Biomedical subjects

J Toyama

Publications and source records attributed to J Toyama.

At least 37 records · Page 2Linked to original sources

PubMed1999

Orientation change of cardiocytes induced by cyclic stretch stimulation: time dependency and involvement of protein kinases.

UNLABELLED: Mechanical stress has been implicated as one of the growth regulators in the heart. We investigated the effect of cyclic stretch stimulation on morphology and orientation of cultured cardiocytes. Embryonic rat (17 days postcoital) cardiomyocytes cultured on silicone dishes were cyclically stretched to 120% in length at a frequency of 30 cycles/min. After 12 h, in the initial stage of cultivation, cardiocytes and intracellular myofibrils oriented parallel to the stretch direction. When the stretch stimulus was prolonged to 24-48 h, myofibrils that oriented perpendicular to the stretch direction emerged. Furthermore, when the cells were stretched only in the later stage (after 24 h of cultivation), both cells and myofibrils tended to orient perpendicular to the stretch direction. Next we examined the effects of chemical compounds on these phase-related changes in myofibril orientation. None of the drugs tested (H-7, HA-1004, staurosporine, herbimycin A, genistein, GdCl3, and EGTA) blocked the parallel orientation of myofibrils induced by the initial-stage stretch. By contrast, H-7, staurosporine, herbimycin A, and genistein did inhibit almost completely the perpendicular orientation of the myofibrils induced by the late-stage stretch, but HA-1004, GdCl3, or EGTA did not. Immunoblotting study using anti-phsophotyrosine antibody indicated that tyrosine phosphorylation of a protein of about 125 kDa was enhanced in a time-dependent manner by the late-stage stretch, but not by the initial-stage stretch. IN CONCLUSION: the alignment change induced by cyclic stretch depends on the stage of cultivation: with stretch in the initial stage (within 12 h), cells and myofibrils orient parallel to the stretch; with stretch in the later stage (after 24 h), they orient perpendicular to the stretch. The effect of stretch in the later stage is likely mediated by protein kinase C and tyrosine kinase pathways.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

Hypoxia inhibits the changes in action potentials and ion channels during primary culture of neonatal rat ventricular myocytes.

Action potentials of rat ventricular myocytes are progressively shortened after birth within several weeks mainly due to a progressive increase in transient outward potassium current (I(to)). On the supposition that an elevation in blood oxygen after birth may contribute to such developmental change, we studied effects of long-term exposure to hypoxia on changes in cardiac action potentials and I(to). Single ventricular myocytes isolated from day-old neonatal rat hearts were cultured in normoxic condition (21% O(2)) for 15 days and served as control. To test the influence of long-term exposure to hypoxia, O(2)tension was reduced to 7.5% at day 6 during culture. In 15-day cells cultured in normoxia, action potential duration (APD) was shortened by 44% (n=11) compared with 5-day cells (n=10); cell capacitance was increased to 2.0-fold. I(to)density was increased by 189-265% (n=11) at voltage levels from -20 to 50 mV without any changes in the kinetics of current inactivation. In 15-day cells cultured in hypoxia, APD was shortened only by 16% (n=6) from control; the increment of cell capacitance was 2.1-fold (n=6). The I(to)increment was limited to 53% (n=8); both inactivation and its recovery of the current was apparently slowed due to the amplification of the slower component. These results suggest that the developmental augmentation of I(to)expression during culture requires oxygen and the increase in I(to)and cell hypertrophy are likely regulated independently.

Action Potentials↗

PubMed1999

Paracrine hypertrophic factors from cardiac non-myocyte cells downregulate the transient outward current density and Kv4.2 K+ channel expression in cultured rat cardiomyocytes.

