Research Achievements - Original paper -
-
Comparative Study of Transcriptome in the Hearts Isolated from Mice, Rats, and Humans
Okada D.
Biomolecules ( Biomolecules ) 12 ( 6 ) 2022.06 [Refereed]
Research paper (journal)
-
Okamoto Y.
Biomolecules ( Biomolecules ) 12 ( 5 ) 2022.05 [Refereed]
Research paper (journal)
-
Nomura K.
Environmental Health and Preventive Medicine ( Environmental Health and Preventive Medicine ) 26 ( 1 ) 2021.12 [Refereed]
Research paper (journal)
-
Fukuda Y.
Biology of Reproduction ( Biology of Reproduction ) 105 ( 1 ) 258 - 266 2021.07 [Refereed]
Research paper (journal)
<jats:title>Abstract</jats:title>
<jats:p>To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.</jats:p> -
Takahashi K.
Heliyon ( Heliyon ) 7 ( 6 ) 2021.06 [Refereed]
Research paper (journal)
-
Requirement of the Ca<sup>2+</sup> channel β<inf>2</inf> subunit for sympathetic PKA phosphorylation
Murakami M.
Journal of Pharmacological Sciences ( Journal of Pharmacological Sciences ) 145 ( 3 ) 253 - 261 2021.03 [Refereed]
Research paper (journal)
-
Detection of pathologic heart murmurs using a piezoelectric sensor
Takahashi K.
Sensors (Switzerland) ( Sensors (Switzerland) ) 21 ( 4 ) 1 - 12 2021.02 [Refereed]
Research paper (journal)
-
Takagi D.
Journal of Physiological Sciences ( Journal of Physiological Sciences ) 70 ( 1 ) 2020.02 [Refereed]
Research paper (journal)
-
Okamoto Y.
Journal of Biological Chemistry ( Journal of Biological Chemistry ) 294 ( 44 ) 16049 - 16061 2019.11 [Refereed]
Research paper (journal)
-
Umehara S.
International Journal of Molecular Sciences ( International Journal of Molecular Sciences ) 20 ( 12 ) 2019.06 [Refereed]
Research paper (journal)
-
Medaka as a model for ECG analysis and the effect of verapamil
Yonekura M.
Journal of Pharmacological Sciences ( Journal of Pharmacological Sciences ) 137 ( 1 ) 55 - 60 2018.05 [Refereed]
Research paper (journal)
-
Stromal interaction molecule 1 haploinsufficiency causes maladaptive response to pressure overload
Ohba T.
PLoS ONE ( PLoS ONE ) 12 ( 11 ) 2017.11 [Refereed]
Research paper (journal)
-
In vitro effect of nicorandil on the carbachol-induced contraction of the lower esophageal sphincter of the rat.
Tomonori Shimbo, Takeshi Adachi, Susumu Fujisawa, Mai Hongoh, Takayoshi Ohba, Kyoichi Ono
Journal of pharmacological sciences 131 ( 4 ) 267 - 74 2016.08 [Refereed]
Research paper (journal)
The lower esophageal sphincter (LES) is a specialized region of the esophageal smooth muscle that allows the passage of a swallowed bolus into the stomach. Nitric oxide (NO) plays a major role in LES relaxation. Nicorandil possesses dual properties of a NO donor and an ATP-sensitive potassium channel (KATP channel) agonist, and is expected to reduce LES tone. This study investigated the mechanisms underlying the effects of nicorandil on the LES. Rat LES tissues were placed in an organ bath, and activities were recorded using an isometric force transducer. Carbachol-induced LES contraction was significantly inhibited by KATP channel agonists in a concentration-dependent manner; pinacidil >> nicorandil ≈ diazoxide. Nicorandil-induced relaxation of the LES was prevented by pretreatment with glibenclamide, whereas N(G)-nitro-l-arginine methyl ester (l-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and iberiotoxin were ineffective at preventing nicorandil-induced LES relaxation. Furthermore, nicorandil did not affect high K(+)-induced LES contraction. Reverse-transcription polymerase chain reaction analysis and immunohistochemistry revealed expression of KCNJ8 (Kir6.1), KCNJ11 (Kir6.2), ABCC8 (SUR1) and ABCC9 (SUR2) subunits of the KATP channel in the rat lower esophagus. These findings indicate that nicorandil causes LES relaxation chiefly by activating the KATP channel, and that it may provide an additional pharmacological tool for the treatment of spastic esophageal motility disorders.
-
Age-related effects of dexmedetomidine on myocardial contraction and coronary circulation in isolated guinea pig hearts.
Maiko Hongo, Susumu Fujisawa, Takeshi Adachi, Tomonori Shimbo, Shigehiro Shibata, Takayoshi Ohba, Kyoichi Ono
Journal of pharmacological sciences 131 ( 2 ) 118 - 25 2016.06 [Refereed]
Research paper (journal)
Dexmedetomidine is a selective α2 adrenergic agonist. Although dexmedetomidine is widely used for sedation and analgesia, it frequently produces hypotension and bradycardia. The present study aimed to evaluate the effects of dexmedetomidine on cardiac function and coronary circulation using Langendorff-perfused guinea pig hearts. Coronary perfusion pressure (CPP) and left ventricular pressure (LVP) were continuously monitored, and electric field stimulation (EFS) was applied to stimulate sympathetic nerve terminals. Dexmedetomidine almost completely inhibited the EFS-induced increase in LVP at all ages. The effect of dexmedetomidine on coronary artery resistance varied according to postnatal age, i.e., dexmedetomidine had little effect on CPP in young hearts (<4 weeks) but increased CPP by 10 mmHg at 4-8 weeks and by 15 mmHg at >8 weeks. The increase in CPP in adult hearts was inhibited by imiloxan, an α2B antagonist, and prazosin, an α1 antagonist. The results suggest that dexmedetomidine acts on α2 adrenergic receptors at sympathetic nerve terminals to suppress the release of norepinephrine. In addition, the findings suggest that dexmedetomidine directly affects α1 adrenoceptors and/or α2B adrenoceptors on coronary smooth muscles to increase CPP. The age-related changes in α adrenoceptor subtypes may be linked to the cardiodepressant effects of dexmedetomidine.
-
Modified sympathetic nerve regulation in AKAP5-null mice.
Chong Han, Hirofumi Tomita, Takayoshi Ohba, Kimitaka Nishizaki, Yoshiki Ogata, Yasushi Matsuzaki, Daisuke Sawamura, Teruyuki Yanagisawa, Tomohiro Osanai, Tadaatsu Imaizumi, Atsushi Matsubara, Takeshi Adachi, Kyoichi Ono, Ken Okumura, Manabu Murakami
Biochemical and biophysical research communications 469 ( 4 ) 897 - 902 2016.01 [Refereed]
Research paper (journal)
Genetic analyses have revealed an important association between A-kinase anchoring proteins (AKAPs) and the intracellular calcium modulating system. AKAP5, also known as AKAP79/150, is an anchoring protein between PKA and voltage-dependent calcium channels, ryanodine receptor-2, phospholamban and other molecules. The aim of the present study was to elucidate the physiological importance of AKAP5 in the creation of cardiac rhythm using AKAP5-null mice. ECG analysis showed a normal sinus rhythm and a decreased responsiveness to isoproterenol in AKAP5-null mice compared with wild-type mice. Analysis of heart rate variability revealed that the R-R interval was unstable in AKAP5-null mutants and that the low-frequency components had decreased, indicating that the tonus of the sympathetic nervous system was affected. Furthermore, the atrium of the AKAP5-null mice showed a decreased positive inotropic response to isoproterenol, indicating the involvement of AKAP5 in a PKA-dependent pathway. Thus, our present study revealed that AKAP5 plays a significant role in the regulation of sympathetic nerve activities.
-
Involvement of the orexin system in sympathetic nerve regulation.
Manabu Murakami, Takayoshi Ohba, Testuya Kushikata, Hidetoshi Niwa, Akira Kurose, Tadaatsu Imaizumi, Hiroyuki Watanabe, Teruyuki Yanagisawa, Shigeyuki Nakaji, Kyoichi Ono, Kazuyoshi Hirota
Biochemical and biophysical research communications 460 ( 4 ) 1076 - 81 2015.05 [Refereed]
Research paper (journal)
Orexin, also known as hypocretin, is a secreted neuropeptide implicated in the regulation of sleep and food intake. In the present study, we examined the importance of orexin in regulation of the sympathetic nervous system using an orexin/ataxin-3 transgenic (OXTg) rat, which has a minimal number of orexin neurons. RT-PCR analysis identified expression of prepro-orexin and orexin receptor-1 (OX1R) in the superior cervical ganglion (SCG), and expression of another receptor (OX2R) was marginal in the wild-type rat. The orexin/ataxin-3 transgenic rat showed increased expression of OX1R and OX2R, whereas expression of prepro-orexin was undetectable, suggesting a compensatory increase in both receptors. In the ECG recording (R-R interval), orexin/ataxin-3 transgenic rats showed decreased responsiveness to the β-adrenergic blocker propranolol. Furthermore, OXTg rats had deteriorated R-R interval regulation, indicating involvement of the orexin system in sympathetic nerve regulation. This was accompanied by decreased baroreflex and responsiveness to β-adrenergic blocker in blood pressure recording, also suggesting involvement of the orexin system in sympathetic nerve regulation. Histological examination revealed hypotrophic changes in the transgenic heart, suggesting involvement of the orexin system in cardiac development. Taken together, our present results indicate involvement of the orexin system in sympathetic nerve control.
