolin do not compete with each other to exert their effects on adenylate cyclase. Considering that synergism was also observed in the FP/salmeterol combination, these non-genomic aspects may play therapeutic roles in mucus congestive airway diseases in addition to the genomic ones that are generally recognized.
American Journal of Respiratory Cell and Molecular Biology 07/2012; · 5.13 Impact Factor
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Yoshitaka Hibino,
Masahiro Morise, Yasushi Ito,
Takefumi Mizutani,
Tadakatsu Matsuno,
Satoru Ito,
Naozumi Hashimoto,
Mitsuo Sato,
Masashi Kondo,
Kazuyoshi Imaizumi,
Yoshinori Hasegawa
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ABSTRACT: To investigate the effects of capsaicinoids on airway anion transporters, we recorded and analyzed transepithelial currents in human airway epithelial Calu-3 cells. Application of capsaicin (100 μM) attenuated vectorial anion transport, estimated as short-circuit currents (I(SC)), before and after stimulation by forskolin (10 μM) with concomitant reduction of cytosolic cyclic AMP (cAMP) levels. The capsaicin-induced inhibition of I(SC) was also observed in the response to 8-bromo-cAMP (1 mM, a cell-permeable cAMP analog) and 3-isobutyl-1-methylxanthine (1 mM, an inhibitor of phosphodiesterases). The capsaicin-induced inhibition of I(SC) was attributed to suppression of bumetanide (an inhibitor of the basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1)- and 4,4'-dinitrostilbene-2,2'-disulfonic acid (an inhibitor of basolateral HCO(3)(-)-dependent anion transporters)-sensitive components, which reflect anion uptake via basolateral cAMP-dependent anion transporters. In contrast, capsaicin potentiated apical Cl(-) conductance, which reflects conductivity through the cystic fibrosis transmembrane conductance regulator, a cAMP-regulated Cl(-) channel. All these paradoxical effects of capsaicin were mimicked by capsazepine. Forskolin application also increased phosphorylated myosin phosphatase target subunit 1, and the phosphorylation was prevented by capsaicin and capsazepine, suggesting that these capsaicinoids assume aspects of Rho kinase inhibitors. We also found that the increments in apical Cl(-) conductance were caused by conventional Rho kinase inhibitors, Y-27632 (20 μM) and HA-1077 (20 μM), with selective inhibition of basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1. Collectively, capsaicinoids inhibit cAMP-mediated anion transport through down-regulation of basolateral anion uptake, paradoxically accompanied by up-regulation of apical cystic fibrosis transmembrane conductance regulator-mediated anion conductance. The latter is mediated by inhibition of Rho-kinase, which is believed to interact with actin cytoskeleton.
American Journal of Respiratory Cell and Molecular Biology 01/2011; 45(4):684-91. · 5.13 Impact Factor
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ABSTRACT: The present study concerns previously unreported effects of menthol, a cyclic terpene alcohol produced by the peppermint herb, on anion transporters in polarized human airway Calu-3 epithelia. Application of menthol (0.01-1mM) attenuated transepithelial anion transport, estimated as short-circuit currents (I(SC)), after stimulation by forskolin (10microM) but not before. In contrast, menthol potentiated forskolin-stimulated and -unstimulated apical Cl(-) conductance, which reflected the cystic fibrosis transmembrane conductance regulator (CFTR: the cAMP-regulated Cl(-) channel)-mediated conductance, without correlation to changes in cytosolic cAMP levels. These results indicate that menthol-induced attenuation of forskolin-induced I(SC) despite CFTR up-regulation was due to cAMP-independent inhibition of basolateral anion uptake, which is the rate-limiting step for transepithelial anion transport. Analyses of the responsible basolateral anion transporters revealed that forskolin increased both bumetanide (an inhibitor of the basolateral Na(+)-K(+)-2Cl(-) cotransporter [NKCC1])- and DNDS (an inhibitor of basolateral HCO(3)(-)-dependent anion transporters [NBC1/AE2])-sensitive I(SC) in the control whereas only the former was prevented by the application of menthol. Neither the bumetanide- nor DNDS-sensitive component was, however, reduced by menthol without forskolin. These heterologous effects of menthol were reproduced by latrunculin B, an inhibitor of actin polymerization. F-actin staining showed that menthol prevented forskolin-stimulated rearrangements of actin microfilaments without affecting the distribution of forskolin-unstimulated microfilaments. Collectively, menthol functions as an activator of CFTR and prevents activation of NKCC1 without affecting NBC1/AE although all of these transporters are commonly cAMP-dependent. The heterologous effects may be mediated by the actin cytoskeleton, which interacts with CFTR and NKCC1.