OBJECTIVES: Cardiac hypertrophy is characterized by a prolongation of action potential duration (APD) and a reduction of outward K+ currents, primarily the transient outward current (Ito). Since the interaction between cardiac non-myocyte cells (NMCs) and cardiomyocytes (MCs) plays a critical role during the process of myocardial hypertrophy, in the present study, we investigated the effects of NMCs on cell growth and K+ channel expression in cultured newborn rat ventricular cells. METHODS: Single MCs were isolated from day-old Wistar rat ventricles and cultured for a period of five days. The effects of NMCs were examined by MC-NMC co-culture or incubating pure MCs in NMC-conditioned growth medium (NCGM). Whole-cell voltage-clamp recording and Western blot analysis using a polyclonal antibody against rat Kv4.2 channel protein were performed. RESULTS: A marked increase in surface area and total cell protein concentration of MCs was observed in the MC-NMC co-culture. In the pure MC culture, this hypertrophic effect could be mimicked by a 72-h addition of NCGM, with a significant prolongation of APD25 (APD at 25% repolarization) and a 42% decrease in Ito density (at +30 mV). The rates of inactivation and recovery from inactivation of Ito were unchanged. In the NCGM-treated MC culture, Western blots of MC proteins also showed a 36% reduction of the Kv4.2 K+ channel protein level. In addition, the NCGM-induced MC hypertrophy was partially inhibited by anti-insulin-like growth factor-1 (IGF-1) antibody, while it revealed no effects on Ito density and Kv4.2 channel expression. CONCLUSIONS: These findings first demonstrate that some paracrine hypertrophic factors released from cardiac NMCs, although unidentified, downregulate cardiac K+ channel expression.

Animals↗

PubMed1999

Heterogeneous distribution of the two components of delayed rectifier K+ current: a potential mechanism of the proarrhythmic effects of methanesulfonanilideclass III agents.

OBJECTIVE: To elucidate the regional difference of the K+ current blocking effects of methanesulfonanilide class III agents. METHODS: Regional differences in action potential duration (APD) and E-4031-sensitive component (IKr) as well as -insensitive component (IKs) of the delayed rectifier K+ current (IK) were investigated in enzymatically isolated myocytes from apical and basal regions of the rabbit left ventricle using the whole-cell clamp technique. RESULTS: At 1 Hz stimulation, APD was significantly longer in the apex than in the base (223.1 +/- 10.6 vs. 182.7 +/- 14.5 ms, p < 0.05); application of 1 microM E-4031 caused more significant APD prolongation in the apex than in the base (32.5 +/- 6.4% vs. 21.0 +/- 8.8%, p < 0.05), resulting in an augmentation of regional dispersion of APD. In response to a 3-s depolarization pulse to +40 mV from a holding potential of -50 mV, both IK tail and IKs tail densities were significantly smaller in apical than in basal myocytes (IK: 1.56 +/- 0.13 vs. 2.09 +/- 0.21 pA/pF, p < 0.05; IKs: 0.40 +/- 0.15 vs. 1.43 +/- 0.23, p < 0.01), whereas IKr tail density was significantly greater in the apex than in the base (1.15 +/- 0.13 vs. 0.66 +/- 0.11 pA/pF, p < 0.01). The ratio of IKs/IKr for the tail current in the apex was significantly smaller than that in the base (0.51 +/- 0.21 vs. 3.09 +/- 0.89; p < 0.05). No statistical difference was observed in the voltage dependence as well as activation and deactivation kinetics of IKr and IKs between the apex and base. Isoproterenol (1 microM) increased the time-dependent outward current of IKs by 111 +/- 8% during the 3-s depolarizing step at +40 mV and its tail current by 120 +/- 9% on repolarization to the holding potential of -50 mV, whereas it did not affect IKr. CONCLUSIONS: The regional differences in IK, in particular differences in its two components may underlie the regional disparity in APD, and that methanesulfonanilide class III antiarrhythmic agents such as E-4031 may cause a greater spatial inhomogeneity of ventricular repolarization, leading to re-entrant arrhythmias.

Action Potentials↗

PubMed1999

Arrhythmogenic changes in action potential configuration in the ventricle induced by DC shocks.