-
Modified autonomic regulation in mice mutated in the β4 subunit of the lh/lh calcium channel.
Manabu Murakami, Takashi Suzuki, Tsai-Wen Wu, Kenji Kuwasako, Eiki Takahashi, Hiroyuki Watanabe, Agnieszka M Murakami, Ichiro Miyoshi, Teruyuki Yanagisawa, Hironobu Sasano, Kyoichi Ono, Takayoshi Ohba
Biochemical and biophysical research communications 461 ( 2 ) 200 - 5 2015.05 [Refereed]
Research paper (journal)
Genetic analyses have revealed an important association between P/Q-type calcium channel activities and hereditary neurological disorders. The P/Q-type channels are composed principally of heterologous multimeric subunits including CaV2.1 and CaVβ4. Of these, the β4 subunit is thought to play a significant role in channel physiology, because a mouse line mutant in that subunit (the lethargic mouse: lh) exhibits a severe ataxic phenotype. The aim of the present study was to elucidate the physiological importance of the β4 subunit. ECG analysis showed that the T wave was high in 8-week-old lh mutants; this may be associated with hyperkalemia. Upon pharmacological ECG analysis, 2-3-week-old lh mutants exhibited reduced responses to a β-blocker and a muscarinic receptor antagonist. Analysis of heart rate variability revealed that the R-R interval was unstable in lh mutants and that both the low- and high-frequency components had increased in extent, indicating that the tonus of both the sympathetic and parasympathetic nervous systems was modified. Thus, our present study revealed that the β4 subunit played a significant role in regulation of sympathetic and parasympathetic nerve activities.
-
Apnea during Cheyne-Stokes-like breathing detected by a piezoelectric sensor for screening of sleep disordered breathing
Takashi Koyama, Shinichi Sato, Takashi Kanbayashi, Hideaki Kondo, Hiroyuki Watanabe, Seiji Nishino, Tetsuo Shimizu, Hiroshi Ito, Kyoichi Ono
SLEEP AND BIOLOGICAL RHYTHMS ( WILEY-BLACKWELL ) 13 ( 1 ) 57 - 67 2015.01 [Refereed]
Research paper (journal)
A simplified diagnostic/monitoring instrument for use in primary screening for sleep-disordered breathing (SDB) has been desired. This study was designed to assess the validity of a newly developed piezoelectric sensor as a simple and noninvasive tool for primary screening for sleep-disordered breathing. Forty-three consecutive patients suspected of having sleep-disordered breathing and 10 healthy volunteers were enrolled. Breathing movement was detected with the piezoelectric sensor (180 x 30 x 1mm), which was placed under a bed sheet under patients, and simultaneous polysomnographic recordings were obtained. We counted the number of apneas of >10s, irrespective of central or obstructive, that appeared during Cheyne-Stokes-like breathing with a waxing and waning pattern with an amplitude ratio of >10 in the piezoelectric sensor signal. The correlation coefficient between the number of apneas and apnea-hypopnea index (AHI) was 0.74 (P < 0.0001). The sensitivity/specificity of the number of apneas for distinguishing non-SDB patients from mild SDB patients (AHI 5) were 92.1/60.0% and those for separating moderate to severe (AHI 15) and severe (AHI 30) SDB patients from the other patients were 96.9/100% and 93.8/86.5%, respectively. There were no complaints from patients about the use of the piezoelectric sensor. The results indicate that the number of apneas detected by this piezoelectric sensor during Cheyne-Stokes-like breathing is a potential new index for primary screening for SDB at least for cardiac patients. Thus, the piezoelectric-sensor system might be useful for simple, noninvasive and comfortable SDB screening at home, hospitals and health care facilities.
-
Pathological impact of hyperpolarization-activated chloride current peculiar to rat pulmonary vein cardiomyocytes.
Yosuke Okamoto, Koichi Kawamura, Yuta Nakamura, Kyoichi Ono
Journal of molecular and cellular cardiology 66 53 - 62 2014.01 [Refereed]
Research paper (journal)
Pulmonary veins (PVs) are believed to be a crucial origin of atrial fibrillation. We recently reported that rat PV cardiomyocytes exhibit arrhythmogenic automaticity in response to norepinephrine. Herein, we further characterized the electrophysiological properties underlying the potential arrhythmogenicity of PV cardiomyocytes. Patch clamping studies revealed a time dependent hyperpolarization-activated inward current in rat PV cardiomyocytes, but not in left atrial (LA) myocytes. The current was Cs(+) resistant, and was not affected by removal of external Na(+) or K(+). The current was inhibited with Cd(2+), and the reversal potential was sensitive to changes in [Cl(-)] on either side of the membrane in a manner consistent with a Cl(-) selective channel. Cl(-) channel blockers attenuated the current, and slowed or completely inhibited the norepinephrine-induced automaticity. The biophysical properties of the hyperpolarization-activated Cl(-) current in rat PVs were different from those of ClC-2 currents previously reported: (i) the voltage-dependent activation of the Cl(-) current in rat PVs was shifted to negative potentials as [Cl(-)]i increased, (ii) the Cl(-) current was enhanced by extracellular acidification, and (iii) extracellular hyper-osmotic stress increased the current, whereas hypo-osmotic cell swelling suppressed the current. qPCR analysis revealed negligible ClC-2 mRNA expression in the rat PV. These findings suggest that rat PV cardiomyocytes possess a peculiar voltage-dependent Cl(-) channel, and that the channel may play a functional role in norepinephrine-induced automaticity.
-
Effects of propofol on electrocardiogram measures in mice.
Tomohiro Shintaku, Takayoshi Ohba, Hidetoshi Niwa, Tetsuya Kushikata, Kazuyoshi Hirota, Kyoichi Ono, Yasushi Matsuzaki, Tadaatsu Imaizumi, Kenji Kuwasako, Daisuke Sawamura, Manabu Murakami
Journal of pharmacological sciences 126 ( 4 ) 351 - 8 2014 [Refereed]
Research paper (journal)
We investigated the anesthetic effects of propofol on the electrocardiogram (ECG) in mice. We also compared the effects of isoflurane (2%) inhalation anesthesia, intraperitoneal propofol (50 or 100 mg/kg), and pentobarbital (50 mg/kg) on ECG in mice. Isoflurane inhalation and pentobarbital anesthesia were both associated with an acceptable heart rate (HR) range (ca. 450 - 500 bpm). In contrast, high-dose propofol anesthesia significantly decreased the HR. Importantly, propofol anesthesia led to significantly reduced responses to propranolol, a β-blocker, suggesting that it affects sympathetic tonus and is not suitable for the evaluation of cardiovascular or sympathetic function. Propofol also reduced the response to atropine, indicative of suppression of mouse parasympathetic nerve activity. Our data suggest that propofol anesthesia should not be the first choice for cardiovascular analysis in mice.
-
Inhalation anesthesia is preferable for recording rat cardiac function using an electrocardiogram.
Manabu Murakami, Hidetoshi Niwa, Tetsuya Kushikata, Hiroyuki Watanabe, Kazuyoshi Hirota, Kyoichi Ono, Takayoshi Ohba
Biological & pharmaceutical bulletin 37 ( 5 ) 834 - 9 2014 [Refereed]
Research paper (journal)
The effects of inhalation anesthesia (2% isoflurane, sevoflurane, or enflurane) and intraperitoneal anesthesia with pentobarbital (65 mg/kg) were compared in rats using an electrocardiogram (ECG) and determination of blood oxygen saturation (SPO2) levels. Following inhalation anesthesia, heart rate (HR) and SPO2 were acceptable while pentobarbital anesthesia decreased HR and SPO2 significantly. This indicates that inhalation anesthesia is more preferable than pentobarbital anesthesia when evaluating cardiovascular factors. Additionally, pentobarbital significantly increased HR variability (HRV), suggesting a regulatory effect of pentobarbital on the autonomic nervous system, and resulted in a decreased response of the baro-reflex system. Propranolol or atropine had limited effects on ECG recording following pentobarbital anesthesia. Taken together, these data suggest that inhalation anesthesia is suitable for conducting hemodynamic analyses in the rat.
-
Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy.
Hironori Kiso, Takayoshi Ohba, Kenji Iino, Kazuhiro Sato, Yutaka Terata, Manabu Murakami, Kyoichi Ono, Hiroyuki Watanabe, Hiroshi Ito
Biochemical and biophysical research communications 436 ( 3 ) 514 - 8 2013.07 [Refereed]
Research paper (journal)
BACKGROUND: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. METHODS AND RESULTS: In cultured neonatal rat cardiomyocytes, a 48 h treatment with 10nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. CONCLUSIONS: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression.
-
The mechanism of increased postnatal heart rate and sinoatrial node pacemaker activity in mice.