European journal of pharmacology 03/2010; 635(1-3):204-11. · 2.59 Impact Factor
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ABSTRACT: We analyzed the mechanisms underlying the ion transport induced by tert-butyl hydroperoxide (t-BOOH), a membrane-permeant oxidant that has been widely used as a model of oxidative stress, in human airway epithelial cells (Calu-3). We found that t-BOOH induced a short-circuit current that was composed of two distinct components, a peaked component (PC) and a sustained component (SC). Both components were reduced by the presence of H-89 (N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline) [10 microM, a protein kinase A (PKA) inhibitor] and clofilium (100 microM, a cAMP-dependent K+ channel inhibitor) but not by charybdotoxin (50 nM, a human intermediate conductance Ca2+-activated K+ channel inhibitor), suggesting that both PC and SC were generated through a common PKA-dependent/Ca2+-independent pathway. Notwithstanding, analyses of the physiological properties revealed that PC and SC were attributable to different pathways. PC, but not SC, was correlated with apical membrane Cl- conductance and was inhibited by the cyclooxygenase (COX)-2 inhibitor NS-398 (N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide; 10 microM). In contrast, SC, but not PC, was composed of a component sensitive to bumetanide (50 microM), an inhibitor of the basolateral Na+-K+-2Cl- cotransporter (NKCC1), and was abolished by the cytoskeleton dysfunction induced by cytochalasin D (10 microM) and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide (Y-27632; 20 microM). Collectively, t-BOOH induces PKA-related anion secretion through two independent pathways: rapid activation of apical anion efflux through a COX-2-dependent/cytoskeleton-independent pathway and relatively delayed activation of NKCC1 for basolateral anion uptake through a COX-2-independent/cytoskeleton-dependent pathway.
Journal of Pharmacology and Experimental Therapeutics 08/2008; 327(2):453-64. · 3.83 Impact Factor
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ABSTRACT: Platelet-derived growth factor (PDGF), which is released from eosinophils and fibroblasts, may be implicated in the pathophysiology of bronchial asthma. To examine the involvement of airway inflammation in beta-adrenergic desensitization, the present study was designed to determine whether pre-exposure to PDGF deteriorates beta-adrenoceptor function in airway smooth muscle. We focused on Ca(2+) signaling as an intracellular mechanism involved in this phenomenon. Isometric tension and F(340)/F(380) (an indicator of intracellular Ca(2+) concentration) induced by isoprenaline and other cAMP-related agents were simultaneously measured before and after exposure to PDGF in fura-2-loaded guinea-pig tracheal smooth muscle. Indomethacin was applied throughout the experiments to abolish prostaglandin synthesis by PDGF. After exposure of the tissues to 10 ng/ml PDGF for 15 min, the effects of isoprenaline, a beta-adrenoceptor agonist, and forskolin, a direct inhibitor of adenylyl cyclase, against methacholine-induced contraction were markedly reduced with increasing F(340)/F(380). However, in the presence of verapamil, an inhibitor of voltage-dependent Ca(2+) channels, the reduced responsiveness to isoprenaline and forskolin induced by pre-exposure to PDGF was reversed with reducing F(340)/F(380). Reduced responsiveness to isoprenaline by PDGF was also not observed in the presence of Ca(2+)-free solution. The inhibitory effects of db-cAMP, an analogue of cAMP, and theophylline, a nonselective inhibitor of phosphodiesterase, were not attenuated by PDGF. In conclusion, pre-exposure to PDGF causes impairment of the beta-adrenoceptors/adenylyl cyclase processes in airway smooth muscle that is independent of cyclooxygenase synthesis by PDGF. Ca(2+) mobilization by Ca(2+) influx through voltage-dependent Ca(2+) channels is involved in this heterologous desensitization of beta-adrenoceptors.
