Biomedical subjects

R Sato

Publications and source records attributed to R Sato.

At least 145 records · Page 8Linked to original sources

PubMed1997

Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes.

Muscarinic receptor-linked G protein, Gi, can directly activate the specific K+ channel (IK(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of Gi to the K+ channel in the ventricle has not been well defined. G protein regulation of K+ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 microM acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 +/- 12.1% of control (at 90% repolarization, mean +/- SD, n = 8) and increased the whole-cell membrane current conductance without prior beta-adrenergic stimulation in human ventricular myocytes. The ACh effect was reversed by atropine (1 microM). In excised inside-out patch configurations, application of GTPgammaS (100 microM) to the bath solution (internal surface) caused activation of IK(ACh) and/or the background inwardly-rectifying K+ channel (IK1) in ventricular cell membranes. IK(ACh) exhibited rapid gating behavior with a slope conductance of 44 +/- 2 pS (n = 25) and a mean open lifetime of 1.8 +/- 0.3 msec (n = 21). Single channel activity of GTPgammaS-activated IK1 demonstrated long-lasting bursts with a slope conductance of 30 +/- 2 pS (n = 16) and a mean open lifetime of 36.4 +/- 4.1 msec (n = 12). Unlike IK(ACh), G protein-activated IK1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 microM in IK(ACh) and 1.2 microM in IK1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic receptor-linked PTX-sensitive G protein, Gi, is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action potential by activating IK(ACh) and IK1 via muscarinic receptor-linked Gi proteins in human ventricular myocytes.

Cells, Cultured↗

PubMed1997

Different electrophysiological character of I-, ClO4-, and SCN- in the transport by Na+/I- symporter.

The electrophysiological characteristics of the Na+/I- symporter were examined using the Chinese hamster ovary (CHO) cell line, which was transfected with the rat Na+/I- symporter gene and stably expressed the Na+/I- symporter. In this cell line, iodide uptake was dependent on Na+, and kinetic studies revealed that the K(m) for iodide was 35 microM, similar to that of FRTL-5 cells. The maximal velocity at the cell protein level was 6- to 10-fold higher than in FRTL-5 cells. ClO-4 and SCN- dose-dependently inhibited iodide uptake in a competitive manner. Electrophysiological characteristics were examined using the whole-cell patch-clamp technique. The holding current at-40 mV rapidly shifted inwardly when the cells were perfused with 1 mEq I- or SCN-. The inward current induced by 1 mEq I- did not increase when bathing solution was replaced with a Tyrode solution with 10 mEq I-, indicating that 1 mEq I- was a saturating amount. The inward current induced by 1 mEq I- increased 1.5-fold by changing the bathing solution to a Tyrode solution containing 1 mEq I- and 1 mEq SCN-. The inward current induced by 0.5 mEq SCN- decreased when the bathing solution was changed to a Tyrode solution containing 0.5 mEq SCN- and 10 mEq I-. These findings indicated that the I- ion and the SCN- ion were carried by the NA+/ I- symporter with at least two Na+ ions. The current induced by the transport of SCN- was larger than that induced by the transport of I-, possibly because the number of Na+ ions that was carried with one SCN- ion was larger than the number of Na+ ions carried with one I- ion. Surprisingly, the perfusion of ClO-4 did not induce an inward current, indicating that ClO-4 bound to the Na+/I- symporter, but was not carried by it, or that one ClO-4 ion was carried with one Na+ ion.

Animals↗

PubMed1997

Responses of catecholamines, renin-angiotensin system, and atrial natriuretic peptide to exercise in untrained men and women.

1. Plasma norepinephrine (NE), epinephrine (E), renin activity (PRA), angiotensin II (ATII), aldosterone (ALD), and atrial natriuretic peptide (ANP) were measured in 20 male and 15 female subjects during submaximal treadmill test. 2. Exercise duration was not different between the two groups (male vs. female: 13.4 +/- 0.8 min vs. 11.6 +/- 0.7 min, ns). Female subjects had higher heart rate during exercise, while systolic blood pressure at peak exercise was higher in male subjects. 3. Plasma NE, E, ANP, and ATII responses were comparable between male and female subjects, but PRA both at rest and during exercise and ALD at rest were significantly higher in male subjects. 4. Cardiac responses to submaximal exercise were different between male and female subjects, but neurohormonal responses were comparable between the two groups except for the high PRA at rest and during exercise and high plasma ALD at rest in male subjects.

Adult↗

PubMed1997

Sematilide blocks the inward rectifier potassium channel in isolated guinea pig ventricular myocytes.