Failure of defibrillation by direct current (DC) shocks is the result in part of new ventricular tachyarrhythmias induced by the shocks. We investigated the arrhythmogenic substrate produced by the shocks. Fluorescent action potential (AP) signals were recorded from rabbit hearts perfused in vitro with the use of our original optical recording system. Localized application of 10-ms shocks (S2) during the plateau phase of APs by basic stimuli (S1) caused field intensity (FI)-dependent changes in APs: (a) S2 > 7 V/cm caused additional depolarization, giving rise to a prolongation of AP duration (APD); (b) With S2 > 20 V/cm, terminal repolarization was inhibited, and subsequent postshock S1 APs for 1 to 5 min were characterized by decreases in the maximum diastolic potential and amplitude of APs; and (c) S2 > 30 V/cm often resulted in a prolonged refractoriness, oscillation of membrane potential leading to ventricular tachycardia or fibrillation (VT/VF). The right ventricle was more susceptible than other regions for the aftereffects of high-intensity shocks. Using an 8-channel recording system, we compared the effect of 10-ms monophasic (M) and 5/5-ms biphasic (B) shocks applied to the whole ventricles with FI of 1 to 20 V/cm at the signal recording sites. B shocks were less potent than M shocks in the FI-dependent action potential duration (APD) prolongation, and in the shock-induced enhancement of APD dispersion. Incidence and duration of VT/VF induced by M shocks were significantly greater than those by B shocks. These findings suggest that DC shocks will cause two types of arrhythmogenic substrate: one induced at sites of high FI, and the other at sites with moderate FI. The former would produce local block or focal repetitive excitation due to prolonged depolarization and oscillation of membrane potential, and the latter circuitous movement of wavefronts through an enhancement of spatial inhomogeneity of repolarization.

Animals↗

PubMed1999

Sudden death in the working population: a collaborative study in central Japan.

AIM: Few epidemiological data are available describing the sudden death of persons in their prime. This study aims to elucidate when and how sudden death occurs among employees. METHODS: A total of 196775 employees from 10 workplaces in Central Japan were surveyed for non-traumatic sudden death during 1989-1995. Demographic data and information regarding onset were collected by their workplace healthcare professionals. RESULTS: We identified 251 male and 13 female cases of sudden death. The annual incidence was 21.9 (for men) and 5.7 (for women) per 100000 population. Sudden death occurred more frequently in April when the new business year starts (risk ratio [95% confidence interval], 1.62 [0.94-2.79]) than in other months, without seasonality. Sudden death peaked on Sundays (risk ratio, 1.90 [1.20-2.99]) and Saturdays (risk ratio, 1.36 [0.83-2.21]) as compared with weekdays, and was likely to occur in the small hours (risk ratio, 1.71 [0.94-3.10] at 00-0300 h and 1.47 [0.79-2.72] at 0300-0600 h vs at 0900-1200 h. Only 17% of employees died at work, which was significantly less than expected (P<0.001). CONCLUSION: These findings differed from those of elderly people and suggest that sudden death of persons in their prime is related to occupational stress and its relief.

Adult↗

PubMed1999

Localization of precise origin of idiopathic ventricular tachycardia from the right ventricular outflow tract by a 12-lead ECG: a study of pace mapping using a multielectrode "basket" catheter.

Radiofrequency catheter ablation guided by pace-mapping techniques has proven effective in eliminating idiopathic ventricular tachycardia (VT) originating from the right ventricular outflow tract (RVOT). A method for rapidly identifying the origin of VT from 12-lead electrocardiogram (ECG) findings would be helpful for the catheter ablation procedure. The purpose of this study is to precisely localize the origin of idiopathic VT from the RVOT by a 12-lead ECG from a study of multipoint pace mapping. In one patient with premature ventricular complex (PVC) and 3 with VT, a "basket" catheter was deployed in the RVOT for bipolar pacing from 56 sites in the endocardium of RVOT. Under fluoroscopy the pacing sites were classified into the septum and free wall. We investigated the QRS morphology in leads, I, II, and III; the depth of the QS wave in leads aVR and aVL; and the height of the initial r wave in leads V1 and V2. Pacing was captured in 30-47 of 56 sites (54%-84%). As the pacing sites changed from the anterior to posterior of the septum, the QS notch (-) type in lead I shifted through rs to R, while a shift from R type to rR' or Rr' was noted in leads II and III. As the pacing sites changed from the anterior to posterior of the free wall, lead I showed a shift from the QS notch (+) type to R, while a shift from rR' to Rr' (or rR' unchanged) was found in leads II and III. The depth of the QS wave in leads aVR and aVL showed a tendency for aVR to be deeper than aVL toward the posterolateral attachment of both the septum and free wall, whereas aVL tended to be deeper than aVR toward the anterior attachment. The initial r waves in V1 and V2 became greater as the pacing site was positioned at a higher or more posterior location. These findings may provide more precise and clinically useful diagnostic information on the site of the origin of idiopathic VT originating from the RVOT by a 12-lead ECG.

Adult↗

PubMed1999

beta-adrenergic modulation of L-type Ca2+-channel currents in early-stage embryonic mouse heart.