European Journal of Pharmacology 07/2008; 591(1-3):259-65. · 2.52 Impact Factor
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ABSTRACT: Reactive oxidant species are implicated in the chronic airway inflammation related to asthma and chronic obstructive pulmonary disease. This study was designed to determine mechanisms underlying contraction induced by hydrogen peroxide (H(2)O(2)), a clinical marker of oxidative stress, in airway smooth muscle. Isometric tension and fluorescent intensities of fura-2, an index of intracellular Ca(2+) concentrations ([Ca(2+)](i)), were measured in epithelium-denuded tracheal smooth muscle tissues isolated from guinea pigs. H(2)O(2) (0.01-1 mM) caused contraction with an augmentation of [Ca(2+)](i) in a concentration-dependent manner in the normal physiological solution containing 2.4 mM of extracellular Ca(2+) concentrations. The contractile force and [Ca(2+)](i) by H(2)O(2) (1 mM) were approximately half of those in response to 1 microM methacholine. However, contraction by H(2)O(2) was not generated under the condition that extracellular Ca(2+) concentrations were less than 0.15 mM. Verapamil (10 microM), an inhibitor of voltage-operated Ca(2+) channels, partially but significantly inhibited the H(2)O(2)-induced contraction. In contrast, SKF-96365 (1-{beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride) (100 microM), a non-selective inhibitor of Ca(2+) channels, completely abolished both the contraction and the increase in [Ca(2+)](i) elicited by H(2)O(2). Moreover, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) (0.03-10 microM), an inhibitor of Rho-kinase, caused a concentration-dependent inhibition of the H(2)O(2)-induced contraction. In conclusion, both the Ca(2+) influx from the extracellular side and the Ca(2+) sensitization by Rho-kinase are involved in the regulation of airway smooth muscle tone induced by H(2)O(2). An inhibition of the Rho/Rho-kinase pathway may be beneficial for the treatment of airflow limitation mediated by oxidative stress.
European Journal of Pharmacology 03/2007; 556(1-3):151-6. · 2.52 Impact Factor
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ABSTRACT: In the present study, we investigated whether extracellular sphingosine 1-phosphate (S1P) is involved in airway hyper-reactivity in bronchial asthma. The effects of S1P on the response to methacholine was examined in the fura-2-loaded strips of guinea pig tracheal smooth muscle using simultaneous recording of the isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F(340)/F(380)). A 15-min pretreatment with S1P (>100 nM) markedly enhanced methacholine-induced contraction without elevating F(340)/F(380). This effect of S1P was suppressed in the presence of Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide], a selective inhibitor of Rho-kinase, in a concentration-dependent manner. Moreover, pretreatment with pertussis toxin caused an inhibition in S1P-induced hyper-reactivity to methacholine in a time- and concentration-dependent manner. In contrast, although S1P-induced Ca(2+) mobilization was attenuated by SKF96365 and verapamil, the subsequent response to methacholine was unaffected. A 15-min pretreatment with lower concentrations of S1P (<100 nM), which is clinically attainable, did not increase methacholine-induced contraction. However, when the incubation was lengthened to 6 h, S1P (<100 nM) enhanced the subsequent response to methacholine. Next, application of S1P to cultured human bronchial smooth muscle cells increased the proportion of active RhoA (GTP-RhoA) and phosphorylation of myosin phosphatase target subunit 1 (MYPT1). This phosphorylation of MYPT1 was significantly inhibited by application of Y-27632 and by pretreatment with pertussis toxin. Our findings demonstrate that exposure of airway smooth muscle to S1P results in airway hyper-reactivity mediated by Ca(2+) sensitization via inactivation of myosin phosphatase, which links G(i) and RhoA/Rho-kinase processes.
Journal of Pharmacology and Experimental Therapeutics 02/2007; 320(2):766-73. · 3.83 Impact Factor
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ABSTRACT: Enhanced proliferation of smooth muscle cells contributes to airway remodeling of bronchial asthma. Recently, statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, have been shown to inhibit proliferation of both vascular and airway smooth muscle cells independently of lowering cholesterol. However, the mechanisms remain to be elucidated. The purpose of this study was to determine molecular processes by which statins inhibit proliferation of human bronchial smooth muscle cells. Simvastatin (0.1-1.0 muM) significantly inhibited cell proliferation and DNA synthesis induced by FBS in a concentration-dependent manner. The inhibitory effects of simvastatin were antagonized by mevalonate and geranylgeranylpyrophosphate, whereas the effects were not affected by squalene and farnesylpyrophosphate. The antiproliferative effects of simvastatin were mimicked by GGTI-286, a geranylgeranyltransferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho, and Y-27632, an inhibitor of Rho-kinase, a target protein of RhoA. Western blot analysis showed that the level of membrane localization of RhoA (active Rho) was markedly increased by FBS, and that the level of active RhoA increased by FBS was reduced by simvastatin. Moreover, the inhibitory effect of simvastatin on FBS-induced RhoA activation was also antagonized by geranylgeranylpyrophosphate, but not by farnesylpyrophosphate. Because these isoprenoids are required for prenylation of small G proteins RhoA and Ras, respectively, the present results demonstrate that an inhibition in airway smooth muscle cell proliferation by simvastatin is due to prevention of geranylgeranylation of RhoA, not farnesylation of Ras. Therefore, statins may have therapeutic potential for prohibiting airway remodeling in bronchial asthma.