Takeshi Adachi, Shigehiro Shibata, Yosuke Okamoto, Shinichi Sato, Susumu Fujisawa, Takayoshi Ohba, Kyoichi Ono
The journal of physiological sciences : JPS 63 ( 2 ) 133 - 46 2013.03 [Refereed]
Research paper (journal)
Heart rate (HR) of mammalian species changes postnatally, i.e., HR of large animals including humans decreases, while HR in small animals such as mice and rats increases. To clarify cellular mechanisms underlying the postnatal HR changes, we performed in vivo HR measurement and electrophysiological analysis on sinoatrial node (SAN) cells in mice. The in vivo HR was ~320 beats min(-1) (bpm) immediately after birth, and increased with age to ~690 bpm at postnatal day 14. Under blockage of autonomic nervous systems, HR remained constant until postnatal day 5 and then increased day by day. The spontaneous beating rate of SAN preparation showed a similar postnatal change. The density of the L-type Ca(2+) current (LCC) was smaller in neonatal SAN cells than in adult cells, accompanied by a positive shift of voltage-dependent activation. Thus, the postnatal increase in HR is caused by both the increased sympathetic influence and the intrinsic activity of SAN cells. The different conductance and kinetics of LCC may be involved in the postnatal increase in pacemaker activity.
-
Cytosolic Ca2+-induced apoptosis in rat cardiomyocytes via mitochondrial NO-cGMP-protein kinase G pathway.
Kazuhiko Seya, Kyoichi Ono, Susumu Fujisawa, Ken Okumura, Shigeru Motomura, Ken-ichi Furukawa
The Journal of pharmacology and experimental therapeutics 344 ( 1 ) 77 - 84 2013.01 [Refereed]
Research paper (journal)
Previously, we showed that in adult rat cardiomyocytes, nitric oxide (NO) donors stimulate mitochondrial cGMP production, followed by cytochrome c release, independently of the mitochondrial permeable transition pore. We investigated whether mitochondrial cGMP-induced cytochrome c release from cardiac mitochondria is Ca(2+)-sensitive. Mitochondria and primary cultured cardiomyocytes were prepared from left ventricles of male Wistar rats. The cytosolic Ca(2+) concentration was adjusted with Ca(2+)-EGTA buffers. Cytochrome c released from mitochondria was measured by Western blotting. Cardiomyocyte apoptosis was assessed by Annexin V staining. Cytochrome c release from cardiac mitochondria was evoked by buffered Ca(2+) (1 μM); this was inhibited by NO-cGMP pathway inhibitors such as N(G)-monomethyl-l-arginine monoacetate (inhibitor of NO synthase), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NO scavenger), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, NO-sensitive guanylyl cyclase inhibitor) and voltage-dependent anion channel (VDAC) inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, but not by cyclosporin A (mitochondrial permeable transition pore inhibitor). Furthermore, this release was significantly and dose dependently inhibited by 0.3-3 μM KT5823 (protein kinase G inhibitor). At the cellular level, intracellular perfusion of cardiomyocytes with buffered Ca(2+) (1 μM) also induced apoptosis, which was inhibited in the presence of ODQ. A membrane-permeable cGMP analog, 8-Br-cGMP, but not cGMP itself, mimicked buffered Ca(2+) actions in both cardiac mitochondria and cardiomyocytes. We further confirmed an increase in protein kinase G activity by adding cGMP in mitochondrial protein fraction. Our results suggest that mitochondrial NO-cGMP pathway-induced cytochrome c release from cardiac mitochondria, triggered by increased cytosolic Ca(2+), occurs through VDAC via the stimulation of an undiscovered mitochondrial protein kinase G.
-
Enhancement of liver regeneration by adenosine triphosphate-sensitive K⁺ channel opener (diazoxide) after partial hepatectomy.
Yasuhiko Nakagawa, Masato Yoshioka, Yuki Abe, Hiroshi Uchinami, Takayoshi Ohba, Kyoichi Ono, Yuzo Yamamoto
Transplantation 93 ( 11 ) 1094 - 100 2012.06 [Refereed]
Research paper (journal)
BACKGROUND: Enhancement of liver regeneration is a matter of importance after partial liver transplantation including small-for-size grafting. Mitochondrial adenosine triphosphate (ATP)-sensitive K⁺ (mitoKATP) channel plays an important role in mitochondrial bioenergetics, which is a prerequisite for liver regeneration. However, the ATP-sensitive K⁺ (KATP) channel in hepatocytes is incompletely understood. We investigated the KATP channel in hepatocytes and examined the effects of diazoxide, a potent KATP channel opener, on liver regeneration using a rat model. METHODS: Using rat primary hepatocytes, expression and localization of KATP channel subunits, Kir6.x and sulfonylurea receptor (SUR)x, were studied by polymerase chain reaction, Western blotting, and immunostaining. To investigate the role of KATP channel openers in liver regeneration, we allocated rats into four groups: control (vehicle) (n=24), diazoxide (n=24), vehicle plus channel blocker (n=6), and diazoxide plus channel blocker (n=6) groups. After 70% partial hepatectomy, hepatic tissue ATP levels, liver-to-body weight ratio, and proliferation rate of hepatocytes were examined. RESULTS: KATP channel subunits, Kir6.1 and SUR1, were detected on hepatic mitochondria. During liver regeneration, liver-to-body weight ratio, proliferation rate of hepatocytes, and the hepatic ATP level were significantly higher in the diazoxide group than the control group at 2 days after partial hepatectomy. These effects of diazoxide were neutralized by a KATP channel blocker. CONCLUSIONS: We demonstrated the existence of a mitoKATP channel in hepatocytes composed of Kir6.1 and SUR1. Diazoxide could enhance liver regeneration by keeping a higher ATP content of the liver tissue. These results suggest that diazoxide will sustain the mitochondrial energetics through the mitoKATP channel opening.
-
Arrhythmogenic coupling between the Na+ -Ca2+ exchanger and inositol 1,4,5-triphosphate receptor in rat pulmonary vein cardiomyocytes.
Yosuke Okamoto, Makoto Takano, Takayoshi Ohba, Kyoichi Ono
Journal of molecular and cellular cardiology 52 ( 5 ) 988 - 97 2012.05 [Refereed]
Research paper (journal)
Atrial fibrillation, the most common sustained arrhythmia, is believed to be triggered by ectopic electrical activity originating in the myocardial sleeves surrounding the pulmonary veins (PVs). It has been reported that myocardial sleeves have the potential to generate automaticity in response to norepinephrine. This study investigated the cellular mechanisms underlying norepinephrine-induced automaticity in PV cardiomyocytes isolated from rats. Application of 10 μM norepinephrine to PV cardiomyocytes induced repetitive and transient increases in intracellular Ca(2+) concentrations. The Ca(2+) transient was accompanied by depolarization, and induced automatic rhythmic action potentials at approximately 4Hz in perforated patch clamp preparations in 27% of myocytes were observed. When the recording mode was switched from current-clamp to voltage-clamp mode during the continuous presence of automaticity, an oscillatory current was observed. The oscillatory current was always inward, irrespective of the membrane potential, indicating that the current was derived mainly from the Na(+)-Ca(2+) exchanger (NCX). The norepinephrine-induced automaticity was suppressed by blocking either the β(1)- or α(1)-adrenoceptor. Additionally, this automaticity was blocked by inhibitors of phospholipase C and the inositol 1,4,5-triphosphate receptor (IP(3)R) but not by a protein kinase C inhibitor. We observed that the transverse-tubule system was enriched in cardiomyocytes in the PV, in contrast to those of the atrium, and that the NCX and IP(3)R were co-localized along transverse tubules. These findings suggest that a functional coupling between the NCX and IP(3)R causes arrhythmic excitability of the PV during the presence of combined β(1)- and α(1)-adrenoceptor stimulation.
-
Direct effects of esmolol and landiolol on cardiac function, coronary vasoactivity, and ventricular electrophysiology in guinea-pig hearts.
Shigehiro Shibata, Yosuke Okamoto, Shigeatsu Endo, Kyoichi Ono
Journal of pharmacological sciences 118 ( 2 ) 255 - 65 2012 [Refereed]
Research paper (journal)
The ultra-short acting, selective β(1)-adrenergic antagonists landiolol and esmolol are widely used perioperatively; however, little is known about their acute direct actions on the heart. The current study utilized the Langendorff perfused heart system to measure changes in cardiac function and hemodynamics in response to each drug. Furthermore, electrophysiological analysis was performed on isolated ventricular myocytes. Direct application of esmolol significantly decreased systolic left ventricular pressure and heart rate at concentrations > 10 µM, while it dose-dependently increased coronary perfusion pressure. Esmolol also shortened the action potential duration (APD) in a concentration-dependent manner, an action maintained even when the delayed rectifier K(+) current or ATP sensitive K(+) current was blocked. Moreover, esmolol inhibited both the inward rectifier K(+) current (I(K1)) and the L-type Ca(2+) current (I(CaL)) and increased the outward current dose-dependently. In contrast, landiolol had minimal cardiac effects. In the Kyoto Model computer simulation, inhibition of either I(K1) or I(CaL) alone failed to shorten the APD; however, an additional increase in the time-independent outward current caused shortening of the APD, equal to that induced by esmolol. In conclusion, esmolol directly inhibits cardiac performance significantly more so than landiolol, an effect revealed to be at least in part mediated by esmolol-induced APD shortening.