Little information is available concerning the modulation of cardiac function by beta-adrenergic agonists in early-stage embryonic mammalian heart. We have examined the effects of isoproterenol (Iso) on the spontaneous beating rate and action potential (AP) configuration in embryonic mouse hearts at 9.5 days postcoitum (dpc), just 1 day after they started to beat. Iso (3 microM) increased the spontaneous beating rate in whole hearts, dissected ventricles, and isolated ventricular myocytes. In ventricular myocytes, Iso also increased the slope of the pacemaker potential and the action potential duration but decreased the maximum upstroke velocity. In whole cell voltage-clamp experiments, the Ca2+-channel currents were measured as Ba2+ currents (IBa). In 9.5-dpc myocytes, IBa was enhanced significantly from -4.7 +/- 0.9 to -6.7 +/- 1.2 pA/pF (by 52.4 +/- 14.8%, n = 10) after the application of Iso. Propranolol (3 microM) reversed the effect of Iso. Forskolin (For, 10 microM) produced an increase in IBa by 95.5 +/- 18.8% (n = 8). In ventricular myocytes at a late embryonic stage (18 dpc), 3 microM Iso caused an appreciably greater increase in IBa from -6.2 +/- 0.5 to -14.5 +/- 2.2 pA/pF (by 137.8 +/- 33.0%, n = 8), whereas the increase in IBa by 10 microM For (by 120.0 +/- 23.0%, n = 7) was comparable to that observed in the early stage (9.5 dpc). These results indicate that the L-type Ca2+-channel currents are modulated by beta-adrenergic receptors in the embryonic mouse heart as early as 9.5 dpc, probably via a cAMP-dependent pathway.

Action Potentials↗

PubMed1999

Downregulation of voltage-gated K(+) channels in rat heart with right ventricular hypertrophy.

The effects of myocardial hypertrophy on mRNA expression levels of voltage-gated K(+) channels were investigated using monocrotaline (MCT)-induced pulmonary hypertensive rats. The ratio of right ventricle weight to left ventricle plus septum weight on day 28 was increased significantly compared with control rats [control vs. MCT: 0.27 +/- 0.01 vs. 0.58 +/- 0.03 ms (n = 8-13); P < 0.05]. Electrocardiograms showed that QRS duration [control vs. MCT: 26.4 +/- 2.6 ms vs. 31.5 +/- 5.8 ms (n = 6); P < 0.05], Q-T interval [control vs. MCT: 100.8 +/- 8.9 ms vs. 110.0 +/- 4.2 ms (n = 6); P < 0.05] and corrected Q-T interval [Q-T(c); control vs. MCT: 8.4 +/- 0. 7 ms vs. 10.2 +/- 0.4 ms (n = 6); P < 0.05] were prolonged significantly on day 28. mRNA levels of Kv1.2, 1.5, 2.1, 4.2, and 4. 3 for day 28 assessed by ribonuclease protection assays were decreased significantly from control by 60 +/- 10, 76 +/- 3, 58 +/- 5, 81 +/- 5, and 45 +/- 12%, respectively (n = 3; P < 0.005), and Kv1.4 mRNA level for day 28 was unaffected [Kv1.4, control vs. MCT: 1.0 +/- 0.28 vs. 0.88 +/- 0.44 (arbitrary units) (n = 3); not significant (NS)]. On the other hand, there was no significant difference between control and MCT rats in mRNA levels of these Kv channels for day 14 [Kv1.2 (control vs. MCT): 1.0 +/- 0.25 vs. 0.87 +/- 0.18 (n = 3), NS; Kv1.4: 1.0 +/- 0.22 vs. 1.27 +/- 0.37 (n = 3), NS; Kv1.5: 1.0 +/- 0.16 vs. 0.91 +/- 0.28 (n = 3), NS; Kv2.1: 1.0 +/- 0.26 vs. 0.99 +/- 0.25 (n = 3), NS; Kv4.2: 1.0 +/- 0.15 vs. 1.22 +/- 0.28 (n = 3), NS; Kv4.3: 1.0 +/- 0.20 vs. 1.21 +/- 0.28 (n = 3), NS]. These findings suggest that altered ventricular repolarization at the advanced stage of hypertrophy may be the result of an inhibition of gene expression of multiple types of voltage-gated K(+) channels.

Animals↗

PubMed1999

[The 13C-urea breath test efficacy in determining Helicobacter pylori eradication].