American Journal of Respiratory Cell and Molecular Biology 01/2007; 35(6):722-9. · 5.13 Impact Factor
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ABSTRACT: In guinea pigs, it is well-known that mechanical stretch of airway smooth muscle exhibits spontaneous tone which is mediated by cyclooxygenase (COX) activation. We tested the hypothesis that this spontaneous contraction of airway smooth muscle is mediated by stretch-activated non-selective cation channels and the Rho/Rho-kinase pathway, as well as COX-2 using a pharmacological approach. Isometric force and intracellular Ca(2+) concentrations ([Ca(2+)](i)) were assessed in isolated guinea pig tracheal smooth muscle tissues. The samples were stretched to a given level and the muscle behavior was monitored under isometric conditions. We observed an increase in [Ca(2+)](i) and subsequent force generation over a 15-min period. The augmented [Ca(2+)](i) and spontaneous contraction due to the stretch were markedly attenuated by application of Gd(3+), an inhibitor of stretch-activated channels, and removal of extracellular Ca(2+). In contrast, nifedipine only had a mild inhibitory effect on the contraction. (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide (Y-27632; a Rho-kinase inhibitor) abolished the spontaneous contraction with no changes in [Ca(2+)](i). Simvastatin, which down-regulates Rho activity, also significantly inhibited the contraction. Moreover, indomethacin, an inhibitor of COX-1 and -2, and N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398; a COX-2 inhibitor) abolished the stretch-induced contraction without affecting [Ca(2+)](i), whereas the inhibitory effect of 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC560; a COX-1 inhibitor) on the contraction was much less. These findings demonstrated that Ca(2+) entry via stretch-activated channels, the Rho/Rho-kinase pathway, and COX-2 are involved in the mechanotransduction in guinea pig tracheal smooth muscle. Additionally, while the Rho/Rho-kinase pathway and COX-2 regulate the spontaneous contraction independently of [Ca(2+)](i), COX-1 is not involved in the stretch-induced force generation.
European Journal of Pharmacology 01/2007; 552(1-3):135-42. · 2.52 Impact Factor
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ABSTRACT: The present study concerns intriguing effects of hydrogen peroxide (H2O2) on cAMP-mediated anion secretion in polarized human airway epithelia. Although H2O2 applied to the apical and basolateral membrane increases short-circuit currents (ISC) with analogous properties, it has opposite effects on subsequent cAMP-activated ISC responses. Namely, forskolin (FK)-induced ISC responses were down-regulated by the apical presence of H2O2, whereas they were up-regulated by its basolateral presence. Despite this contrasting effect, oxidative stimuli from either aspect of the monolayer hindered FK-induced increments in cytosolic cAMP levels and apical membrane Cl- conductance. The site-dependent effects of H2O2 were reproduced in the responses to 8-bromo-cAMP. Estimation of the anionic composition of the ISC revealed that the FK up-regulated both bumetanide [an Na+-K+-2Cl- cotransporter (NKCC1) inhibitor]-sensitive and 4,4'-dinitrostilbene-2,2'-disulfonic acid [an HCO3--dependent anion transporter (NBC1/AE2) inhibitor]-sensitive ISC in the control, whereas the up-regulation evidently favored bumetanide-sensitive ISC in the basolateral presence of H2O2. The FK-induced NKCC1 augmentation after exposure to basolateral H2O2 was counteracted by cytochalasin D, an inhibitor of microfilament function, but not by charybdotoxin, a blocker of the intermediate conductance Ca2+-activated K+ channel, whose activation could be related to NKCC1-mediated Cl- secretion. These observations suggest that basolaterally but not apically applied H2O2 potentiates subsequent cAMP-mediated Cl- secretion by an increase in Cl- uptake via basolateral NKCC1, whose sensitivities to cAMP/protein kinase A are up-regulated, overcoming the H2O2-induced inhibition of cAMP-mediated apical anion conductance. The basolateral membrane-specific effects of H2O2 may be relevant to the basolateral cytoskeleton, which is believed to interact with NKCC1.
