Biomedical subjects

K Kiuchi

Publications and source records attributed to K Kiuchi.

At least 73 records · Page 4Linked to original sources

PubMed1994

Decrease in myocardial ryanodine receptors and altered excitation-contraction coupling early in the development of heart failure.

BACKGROUND: Rapid ventricular pacing for 1 day reduced myocardial contractile function without inducing heart failure in conscious, chronically instrumented dogs. After 4 to 7 weeks of pacing, myocardial contractility was depressed further and overt signs of congestive heart failure, eg, ascites, dyspnea, and edema, were evident. METHODS AND RESULTS: The mechanical restitution response, a physiological index of calcium release, was depressed at 1 day of rapid ventricular pacing. Postextrasystolic potentiation was also depressed by a similar amount, 14 +/- 3%, at 1 day after pacing. The response to isoproterenol 0.2 microgram/kg per minute was depressed by a significantly greater amount (P < .05), 52 +/- 7%, at 1 day after pacing. 3H-ryanodine receptor binding fell from 1013 +/- 25 to 808 +/- 42 fmol/mg after 1 day of pacing and remained depressed at similar levels (782 +/- 61 fmol/mg) at 4 to 7 weeks when heart failure was manifest. Ryanodine receptor affinity was unchanged from control values. Neither dihydropyridine binding nor affinity for 3H-PN200-110 was changed from control levels. Within 5 days after recovery from 1 day of pacing, physiological responses to isoproterenol, postextrasystolic potentiation, and mechanical restitution recovered, as did 3H-ryanodine binding density. CONCLUSIONS: These findings suggest that the changes in excitation-contraction coupling and potentially the sarcoplasmic reticulum calcium release channel occur early in the development of heart failure and therefore may be important in the pathogenesis of the contractile abnormalities in this disease state.

Animals↗

PubMed1994

Downregulation of adenylylcyclase types V and VI mRNA levels in pacing-induced heart failure in dogs.

We have shown that the heart expresses two distinct forms of adenylylcyclase mRNA, types V and VI. In this study we have characterized the expression of these two mRNA species in heart failure generated by overdrive pacing at a rate of 240 beats/min. After 4 wk, left ventricular end-diastolic pressure and heart rate increased significantly with the appearance of signs of heart failure, i.e., edema, ascites, and exercise intolerance. Basal as well as forskolin-stimulated adenylylcyclase activities decreased significantly, which was accompanied by a reduction in the steady state mRNA levels of adenylylcyclase types V and VI. These data suggest that in this model of cardiomyopathy, the downregulation of adenylylcyclase catalytic activity results, at least in part, from a reduction in the steady state levels of types V and VI adenylylcyclase mRNA levels.

Adenylyl Cyclases↗

PubMed1993

The phosphorylation and DNA binding of the DNA-binding domain of the orphan nuclear receptor NGFI-B.

NGFI-B is an orphan member of the nuclear receptor superfamily encoded by an immediate-early gene. It is rapidly synthesized and phosphorylated in PC12 cells in response to nerve growth factor (NGF) and other agents and is differentially phosphorylated dependent upon the inducing stimulus. The DNA-binding domain (DBD) of NGFI-B has been expressed in bacteria and purified. The purified protein is phosphorylated by protein kinase A or by extracts from NGF-treated PC12 cells. The phosphorylated residues within the DBD have been identified as Ser-340 and Ser-350. The use of mutants in which either or both of these residues were replaced with alanines revealed that phosphorylation of Ser-350, located within the "A box," a motif necessary for DNA binding by NGFI-B, resulted in a decrease in binding to the NGFI-B response element, while phosphorylation of Ser-340 had little or no effect. These findings demonstrate that phosphorylation of a nuclear receptor DBD results in a change in DNA binding and provides another potential mechanism for regulating NGFI-B activity.

Amino Acid Sequence↗

PubMed1993

Regulatory mechanism of dopamine biosynthesis in the striatum of transgenic mice carrying human tyrosine hydroxylase gene.

