Research Achievements - Original paper -
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Regulatory role of neuron-restrictive silencing factor in expression of TRPC1.
Takayoshi Ohba, Hiroyuki Watanabe, Yoichiro Takahashi, Takashi Suzuki, Ichiro Miyoshi, Shinnsuke Nakayama, Eisaku Satoh, Kenji Iino, Hironobu Sasano, Yasuo Mori, Sadao Kuromitsu, Keiichi Imagawa, Yoshihiko Saito, Toshihiko Iijima, Hiroshi Ito, Manabu Murakami
Biochemical and biophysical research communications 351 ( 3 ) 764 - 70 2006.12 [Refereed]
Research paper (journal)
Neuron-restrictive silencer factor (NRSF) binds its consensus element to repress the transcription of various genes. The dominant-negative form (dnNRSF) has a hypertrophic effect on cardiogenesis through an unidentified mechanism. We examined the involvement of transient receptor potential (TRP) channel proteins, using transgenic mice overexpressing dnNRSF (dnNRSF mice). Electrophoretic mobility-shift assays revealed an interaction between NRSF and a neuron-restrictive silencer element-like sequence in intron 4 of TRPC1 genomic DNA. According to RT-PCR and Western analyses, TRPC1 was up-regulated in dnNRSF mouse heart. Transient overexpression of TRPC1 in HEK 293T cells increased the activity of the nuclear factor in activated T cells (NFAT) promoter and stimulated store-operated Ca(2+) channel (SOCC)-mediated Ca(2+) entry. Transfection of TRPC1 into primary cardiomyocytes increased NFAT activity, indicating a major role for TRPC1 in NFAT activation. Our findings strongly suggest that NRSF regulates TRP1 gene expression and causes changes in the levels of calcium entry through SOCCs.
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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.
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Increased expression of transient receptor potential C1 in rats with hypertensive hypertrophy
Takayoshi Ohba, Hiroyuki Watanabe, Manabu Murakami,Yoichiro Tkahashi,Hiroshi Ito
秋田医学 32 201 - 207 2006.03 [Refereed]
Research paper (journal)
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The effect of Unoprostone on store operated Ca entry in human aortic smooth muscle cells
Watanabe Akiko, Watanabe Hiroyuki, Ono Kyoichi, Ohba Takayoshi, Murakami Manabu, Hasegawa Hitoshi, Sasaki Masahiro, Iijima Toshihiko, Yoshitomi Takeshi, Ito Hiroshi
秋田医学 32 ( 1 ) 7 - 14 2005.05 [Refereed]
Research paper (journal)
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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.
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Low Molecular Weight Heparin Prevents Cardiovascular Remodeling Induced by the Long-Term Inhibition of Nitric Oxide Synthase with N-Nitro-L-Arginine Methyl Ester in Rat Hearts
HASEGAWA Hitoshi,SAITO Takashi,FUJIWARA Yoshimasa,KUROKAWA Fukiko,WATANABE hiroyuki
秋田医学 31 221 - 230 2004.12 [Refereed]
Research paper (journal)
To evaluate the effect of low molecular weight heparin (LMWH) oncardiovascular remodeling induced by long term inhibition of nitric oxide synthase withN-nitro-L-arginine methyl ester (L-NAME), 40 male Sprague-Dawley rats were randomlydivided into four groups: the control group (n=10) that received 0.9% saltsolution; the L-NAME group (n=10) that received 30 mg/kg/day of L-NAME, theLMWH3000 group (n=10) that received 30 mg/kg/day of L-NAME and 4 mg/kg/dayof LMWH (molecular weight (M.W.) of 3000) and the LMWH6000 group (n= 10)that received 30 mg/kg/day of L-NAME and 4 mg/kg/day of LMWH (M.W. 6000).All agents, including saline, were administered intraperitoneally by implantation ofosmotic mini pumps. Systolic blood pressure was measured by the tail-cuff method ontreatment days 1,7, 14, and 28, and histological examination of harvested cardiac tissueand coronary arteries was performed on day 28. Systolic blood pressure was greater inthe L-NAME, LMWH3000 and LMWH6000 groups when compared with the controlgroup. There were no significant differences in activated coagulation time (ACT)when comparing the four groups. Medial smooth muscle cell proliferation and hypertrophyand increases in perivascular and myocardial fibrosis were observed in the L-NAMEgroup compared with the control group. Further, these changes were inhibited by thecoadministration of LMWH3000 or LWMH6000. This study demonstrates that LMWHprevents cardiovascular remodeling induced by L-NAME in rat hearts independent of itsanticoagulant activity. Low molecular weight heparin prevents cardiovascular remodeling
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Determinants of severity in dilated cardiomyopathy: The gap of tricuspid and mitral valve opening
IZUMI Manabu,KIBIRA Satoshi,WATANABE Hiroyuki,TSUYA Hiroyuki
秋田医学 31 201 - 209 2004.12 [Refereed]
Research paper (journal)
Objectives: The purpose of this study is to analyze factors that influence the gap of
opening timing between the mitral valve (MV) and the tricuspid valve (TV) in
patients with dilated cardiomyopathy (DCM).