Journal of Pharmacology and Experimental Therapeutics 08/2006; 318(1):296-303. · 3.83 Impact Factor
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ABSTRACT: Properties of smooth and cardiac L-type Ca2+ channels differ prominently in several physiological aspects, including sympathetic modulation. To assess the possible underlying mechanisms, we applied the whole cell patch-clamp technique to guinea pig detrusor smooth muscle cells, in which only L-type Ca2+ channel currents are observed in practice. During depolarization to large positive potentials, the conformation of the majority of L-type Ca2+ channels is converted from the normal (O1) to a second open state (O2), which undergoes little inactivation during depolarization. Extracellular application of genistein, a known tyrosine kinase inhibitor, significantly attenuated the voltage-dependent conversion of Ca2+ channels to O2, accompanied by reduction of availability, whereas genistin, an inactive analog, had little effect. In the absence of ATP in the patch pipette, intracellular application of either genistein or tyrphostin-47 suppressed the conversion to O2. Computer calculation revealed that the acceleration of the O1 to an inactivated state qualitatively reconstructs the unique effects of PTK inhibitors antagonized by ATP. We concluded that under normal conditions smooth muscle L-type Ca2+ channels are already modulated by tyrosine-kinase and ATP-related mechanism(s) and thereby easily achieve the second conversion, which yields voltage-dependent modulation of L-type Ca2+ current analogous to that in cardiac myocytes during beta-adrenoceptor stimulation.
The FASEB Journal 08/2006; 20(9):1492-4. · 5.71 Impact Factor
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ABSTRACT: We examined the mechanisms underlying anion secretion mediated by protease-activated receptor 2 (PAR2) and its role in the regulation of ion transport, using polarized human airway Calu-3 cells. PAR2 stimulation by trypsin and a PAR2-activating peptide (PAR2AP), especially from the basolateral aspect, caused transient Cl(-) secretion due to cytosolic Ca(2+) mobilization. Antagonists of PI-PLC (U73122, ET-18-OCH(3)) and inositol 1,4,5-triphosphate (xestospongin C (Xest C)) were without effect on the PAR2AP-mediated Cl(-) secretion, whereas it was attenuated by D609 (a PC-PLC inhibitor) and phorbol 12-myristate 13 acetate (PMA, a PKC activator). Even 30 min after removal of PAR2AP after a 10-min-exposure, cells were still poorly responsive to PAR2 stimulation, but the reduced responsiveness was upregulated by a PKC inhibitor, GF109203X (GFX). Pretreatment with PAR2AP did not affect responses to anion secretagogues, such as isoproterenol, forskolin, thapsigargin, 1-ethyl-2-benzimdazolinone, and adenosine, but ATP-induced responses were significantly reduced. Nystatin permeabilization studies revealed that the presence of PAR2AP prevented ATP-induced increments in basolateral membrane K(+) conductance without affecting apical membrane Cl(-) conductance. ATP-elicited Ca(2+) mobilization, which was sensitive to D609 and PMA, was inhibited by the pretreatment with PAR2AP, and this inhibition was blunted by the presence of GFX. Collectively, stimulation of PAR2 generates a brief response of Cl(-) secretion through PC-PLC-mediated pathway, followed by not only auto-desensitization of PAR2 itself but also cross-desensitization of a PC-PLC-coupled purinoceptor. The two types of desensitization seem likely to have PKC-mediated downregulation of PC-PLC in common.