-
Cardiac T-type Ca(2+) channels in the heart.
Kyoichi Ono, Toshihiko Iijima
Journal of molecular and cellular cardiology 48 ( 1 ) 65 - 70 2010.01 [Refereed] [Invited]
Research paper (journal)
Two different Ca(2+) channels exist in cardiac myocytes. While the L-type Ca(2+) channel is ubiquitous and the main source of Ca(2+) for excitation-contraction coupling and pacemaker activity, the functional role of the T-type Ca(2+) channel is diverse and depends on mammalian species, heart region, age and various cardiac diseases. Two isoforms of T-type Ca(2+) channel proteins in the heart, Ca(V)3.1 and Ca(V)3.2, are functionally expressed in embryonic hearts, but markedly diminish during development. In the adult heart, the T-type Ca(2+) channel is almost undetectable in ventricular myocytes and is most prevalent in the conduction system, playing a functional role in facilitating pacemaker depolarization of the sinoatrial node. Interestingly, the T-type Ca(2+) channel is re-expressed in atrial and ventricular myocytes under various pathological conditions such as hypertrophy and heart failure, and contributes to abnormal electrical activity and excitation-contraction coupling, but the T-type channel provides a smaller contribution to the trigger for Ca(2+) release than does the L-type Ca(2+) channel. Instead, the T-type Ca(2+) channel has been shown to play a crucial role in the process of pathological cardiac hypertrophy. Increased Ca(2+) influx via Ca(V)3.2, the T-type Ca(2+) channel, induces calcineurin/NFAT (nuclear factor of activated T-cell) hypertrophic signaling. Furthermore, new evidence has been accumulating on the regulatory mechanism of T-type Ca(2+) channel expression, including the neuron restrictive silencer element-neuron restrictive silencer factor (NRSE-NRSF) system, mitogen activated protein (MAP) kinases and cardiac homeobox transcription factor Csx/Nkx2.5. This review summarizes our present knowledge regarding cardiac T-type Ca(2+) channels, and discusses their pathophysiological significance in the heart.
-
Assessment of a new piezoelectric transducer sensor for noninvasive cardiorespiratory monitoring of newborn infants in the NICU.
Shinichi Sato, Wako Ishida-Nakajima, Akira Ishida, Masanari Kawamura, Shinobu Miura, Kyoichi Ono, Nobuya Inagaki, Goro Takada, Tsutomu Takahashi
Neonatology 98 ( 2 ) 179 - 90 2010 [Refereed]
Research paper (journal)
BACKGROUND: Electrocardiogram (ECG) and impedance pneumography (IPG), the most widely used techniques for cardiorespiratory monitoring in the neonatal intensive care unit (NICU), have the disadvantage of causing skin damage when used for very premature newborn infants. To prevent skin damage, we designed a new piezoelectric transducer (PZT) sensor. OBJECTIVE: To assess the potential of the PZT sensor for cardiorespiratory monitoring in the NICU. METHODS: The PZT sensor was placed under a folded towel under a neonate to detect an acoustic cardiorespiratory signal, from which heart rate (HR) and breathing rate (BR) were calculated, together with simultaneous ECG/IPG recording for 1-9 days for long and brief (1-min) assessment. RESULTS: The brief assessment showed average correlation coefficients of 0.92 +/- 0.12 and 0.95 +/- 0.02 between instantaneous HRs/BRs detected by the PZT sensor and ECG/IPG in 27 and 11 neonates examined. During the long assessment, the HR detection rate by the PZT sensor was approximately 10% lower than that by ECG (82.6 +/- 12.9 vs. 91.8 +/- 4.1%; p = 0.001, n = 27), although comparable (90.3 +/- 4.1 vs. 92.5 +/- 3.4%, p = 0.081) in approximately 70% (18/27) of neonates examined; BR detection rate was comparable between the PZT sensor and IPG during relatively stable signal conditions (95.9 +/- 4.0 vs. 95.3 +/- 3.5%; p = 0.38, n = 11). The PZT sensor caused neither skin damage nor body movement increase in all neonates examined. CONCLUSION: The PZT sensor is noninvasive and does not cause skin irritation, and we believe it does provide a reliable, accurate cardiorespiratory monitoring tool for use in the NICU, although the issue of mechanical-ventilation noise remains to be solved.
-
Rapid increase to double breathing rate appears during REM sleep in synchrony with REM - a higher CNS control of breathing? -.
Shinichi Sato, Takashi Kanbayashi, Hideaki Kondo, Namiko Matsubuchi, Kyoichi Ono, Tetsuo Shimizu
Advances in experimental medicine and biology 669 249 - 52 2010 [Refereed]
Research paper (journal)
Breathing rate (BR) during rapid eye movement (REM) sleep is known to fluctuate largely, while increases in BR during REM sleep reported were small. In our mice experiments, we found that mice exhibit a rapid increase in instantaneous BR (RIBR) of >2 fold during natural sleep with accompanying atonia, laying their sides down. The RIBR was further found in a sleeping mouse attached with EEG electrodes when the EEG amplitude and delta wave power were lower. Therefore, it is likely that mice show RIBRs during REM sleep. Interestingly, similar RIBRs accompanied by atonia and REM burst during REM sleep were also found in humans by standard polysomnographic studies in 11 healthy volunteers (age: 22.3 +/- 2.8) with BR measurement by nasal/oral airflow sensors and chest/abdomen belt sensors. All subjects underwent RIBR of doubled BR at least once a night. As SpO(2) before RIBRs was a level not effective to be a respiratory stimulant (96.7 +/- 1.6 %, n = 63), the RIBR seems to be controlled by higher central nervous system rather than autonomic nervous system control on response to central and peripheral chemical sensors. In fact, tachypnea with suppressed amplitude during RIBR resulted in a slight fall in SpO(2) (96.4 +/- 1.7 %, p = 0.0007). In the present study, RIBRs accompanied by atonia and REM were not necessarily consistent in change in rate and/or amplitude, therefore, these various pattern of RIBRs may be potential indices of dreams with various emotional contents. Analysis of instantaneous BR, thus, may be a helpful tool for understanding the neural control of breathing during REM sleep.
-
Essential role of STIM1 in the development of cardiomyocyte hypertrophy.
Takayoshi Ohba, Hiroyuki Watanabe, Manabu Murakami, Takako Sato, Kyoichi Ono, Hiroshi Ito
Biochemical and biophysical research communications 389 ( 1 ) 172 - 6 2009.11 [Refereed]
Research paper (journal)
Store-operated Ca(2+) entry (SOCE) through transient receptor potential (TRP) channels is important in the development of cardiac hypertrophy. Recently, stromal interaction molecule 1 (STIM1) was identified as a key regulator of SOCE. In this study, we examined whether STIM1 is involved in the development of cardiomyocyte hypertrophy. RT-PCR showed that cultured rat cardiomyocytes constitutively expressed STIM1. Endothelin-1 (ET-1) treatment for 48h enhanced TRPC1 expression, SOCE, and nuclear factor of activated T cells activation without upregulating STIM1. However, the knockdown of STIM1 suppressed these effects, thereby preventing a hypertrophic response. These results suggest that STIM1 plays an essential role in the development of cardiomyocyte hypertrophy.
-
The pathological role of transient receptor potential channels in heart disease.
Hiroyuki Watanabe, Manabu Murakami, Takayoshi Ohba, Kyoichi Ono, Hiroshi Ito
Circulation journal : official journal of the Japanese Circulation Society 73 ( 3 ) 419 - 27 2009.03 [Refereed]
Research paper (journal)
Transient receptor potential (TRP) channels are expressed in almost every human tissue, including the heart and vasculature. Most are permeable to Ca(2+) and play unique roles as multifunctional cellular sensors. Their involvement in many fundamental cell functions (eg, contraction, proliferation, and cell death) has made investigating their roles in human disease an urgent priority for medical science. This review presents an overview of current knowledge about the pathological role of TRP channels in heart disease and highlights some TRP channels with anticipated roles in disease. Evidence suggests that (a) upregulation of TRPC channels is involved in the development of cardiac hypertrophy and heart failure; (b) TRPC1, TRPC6, and TRPV2 play a role in the pathogenesis of cardiomyopathy associated with muscular dystrophy; (c) TRPC6 or TRPM4 is involved in the delayed after-depolarization; (d) TRPP2 is involved in the normal development of the interventricular and interatrial septa; and (e) neuronal TRPV1 acts as a detector of pain-producing stimuli. Ultimately, TRP channels might become novel pharmacological targets in the treatment of human heart disease.
-
Molecular and electrical remodeling of L- and T-type Ca(2+) channels in rat right atrium with monocrotaline-induced pulmonary hypertension.