We investigated the regulatory mechanism of dopamine biosynthesis in the striatum of transgenic mice carrying multiple copies of human tyrosine hydroxylase (TH). The in vitro TH activity of transgenic striatum at pH 7.0 was approximately 2.8-fold higher than that of non-transgenic striatum. This augmentation is similar to that of the in vitro TH activity at pH 6.0, indicating that the expression of human TH in transgenic striatum induced little change in the phosphorylation level of TH. L-3,4-Dihydroxyphenylalanine (DOPA) formation in striatal slices of transgenic mice was approximately 2.7-fold higher than that of non-transgenic mice. The addition of 0.5 mM (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) to the incubation medium brought a negligible increase in DOPA formation in both cases. These results suggest that 6R-BH4 is not the limiting factor of TH in situ both in the transgenic and non-transgenic mice.

Animals↗

PubMed1993

Effects of a thromboxane synthetase inhibitor, Y-20811, on infarct size, neutrophil accumulation, and arrhythmias after coronary artery occlusion and reperfusion in dogs.

To examine effects of a new thromboxane synthetase inhibitor, Y-20811, on infarct size, neutrophil accumulation, and arrhythmias, coronary artery was occluded for 90 min and reperfused for 6 h in anesthetized dogs. Y-20811 administered intravenously (i.v.) 30 min before occlusion decreased serum thromboxane B2 (TBX2) formation by 98% 30 min later and by 79% at 6 h after reperfusion. Ventricular fibrillation (VF) developed in 1 of 15 control and 3 of 10 treated dogs during occlusion (p = NS), whereas after reperfusion it occurred in 7 of 14 control and none of seven treated dogs (p < 0.05). The number of arrhythmias during the first hour of reperfusion was significantly reduced in treated dogs (134 +/- 74 beats/min in control vs. 14 +/- 4 beats/min in treated dogs, p < 0.05). Hemodynamics, area at risk, and collateral flow to the ischemic region were similar for the two groups. The extent of myocardial necrosis was 28.0 +/- 10.0% (n = 7) of the area at risk in control dogs and 27.6 +/- 6.2% (n = 7) in treated dogs (p = NS). The relation between the ratio of myocardial necrosis to area at risk and collateral flow was similar. The degree of neutrophil accumulation did not differ but correlated with infarct size (r = 0.85). Thus, Y-20811 reduced reperfusion arrhythmias but failed to limit infarct size and neutrophil accumulation after coronary artery occlusion/reperfusion in dogs.

Animals↗

PubMed1993

Effects of coronary arterial reperfusion on beta-adrenergic receptor-adenylyl cyclase coupling.

The effects of 1 h of coronary arterial occlusion (CAO) followed by 15 min reperfusion (CAR) were examined in nine conscious dogs. Ischemia was verified by decreased regional blood flow (radioactive microspheres) and loss of systolic regional wall motion in the ischemic zone. beta-Adrenergic receptor density assessed by 125I-labeled cyanopindolol binding in a crude membrane fraction tended to decrease but was not significantly different. However, adenylyl cyclase activity and the guanine nucleotide stimulatory protein (Gs) were reduced in ischemic subendocardium compared with nonischemic subendocardium. The fraction of beta-adrenergic receptors binding agonist with high affinity increased in ischemic subendocardial and subepicardial layers. Compared with prior data in experiments with 1 h CAO without CAR, the increase in beta-adrenergic receptor density that occurs with myocardial ischemia is rapidly reversed with CAR of 15 min duration, while the decreased fraction of receptors binding agonist with high affinity was reversed to an increase in high-affinity receptors. The global decreases in adenylyl cyclase and Gs, which have been observed with simple CAO, persist but are observed selectively in the previously ischemic subendocardium after CAR. Thus both CAO and CAR affect beta-adrenergic receptors and adenylyl cyclase differently. During CAR, increased numbers of beta-adrenergic receptors binding agonist with high affinity occur potentially as a compensatory mechanism in the face of persistent reductions in adenylyl cyclase activity and Gs.