British Journal of Pharmacology 11/2005; 146(3):397-407. · 4.41 Impact Factor
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Shinsuke Nakayama,
Susumu Ohya,
Hong-Nian Liu,
Toshiya Watanabe,
Shinji Furuzono,
Jing Wang,
Yuji Nishizawa,
Masahiro Aoyama,
Naruhiko Murase,
Tatsuaki Matsubara, Yasushi Ito,
Yuji Imaizumi,
Shunichi Kajioka
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ABSTRACT: Appropriate gastrointestinal motility is essential to properly control the body energy level. Intracellular Ca2+ ([Ca2+]i) oscillations in interstitial cells of Cajal (ICCs; identified with c-Kit immunoreactivity) are considered to be the primary mechanism for the pacemaker activity in gastrointestinal motility. In the present study, RT-PCR examinations revealed predominant expression of the type 1 isoform of sulphonylurea receptors (SUR1) in ICCs of the mouse ileum, but expression of SUR2 was predominant in smooth muscle. In cell clusters prepared from the same tissue, smooth muscle contractility and pacemaker [Ca2+]i activity in ICCs were found to be differentially modulated by K(ATP) channel openers and sulphonylurea compounds, in accordance with the expression of SUR isoforms. 1 microM cromakalim nearly fully suppressed the mechanical activity in smooth muscle, whereas ICC pacemaker [Ca2+]i oscillations persisted. Greater concentrations (approximately 10 microM) of cromakalim attenuated pacemaker [Ca2+]i oscillations. This effect was not reversed by changing the reversal potential of K+, but was prevented by glibenclamide. Diazoxide at 30 muM terminated ICC pacemaker [Ca2+]i oscillations, but again treatment with high extracellular K+ did not restore them. These results suggest that SUR can modulate pacemaker [Ca2+]i oscillations via voltage-independent mechanism(s), and also that intestinal pacemaking and glucose control are closely associated with SUR.
Journal of Cell Science 10/2005; 118(Pt 18):4163-73. · 6.11 Impact Factor
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ABSTRACT: The Fas/Fas ligand (FasL) system is a major regulator of apoptosis. Chemotherapeutic drugs have been shown to induce Fas expression on the surface of lung cancer cells, and cancer cell apoptosis. However, this mechanism is not considered to be associated with Fas expressed on lung cancer cells. Soluble Fas and FasL concentrations are reportedly elevated in the peripheral blood of patients with lung cancer, but the roles of circulating soluble Fas and FasL in that disease have not been clarified.
We measured the circulating soluble Fas and FasL levels in 21 patients with small cell lung cancer (SCLC), and 12 healthy matched controls, in order to examine whether such ligands could provide any important information and/or reveal any new clinical features of SCLC.
In the CR patients, the neuronal specific enolase (NSE), soluble Fas and soluble FasL concentrations were 21.26+/-3.65 ng/ml, 3.58+/-0.19 ng/ml and 0.50+/-0.15 ng/ml, while in the partial response (PR)/no change (NC)/progressive disease (PD) group of patients they were 33.96+/-7.86 ng/ml, 5.29+/-0.29 ng/ml and 0.59+/-0.07 ng/ml, respectively. The NSE, soluble Fas and soluble FasL concentrations were all elevated in the PR/NC/PD patients, however, significant differences were only seen in Fas concentration between CR and PR/NC/PD patients and CR patients and the controls (p<0.001).
Serum soluble Fas and FasL play important roles in the proliferation and metastasis of SCLC, as well as in the cytotoxic reaction and apoptosis induced by anticancer drugs in SCLC. Further study of the mechanisms and participation of circulating soluble Fas and FasL is necessary to develop treatment strategies for SCLC.
Cancer Detection and Prevention 01/2005; 29(2):175-80. · 2.52 Impact Factor
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ABSTRACT: The present study concerns the involvement of the ceramide produced through sphingomyelinase (SMase)-mediated catalysis in airway anion secretion of Calu-3 cells. Short-circuit current (Isc) measurement revealed that isoproterenol (ISO, 0.1 microM)-induced anion secretion was prevented by pretreatment with SMase (0.3 U/ml, for 30 min) from the basolateral but not the apical side, although basal and 1-ethyl-2-benzimidazolinone (1-EBIO, a Ca2+-activated K+ channel opener)-induced Isc were unaffected. The effects of SMase were reproduced in responses to forskolin (20 microM) or 8-bromo-cAMP (2 mM). C2-ceramide, a cell-permeable analog, also repressed the 8-bromo-cAMP-induced responses. Nystatin permeabilization studies confirmed that the SMase- and C2-ceramide-induced repressions were due to hindrance of augmentation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated conductance across the apical membrane. Further, SMase failed to influence K+ conductance across the basolateral membrane. These results suggest that the ceramide originating from basolateral sphingomyelin acts on activated CFTR from the cytosolic side, hindering anion secretion.