Background: The DCM patients with heart failure often show shorter left ventricular
(LV) isovolumic relaxation time (IRT) and longer right ventricular (RV) IRT, which
may influence the gap of opening timing between TV and MV.
Method: We evaluated consecutive 34 patients with DCM. The time between QRS
initial and TV opening (Q-T time), and that between QRS initial and MV opening (QM
time) were measured by pulsed Doppler recording of each ventricular inflow. The
time interval between MV and TV opening (M-T time) was determined by subtracting
Q-M time from Q-T time. In addition, the other Doppler time intervals including preejectional
period, ejection time, IRT were determined. We analyzed the relation
between Doppler time intervals and factors including NYHA classification and rightsided
cardiac pressure.
Result: There is significant relationship (p <0.001) between M-T time and PCWP
(r=0.65), and NYHA (rs=0.67), respectively. The Ll IRT determined by subtraction
of LV IRT from RV IRT, correlated significantly with M-T time (r=0.83, p<O.OOl),
however, the difference between LV and RV in PEP and ET did not correlated
significantly with M-T time.
Conclusion: The gap of opening timing between TV and MV, determined as M-T time,
correlates strongly with LlIRT and also the severity of congestive heart failure in DCM
patients. -
Cell swelling， heat and chemical agonists use distinet pathways for the activation of the cation channel TRPV4
Vriens J， Watanabe H et. Al
Proc Natl Acad Sci USA 2004 101 ( 1 ) 396 - 401 2004.01 [Refereed]
Research paper (journal) Domestic Co-author
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Reduction of ST elevation in repeated coronary occlusion model depends on both altered metabolic response and conduction property.
Takashi Saito, Hiroto Miura, Yutaka Kimura, Hiroyuki Watanabe, Akira Nakagomi, Yoshikazu Tamura, Hitoshi Hasegawa, Satoshi Kibira, Mamoru Miura
International journal of cardiology 92 ( 2-3 ) 219 - 27 2003.12 [Refereed]
Research paper (journal)
The aim of this study was to elucidate the mechanisms of altered electrical response to ischemia in repeated coronary occlusion model. To test its dependence on metabolic response, extracellular K+ concentration (eKC), myocardial pH and PCO2 were simultaneously measured with epicardial ECG during three consecutive 4 min of left anterior descending coronary artery (LAD) occlusion separated by 15 min of reperfusion in canine hearts. ECG changes induced by infusion of high K+-buffer (10 mM) into the coronary arterial bed via carotid artery-LAD bypass (referred to as high K+-challenges: HKC) were also tested prior to (the first HKC), and during each reperfusion period (the second to the fourth HKC). ST elevation was significantly reduced in subsequent occlusions (3.14 +/- 0.48 and 2.98 +/- 0.47 mV in the second and third occlusion, both P<0.05, compared to 4.91 +/- 0.78 mV in the first). This was accompanied by significant attenuation of the changes in eKC, tissue pH and PCO2. ST elevation induced by HKC also significantly reduced after repeated occlusion (4.09 +/- 0.79 mV in the fourth HKC vs. 5.64 +/- 0.68 mV in the first, P<0.05) in spite of the identical changes in eKC during HKC. This progressive decrease in ST changes by HKC was rather consistent with augmented conduction delay (86.4 +/- 7.1% increase in activation time in the fourth vs. 54.3 +/- 3.4% in the first, P<0.01). These findings indicate that repeated ischemia induces altered electrical response to subsequent ischemia based on both attenuated metabolic response and altered conduction property.