Helicobacter pylori eradication therapy is useful and requires more precise determination of actual eradication. Patients often become positive for Helicobacter pylori again after presumed eradication. Reinfection is thought rare and patients with positive reconversion may be false-negative at determination. After a year, positive reconversion was 26.8% in our work after determination by culture and histopathological methods and 4.3% after these were combined with urea breath test (UBT). The positive reconversion rate is significantly lower after combination with UBT, suggesting the usefulness of UBT in determining Helicobacter pylori eradication. This may be because UBT includes no biopsy, eliminating sampling error, and highly sensitive UBT detects traces of Helicobacter pylori. The UBT is thus expected to become widely used to determine Helicobacter pylori eradication.

Breath Tests↗

PubMed1998

Na+ channel blocking effects of cibenzoline on guinea-pig ventricular cells.

The effects of cibenzoline on transmembrane action potentials were examined in right ventricular papillary muscles and in single ventricular myocytes isolated from guinea-pig hearts. In papillary muscles, cibenzoline > or = 3 microM caused a significant decrease in the maximum upstroke velocity (Vmax) of the action potential without affecting the action potential duration. The inhibition of Vmax was enhanced at higher stimulation frequencies. In the presence of cibenzoline, trains of stimuli at rates > or = 0.2 Hz led to a use-dependent inhibition of Vmax. The time constant for Vmax recovery (tauR) from the use-dependent block was 26.2 s. The use-dependent block of Vmax with cibenzoline was enhanced and tauR was shortened when the resting potential was depolarized by high (8, 10 mM) [K+]o. The curve relating membrane potential and Vmax in single myocytes was shifted by cibenzoline (10 microM) in a hyperpolarizing direction by 7.1 mV. In myocytes treated with cibenzoline (10 microM), a 10-ms conditioning clamp to 0 mV caused a significant decrease in Vmax of the subsequent test action potential; the Vmax inhibition was enhanced modestly in association with a prolongation of the 0 mV clamp pulse duration. In the presence of cibenzoline (3 microM), application of a train of depolarizing pulses (10 ms, 200 ms) to myocytes from the resting level (-80 mV) to 0 mV resulted in a progressive Vmax reduction in a pulse number-dependent manner. Unlike glibenclamide (30 microM), cibenzoline (10 microM) did not prevent the hypoxia-induced shortening of action potential duration in papillary muscles. These findings indicate that the onset and offset kinetics of use-dependent Na+ channel block by cibenzoline are slow. Given its state dependence, cibenzoline may be a blocker of activated Na+ channels. The inhibitory action of this compound on the ATP-sensitive K+ current (I(K), ATP) would be minimal or negligible at concentrations causing sufficient Na+ channel block.

Animals↗

PubMed1998

Familial neuronal migration disorder: subcortical laminar heterotopia in a mother and pachygyria in the son.

We describe clinical manifestations and magnetic resonance imaging (MRI) findings in a man and his mother who were diagnosed as having a neuronal migration disorder. The son had severe psychomotor retardation and the mother had intractable seizures and mild psychomotor retardation. MRI demonstrated moderate pachygyria in the son and subcortical heterotopia in the mother. In both patients, the frontal parts of the brain were characteristically more affected than any other areas. A dominant pattern of inheritance in the family suggests a genetic role in the underlying cause of the migration disorder. The difference in severity between the two patients also suggests an X-linked dominant inheritance. Our family fits the condition of X-linked lissencephaly.

Adult↗

PubMed1998

Regulation of cardiac Kv1.5 K+ channel expression by cardiac fibroblasts and mechanical load in cultured newborn rat ventricular myocytes.