Biochemical and Biophysical Research Communications 12/2004; 324(2):901-8. · 2.48 Impact Factor
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Masahiro Aoyama,
Aki Yamada,
Jing Wang,
Susumu Ohya,
Shinji Furuzono,
Takayo Goto,
Shingo Hotta, Yasushi Ito,
Tatsuaki Matsubara,
Kaoru Shimokata,
S R Wayne Chen,
Yuji Imaizumi,
Shinsuke Nakayama
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ABSTRACT: Intracellular Ca(2+) ([Ca(2+)](i)) oscillations seen in interstitial cells of Cajal (ICCs) are considered to be the primary pacemaker activity in the gut. Here, we show evidence that periodic Ca(2+) release from intracellular Ca(2+) stores produces [Ca(2+)](i) oscillations in ICCs, using cell cluster preparations isolated from mouse ileum. The pacemaker [Ca(2+)](i) oscillations in ICCs are preserved in the presence of dihydropyridine Ca(2+) antagonists, which suppress Ca(2+) activity in smooth muscle cells. However, applications of drugs affecting either ryanodine receptors or inositol 1,4,5-trisphosphate receptors terminated [Ca(2+)](i) oscillations at relatively low concentrations. RT-PCR analyses revealed a predominant expression of type 3 RyR (RyR3) in isolated c-Kit-immunopositive cells (ICCs). Furthermore, we demonstrate that pacemaker-like global [Ca(2+)](i) oscillation activity is endowed by introducing RyR3 into HEK293 cells, which originally express only IP(3)Rs. The reconstituted [Ca(2+)](i) oscillations in HEK293 cells possess essentially the same pharmacological characteristics as seen in ICCs. The results support the functional role of RyR3 in ICCs.
Journal of Cell Science 07/2004; 117(Pt 13):2813-25. · 6.11 Impact Factor
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ABSTRACT: Airway mucociliary clearance is subject to the autocrine/paracrine regulation of extracellular nucleotides released from the airway epithelial cells. The present study was performed in pursuit of effective modulators of ATP release under physiologic conditions in polarized human airway epithelial cells (Calu-3). Neither isoproterenol, forskolin, nor ionomycin augmented extracellular ATP release detected by luciferase assay. However, direct activation of the human intermediate conductance, Ca(2+)-activated K(+) channel (hIK-1) by 1-ethyl-2-benzimdazolinone (1-EBIO, 1 mM) and chlorzoxazone (CZ, 1 mM) increased ATP release predominantly in the apical compartment. Measurement of fluo-3 signals revealed that 1-EBIO- and CZ-stimulated cytosolic Ca(2+) mobilization was suppressed by the presence of MRS-2179, a specific P2Y(1) receptor antagonist. The hIK-1-mediated ATP release was inhibited by a hIK-1 blocker (charybdotoxin), and an Na(+)-K(+)-2Cl(-) cotransport blocker (bumetanide) without interruption by GdCl(3), an inhibitor of stretch-activated nonselective cation (SA) channels, or glybenclamide, a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR). These results suggest that a cell volume decrease via the hIK-1-mediated KCl loss and the resultant induction of a regulatory volume increase via the Na(+)-K(+)-2Cl(-) transporter may trigger release of ATP, which causes P2Y(1)-mediated Ca(2+) mobilization, through mechanisms unrelated to the CFTR and SA channels.
American Journal of Respiratory Cell and Molecular Biology 04/2004; 30(3):388-95. · 5.13 Impact Factor
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ABSTRACT: The present study investigated mechanisms underlying apical and basolateral P2Y(1)-mediated Cl(-) secretion in human airway epithelial cells. Apical and basolateral ATP induced short-circuit currents (I(sc)) with different properties via P2Y(1) receptors. The former comprised an immediate rise followed by a slow attenuation, whereas the latter was a transient rise with a higher peak and shorter duration (< 2 min). The actions of ATP were simulated by those of ADP, ADPbetaS, and ATPgammaS. Antagonists of phosphatidylinositol-phospholipase C (U73122, ET-18-OCH(3)) were without any effect on the bilateral ATP-induced I(sc), which were, in contrast, attenuated by a phosphatidylcholine-phospholipase C inhibitor (D609) and an adenylate cyclase inhibitor (SQ22536). The responses to ATP from either aspect were also sensitive to an intracellular Ca(2+) chelator, 1,2-bis (o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester, or a Ca(2+)-activated K(+) channel inhibitor, charybdotoxin, although differential Ca(2+) signals were concomitant with each reaction. Nystatin permeabilization studies revealed a good correlation between the I(sc) and the basolateral K(+) current rather than the apical Cl(-) current under ATP-stimulated conditions. In conclusion, apical and basolateral P2Y(1) receptors couple with both phosphatidylcholine-phospholipase C and adenylate cyclase, leading to Cl(-) secretion, whose rate is essentially regulated by the Ca(2+)-activated K(+) channel-mediated K(+) conductance. This suggests the importance of this channel in airway mucociliary clearance.