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Pitavastatin inhibits upregulation of intermediate conductance calcium-activated potassium channels and coronary arteriolar remodeling induced by long-term blockade of nitric oxide synthesis.
Yutaka Terata, Takashi Saito, Yoshimasa Fujiwara, Hitoshi Hasegawa, Hiroto Miura, Hiroyuki Watanabe, Yoshikatsu Chiba, Satoshi Kibira, Mamoru Miura
Pharmacology 68 ( 4 ) 169 - 76 2003.08 [Refereed]
Research paper (journal)
UNLABELLED: We have reported that intermediate conductance Ca(2+)-activated K(+) channels (ImK) showed augmented expression in angiotensin II (AII) type 1 receptor-dependent manner in post-ischemic rat heart. ImK has tyrosine phosphorylation sequence in the C-terminus and motifs for NFkappaB and AP1 in the promoter. While statin inhibits AII-mediated vascular remodeling via anti-inflammatory effect independent of cholesterol lowering. To test the possible effect of statin on expression of ImK, Wistar-Kyoto rats received L-nitro-arginine methyl ester (LNAME: 1 mg/ml in drinking water) for 4 weeks in group L. While in L+P group, rats received both LNAME and pitavastatin (PTV: 1 mg/kg/day in drinking water). Temporal profile of ImK mRNA was examined by RT-PCR using specific primers for ImK. RESULTS: Long-term LNAME administration produced significant hypertension and resulted in marked microvascular remodeling characterized by medial thickening and perivascular fibrosis of coronary arterioles (100-200 microm in diameter). RT-PCR revealed significant up-regulation of ImK mRNA with two distinct peaks in L group in the early phase (days 3-7) and the late phase (4 weeks). PTV partially inhibited a rise in systolic blood pressure, but completely abolished the first peak of ImK upregulation (0.76 +/- 0.04 vs. 3.96 +/- 1.43 folds at day 7, p < 0.001). Co-treatments with PTV also significantly inhibited medial thickening and perivascular fibrosis. These findings indicate that statin inhibits microvascular remodeling induced by chronic inhibition of NO synthesis through the action independent of cholesterol lowering.
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The TRPV4 channel: structure-function relationship and promiscuous gating behaviour.
Bernd Nilius, Hiroyuki Watanabe, Joris Vriens
Pflugers Archiv : European journal of physiology 446 ( 3 ) 298 - 303 2003.06 [Refereed]
Research paper (journal)
Transient receptor potential (TRP) channels provide an enormous variability of Ca(2+) influx mechanisms triggered by a wide range of stimuli. In this review, we discuss the activation properties of the Ca(2+)- and Mg(2+)-permeable TRP channel of the vanilloid subfamily TRPV4. This channel is activated by various physical and chemical stimuli, such as cell swelling, heat, phorbols and, probably, by endogenous ligands, which are able to induce Ca(2+) entry. Not much is known about the regulation of this channel. We will refer only to a mechanism of Ca(2+)-dependent inhibition of TRPV4. Possible functional roles of this channel will be correlated with its observed expression pattern. Finally, we discuss the structural determinants of TRPV4 channel function.
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Modulation of TRPV4 gating by intra- and extracellular Ca2+.
Hiroyuki Watanabe, Joris Vriens, Annelies Janssens, Robert Wondergem, Guy Droogmans, Bernd Nilius
Cell calcium 33 ( 5-6 ) 489 - 95 2003.05 [Refereed]
Research paper (journal)
We have studied the modulation of gating properties of the Ca2+-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4alphaPDD transiently activated a current through TRPV4 in the presence of extracellular Ca2+. Increasing the concentration of extracellular Ca2+ ([Ca2+](e)) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca2+](e). It was also much slower in the presence of [Ca2+](e) in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797), were constitutively open. In conclusion, gating of the 4alphaPDD-activated TRPV4 channel depends on both extra- and intracellular Ca2+, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.