Takashi Koyama, Kyoichi Ono, Hiroyuki Watanabe, Takayoshi Ohba, Manabu Murakami, Kenji Iino, Hiroshi Ito
Circulation journal : official journal of the Japanese Circulation Society 73 ( 2 ) 256 - 63 2009.02 [Refereed]
Research paper (journal)
BACKGROUND: Atrial arrhythmia is often encountered in chronic pulmonary disease with pulmonary hypertension (PH), but few studies have investigated the electrical remodeling of atrial Ca(2+) channels under PH. METHODS AND RESULTS: Wistar rats were injected with monocrotaline (MCT), resulting in PH with right atrial and ventricular hypertrophy. The L-type Ca(2+) channel current density was significantly decreased in right atrial cells of MCT-treated rats, accompanied by a significant reduction in mRNA expression of the CaV1.2 (alpha(1C)) subunit and accessory beta(2) subunit. Conversely, the low voltage-activated Ca(2+) current was more marked in the right atrial cells of MCT-treated rats than in those of control rats. The current-voltage relationship and the time course of inactivation closely resembled those of T-type Ca(2+) channels, although the current was only slightly inhibited by 10-100 micromol/L Ni(2+). No significant differences were observed in the mRNA expression levels of CaV3.1 (alpha(1G)) and CaV3.2 (alpha(1H)) or the protein level of the CaV3.1 subunit. In left atrial cells, the electrophysiological molecular properties of Ca(2+) channels were unaffected by MCT treatment. CONCLUSIONS: PH causes right atrial hypertrophy, associated with alteration of the electrophysiological molecular properties of Ca(2+) channels.
-
Identification and physiological activity of survival factor released from cardiomyocytes during ischaemia and reperfusion.
Yoichi Mizukami, Kyoichi Ono, Cheng-Kun Du, Toshihiko Aki, Naoya Hatano, Yosuke Okamoto, Yasuhiro Ikeda, Hiroshi Ito, Kimikazu Hamano, Sachio Morimoto
Cardiovascular research 79 ( 4 ) 589 - 99 2008.09 [Refereed]
Research paper (journal)
AIMS: We carried out a screening of survival factors released from cells exposed to simulated ischaemia and reperfusion (sI/R) using the embryonic rat heart-derived cell line, H9c2 cells, and examined the physiological role of the identified factor. METHOD AND RESULTS: The culture medium supernatant of H9c2 cells exposed to sI/R was separated by column chromatography and the fractions examined for survival activity. The protein with survival activity was identified by mass spectrometry, and its physiological role was examined in the models of ischaemia. Cell survival activity was detected in at least three fractions of the cell supernatant collected during sI/R and subjected to a series of column chromatographic steps. Among the proteins measured by mass spectrometry and western blotting, a p36 protein identified as a glycolytic enzyme, lactate dehydrogenase muscle subunit (M-LDH), showed strong survival activity. H(2)O(2)-induced intracellular calcium overload in H9c2 cells and irregular Ca(2+) transients in adult rat cardiomyocytes were both found to be inhibited by pretreatment with M-LDH. M-LDH also lowered the frequency and amplitude of early afterdepolarizations induced by H(2)O(2) in adult rat cardiomyocytes and suppressed the ischaemia-reperfusion-induced reduction of cardiac output from mouse working heart preparations. M-LDH was found to increase the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), which plays a role in H9c2 cell survival. CONCLUSION: M-LDH released from cardiomyocytes after hypoxia and reoxygenation has a role in protecting the heart from oxidative stress-induced injury through an intracellular signal transduction pathway involving ERK1/2.
-
Modified sympathetic nerve system activity with overexpression of the voltage-dependent calcium channel beta3 subunit.
Manabu Murakami, Takayoshi Ohba, Feng Xu, Eisaku Satoh, Ichiro Miyoshi, Takashi Suzuki, Yoichirou Takahashi, Eiki Takahashi, Hiroyuki Watanabe, Kyoichi Ono, Hironobu Sasano, Noriyuki Kasai, Hiroshi Ito, Toshihiko Iijima
The Journal of biological chemistry 283 ( 36 ) 24554 - 60 2008.09 [Refereed]
Research paper (journal)
N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory beta3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a beta3-overexpressing transgenic (beta3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of beta3-null and CaV2.2 (alpha1B)-null mice. The beta3-Tg mice showed increased expression of the beta3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The beta3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the beta3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the beta3-Tg mice, whereas beta3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the beta3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of beta3-Tg mice revealed increased calcium channel currents, especially N- and L-type currents. These results identify a determining role for the beta3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.
-
Amlodipine inhibits cell proliferation via PKD1-related pathway.
Takayoshi Ohba, Hiroyuki Watanabe, Manabu Murakami, Milena Radovanovic, Kenji Iino, Masaru Ishida, Shinya Tosa, Kyoichi Ono, Hiroshi Ito
Biochemical and biophysical research communications 369 ( 2 ) 376 - 81 2008.05 [Refereed]
Research paper (journal)
Human coronary artery smooth muscle cell (hCASMC) proliferation is involved in the progression of coronary artery disease. Amlodipine, a widely used antihypertensive drug, exerts antiproliferative effects by increasing the expression of p21((Waf1/Cip1)). Polycystic kidney disease 1 (PKD1) is also involved in cell cycle inhibition via p21((Waf1/Cip1)) up-regulation. We clarified the involvement of PKD1-related signaling on hCASMCs. Cultured hCASMCs, which constitutively express PKD1, were stimulated with 5% serum. Amlodipine increased p21((Waf1/Cip1)) expression in a dose- and time-dependent manner, resulting in reduced hCASMC proliferation. The inhibitory effect of amlodipine was mimicked by overexpression of PKD1 and was reversed by a dominant-negative version of PKD1 (R4227X). Immunoblot analysis showed that phosphorylated JAK2 was increased by amlodipine treatment or PKD1 overexpression. A luciferase assay revealed that the overexpression of PKD1 induced STAT1 enhancer activity. These data suggest that PKD1 contributes to the antiproliferative effect of amlodipine on hCASMCs via JAK/STAT signaling and p21((Waf1/Cip1)) up-regulation.
-
Involvement of transient receptor potential canonical 1 (TRPC1) in angiotensin II-induced vascular smooth muscle cell hypertrophy.
Yoichiro Takahashi, Hiroyuki Watanabe, Manabu Murakami, Takayoshi Ohba, Milena Radovanovic, Kyoichi Ono, Toshihiko Iijima, Hiroshi Ito
Atherosclerosis 195 ( 2 ) 287 - 96 2007.12 [Refereed]
Research paper (journal)
Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) hypertrophy as one of the major events leading to atherosclerosis. Increased Ca(2+) entry is an important stimulus for VSMC hypertrophy, but the association with Ang II remains to be determined. Transient receptor potential canonical 1 (TRPC1) forms store-operated Ca(2+) (SOC) channels that are involved in Ca(2+) homeostasis. Our aim was to ascertain the potential involvement of TRPC1 in Ang II-induced VSMC hypertrophy. For this purpose, we used cultured human coronary artery smooth muscle cells (hCASMCs). Store-operated Ca(2+) entry (SOCE) increased in the Ang II-induced hypertrophied cells, and SOC channel blocker inhibited the Ang II-induced hypertrophic response. Although hCASMCs constitutively expressed TRPC1, C3, C4, C5, and C6, only TRPC1 increased in response to Ang II stimulation. TRPC1 siRNA decreased SOCE and prevented Ang II-induced hypertrophy. We found NF-kappaB binding sites in the 5'-regulatory region of the human TRPC1 gene. An electrophoretic mobility shift assay showed that Ang II increased the TRPC1 promoter's NF-kappaB binding activity. Co-treatment with NF-kappaB decoy oligonucleotides not only reduced TRPC1 expression, but also inhibited the hypertrophic responses. In conclusion, our data suggest that Ang II and subsequent NF-kappaB activation induces hCASMC hypertrophy through an enhancement of TRPC1 expression.
-
Characterization of SN-6, a novel Na+/Ca2+ exchange inhibitor in guinea pig cardiac ventricular myocytes.
Chun-Feng Niu, Yasuhide Watanabe, Kyoichi Ono, Takahiro Iwamoto, Kanna Yamashita, Hiroshi Satoh, Tuyoshi Urushida, Hideharu Hayashi, Junko Kimura
European journal of pharmacology 573 ( 1-3 ) 161 - 9 2007.11 [Refereed]
Research paper (journal)
We examined the effect of SN-6, a new benzyloxyphenyl Na(+)/Ca(2+) exchange (NCX) inhibitor on the Na(+)/Ca(2+) exchange current (I(NCX)) and other membrane currents in isolated guinea pig ventricular myocytes using the whole-cell voltage-clamp technique. SN-6 suppressed I(NCX) in a concentration-dependent manner. The IC(50) values of SN-6 were 2.3 microM and 1.9 microM for the outward and inward components of the bi-directional I(NCX), respectively. On the other hand, SN-6 suppressed the outward uni-directional I(NCX) more potently (IC(50) value of 0.6 microM) than the inward uni-directional I(NCX). SN-6 at 10 microM inhibited the uni-directional inward I(NCX) by only 22.4+/-3.1%. SN-6 and KB-R7943 suppressed I(NCX) more potently when intracellular Na(+) concentration was higher. Thus, both drugs inhibit NCX in an intracellular Na(+) concentration-dependent manner. Intracellular application of trypsin via a pipette solution did not change the blocking effect of SN-6 on I(NCX). Therefore, SN-6 is categorized as an intracellular-trypsin-insensitive NCX inhibitor. SN-6 at 10 microM inhibited I(Na), I(Ca), I(K) and I(K1) by about 13%, 34%, 33% and 13%, respectively. SN-6 at 10 microM shortened the action potential duration at 50% repolarization (APD(50)) by about 34%, and that at 90% repolarization (APD(90)) by about 25%. These results indicate that SN-6 inhibits NCX in a similar manner to that of KB-R7943. However, SN-6 at 10 microM affected other membrane currents less potently than KB-R7943.