American Journal of Respiratory Cell and Molecular Biology 04/2004; 30(3):411-9. · 5.13 Impact Factor
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ABSTRACT: The human respiratory tract is constantly exposed to polycyclic aromatic hydrocarbons (PAHs) through inhalation of atmospheric pollutants. We examined the effects of three PAHs (benzo[a]pyrene, anthracene, and fluoranthene) on the airway ion transport, which is essential for lung defense and normal airway function, using human airway epithelia (Calu-3). These three PAHs had no significant effect on the basal short-circuit current (I(sc)). However, fluoranthene (1-100 microM) applied in the apical compartment potentiated I(sc) in response to cAMP-related agents (isoproterenol, forskolin, and 8-bromo-cAMP). The effects of fluoranthene were unaffected by ellipticine, a PAH receptor antagonist. Estimation of the anionic composition of I(sc) revealed that isoproterenol increased both HCO(3)(-) and Cl(-) transport in the control, whereas it potentiated only Cl(-) transport in the presence of fluoranthene. The fluoranthene-induced modulations of these anion transporters were counteracted by charybdotoxin (ChTx, a hIK-1 channel blocker). Fluoranthene gradually augmented the ChTx-sensitive K(+) current (I(K)) across the basolateral membrane, accompanied by a sustained increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)). In the presence of fluoranthene, however, a much larger hIK-1-dependent I(K) was identified by the application of 8-bromo-cAMP without concomitant elevation of [Ca(2+)](i). These results suggest that fluoranthene switches from cAMP-dependent HCO(3)(-) secretion to Cl(-) secretion through the hIK-1 channel, whose sensitivity to protein kinase A may be up-regulated by the sustained [Ca(2+)](i) elevation produced by this chemical.
Journal of Pharmacology and Experimental Therapeutics 03/2004; 308(2):651-7. · 3.83 Impact Factor
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ABSTRACT: 1. Lysophosphatidylcholine (Lyso-PC), which is synthesized by phospholipase A2, is generally considered to induce adhesion molecules. However, little is known about the involvement of Lyso-PC in the pathogenesis of bronchial asthma. The present study was designed to examine whether pre-exposure to Lyso-PC causes eosinophil recruitment and an increase in resistance in airways. 2. Eosinophils in bronchoalveolar lavage fluid (BALF) and the airway walls were enumerated after inhalation of 0.5 mg/mL Lyso-PC to guinea-pigs for 10 min. Respiratory resistance (Rrs) was recorded continuously over 6 h after inhalation of an equi-dose of Lyso-PC for an equivalent period. 3. The proportion of eosinophils was increased from 10.7 +/- 3.3 to 27.5 +/- 3.1% (P < 0.0001) in BALF 6 h after inhalation of Lyso-PC, whereas the proportion of neutrophils and lymphocytes was not increased. Histological examination also showed uniform distribution of eosinophils in the airway wall of bronchi and bronchioles 6 h after inhalation of Lyso-PC. The number of eosinophils (/10 h.p.f.) in the bronchi and bronchioles was increased from 43.5 +/- 16.8 to 154.8 +/- 21.7 (P < 0.0001) and from 34.8 +/- 0.7 to 106.0 +/- 26.6 (P < 0.01), respectively. This eosinophil infiltration was similarly observed 24 h later. 4. Next, we examined the effects of eosinophil infiltration induced by Lyso-PC on Rrs. Inhalation of Lyso-PC caused a slow increase in Rrs and the percentage increase in Rrs was 19.8 +/- 1.9% (P < 0.0001) 6 h later. Eosinophil infiltration and an increase in Rrs did not occur after inhalation of physiological saline. These phenomena induced by Lyso-PC were diminished by pretreatment with dexamethasone (6 micro g/kg per day for 3 days). 5. Lysophosphatidylcholine causes eosinophil infiltration and a subsequent increase in resistance in airways. Our results indicate that Lyso-PC may be involved in the pathophysiology of bronchial asthma.
Clinical and Experimental Pharmacology and Physiology 03/2004; 31(3):179-84. · 1.85 Impact Factor