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TNF-alpha rapidly antagonizes the beta-adrenergic responses of the chloride current in guinea-pig ventricular myocytes.
Kenji Iino, Hiroyuki Watanabe, Takashi Saito, Satoshi Kibira, Toshihiko Iijima, Mamoru Miura
Circulation journal : official journal of the Japanese Circulation Society 67 ( 4 ) 347 - 53 2003.04 [Refereed]
Research paper (journal)
The purpose of this study was to test the hypothesis that tumor necrosis factor-alpha (TNF-alpha) rapidly antagonizes the beta-adrenergic responses of the chloride current and to clarify the intracellular mechanisms responsible for the anti-adrenergic action. The whole-cell patch-clamp technique was used to monitor the anti-adrenergic effects of TNF-alpha on the cAMP-dependent chloride current (I(Cl)) recorded from isolated guinea-pig ventricular myocytes. Ramp pulses (+/-120 mV; dv/dt = +/-0.4 V/s) were applied from the holding potential of -40 mV. TNF-alpha rapidly (<15 min) inhibited the isoproterenol (Iso, 0.1 micromol/L)-induced I(Cl) in a concentration-dependent manner (30-1,000 U/ml, IC (50) = 144 U/ml, n=30). The inhibitory action of TNF-alpha was also observed when I(Cl) had been previously stimulated by 1 micromol/L forskolin (n=5). Prior exposure of myocytes to 5 microg/ml pertussis toxin (PTX) hardly affected the anti-adrenergic action of TNF-alpha (n=4). However, when I(Cl) was induced by both 8-bromo-cAMP (100 micromol/L) and isobutylmethylxanthine (0.1 mmol/L), TNF-alpha (1,000 U/ml) failed to decrease I(Cl) amplitude (n=5). Prior exposure of myocytes to 5 mg/ml pertussis toxin (PTX) hardly affected the anti-adrenergic action of TNF-alpha (n=4). Furthermore, despite of the presence of nitro-L-arginine methyl ester (0.1 mmol/L), a nitric oxide synthase (NOS) inhibitor, TNF-alpha reversed the Iso-induced increase in I(Cl) (n=5). These results suggest that TNF-alpha rapidly antagonizes the beta-adrenergic responses of I(Cl) by reducing cAMP concentration. This anti-adrenergic action is mediated by neither the PTX-sensitive G proteins regulatory pathway nor constitutive NOS activation.
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Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels
Watanabe H et. Al
Nature 424 ( 6947 ) 434 - 438 2003.01 [Refereed]
Research paper (journal) Domestic Co-author
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Beneficial effect of dual-chamber pacing for a left mid-ventricular obstruction with apical aneurysm.
Hiroyuki Watanabe, Satoshi Kibira, Takashi Saito, Hiroshi Shimizu, Toyohiko Abe, Ichirota Nakajima, Kenji Iino, Mamoru Miura
Circulation journal : official journal of the Japanese Circulation Society 66 ( 10 ) 981 - 4 2002.10 [Refereed]
Research paper (journal)
Sustained ventricular tachycardia (VT) developed in a 63-year-old woman. The 2-dimensional echocardiogram revealed left mid-ventricular obstructive hypertrophy and a discrete apical chamber. A continuous wave Doppler signal across the mid-ventricular narrowing exhibited early systolic ejection flow and diastolic paradoxical jet flow from the apex to the basal chamber, implying a significant systolic and diastolic intraventricular gradient with a high apical pressure. The left ventriculogram confirmed a mid-ventricular obstruction with an apical aneurysm. Invasive assessment of intraventricular pressure showed a peak-to-peak gradient greater than 100 mmHg. Treatment with antiarrhythmic agents could not prevent the VT, but dual-chamber pacing reduced the intraventricular pressure gradient and suppressed the VT completely. Continuous wave Doppler showed that the early systolic ejection flow from the apex had disappeared, that there was isovolumetric relaxation flow toward the apex and that there was attenuation of the diastolic paradoxical jet flow toward the basal chamber. Such findings by continuous wave Doppler can be useful in pacing therapy for evaluating changes in the severity of mid-ventricular obstruction.