1. In whole-cell patch recording, the relative potency of the blocking action of sematilide on IK1 was found to be constant at each potential level of IK1 activation. Under more acidic condition, the degree of block was decreased. These results strongly suggested that the neutral form of sematilide may penetrate the cardiac cell membrane via hydrophobic pathway. 2. In cell-attached patches, sematilide prolonged the interburst interval and reduced the opening probabilities of the IK1 channel without affecting either the mean open time or the mean closed time within a burst.

Animals↗

PubMed1997

The blocking mechanism of sodium currents by a new class I antiarrhythmic drug, Ro 22-9194, in isolated guinea-pig ventricular myocytes.

1. Ro 22-9124 produced tonic block of sodium currents (INa) with a Hill coefficient of 1.01 and a dissociation constant of 0.12 microM at a holding potential of - 100 mV, and use-dependent block at 2 Hz in conjunction with an increase in pulse duration (5-300 msec). 2. Ro 22-9194 blocks INa, at least partially, due to binding to the activated state. 3. The course of recovery from the use-dependent block, which was not affected by the membrane potential, was 4.0 sec, suggesting that Ro 22-9194 belongs to the intermediate kinetic class I drugs and it probably acts via a hydrophobic pathway.

Animals↗

PubMed1997

Influence of extracellular H+ and Ca2+ on Ro 22-9194-induced block of sodium current in cardiac myocytes.

1. Ro 22-9194 reduced the Na current in ventricular myocytes in either a tonic block or phasic block manner. 2. Ro 22-9194 had a higher affinity to the inactivated state (Kdi = 10.3 microM) than to the rested state (Kdrest = 180 microM). 3. Extracellular acidification enhanced the tonic block but reduced the phasic block. 4. Elevation of extracellular Ca2+ inhibited the enhancing effects of extracellular acidification. 5. These findings suggest that Ro 22-9194 strongly inhibits Na+ channels of the ventricular myocytes of the diseased hearts, characterized by the depolarized cell membranes and by acid conditions.

Animals↗

PubMed1997

A novel mutation causing complete deficiency of thyroxine binding globulin.

OBJECTIVE: Thyroxine binding globulin (TBG) is a serum protein that transports thyroxine. Three naturally occurring mutations have been reported to produce complete deficiency of TBG (TBG-CD). The first to be reported was TBG-CD5 in caucasian families of French-Canadian origin and consists of substitutions in exons 2 and 3. TBG-CD of English ethnic origin (TBG-CD6) is characterized by a thymine deletion in codon 165 (exon 1). In Japanese families with TBG-CD (TBG-CDJ), a variant has been characterized with a deletion of the first base of the codon for amino acid 352 (exon 4) in the common type TBG. In this communication we report a new type of TBG-CD in a family of Japanese ethnic origin that is characterized by a single nucleotide substitution in place of two nucleotides in exon 1. This is an uncommon mutation which we have been unable to find in other genes. DESIGN: Exons of the TBG gene amplified by the polymerase chain reaction (PCR) were subcloned and sequenced. To examine for the presence of the same mutation in potentially affected individuals, we performed PCR using primer-directed mutagenesis or allele-specific amplification. PATIENTS: The index case was of Japanese ethnic origin, and was diagnosed as having TBG deficiency on the basis of undetectable serum TBG. The patient consented to this evaluation and the protocol was in accordance with IRB standards. MEASUREMENTS: Serum thyroid hormones, thyrotrophin binding inhibitory immunoglobulin and TBG concentrations were measured by conventional radio-immunoassay. Genomic DNA was extracted from white blood cells. RESULTS: In the index case exons 2, 3 and 4 were normal, but nucleotides 144 (cytosine) and 145 (thymine) in exon 1 were substituted with a single base (adenine) which induced a frame shift in the reading frame, resulting in an early stop codon at codon 51. The patient and his daughters were confirmed as having this mutation using primer-directed mutagenesis or allele-specific amplification. CONCLUSIONS: We have described a novel mutation in the TBG gene in a Japanese family. This results in a frame shift and premature stop codon, and was associated with undetectable serum TBG in the index case.

Exons↗

PubMed1997

Role of H-ras gene in chronic liver damage in mice. By using transgenic mice carrying a human C-H-ras proto-oncogene without mutations.

Hepatic tumors including hepatocellular carcinoma were generated by carbon tetrachloride in transgenic mice carrying a human c-H-ras gene (rasH2 mice). RasH2 mice express 2 to 3 times more ras protein (ras p21) in the liver than do non-Tg mice. When carbon tetrachloride was administered, the rasH2 mice produced about 5 times as many hepatic tumors than did the non-transgenic mice. However, neither the 10-100 times higher ras p21 expression required for murine fibroblast transformation by itself nor the mutational activation of the H-ras gene was observed in carbon tetrachloride-induced hepatic tumors. These results show that H-ras proto-oncogene expression in the murine liver, even if it is not high enough to transform cells, also causes liver tumors when CC1(4) are repeatedly given.