-
Functional role of stromal interaction molecule 1 (STIM1) in vascular smooth muscle cells.
Yoichiro Takahashi, Hiroyuki Watanabe, Manabu Murakami, Kyoichi Ono, Yoshiko Munehisa, Takashi Koyama, Kiyoshi Nobori, Toshihiko Iijima, Hiroshi Ito
Biochemical and biophysical research communications 361 ( 4 ) 934 - 40 2007.10 [Refereed]
Research paper (journal)
We investigated the functional role of STIM1, a Ca(2+) sensor in the endoplasmic reticulum (ER) that regulates store-operated Ca(2+) entry (SOCE), in vascular smooth muscle cells (VSMCs). STIM1 was mainly localized at the ER and plasma membrane. The knockdown of STIM1 expression by small interfering (si) RNA drastically decreased SOCE. In contrast, an EF-hand mutant of STIM1, STIM1(E87A), produced a marked increase in SOCE, which was abolished by co-transfection with siRNA to transient receptor potential canonical 1 (TRPC1). In addition, transfection with siRNA against STIM1 suppressed phosphorylation of cAMP-responsive element binding protein (CREB) and cell growth. These results suggest that STIM1 is an essential component of SOCE and that it is involved in VSMC proliferation.
-
Essential role of the N-terminus of murine Orai1 in store-operated Ca2+ entry.
Yoichiro Takahashi, Manabu Murakami, Hiroyuki Watanabe, Hitoshi Hasegawa, Takayoshi Ohba, Yoshiko Munehisa, Kiyoshi Nobori, Kyoichi Ono, Toshihiko Iijima, Hiroshi Ito
Biochemical and biophysical research communications 356 ( 1 ) 45 - 52 2007.04 [Refereed]
Research paper (journal)
Store-operated Ca(2+) entry (SOCE) is a physiologically important process that is triggered by intracellular Ca(2+) depletion. Recently, human Orai1 (the channel-forming subunit) and STIM1 (the calcium sensor) were identified as essential molecules for SOCE. Here, we report the cloning and functional analysis of three murine orthologs of Orai1, termed Orai1, 2, and 3. Among the genes identified, Orai1 contains a distinctive proline- and arginine-rich N-terminal cytoplasmic sequence. Co-expression of STIM1 with Orai1 produced a marked effect on SOCE, while co-expression with Orai2 or Orai3 had little effect. Expression of Orai1 without its N-terminal tail had a marginal effect on SOCE, while chimeric Orai2 containing the Orai1 N-terminus produced a marked increase in SOCE. In addition, a truncated version of Orai1 containing the N-terminus without the pore-forming transmembrane domain had a dominant negative effect on SOCE. These results reveal the essential role of Orai1 and its N-terminal sequence in SOCE.
-
Modified sympathetic regulation in N-type calcium channel null-mouse.
Manabu Murakami, Takayoshi Ohba, Tsai-Wen Wu, Susumu Fujisawa, Takashi Suzuki, Yoichiro Takahashi, Eiki Takahashi, Hiroyuki Watanabe, Ichiro Miyoshi, Kyoichi Ono, Hironobu Sasano, Hiroshi Ito, Toshihiko Iijima
Biochemical and biophysical research communications 354 ( 4 ) 1016 - 20 2007.03 [Refereed]
Research paper (journal)
To elucidate the physiological importance of neuronal (N)-type calcium channels in sympathetic controls, we analyzed N-type channel-deficient (NKO) mice. Immunoprecipitation analysis revealed increased interaction between beta3 (a major accessory subunit of N-type channels) and R-type channel-forming CaV2.3 in NKO mice. R-R intervals in NKO ECG recordings were elongated and fluctuating, suggesting disturbed sympathetic tonus. N-type channel inhibitors elongated the R-R interval in control mice, whereas R-type channel blocking with SNX-482 significantly affected NKO but not control mice, indicating a compensatory role for R-type channels. Echocardiography and Langendorff heart analysis confirmed a major role for R-type channels in NKO mice. Combined, our biochemical and physiological analyses strongly suggest that the remaining sympathetic tonus in NKO mice is dependent on R-type calcium channels.
-
Upregulation of TRPC1 in the development of cardiac hypertrophy.
Takayoshi Ohba, Hiroyuki Watanabe, Manabu Murakami, Yoichiro Takahashi, Kenji Iino, Sadao Kuromitsu, Yasuo Mori, Kyoichi Ono, Toshihiko Iijima, Hiroshi Ito
Journal of molecular and cellular cardiology 42 ( 3 ) 498 - 507 2007.03 [Refereed]
Research paper (journal)
The importance of Ca(2+) entry in the cardiac hypertrophic response is well documented, but the actual Ca(2+) entry channels remain unknown. Transient receptor potential (TRP) proteins are thought to form either homo- or heteromeric Ca(2+) entry channels that are involved in the proliferation and differentiation of various cells. The purpose of this study was to explore the potential involvement of TRP channels in the development of cardiac hypertrophy. The mRNA and protein expression of several TRP channel subunits were evaluated using hearts from abdominal aortic-banded (AAB) rats. Although TRPs C1, C3, C5, and C6 were constitutively expressed, only TRPC1 expression was significantly increased in the hearts of AAB rats compared to sham-operated rats. Using primary cultures of neonatal rat cardiomyocytes, we detected increases in the expression of TRPC1, brain natriuretic peptide (BNP), and atrial natriuretic factor (ANF), as well as increases in store-operated Ca(2+) entry (SOCE) and cell surface area, following endothelin-1 (ET-1) treatment. Silencing of the TRPC1 gene via small interfering RNA (siRNA) attenuated SOCE and prevented ET-1-, angiotensin-II (AT II)-, and phenylephrine (PE)-induced cardiac hypertrophy. In HEK 293T cells, overexpression of TRPC1 augmented SOCE, leading to an increase in nuclear factor of activated T cells (NFAT) promoter activity, while co-transfection with dominant-negative forms of TRPC1 suppressed it. In conclusion, TRPC1 functions in Ca(2+) influx, and its upregulation is involved in the development of cardiac hypertrophy; moreover, it plays an important role in the regulation of the signaling pathways that govern cardiac hypertrophy. These findings establish TRPC1 as a functionally important regulator of cardiac hypertrophy.
-
[T-type calcium channel].
Kyoichi Ono
Nihon yakurigaku zasshi. Folia pharmacologica Japonica 128 ( 3 ) 185 - 7 2006.09 [Refereed] [Invited]
Research paper (journal)
-
Identification of a cardiac isoform of the murine calcium channel alpha1C (Cav1.2-a) subunit and its preferential binding with the beta2 subunit.
Manabu Murakami, Takayoshi Ohba, Yoichiro Takahashi, Hiroyuki Watanabe, Ichiro Miyoshi, Shinsuke Nakayama, Kyoichi Ono, Hiroshi Ito, Toshihiko Iijima
Journal of molecular and cellular cardiology 41 ( 1 ) 115 - 25 2006.07 [Refereed]
Research paper (journal)
We describe a cardiac muscle isoform of the voltage-dependent calcium channel alpha1 subunit, which corresponds to the rabbit ortholog of alpha1C-a (Cav1.2a). We also cloned smooth muscle isoforms alpha1C-b (Cav1.2b) and alpha1C-d (Cav1.2d). Differences among these three isoforms lie within the N-terminal region (exon 1A or 1B), the sixth transmembrane segment of domain I (exon 8A or 8B), and the use of exon 10, which forms the intracellular loop between transmembrane domains I and II. Two-hybrid analysis revealed interactions among the three alpha1 isoforms and beta subunits. In vitro overlay and immunoprecipitation analyses revealed preferential binding between alpha1C-a and beta2, which is also expressed at a high level in the heart.
-
Involvement of voltage-dependent Ca(2+) channel beta(3) subunit in the autonomic control of heart rate variability.