Adenylyl Cyclases↗

PubMed1993

Depressed beta-adrenergic receptor- and endothelium-mediated vasodilation in conscious dogs with heart failure.

Peripheral vasodilator responsiveness was examined in pacing-induced heart failure (HF) in 11 conscious dogs chronically instrumented for measurement of systemic (total peripheral resistance [TPR]) and local (iliac blood flow) vascular resistance. Dose responses to isoproterenol (ISO), acetylcholine (ACh), and nitroglycerin (NTG) were examined in the same dogs before pacing (control) and after 4 to 7 weeks of rapid ventricular pacing, which induced congestive HF, characterized by increased left ventricular end-diastolic pressure (6.7 +/- 0.4 [control] versus 28 +/- 1.5 [HF] mm Hg) and decreased cardiac output (-30 +/- 5%) and left ventricular dP/dt (-53 +/- 3%), as well as ascites and peripheral edema. In the control state, TPR fell by 57 +/- 2% in response to ISO (100 ng/kg), by 61 +/- 3% in response to ACh (3 micrograms/kg), and by 55 +/- 2% in response to NTG (10 micrograms/kg). In HF, smaller decreases (P < .05) in TPR were observed with the same doses of ISO (-50 +/- 2%) and ACh (-49 +/- 2%) but not with NTG (-58 +/- 3%). Depressed responses to systemic ISO and ACh, but not NTG, were observed in HF in the presence of ganglionic blockade and also after local administration of smaller doses of the drugs in the absence of ganglionic blockade, but where systemic effects were not elicited. Inhibition of nitric oxide synthase increased TPR to a greater degree before HF (+154 +/- 28% [control]) than after (+80 +/- 22% [HF]) and eliminated the depressed responses to ACh but not to ISO. beta-Adrenergic receptor density, as determined by 125I-cyanopindolol binding in membrane preparations from mesenteric vessels was significantly decreased after HF (130 +/- 3 [control] versus 100 +/- 8 [HF] fmol/mg, P < .05) without any change in affinity. Thus, peripheral vascular beta-adrenergic receptor downregulation occurs in HF, independent of altered endothelium-mediated peripheral vasodilation.

Acetylcholine↗

PubMed1993

Myocardial beta-adrenergic receptor function during the development of pacing-induced heart failure.

The development of pacing-induced heart failure was studied in chronically instrumented, conscious dogs paced at a rate of 240 beats/min for 1 d (n = 6), 1 wk (n = 6), and 3-4 wk (n = 7). Left ventricular (LV) dP/dt was decreased (P < 0.0125) at 1 d, LV end-diastolic pressure and heart rate were increased (P < 0.0125) at 1 wk, but clinical signs of heart failure were only observed after 3-4 wk of pacing. Plasma norepinephrine rose (P < 0.0125) after 1 d of pacing, whereas LV norepinephrine was reduced (P < 0.0125) only after 3-4 wk of pacing. Both the fraction of beta-adrenergic receptors binding agonist with high affinity and adenylyl cyclase activity decreased (P < 0.0125) after 1 d of pacing. Total beta-adrenergic receptor density was not changed at any time point, but beta 1-adrenergic receptor density was decreased (P < 0.0125) after 1 wk. The functional activity of the guanine nucleotide binding protein, Gs, was not reduced, but the Gi alpha 2 isoform of the alpha subunit of the GTP-inhibitory protein rose after 3-4 wk of pacing. Thus, myocardial beta-adrenergic signal transduction undergoes change shortly (1d) after the initiation of pacing, before heart failure develops. The mechanism of beta-adrenergic receptor dysfunction in pacing-induced heart failure is characterized initially by elevated plasma levels of catecholamines, uncoupling of beta-adrenergic receptors, and a defect in the adenylyl cyclase catalytic unit. Selective down-regulation of beta 1-adrenergic receptors, increases in Gi alpha 2, and decreases in myocardial catecholamine levels occur as later events.

Adenylyl Cyclases↗

PubMed1993

Relationships of condylar path angle with malocclusion and temporomandibular joint disturbances.