Animals↗

PubMed1997

Inhibitory effects of efonidipine hydrochloride on contraction induced by several vasoconstrictors in porcine coronary artery: comparison with effects of nifedipine and nisoldipine.

We studied the effects of efonidipine hydrochloride (efonidipine), a 1,4-dihydropyridine derivative, on contractions induced by high-K+ solution (high K+), serotonin (5-HT), U46619, which is a stable analog of thromboxane A2, and endothelin-1 (ET-1) in comparison with those of nifedipine and nisoldipine in porcine coronary arteries. The effects of the drugs were compared after 1- and 3-h incubations. Efonidipine, nifedipine, and nisoldipine each inhibited the contractions induced by these vasoconstrictors. The inhibition of high-K(+)- and 5-HT-induced contractions by efonidipine, but not by nifedipine and nisoldipine, increased when the incubation time was prolonged, whereas the inhibition of U46619- and ET-1-induced contractions was not altered. The potency of efonidipine on U46619- and ET-1-induced contractions was greater than that of nifedipine and equivalent to that of nisoldipine. Thus the inhibitory effect of efonidipine on U46619- and ET-1-induced contractions seems to be stronger than its effects on high-K(+)- or 5-HT-induced contractions, in contrast to the effects of other dihydropyridines. In an additional series of experiments, efonidipine did not inhibit U46619-induced contractions in Ca2(+)-free solution or in the presence of nifedipine. Moreover, efonidipine did not inhibit the specific binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to porcine coronary arterial membrane. Therefore we think that the inhibitory effect of efonidipine on contractions induced by vasoconstrictors was caused by blockade of Ca2+ influx through L-type Ca2+ channels. However, some unknown mechanism(s) in addition to this effect on Ca2+ channels may contribute to the effect of efonidipine on U46619- and ET-1-induced contractions.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1997

Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta.

We have isolated a cDNA for a cytochrome P450, cinnamate 4-hydroxylase (C4H), of Arabidopsis thaliana using a C4H cDNA from mung been as a hybridization probe. The deduced amino acid sequence is 84.7% identical to that of mung bean C4H and therefore was designated CYP73A5. The CYP73A5 protein was expressed in insect cells using the baculovirus expression system and when reconstituted with lipid and NADPH-cytochrome P450 reductase resulted in C4H activity with a specific activity of 68 nmol min-1 nmol-1 P450. Southern blot analysis revealed that CYP73A5 is a single-copy gene in Arabidopsis. C4H (CYP73A5) expression was apparently coordinated in Arabidopsis with both PAL1 and 4CL in response to light and wounding. Although the light induction of CHS followed a time course similar to that observed with C4H, no induction of CHS was detected upon wounding. On the other hand, the C4H expression patterns exhibited no significant coordination with those of PAL2 and PAL3. A C4H promoter region of 907 bp contained all of the three cis-acting elements (boxes P, A, and L) conserved among the PAL and 4CL genes so far reported as controlling expression.

Amino Acid Sequence↗

PubMed1997

Intestinal distribution and intraluminal localization of orally administered Clostridium butyricum in rats.

Clostridium butyricum has been used as a probiotic in animals and humans for years, however, its fate in the intestine has not been clarified yet. We investigated the intestinal fate of C. butyricum using a selective medium and a monoclonal antibody after orally administering C. butyricum spores to rats. The number of C. butyricum, both viable and dead cells, in the intestinal contents were counted by enzyme-linked immunosorbent assay (ELISA) at various times after a single oral administration. The total viable number of C. butyricum was counted using a selective medium, and viable resting spores were selectively detected by treating the samples with ethanol. To investigate the intraluminal localization of the C. butyricum cells, frozen intestinal tracts were imprinted onto slides and stained with immunogold-silver. Total viable spores exceeded the number of viable resting spores by more than 10-fold from the proximal to middle of the small intestine 30 min after administration. Vegetative cells of C. butyricum were first detected in the distal small intestine after 2 hr, and vegetative growth was observed from the cecum to the colon 5 hr after administration. Dead vegetative cells were detected 9 hr after administration, and C. butyricum cells were not detected in the intestine after 3 days. The C. butyricum cells in the intestinal imprints were stained specifically by immunogold-silver staining, and proliferative cells were observed in the cecum after 3 hr. These results suggest that the administered C. butyricum germinated in the upper small intestine, grew mainly from the distal small intestine to the colon and were excreted from the rat intestine within 3 days.