Of the six voltage-gated K+ channel alpha subunits detected in rat heart, the Kv1.5 channel is abundantly expressed, and its gene transcription and protein expression are reduced during cardiac remodeling. Since cardiac fibroblasts and mechanical load have been known to play important roles in myocardial hypertrophy, we studied the regulation of Kv1.5 K+ channel protein expression by these factors in cultured newborn rat ventricular myocytes, using immunofluorescent cytochemistry and Western blot analysis. Ventricular cells were isolated from 1-day-old Wistar rats and cultured for a period of 5 days. The effect of cardiac fibroblasts was examined by co-culturing myocytes with fibroblasts or incubating pure myocytes in fibroblast-conditioned growth medium (FCGM) for 72 h. In addition, a 48-h cyclic stretch at 0.5 Hz with 20% elongation in length was applied to pure myocyte cultures to mimic mechanical load. With a polyclonal antibody against rat Kv1.5 K+ channel protein, single cultured myocytes showed a weak and uniform antibody labeling. Co-culturing with fibroblasts or incubating pure myocytes in FCGM both induced a significant increase in myocyte size implying cell hypertrophy, but neither allowed normal expression of the Kv1.5 K+ channel as indicated by almost negative anti-Kv1.5 labeling. Western blots of cell proteins prepared from ventricular myocyte cultures revealed a single protein band at 75 kD recognized by the anti-Kv1.5 antibody and a 45% decrease in Kv1.5 immunoreactive protein level in the FCGM-treated preparations. Application of 1 microM losartan, an angiotensin II type I receptor blocker, significantly attenuated the FCGM-induced myocyte hypertrophy and reduction of Kv1.5 K+ channel expression. On the other hand, although no cell hypertrophy was stimulated by mechanical stretch, intense punctate antibody labeling with a 48% increase in Kv1.5 protein level was observed in the stretched myocytes. These results suggest that the protein expression of cardiac Kv1.5 K+ channel is differentially regulated by cardiac fibroblasts and mechanical load. Some soluble factors produced from cardiac fibroblasts contribute to the depressed Kv1.5 K+ channel expression in myocardial hypertrophy. This channel regulation may be mediated by angiotensin II type I receptor.

Animals↗

PubMed1998

Cell cycle-related changes in the voltage-gated Ca2+ currents in cultured newborn rat ventricular myocytes.

The expression of T-type Ca2+ current (ICa,T) has been reported to change during postnatal heart development and myocardial hypertrophy, which are characterized respectively by the arrest of the cell cycle soon after birth and a switching on of DNA synthesis in the terminally differentiated cardiac myocytes. The hypothesis that there are cell cycle-related changes in cardiac Ca2+ channel expression was tested by performing whole-cell voltage-clamp recording and BromodeoxyUridine (BrdU) immunolabeling to determine the S phase of the cell cycle in the same single cultured newborn rat ventricular cells. Myocytes were isolated from 1-day-old Wistar rats and cultured for 15 days. ICa,T was detected in 27% of the 5-day cultured myocytes. The progressive loss of ICa,T during the period of 15-day incubation, which resembles the developmental changes in vivo, paralleled the decrease in the percentage of cells showing BrdU labeling. At day 5 of cell culture, the fraction of myocytes expressing ICa,T was significantly higher in the BrdU-labeled population (95%) as compared with the non-labeled cells (19%). In addition, a 72-h treatment with 20 microM nickel, an ICa,T blocker, revealed no effect on the percentage of BrdU-positive cells. L-type Ca2+ current (ICa,L) was constantly expressed throughout the 15-day cell culture. The frequency of ICa,L expression was identical between the BrdU-labeled and the non-labeled myocytes, although the latter cell population demonstrated a relatively greater current density. No differences in the inactivating kinetics of ICa,L and their reaction to beta-adrenoceptor stimulation were observed between the two groups. These findings provide convincing evidence for the cell cycle-related expression of cardiac Ca2+ channel. Cardiomyocytes at the S phase of the cell cycle predominantly express ICa,T, while the major properties of ICa,L' are unchanged during the cell cycle. Such a cell cycle-related channel expression may play a critical role in regulating the cardiac electrophysiological properties during heart development and myocardial remodeling.

Animals↗

PubMed1998

Regulation of Kv4.2 and Kv1.4 K+ channel expression by myocardial hypertrophic factors in cultured newborn rat ventricular cells.