A study was conducted to investigate the condylar path angle in relation to Angle's classification, amount of overbite and subjective symptoms. The condylar path angle in Angle's Class I group was similar to that in the Class II group. The angle in the Class III group was smaller than in the other two classes. A tendency for the angle to decrease as the amount of overbite became smaller was observed. The angle in the symptom group was slightly smaller than in the no-symptom group.

Adult↗

PubMed1992

Mechanisms of alpha 1-adrenergic vascular desensitization in conscious dogs.

To investigate the mechanisms of alpha 1-adrenergic vascular desensitization, osmotic minipumps containing either saline (n = 9) or amidephrine mesylate (AMD) (n = 9), a selective alpha 1-adrenergic receptor agonist, were implanted subcutaneously in dogs with chronically implanted arterial and right atrial pressure catheters and aortic flow probes. After chronic alpha 1-adrenergic receptor stimulation, significant physiological desensitization to acute AMD challenges was observed, i.e., pressor and vasoconstrictor responses to the alpha 1-adrenergic agonist were significantly depressed (p < 0.01) compared with responses in the same dogs studied in the conscious state before pump implantation. However, physiological desensitization to acute challenges of the neurotransmitter norepinephrine (NE) (0.1 micrograms/kg per minute) in the presence of beta-adrenergic receptor blockade was not observed for either mean arterial pressure (MAP) (30 +/- 7 versus 28 +/- 5 mm Hg) or total peripheral resistance (TPR) (29.8 +/- 4.9 versus 28.9 +/- 7.3 mm Hg/l per minute). In the presence of beta-adrenergic receptor plus ganglionic blockade after AMD pump implantation, physiological desensitization to NE was unmasked since the control responses to NE (0.1 micrograms/kg per minute) before the AMD pumps were now greater (p < 0.01) than after chronic AMD administration for both MAP (66 +/- 5 versus 32 +/- 2 mm Hg) and TPR (42.6 +/- 10.3 versus 23.9 +/- 4.4 mm Hg/l per minute). In the presence of beta-adrenergic receptor, ganglionic, plus NE-uptake blockade after AMD pump implantation, desensitization was even more apparent, since NE (0.1 micrograms/kg per minute) induced even greater differences in MAP (33 +/- 5 versus 109 +/- 6 mm Hg) and TPR (28.1 +/- 1.8 versus 111.8 +/- 14.7 mm Hg/l per minute). The maximal force of contraction induced by NE in the presence or absence of endothelium was significantly decreased (p < 0.05) in vitro in mesenteric artery rings from AMD pump dogs compared with saline control dogs. Furthermore, alpha 1-adrenergic receptor density, as determined by [3H]prazosin binding in membrane preparations from vessels in the mesentery, was decreased (8.2 +/- 1.0 versus 18.4 +/- 1.4 fmol/mg protein, p < 0.001) without any change in Kd in the AMD pump dogs compared with the saline pump dogs.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic alpha-Agonists↗

PubMed1992

Highly sensitive, specific enzyme-linked immunosorbent assay of neopterin and biopterin in biological samples.

An enzyme immunosorbent assay of neopterin and biopterin on a polystyrene microtiter plate has been developed. A conjugate of neopterin or biopterin to bovine serum albumin was used to raise a specific antiserum against neopterin or biopterin in rabbits. An incubation mixture of the antiserum and samples prepared from human serum underwent another antigen-antibody reaction with the hapten fixed on the microtiter plate. The amount of antibody bound to the fixed hapten, which is inverse to the amount of hapten in the sample, was determined by using anti-rabbit IgG-horseradish peroxidase conjugate in a usual manner by measuring absorbance at 490 nm after reaction with o-phenylenediamine and hydrogen peroxide. The minimal detectable amounts of neopterin and biopterin were approximately 0.1 pmol. The specificity of the assay was so high that the assay system for neopterin completely distinguished it from biopterin, as judged from the cross-reaction of 0.002%, and vice versa. The amounts of neopterin and biopterin in human serum determined by the present method agreed well with those determined by high-performance liquid chromatography. We used the present method to determine the concentrations of neopterin in serum from healthy control subjects and patients with cancers and systemic lupus erythematosus; the results were consistent with literature data.