Tsai-Wen Wu, Kyoichi Ono, Manabu Murakami, Toshihiko Iijima
Pharmacology 76 ( 4 ) 170 - 9 2006 [Refereed]
Research paper (journal)
Noradrenaline release from sympathetic nerve terminals is dependent on Ca(2+) entry through neuronal voltage-gated N-type Ca(2+) channels. The accessory beta(3) subunits of Ca(2+) channels (Ca(V)beta(3)) are preferentially associated with the alpha(1B) subunit to form N-type Ca(2+) channels, and are therefore expected to play a functional role in the stimulation-evoked release of noradrenaline. In this study, we employed Ca(V)beta(3)-null, Ca(V)beta(3)-overexpressing (Ca(V)beta(3)-Tg), and wild-type (WT) mice to investigate the possible roles of Ca(V)beta(3) in the sympathetic regulation of heart rate in vivo. Telemetry was used to monitor the ECG and both time and frequency domain analyses were carried out to evaluate heart rate variability. In the frequency domain analysis, power spectral density of the RR interval series was computed using the fast Fourier transform algorithm. The resting heart rate was increased in Ca(V)beta(3)-Tg mice compared with both Ca(V)beta(3)-null and WT mice. Mice overexpressing Ca(V)beta(3) displayed decreased heart rate variability, which was measured by the time domain analysis of the standard deviation of RR intervals. In the frequency domain analysis, Ca(V)beta(3)-Tg mice showed decreased spectral powers compared with WT and Ca(V)beta(3)-null mice. Pharmacological blockade of beta-adrenergic receptors with metoprolol decreased the heart rate in all genotypes, but the extent of the decrease was most obvious in Ca(V)beta(3)-Tg mice. On the other hand, the spectral powers were decreased in response to parasympathetic blockade (atropine) in WT and Ca(V)beta(3)-Tg mice. These results indicate the functional roles of Ca(V)beta(3) in regulating sympathetic nerve signaling.
-
Pathophysiological significance of T-type Ca2+ channels: properties and functional roles of T-type Ca2+ channels in cardiac pacemaking.
Kyoichi Ono, Toshihiko Iijima
Journal of pharmacological sciences 99 ( 3 ) 197 - 204 2005.11 [Refereed]
Research paper (journal)
Calcium channels are essential for excitation-contraction coupling and pacemaker activity in cardiac myocytes. While L-type Ca(2+) channels (LCC) have been extensively studied, functional roles of T-type channels (TCC) in native cardiac myocytes are still debatable. TCC are activated at more negative membrane potentials than LCC and therefore facilitate slow diastolic depolarization in sinoatrial node cells. Recent studies showed that selective inhibition of TCC produced a marked slowing of the pacemaker rhythm, indicating that contribution of TCC to cardiac automaticity was relatively larger than what had been speculated in previous studies. To re-evaluate TCC, we measured current density and kinetics of TCC in sinoatrial node cells of various mammalian species. Current density of TCC was larger in mice and guinea pigs than in rabbit and porcine sinoatrial node cells. Interestingly, few or no obvious TCC were recorded in porcine sinoatrial node cells. Furthermore, it was demonstrated that TCC could be enhanced by several vasoactive substances, thereby increasing spontaneous firing rate of sinoatrial node cells. TCC may, at least in part, account for different heart rates among various mammalian species. In addition, TCC might be involved in physiological and/or pathophysiological modulations of the heart rate.
-
Thromboxane A2 and prostaglandin F2alpha mediate inflammatory tachycardia.
Koji Takayama, Koh-ichi Yuhki, Kyoichi Ono, Takayuki Fujino, Akiyoshi Hara, Takehiro Yamada, Shuhko Kuriyama, Hideji Karibe, Yuji Okada, Osamu Takahata, Takanobu Taniguchi, Toshihiko Iijima, Hiroshi Iwasaki, Shuh Narumiya, Fumitaka Ushikubi
Nature medicine 11 ( 5 ) 562 - 6 2005.05 [Refereed]
Research paper (journal)
Systemic inflammation induces various adaptive responses including tachycardia. Although inflammation-associated tachycardia has been thought to result from increased sympathetic discharge caused by inflammatory signals of the immune system, definitive proof has been lacking. Prostanoids, including prostaglandin (PG) D(2), PGE(2), PGF(2alpha), PGI(2) and thromboxane (TX) A(2), exert their actions through specific receptors: DP, EP (EP(1), EP(2), EP(3), EP(4)), FP, IP and TP, respectively. Here we have examined the roles of prostanoids in inflammatory tachycardia using mice that lack each of these receptors individually. The TXA(2) analog I-BOP and PGF(2alpha) each increased the beating rate of the isolated atrium of wild-type mice in vitro through interaction with TP and FP receptors, respectively. The cytokine-induced increase in beating rate was markedly inhibited in atria from mice lacking either TP or FP receptors. The tachycardia induced in wild-type mice by injection of lipopolysaccharide (LPS) was greatly attenuated in TP-deficient or FP-deficient mice and was completely absent in mice lacking both TP and FP. The beta-blocker propranolol did not block the LPS-induced increase in heart rate in wild-type animals. Our results show that inflammatory tachycardia is caused by a direct action on the heart of TXA(2) and PGF(2alpha) formed under systemic inflammatory conditions.
-
Effects of PFOS and PFOA on L-type Ca2+ currents in guinea-pig ventricular myocytes.
Kouji Harada, Feng Xu, Kyoichi Ono, Toshihiko Iijima, Akio Koizumi
Biochemical and biophysical research communications 329 ( 2 ) 487 - 94 2005.04 [Refereed]
Research paper (journal)
Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are amphiphiles found ubiquitously in the environment, including wildlife and humans, and are known to have toxic effects on physiological functions of various tissues. We investigated the effects of PFOS and PFOA on action potentials and L-type Ca(2+) currents, I(CaL), in isolated guinea-pig ventricular myocytes using whole-cell patch-clamp recording. In current-clamp experiments, PFOS significantly decreased the rate of spike, action potential duration, and peak potential at doses over 10 microM. In voltage-clamp experiments, PFOS increased the voltage-activated peak amplitude of I(CaL), and shifted the half-activation and inactivation voltages of I(CaL) to hyperpolarization. PFOA had similar effects PFOS, but showed significantly lower potency. These findings are consistent with previous observations for anionic n-alkyl surfactants, suggesting that PFOS and PFOA may change membrane surface potential, thereby eliciting general effects on calcium channels. These findings provide further insights into the mechanisms of PFOA and PFOS toxicities.
-
Genomic organization and functional analysis of murine PKD2L1.
Manabu Murakami, Takayoshi Ohba, Feng Xu, Seiji Shida, Eisaku Satoh, Kyoichi Ono, Ichiro Miyoshi, Hiroyuki Watanabe, Hiroshi Ito, Toshihiko Iijima
The Journal of biological chemistry 280 ( 7 ) 5626 - 35 2005.02 [Refereed]
Research paper (journal)
Mutations in genes that encode polycystins 1 or 2 cause polycystic kidney disease (PKD). Here, we report the genomic organization and functional expression of murine orthologue of human polycystin-2L1 (PKD2L1). The murine PKD2L1 gene comprises 15 exons in chromosome 19C3. Coexpression of PKD2L1 together with polycystin-1 (PKD1) resulted in the expression of PKD2L1 channels on the cell surface, whereas PKD2L1 expressed alone was retained within the endoplasmic reticulum (ER). This suggested that interaction between PKD1 and PKD2L1 is essential for PKD2L1 trafficking and channel formation. Deletion analysis at the cytoplasmic tail of PKD2L1 revealed that the coiled-coil domain was important for trafficking by PKD1. Mutagenesis within two newly identified ER retention signal-like amino acid sequences caused PKD2L1 to be expressed at the cell surface. This indicated that the coiled-coil domain was responsible for retaining PKD2L1 within the ER. Functional analysis of murine PKD2L1 expressed in HEK 293 cells was undertaken using calcium imaging. Coexpression of PKD1 and PKD2L1 resulted in the formation of functional cation channels that were opened by hypo-osmotic stimulation, whereas neither molecule formed functional channels when expressed alone. We conclude that PKD2L1 forms functional cation channels on the plasma membrane by interacting with PKD1. These findings raise the possibility that PKD2L1 represents the third genetic locus that is responsible for PKD.
-
Sevoflurane inhibition of the slowly activating delayed rectifier K+ current in guinea pig ventricular cells.
Shigehiro Shibata, Kyoichi Ono, Toshihiko Iijima
Journal of pharmacological sciences 95 ( 3 ) 363 - 73 2004.07 [Refereed]
Research paper (journal)
Single ventricular cells were enzymatically isolated from guinea pig hearts and the effects of sevoflurane on the delayed rectifier K(+) current were investigated by the patch clamp method. The rapidly (I(Kr)) and slowly activating delayed rectifier K(+) current (I(Ks)) were isolated using chromanol 293B, a selective blocker for I(Ks) or E4031 (N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide dihydrochloride), a blocker for I(Kr). Sevoflurane and halothane decreased I(Ks) in a concentration-dependent manner with an IC(50) value of 0.38 mM for sevoflurane and 1.05 mM for halothane. I(Ks) inhibition was characterized by suppression of maximum conductance with little effect on activation kinetics. Inhibition occurred immediately after anesthetic application and recovered upon wash-out. In contrast to the marked inhibition of I(Ks), I(Kr) was hardly affected by sevoflurane. Under the current clamp, sevoflurane prolonged the action potential duration in a reversible manner and this effect was more marked when I(Kr) was inhibited by E4031. The results suggest that sevoflurane inhibits I(Ks), and not I(Kr), in a concentration-dependent manner at clinically relevant concentrations. The resulting prolongation of ventricular repolarization may partly account for the clinical observation of excessive QT prolongation by these anesthetics.