Postnatal development and myocardial hypertrophy are associated with alterations in cardiac voltage-gated K+ channels. To investigate mechanisms underlying this K+ channel remodeling, expression of Kv4.2 and Kv1.4 K+ channel alpha-subunits was examined in cultured newborn rat ventricular myocytes by Western blot analysis using polyclonal antibodies against each of the subunits. At day 5 of cell culture, Kv1.4 protein was expressed at higher level than Kv4.2; as the age of culture progressed, Kv1.4 was significantly diminished while Kv4.2 increased with time in culture and became the predominant K+ channel protein. Such K+ channel isoform switch from Kv1.4 to Kv4.2 resembles that of the development in vivo. A 72-h treatment with exogenous triiodothyronine (T3, 0.1 microM) to cultured neonatal myocytes enhanced the expression of Kv4.2 by 73% and decreased the Kv1.4 expression by 22%. The effects of T3 were associated with an increase in the protein-to-DNA ratio indicating myocyte hypertrophy. On the other hand, a 72-h treatment with cardiac non-myocyte cell (NMC)-conditioned growth medium (NCGM) or phenylephrine (20 microM) induced similar cell hypertrophy, but in sharp contrast to T3, both markedly suppressed the Kv4.2 channel protein level. In addition, the trophic and the Kv4.2-downregulating effects of NCGM could be mimicked by exogenous endothelin-1 (0.1 microM), a paracrine factor secreted from cardiac NMCs. Our observations for the first time suggest that cardiac Kv4.2 and Kv1.4 K+ channel alpha-subunits are differentially regulated by a variety of myocardial hypertrophic factors. That T3 accelerated the developmental K+ channel isoform switch from Kv1.4 to Kv4.2 in vitro indicates the critical importance of thyroid hormone in postnatal K+ channel remodeling. Cardiac NMCs and alpha-adrenoceptor activation may contribute to the reduced outward K+ channel density in hypertrophied cardiomyocytes.

Adrenergic alpha-Agonists↗

PubMed1998

alpha1-adrenoceptor agonists and IGF-1, myocardial hypertrophic factors, regulate the Kv1.5 K+ channel expression differentially in cultured newborn rat ventricular cells.

Interest has arisen concerning the importance of alpha-adrenergic function and insulin-like growth factor-1 (IGF-1) in cardiac remodelling. The hypothesis that these two factors may underlie the regulation of voltage-gated K+ channel expression in hypertrophied cardiomyocytes was tested by performing Western blot analysis of the Kv1.5 K+ channel alpha-subunit in cultured newborn rat ventricular cells. Myocyte size was quantified by surface area and total cell protein concentration. Cell exposure to the alpha1-adrenoceptor agonist phenylephrine (PE, 20 microM) and IGF-1 (60 ng/ml) for 72 h both induced a significant increase of cell size indicating myocyte hypertrophy, which could be separately blocked by the protein kinase C inhibitor staurosporine (20 nM) and the tyrosine kinase inhibitor genistein (15 microM). Western blots of cell proteins prepared from myocyte cultures showed a single protein band at 75 kD recognized by the anti-Kv1.5 antibody, and demonstrated a 56% reduction in the Kv1. 5 immunoreactive protein level in the PE-treated cell preparations. This suppression was not affected by staurosporine, but was remarkably attenuated by W7 (20 microM), a selective calmodulin antagonist. In contrast to PE, a 48% enhancement of the protein expression of Kv1.5 channel was induced by IGF-1 and this stimulation was specifically blocked by genistein. Our findings suggest that the differential regulation of cardiac Kv1.5 K+ channel expression can be produced by alpha1-adrenoceptor activation and IGF-1 via distinctive signalling pathways. Calmodulin-dependent kinase and tyrosine kinase contribute importantly to the alpha1-adrenoceptor-mediated decrease and the IGF-1-mediated increase in cardiac Kv1.5 K+ channel expression, respectively.

Adrenergic alpha-1 Receptor Agonists↗

PubMed1998

Effects of thyroid status on expression of voltage-gated potassium channels in rat left ventricle.