Biopterins↗

PubMed1991

The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells.

We reported that one of the isoquinolinesulfonamide derivatives, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M. and Hidaka, H. (1990) J. Biol. Chem. 265, 4315-4320). We have now investigated the inhibitory property of a newly synthesized methoxybenzenesulfonamide, KN-93, on CaMKII activity in situ and in vitro. KN-93 elicited potent inhibitory effects on CaMKII phosphorylating activity with an inhibition constant of 0.37 microM but this compound had no significant effects on the catalytic activity of cAMP-dependent protein kinase, Ca2+/phospholipid dependent protein kinase, myosin light chain kinase and Ca(2+)-phosphodiesterase. KN-93 also inhibited the autophosphorylation of both the alpha- and beta-subunits of CaMKII. Kinetic analysis indicated that KN-93 inhibits CaMKII, in a competitive fashion against calmodulin. To evaluate the regulatory role of CaMKII on catecholamine metabolism, we examined the effect of KN-93 on dopamine (DA) levels in PC12h cells. The DA levels decreased in the presence of KN-93. Further, the tyrosine hydroxylase (TH) phosphorylation induced by KCl or acetylcholine was significantly suppressed by KN-93 in PC12h cells while events induced by forskolin or 8-Br-cAMP were not affected. These results suggest that KN-93 inhibits DA formation by modulating the reaction rate of TH to reduce the Ca(2+)-mediated phosphorylation levels of the TH molecule.

Animals↗

PubMed1991

Limited proteolysis of tyrosine hydroxylase by Ca(2+)-activated neutral protease (calpain).

Limited proteolysis of tyrosine hydroxylase (TH) by calpain, Ca(2+)-activated neutral protease, was studied. Cleavage of TH with calpain in vitro produced molecules having Mrs of approximately 57,000 and 56,000 in SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence, Ser-Pro-Arg-Phe-Val, of the 56-kDa species indicated that calpain cleaved off the N-terminal region (residues 1-30) encoded by the first exon including Ser-8 and Ser-19, the phosphorylation sites by proline-directed protein kinase (PDPK) and by Ca2+/calmodulin-dependent protein kinase II (kinase II), respectively, from the native TH. The removal of the N-terminal region from the native molecule induced a slight but significant activation of TH at pH 7.0. The native TH behaved as the tetramer with an Mr of 240,000. In contrast, calpain-cleaved TH showed the monomeric Mr by gel permeation chromatography and increased Ki for catecholamine which inhibits the native TH in competition to the coenzyme, DL-6-methyl-5,6,7,8-tetrahydropterin (6MPH4). These results imply that calpain cleavage would effectively release TH from the feedback inhibition by removal of the N-terminal region resulting in disruption of the quaternary structure.

Adrenal Medulla↗

PubMed1991

A selective Ca2+/calmodulin-dependent protein kinase II inhibitor, KN-62, inhibits the enhanced phosphorylation and the activation of tyrosine hydroxylase by 56 mM K+ in rat pheochromocytoma PC12h cells.

Involvement of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-kinase II) on the phosphorylation of tyrosine hydroxylase (TH, EC.1.14.16.2) in rat pheochromocytoma, PC12h cells was examined using KN-62, 1-[N,O-Bis(5-isoquinolinsulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipe razine, a selective inhibitor of Ca2+/CaM-kinase II. Both the enhanced phosphorylation of TH and the activated L-3,4-dihydroxyphenylalanine (DOPA) formation in the high K+ depolarization were inhibited by 10 microM KN-62. After incubation of PC12h cells with 10 microM KN-62 for 1 hr, the activation of TH with 3 min incubation of 56 mM K+ was reduced to the basal activity. However, KN-62 did not directly affect the activity of purified rat TH at pH 6.0 or 7.0. These results indicate that Ca2+/CaM-kinase II phosphorylates and activates TH of PC12h cells in the high K+ depolarization.