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Molecular determinants of permeation through the cation channel TRPV4.
Thomas Voets, Jean Prenen, Joris Vriens, Hiroyuki Watanabe, Annelies Janssens, Ulrich Wissenbach, Matthias Bödding, Guy Droogmans, Bernd Nilius
The Journal of biological chemistry 277 ( 37 ) 33704 - 10 2002.09 [Refereed]
Research paper (journal)
We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp(672) and Asp(682), as important determinants of the Ca(2+) sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca(2+) permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp(682) but not Asp(672) strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met(680), which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca(2+) permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys(675), had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway.
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L-nitro-arginine inhibits increase in endothelin binding sites induced by ischemia and reperfusion.
Takashi Saito, Etsuko Fushimi, Toshiko Tamura, Yoshimasa Fujiwara, Hiroto Miura, Hiroyuki Watanabe, Satoshi Kibira, Hitoshi Hasegawa, Mamoru Miura
Journal of molecular and cellular cardiology 34 ( 8 ) 1041 - 7 2002.08 [Refereed]
Research paper (journal)
We have demonstrated that ischemia and reperfusion promoted augmented contractile response to endothelin-1 (ET) in coronary arteries in the presence of polymorphonuclear leukocytes (PMN). It has been also reported that ischemia and reperfusion increase ET binding sites in cardiac membrane in isolated rat heart perfused by blood cell-free system. To determine the role of PMN and L-arginine to nitric oxide (NO) pathway in these phenomena, isolated perfused rabbit hearts were subjected to 30 min of global ischemia followed by 30 min of reflow in the absence or presence of PMN and 10(-5)M of L-nitro-arginine (LNA). PMN was prepared with Percoll density gradients from peritoneal exudate elicited by glycogen. PMN activated with 10(-6)M of phorbol myristate acetate or their supernatant were infused into the coronary perfusion circuit after 5 min of reflow. LNA was added to perfusate also after reflow. The effect of superoxide dismutase (SOD: 50 IU/ml) was also determined. After the end of protocols, membrane fraction was isolated from the hearts for (125)I-ET-1 binding assay. ET-1 binding (Bmax) showed a significant increase by ischemia and reperfusion (P<0.01 vs control). That was markedly augmented with addition of activated PMN or their supernatant (both P<0.01), but abolished either by LNA or SOD (P<0.01 and P<0.05, respectively). These results indicate that increase in ET-receptor by ischemia and reperfusion is mediated by free radicals generated via L-arginine to NO pathway.
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The intracellular tyrosine residues of the ATP-gated P2X(1) ion channel are essential for its function.
Emese Toth-Zsamboki, Cecile Oury, Hiroyuki Watanabe, Bernd Nilius, Jos Vermylen, Marc F Hoylaerts
FEBS letters 524 ( 1-3 ) 15 - 9 2002.07 [Refereed]
Research paper (journal)
The four highly conserved intracellular tyrosine residues of the P2X(1) ion channel were mutated into phenylalanine. Simultaneous electrophysiological and calcium measurements in transfected human embryonic kidney (HEK 293) cells indicated that Y362F and Y370F mutants were non-functional, despite their proper plasma membrane expression. The Y16F and Y363F mutants retained 2.2% and 26% of the wild-type P2X(1) activity, respectively. However, no tyrosine phosphorylation was detected on Western blots of P2X(1) immunoprecipitates derived either from HEK 293 cell lysates or from human platelets, expressing P2X(1) endogenously. Thus, Y16, Y362, Y363 and Y370 are required for the appropriate three-dimensional structure and function of the intracellular P2X(1) domains.
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ATP and nitric oxide modulate a Ca(2+)-activated non-selective cation current in macrovascular endothelial cells.