OBJECTIVE: Thyroid hormone modifies cardiac action potentials and outward potassium currents directly and indirectly e.g. through beta-adrenergic signaling pathway. We thus examined the expression of six voltage-gated potassium channel alpha-subunits in the rat left ventricle under hypo- and hyperthyroid status, and tested roles of beta-adrenergic signaling pathway in their expressions under both status. METHODS: Hypothyroidism and hyperthyroidism were induced by administration of methimazole (MMI) for 4 weeks and by injection of L-thyroxine (T4) to the MMI-treated rats for the last 7 days, respectively. To distinguish the effects of T4 and the beta-adrenergic system, propranolol (Pro) was administered to the MMI-treated rats together with T4, and isoproterenol (Iso) was injected to MMI-treated rats for the last 7 days. The mRNA levels of Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3 in the left ventricles were determined by ribonuclease protection assay. RESULTS: MMI treatment induced hypothyroidism and resulted in a significant decrease in the mRNA levels of Kv1.5, Kv2.1 and Kv4.2 (19%, 77% and 61% of control value, respectively; n = 6, p < 0.05). T4 administration induced hyperthyroidism and cardiac hypertrophy, and it increased the Kv1.5 and Kv2.1 mRNA levels over the control value (212% and 140%, respectively; n = 6, p < 0.05). Kv4.2 mRNA level was restored to the control level by T4. In contrast, the Kv1.2 and Kv1.4 mRNA levels increased in hypothyroid rats (161% and 186% of control value, respectively; n = 6, p < 0.01) and decreased in hyperthyroid rats (14% and 33% of control value, respectively; n = 6, p < 0.01). The Kv4.3 mRNA level was not altered by thyroid status. Pro did not inhibit the T4-induced hypertrophy. Iso induced cardiac hypertrophy. Pro or Iso by itself did not alter Kv mRNA levels except for Kv1.2, the message of which was decreased by Iso. CONCLUSION: Thyroid hormone differentially regulates the expression of Kv1.4, Kv1.5, Kv2.1 and Kv4.2 mRNA levels in the rat left ventricle. This effect is not mediated through beta-adrenergic signaling pathway. On the other hand, the reduction in Kv1.2 mRNA level was associated with cardiac hypertrophy induced by T4 or Iso.

Adrenergic beta-Agonists↗

PubMed1998

Relaxant effects of NKH477, a new water-soluble forskolin derivative, on guinea-pig tracheal smooth muscle: the role of Ca2+-activated K+ channels.

1. Mechanisms underlying the bronchorelaxant action of NKH477, a newly developed water-soluble forskolin derivative, were investigated in guinea-pig isolated tracheal smooth muscle. 2. In muscles precontracted with 3 microM histamine, NKH477 (1 nM-1 microM) caused a concentration-dependent decrease of isometric tension, resulting in a complete relaxation at 300 nM. The EC550 for the relaxation was 32.6+/-4.3 nM (n=6). 3. In the presence of 30 or 90 nM iberiotoxin (IbTX), a selective blocker of the large-conductance Ca2+-activated K+ (BK(Ca)) channel, the relaxing action of NKH477 on the histamine-induced contraction was inhibited, giving rise to a parallel shift of the concentration-response curves; the EC50 of NKH477 was increased to 131.4+/-20.4 nM at 30 nM IbTX (n=4), and 125.3+/-12.2 nM at 90 nM IbTX (n=4). 4. Pretreatment of muscles with 30 mM tetraethylammonium (TEA) caused a similar rightward shift of the concentration-response curve to NKH477 with an increase of the EC50 to 139.8+/-18.4 nM (n=5). In contrast, the relaxing action of NKH477 was unaffected by 10 microM glibenclamide, an ATP-sensitive K channel blocker, or by 100 nM apamin, a blocker of small conductance Ca2+-activated K+ channels. 5. In muscles pretreated with 1 microM nifedipine, a blocker of the voltage-dependent Ca2+ channel (VDC), 30-90 nM IbTX did not affect the relaxant effects of NKH477 on the histamine-induced contraction. 6. In muscles precontracted by a K+-rich (40 mM) solution, NKH477 caused only minimal relaxation (19.8+/-1.7%, n=4) even at the highest concentration (1 microM). 7. In experiments to measure the ratio of fura-2 fluorescence signals (R(340/380)) as an index of the intracellular Ca2+ concentration ([Ca2+]i), the application of 100 nM NKH477 or 200 nM isoprenaline to the preparation precontracted by 3 microM histamine resulted in a decrease in [Ca2+]i in association with a decrease in tension. The reduction of [Ca2+]i and tension by NKH477 was 47.0+/-5.6% and 62.8+/-7.0%, respectively (n=5), and that with isoprenaline 60.6+/-7.4% and 67.4+/-6.4%, respectively (n=5). These effects of NKH477 and isoprenaline on [Ca2+]i and tension were inhibited by 30 nM IbTX. The inhibitory action of IbTX was abolished in the presence of 1 microM nifedipine. 8. These results suggest that the bronchorelaxant action of NKH477 may result, at least in part, from activation of BK(Ca) channels, which may cause a hyperpolarization of smooth muscle cell membranes and a secondary decrease in Ca2+ influx through VDCs, leading to a decrease in [Ca2+]i.

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