Administration, Oral↗

PubMed1997

Tonic block of the Na+ current in single atrial and ventricular guinea-pig myocytes, by a new antiarrhythmic drug, Ro 22-9194.

Ro 22-9194 reduced the Na+ current in the atrial myocytes as well as ventricular myocytes in a tonic block fashion. Ro 22-9194 had a higher affinity to the inactivated state Na+ channels (KdI = 3.3 microM in atrial myocytes, KdI = 10.3 microM in ventricular myocytes) than to those in the rested state (KdR = 91 microM in atrial myocytes, KdR = 180 microM in ventricular myocytes), which indicated that Ro 22-9194 had a higher affinity to the Na+ channels in atrial myocytes than in ventricular myocytes. Ro 22-9194 shifted the inactivation curve in the hyperpolarized direction in both atrial and ventricular myocytes. These findings suggest that Ro 22-9194 more strongly inhibited the Na+ channel of the atrial myocytes of the diseased hearts with the depolarized membranes potentials than the Na+ channels in ventricular myocytes.

Animals↗

PubMed1997

Propionyl coenzyme A carboxylase is required for development of Myxococcus xanthus.

A dcm-1 mutant, obtained by transposon mutagenesis of Myxococcus xanthus, could aggregate and form mounds but was unable to sporulate under nutrient starvation. A sequence analysis of the site of insertion of the transposon showed that the insertion lies within the 3' end of a 1,572-bp open reading frame (ORF) designated the M. xanthus pccB ORF. The wild-type form of the M. xanthus pccB gene, obtained from a lambdaEMBL library of M. xanthus, shows extensive similarity to a beta subunit of propionyl coenzyme A (CoA) carboxylase, an alpha subunit of methylmalonyl-CoA decarboxylase, and a 12S subunit of transcarboxylase. In enzyme assays, extracts of the dcm-1 mutant were deficient in propionyl-CoA carboxylase activity. This enzyme catalyzes the ATP-dependent carboxylation of propionyl-CoA to yield methylmalonyl-CoA. The methylmalonyl-CoA rescued the dcm-1 mutant fruiting body and spore development. During development, the dcm-1 mutant cells also had reduced levels of long-chain fatty acids (C16 to C18) compared to wild-type cells.

Amino Acid Sequence↗

PubMed1997

Cardiovascular indexes in the mouse at rest and with exercise: new tools to study models of cardiac disease.

Manipulations of the murine genome that alter cardiovascular function have created the need for methods to study cardiovascular physiology in genetically altered animals in vivo. We adapted chronic physiological measurement techniques to the nonanesthetized, nonrestrained murine model, established strain-specific cardiovascular and metabolic norms, and evaluated responses to anesthesia, exercise, and adrenergic stimulation. Anesthesia resulted in alterations in heart rate (HR), blood pressure (BP), and O2 consumption (V(O2)) and CO2 production (V(CO2)) for up to 6 h postoperatively. There were significant interstrain differences in resting values of HR and BP Graded treadmill exercise resulted in linear increases in HR, V(O2), V(CO2), and respiratory exchange ratio (RER) similar to those seen in larger species. Response to beta-adrenergic stimulation showed a classic sigmoidal dose-response curve; however, there was very little tachycardiac response to vagal blockade, indicating low resting vagal tone. This study demonstrates the feasibility of performing chronic cardiovascular measurements in nonanesthetized mice and stresses the importance of allowing for anesthetic recovery and strain variability. Murine cardiovascular responses to exercise can be reliably measured and are qualitatively similar to those in humans.

Adrenergic Agents↗

PubMed1997

Alterations in ATP-sensitive potassium channel sensitivity to ATP in failing human hearts.

Little is known about the involvement of preexisting heart disease on characteristics of ATP-sensitive K+ channels [I[K(ATP)]] in human heart. We have characterized I[K(ATP)] in isolated cardiac myocytes from patients with congestive heart failure (HF) and compared these channel characteristics with those from donor hearts (healthy control) using the patch-clamp technique. During metabolic inhibition induced by treatment with cyanide (1 mM) and 2-deoxyglucose (10 mM), action potential shortening occurred in atrial myocytes isolated from both HF and donors, but this response was significantly greater and sooner in HF than in donors. The action potential duration at 90% repolarization was 24.7 +/- 4.1% (n = 15) of control in HF, whereas it was 58.7 +/- 5.9% (n = 10, P < 0.001) of control in donors measured at 30-min metabolic inhibition. The shortening of the action potential was partially reversed by glibenclamide (0.5 microM) in both groups. Consistent with the action potential measurements, the whole cell membrane current response to metabolic inhibition, evaluated by the differential current measurement, was sooner and greater in HF than in donors. Single-channel atrial I[K(ATP)] from both HF and donors, recorded in the excised inside-out patch configuration, exhibited bursting opening, conductance, and gating behaviors that did not differ between the two groups. However, the concentration of ATP at half-maximal inhibition of the channel in HF was greater (131.0 microM) than in donors (26.1 microM). We conclude that I[K(ATP)] in cardiac myocytes from patients with HF has channel characteristics substantially similar to those in donors, but that the channel is less sensitive to ATP inhibition in HF than in donors.