Suk H Suh, Hiroyuki Watanabe, Guy Droogmans, Bernd Nilius
Pflugers Archiv : European journal of physiology 444 ( 3 ) 438 - 45 2002.06 [Refereed]
Research paper (journal)
We have studied the properties of a non-selective cation current (NSC(Ca)) in macrovascular endothelial cells derived from human umbilical vein (EA cells) that is activated by an increase of intracellular Ca(2+) concentration, [Ca(2+)](i). Current-voltage relationships are linear and the kinetics of the current is time-independent. Current-[Ca(2+)](i) relationships were fitted to a Ca(2+) binding site model with a concentration for half-maximal activation of 417 +/- 76 nM, a Hill coefficient of 2.3 +/- 0.8 and a maximum current of -23.9 +/- 2.7 pA/pF at -50 mV. The Ca(2+)-activated channel is more permeable to Na(+) than for Cs(+) ( P(Cs)/ P(Na)=0.58, n=7), but virtually impermeable to Ca(2+). Current activation was transient if ATP was omitted from the pipette solution. The maximal currents at 300 and 500 nM [Ca(2+)](i) were smaller than in the absence of ATP, but were not significantly different at 2 microM. The intracellular Ca(2+) concentration for half-maximal activation of the Ca(2+)-activated current was shifted to 811 +/- 12 nM in the absence of ATP. Substitution of ATP by the non-hydrolysable ATP analogue adenylylimidodiphosphate (AMP-PNP) did not affect current activation. Sodium nitroprusside (SNP) decreased NSC(Ca) in a concentration-dependent manner. The nitric oxide (NO) donors S-nitroso- N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) also inhibited NSC(Ca). In contrast, nitro- L-arginine (NLA), which inhibits all NO-synthases, potentiated NSC(Ca), whereas superoxide dismutase (SOD), which inhibits the breakdown of NO, inhibited NSC(Ca). It is concluded that the Ca(2+)-activated non-selective action channel in EA cells is modulated by the metabolic state of the cell and by NO.
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Ca2+ overload evokes a transient outward current in guinea-pig ventricular myocytes.
Ichirota Nakajima, Hiroyuki Watanabe, Kenji Iino, Takashi Saito, Mamoru Miura
Circulation journal : official journal of the Japanese Circulation Society 66 ( 1 ) 87 - 92 2002.01 [Refereed]
Research paper (journal)
There are 2 types of transient outward currents (Ito) in the hearts of various mammals: a 4-aminopyridine (4-AP) sensitive K+ current and a 4-AP resistant Ca2+ activated current, carried by Cl-, (referred to as I(to1) and I(to2), respectively). However, the I(to) has been considered to be absent in guinea-pig ventricular myocytes and so this study tested the hypothesis that I(to1) is generally absent in guinea-pig ventricular myocytes, but I(to2) appears under the condition of Ca2+ overload. Membrane currents were recorded by the whole-cell patch-clamp technique and Ca2+ overload was achieved by adding internal, and eliminating external, Na+ with subsequent enhancement of Ca2+ influx via the Na+-Ca2+ exchange. Under physiological conditions, I(to) could not be elicited by 300 ms-test pulse from -70 mV to 0 mV (n=32). However, under Ca2+ overload, a biphasic current resulting from the overlap of the L-type Ca2+ channel current and Ito was elicited (n=38). This I(to) was resistant to 4-AP (3 mmol/L, n=30) but sensitive to both anthrancene-9-carboxylic acid (9-AC, 3 mmol/L, n=8) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (100 micromol/L, n=3). Replacing K+ with Cs+ on both sides of the membrane failed to abolish I(to) (n=38). I(to) disappeared by lowering the external Cl- (n=3). The amplitude of I(to) was dependent on that of the L-type Ca2+ channel current (n=4). Because Ca2+ release from the sarcoplasmic reticulum was prevented by caffeine (5 mmol/L), I(to) was negligible (n=6). These results suggest that I(to1) is absent, but Ca2+ overload evokes I(to2) in guinea-pig ventricular myocytes.