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

PubMed1991

Expression of two forms of human dopamine-beta-hydroxylase in COS cells.

We previously reported four different cDNA clones encoding human dopamine-beta-hydroxylase (Kobayashi et al., Nucl. Acids Res., 17 (1989) 1089-1102). These clones were different in a 3' untranslated region (types A and B) and/or in 6 nucleotides in mRNAs. The difference at nucleotide 910 caused an amino acid change between Ala (A) and Ser (S) at amino acid residue 304 (DBH/A and DBH/S). We succeeded in expressing both of DBH/A and DBH/S of type A cDNAs in COS cell. Both of the expressed proteins showed enzyme activities and immunoreactivities. The two proteins had similar kinetic constants, but had different homospecific activities (activities per enzyme protein); the homospecific activity of human DBH/S was low, approximately one thirteenth that of human DBH/A.

Animals↗

PubMed1991

Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice.

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.

Adrenal Glands↗

PubMed1991

[Regional myocardial blood flow measurement with radioactive microspheres in dogs with myocardial ischemia].

In order to evaluate the radioactive microsphere technique used in our laboratory for measuring regional myocardial blood flow (RMBF) in dogs with myocardial ischemia, simultaneous blood flow measurements with three different nuclide (141Ce, 51Cr or 113Sn)-labeled microspheres were performed after coronary artery occlusion. There were excellent correlations between all paired RMBF values in 80 samples. The linear regression lines were expressed as: 51Cr = 1.09x 141Ce-0.02 (r = 0.996, SEE = 0.06); 113Sn = 0.99x 141Ce + 0.01 (r = 0.997, SEE = 0.05); 113Sn = 0.90x 51Cr + 0.03 (r = 0.991, SEE = 0.08). The duplicate variability ranged from 5.5 +/- 0.7 to 9.9 +/- 1.1%. When RMBF was measured before and after intravenous administration of nipradilol (0.2 mg/kg), a new beta-blocker, in a dog with myocardial ischemia, RMBF fell in the normal zone (23.6 +/- 1.6%), and did not change in the severely ischemic zone. Thus, this study demonstrated that RMBF can be measured accurately and repeatedly with radioactive microspheres in dogs with regional myocardial ischemia.

Adrenergic beta-Antagonists↗

PubMed1990

Sustained beneficial effects of gallopamil (D600) on size of myocardial infarction 24 hours after coronary artery occlusion in dogs.

To examine whether gallopamil (D600), a methoxy derivative of verapamil, has sustained beneficial effects on the ischemic myocardium, its effects on the size of myocardial infarction determined 6 hours (protocol 1) and 24 hours (protocol 2) after left anterior descending coronary artery occlusion were compared in anesthetized, open-chest dogs. To quantify the extent of the hypoperfused zone, Tc-99m- or In-111-albumin microspheres were injected into the left atrium 1 minute after occlusion. Fifteen minutes after occlusion, dogs were randomly assigned to a control group or a gallopamil-treated group that received immediately after assignment 0.08 mg/kg of gallopamil followed by a continuous infusion of 0.2 mg/kg/hr for 6 hours. Six or 24 hours after occlusion, the left ventricle was cut into 3 mm thick slices for triphenyltetrazolium chloride staining and autoradiography. There were no differences in the extent of the hypoperfused zone among the four groups. In both protocols 1 and 2 the ratio of the extent of myocardial necrosis to the extent of the hypoperfused zone was significantly smaller in the treated groups (56.7 +/- 6.7% [n = 8], p less than 0.01 and 72.3 +/- 5.3% [n = 6], p less than 0.05 for protocols 1 and 2, respectively) than in the control groups (100.7 +/- 6.0% [n = 7] and 95.2 +/- 4.3% [n = 5] for protocols I and II, respectively). Thus gallopamil administered early after coronary artery occlusion had beneficial effects on the ischemic myocardium, which were sustained for at least 24 hours after the onset of infarction.

Animals↗