-
Pacemaker mechanism of porcine sino-atrial node cells.
Kyoichi Ono, Shigehiro Shibata, Toshihiko Iijima
Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi 39 ( 5 ) 195 - 204 2003.10 [Refereed] [Invited]
Research paper (journal)
In cardiac sino-atrial node (SAN) cells, time- and voltage-dependent changes in the gating of various ionic currents provide spontaneous, stable and repetitive firing of action potentials. To address the ionic nature of the species-dependent heart rate, action potentials and membrane currents were recorded in single cells dissociated from the porcine SAN, and compared with those from SAN cells of rabbits, guinea-pigs and mice. The porcine SAN cells exhibited spontaneous activity with a frequency of 60-80 min(-1), which was much slower than that of rabbit SAN cells. Under voltage clamp conditions, depolarization activated the L-type Ca2+ current (I(CaL)) followed by a gradual activation of the delayed rectifier K+ current (I(K)) while hyperpolarization activated the hyperpolarization-activated cation current (I(h)). It was found that the major component of I(K) in porcine SAN is the slowly activating I(K) (I(Ks)), in contrast to SAN cells of the rabbit and other species in which the rapid I(K) (I(Kr)) plays an active role in repolarization and the subsequent pacemaker depolarization. Replacement of rabbit I(Kr) with porcine I(Ks) and a slight modification in the gating parameters and amplitudes of other current systems in the 'Kyoto Model' gave an adequate reconstruction of spontaneous action potentials as well as of the voltage clamp recordings. We conclude that the density and the kinetics of I(K) contribute, in part, to the different heart rates of various species.
-
Identification and characterization of the murine TRPM4 channel.
Manabu Murakami, Feng Xu, Ichiro Miyoshi, Eisaku Sato, Kyoichi Ono, Toshihiko Iijima
Biochemical and biophysical research communications 307 ( 3 ) 522 - 8 2003.08 [Refereed]
Research paper (journal)
The transient receptor potential (TRP) channels form a superfamily with six transmembrane structures, which is common in other types of voltage-dependent channels. The TRP-melastatin (TRPM) subfamily includes the putative tumor-suppressor melastatin, which was originally found as a down-regulated protein in melanoma tumor cell lines. Here, we report a novel TRP-related protein that is a murine orthologue of human TRPM4. The function of the novel murine TRPM4 was studied in HEK-293 cells using a fluorescent calcium indicator, fura-2. The removal and re-introduction of extracellular calcium triggered changes in the intracellular calcium only in cells expressing TRPM4a, which suggests that this novel channel plays a role in the calcium entry process. We also isolated a splice variant of TRPM4 that was proven to be non-functional. Both TRPM4 variants integrated into the plasma membrane. Furthermore, FRET analysis revealed that TRPM4a and TRPM4b localized close together, suggesting a multimerization of the two molecules.
-
Role of individual ionic current systems in the SA node hypothesized by a model study.
Nobuaki Sarai, Satoshi Matsuoka, Shinobu Kuratomi, Kyoichi Ono, Akinori Noma
The Japanese journal of physiology 53 ( 2 ) 125 - 34 2003.04 [Refereed]
Research paper (journal)
This paper discusses the development of a cardiac sinoatrial (SA) node pacemaker model. The model successfully reconstructs the experimental action potentials at various concentrations of external Ca2+ and K+. Increasing the amplitude of L-type Ca2+ current (I(CaL)) prolongs the duration of the action potential and thereby slightly decreases the spontaneous rate. On the other hand, a negative voltage shift of I(CaL) gating by a few mV markedly increases the spontaneous rate. When the amplitude of sustained inward current (I(st)) is increased, the spontaneous rate is increased irrespective of the I(CaL) amplitude. Increasing [Ca2+](o) shortens the action potential and increases the spontaneous rate. When the spontaneous activity is stopped by decreasing I(CaL) amplitude, the resting potential is nearly constant (-35 mV) over 1-15 mM [K+](o) as observed in the experiment. This is because the conductance of the inward background non-selective cation current balances with the outward [K+](o)-dependent K+ conductance. The unique role of individual voltage- and time-dependent ion channels is clearly demonstrated and distinguished from that of the background current by calculating an instantaneous zero current potential ("lead potential") during the course of the spontaneous activity.
-
Role of individual ionic current systems in ventricular cells hypothesized by a model study.
Satoshi Matsuoka, Nobuaki Sarai, Shinobu Kuratomi, Kyoichi Ono, Akinori Noma
The Japanese journal of physiology 53 ( 2 ) 105 - 23 2003.04 [Refereed]
Research paper (journal)
Individual ion channels or exchangers are described with a common set of equations for both the sinoatrial node pacemaker and ventricular cells. New experimental data are included, such as the new kinetics of the inward rectifier K+ channel, delayed rectifier K+ channel, and sustained inward current. The gating model of Shirokov et al. (J Gen Physiol 102: 1005-1030, 1993) is used for both the fast Na+ and L-type Ca2+ channels. When combined with a contraction model (Negroni and Lascano: J Mol Cell Cardiol 28: 915-929, 1996), the experimental staircase phenomenon of contraction is reconstructed. The modulation of the action potential by varying the external Ca2+ and K+ concentrations is well simulated. The conductance of I(CaL) dominates membrane conductance during the action potential so that an artificial increase of I(to), I(Kr), I(Ks), or I(KATP) magnifies I(CaL) amplitude. Repolarizing current is provided sequentially by I(Ks), I(Kr), and I(K1). Depression of ATP production results in the shortening of action potential through the activation of I(KATP). The ratio of Ca2+ released from SR over Ca2+ entering via I(CaL) (Ca2+ gain = approximately 15) in excitation-contraction coupling well agrees with the experimental data. The model serves as a predictive tool in generating testable hypotheses.
-
Cloning and functional expression of a novel splice variant of rat TRPC4.
Eisaku Satoh, Kyoichi Ono, Feng Xu, Toshihiko Iijima
Circulation journal : official journal of the Japanese Circulation Society 66 ( 10 ) 954 - 8 2002.10 [Refereed]
Research paper (journal)
Transient receptor potential protein 4 (TRPC4) has been identified as a candidate for the capacitative calcium entry (CCE) channels, but its functional role is still controversial. Using a RT-PCR technique, a novel isoform of TRPC4, designated rTRPC4gamma, was isolated. It was nearly identical to full-length rTRPC4 (rTRPC4alpha), except that it lacked 53 nucleotides that correspond to the predicted linker between the second and third transmembrane domain of rTRPC4alpha, and its mRNA was expressed in brain and heart. This splice variant encoded a potential protein of 400 residues that consists of an amino-terminal cytoplasmic domain and 2 transmembrane domains by a frameshift mutation. When rTRPC4gamma cDNA was transiently transfected to HEK-293 cells, thapsigargin (TG)-induced Ca2+ entry was suppressed significantly. By contrast, expression of rTRPC4 a did not affect TG-induced Ca2+ entry. To investigate the subcellular localization, plasmids were constructed with green fluorescence protein (GFP) as an amino-terminal fusion to rTRPC4 variants. GFP-rTRPC4gamma fusion protein, unlike GFP-rTRPC4alpha, was localized to the cytoplasm as well as plasma membrane. These results suggest that rTRPC4gamma may play a modulatory role in CCE channel activity in the brain and heart.
-
Modulation by mibefradil of the histamine-induced Ca2+ entry in human aortic endothelial cells.
Kazuto Yazawa, Kyoichi Ono, Toshihiko Iijima
Japanese journal of pharmacology 90 ( 2 ) 125 - 30 2002.10 [Refereed]
Research paper (journal)
The effect of mibefradil, known as a T- and L-type Ca(2+) channel antagonist, on the histamine-induced Cl(-) current and Ca(2+) entry was investigated in human aortic endothelial cells by the fluorescence measurement of intracellular Ca(2+) concentration ([Ca(2+)](i)) combined with the patch clamp method. Mibefradil (10 micro M) inhibited both the Cl(-) current and Ca(2+) entry in a concentration-dependent manner with an IC(50) value of 4.8 and 2.6 micro M for the Cl(-) current and [Ca(2+)](i), respectively. These values were comparable to those reported for the inhibition of the T-type Ca(2+) channel and other Cl(-) channels. The suppression of Ca(2+) entry is not caused by the inhibition of the Cl(-) current and the resulting depolarization since the inhibition was still observed under the voltage clamp condition. These results suggest that mibefradil is a potent blocker not only for the agonist-induced Cl(-) current but also Ca(2+) entry channels in vascular endothelial cells.
-
Time-dependent block of the slowly activating delayed rectifier K+ current by chromanol 293B in guinea-pig ventricular cells
Fujisawa S， Ono K and Iijima T
Br J Pharmacol 129 2000.01 [Refereed]
Research paper (journal)
-
Properties of the delayed rectifier potassium current in porcine sino-atrial node cells
Ono K， Shibata S and Iijima T
J Physiol 524 2000.01 [Refereed]
Research paper (journal)