Action Potentials↗

PubMed1997

Association of granular exocytosis with Ca(2+)-activated K+ channels in human eosinophils.

We studied the mechanism of degranulation caused by Ca(2+)-activated K+ channels (KCa channels) in eosinophils isolated from mildly atopic donors using negative immunoselection. Stimulation of eosinophils with 0.1 microM platelet-activating factor (PAF) caused activation of single channels as recorded by the cell-attached patch-clamp technique. These channels were selectively permeable to K+ because the reversal potential was close to the equilibrium potential for K+. However, the channels were not permeable to Na+ or Cl- as demonstrated by ion substitution experiments. The calcium ionophore A-23187, at 1 microM, increased the K+ channel activity in the presence of Ca2+ in the external perfusate but did not induce channel activity in the absence of Ca2+. Similar results were obtained with another calcium ionophore, ionomycin (1 microM), and the Ca(2+)-releasing agent thapsigargin (10 microM). K+ channels activated by PAF and A-23187 had similar characteristics: two levels of single-channel conductances were observed, 10 +/- 1.5 and 22 +/- 1.7 pS as induced by PAF and 11 +/- 1.3 and 24 +/- 1.9 pS by A-23187; the mean open times of the large-conductance channels were 1.45 +/- 0.3 ms as induced by PAF and 1.26 +/- 0.5 ms by A-23187. These results indicate that PAF activates KCa channels. Both KCa currents and major basic protein release caused by A-23187 were blocked by quinidine. It is suggested that KCa channels are associated with granule secretion in human eosinophils.

Calcimycin↗

PubMed1997

Spasmolytic effect of efonidipine hydrochloride in isolated canine coronary artery: comparison with the effects of nifedipine and nisoldipine.

Spasmolytic effects of efonidipine hydrochloride (efonidipine) on high K(+)-, U46619- and 3,4-diaminopyridine (3,4-DAP)-induced contractions were evaluated in isolated canine coronary, artery, and were compared with the effects of nifedipine and nisoldipine. Efonidipine (0.3-30 nM), nifedipine (1-300 nM) and nisoldipine (0.1-100 nM) each relaxed the contractions induced by high K+ and U46619. However, relaxation produced by efonidipine was slower than that produced by nifedipine or nisoldipine. The rank order of potency of these drugs for U46619-induced contraction was efonidipine > or = nisoldipine > nifedipine, whereas in high K(+)-induced contraction, it was nisoldipine > efonidipine > nifedipine. Thus, the relaxing effect of efonidipine on U46619-induced contraction appeared to be more potent than its effect on high K(+)-induced contractions, when compared with the effects of nifedipine and nisoldipine. These three drugs also suppressed 3,4-DAP-induced rhythmic contractions. However, a marked time-dependent increase in potency was only observed for efonidipine, and was similar to its time-dependent effect on high K(+)- and U46619-induced contractions. Efonidipine did not change the contraction cycle length whilst suppressing the peak contractions. On the other hand, lower concentration of nifedipine at 3 nM and nisoldipine at 1 nM significantly shortened the cycle length. These results suggest that efonidipine may be an effective agent for the treatment of angina pectoris. The high potency of efonidipine for U46619-induced contractions will provide some advantages in the clinical use of this compound on thromboxane A2-mediated coronary vasoconstriction.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1997

Isolation and characterization of chitinase from a flake-chitin degrading marine bacterium, Aeromonas hydrophila H-2330.

A flake-chitin degrading marine bacterium was isolated and identified as Aeromonas hydrophila. This strain secreted five chitinases and an beta-N-acetylglucosaminidase. The main chitinase (Chi-A) was purified and characterized. The optimum pH of Chi-A was 5-8, and the activity was inhibited by Hg2+ and Fe3+. Chi-A was different from chitinases of other Aeromonas species with respect to molecular weight (62,000) and insensitivity to monoiodoacetate. The amino-terminal amino acid sequence showed extensive similarity with chitinases from Gram-negative bacteria.

Aeromonas hydrophila↗