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Biomedical subjects

Y Fukuhara

Publications and source records attributed to Y Fukuhara.

At least 55 records · Page 3Linked to original sources
PubMed1989

Decrease in the fluidity of brush-border membrane vesicles induced by gentamicin. A spin-labeling study.

In our previous paper (Horio et al., Biochim Biophys Acta 858: 153-160, 1986), we reported that the addition of gentamicin in vitro to rabbit renal brush-border membrane vesicles decreases the apparent Vmax of Na+-dependent D-glucose transport without affecting the apparent Km. In the present study, we investigated the effects of gentamicin on the physical state of spin-labeled rabbit renal brush-border membranes, using electron spin resonance spectrometry. Brush-border membrane vesicles were prepared from outer cortex (mainly contains early proximal tubule) and outer medulla (containing primarily late proximal tubule), and the gentamicin toxicities in both preparations were compared. Significant decreases were observed in the membrane fluidity of 5 mM gentamicin-treated brush-border membranes. The fluidity of outer cortical brush-border membranes was affected at both 25 degrees and 35 degrees, whereas that of outer medullary membranes was affected only at 35 degrees. Two different stearic acid spin labels revealed that gentamicin affected the fluidity only in the superficial region of the membranes. We also demonstrated that the gentamicin-induced decreases in Na+-dependent D-glucose transport and in the membrane fluidity were recovered by washing gentamicin-treated brush-border membranes. We suggest that gentamicin binds to the superficial region of brush-border membranes and inhibits Na+-dependent D-glucose transport across brush-border membranes through the decrease in the membrane fluidity.

Animals↗
PubMed1989

Effect of chronic renal failure on the level of albumin messenger RNA.

The effects of chronic renal failure on the level of albumin mRNA and the transcription rate of the albumin gene were studied in seven of eight nephrectomized rats. A paired feeding procedure was employed to eliminate a nutritional difference between sham-operated control and uremic rats. Total RNA was isolated from the livers of control and uremic rats fasted for 24 or 48 hours. The mRNA level was measured by RNA-cDNA dot blot hybridization, and the transcription rate was measured by the "run-on" transcription assay in isolated nuclei. The albumin mRNA levels in uremic rat livers were reduced to 75% at the 24-hour fasted state, and to 45% at 48-hour fasted state compared with those in the respective control groups. There was no difference in the level of beta-actin mRNA between these groups. However, there was no difference in the transcription rate of the albumin gene between control and uremic rats. Northern analysis showed that albumin mRNA isolated from uremic rat liver was identical in size with that from the control. These results suggested that a posttranscriptional process, involving the destabilization of cytoplasmic mRNA, is responsible for the uremia-induced repression of albumin synthesis.

Animals↗
PubMed1989

Furosemide accelerates gentamicin accumulation in cultured renal cells (LLC-PK1 cells).

Furosemide is known to potentiate gentamicin nephrotoxicity. The mechanism of potentiation is unclear. In our previous studies, we demonstrated that furosemide enhanced gentamicin accumulation in rabbit renal tissues when injected as a bolus [Kidney int. 33:363, 1988] or repeatedly subcutaneously [Diuretics II, Elsevier, New York 1987, pp.693]. In this study, we evaluated the effects of furosemide on gentamicin accumulation in cultured renal cells (LLC-PK1) and hepatoma cells (H4IIE). Thus, we excluded effects secondary to furosemide-elicited hemodynamic changes. Seven days after seeding, the culture medium was exchanged for medium containing 1 mM gentamicin alone [G] or 1 mM gentamicin +1 mM furosemide [GF]. In LLC-PK1 cells, gentamicin concentration in [GF] was significantly higher than that in [G], while gentamicin was not detected in H4IIE cells with or without furosemide. We conclude that furosemide can accelerate gentamicin accumulation in renal tissues and potentiate gentamicin nephrotoxicity.

Animals↗
PubMed1989

Binding of hydrochlorothiazide to erythrocytes.

Hydrochlorothiazide (HCT) was administered orally to healthy volunteers and intravenously to rabbits. HCT concentrations in plasma (Cp) and erythrocytes (Ce) were determined by a high-performance liquid chromatographic method. Ce was about 9-fold that of Cp 24 h after the administration to volunteers, and 8-fold 6 h after the administration to rabbits. From the results of the in vitro binding study which was done with rabbit erythrocytes, at least the presence of three kinds of binding site for HCT was expected. The first binding site was characterized by extremely high affinity and very low capacity, and was unaffected by acetazolamide, known as a carbonic anhydrase inhibitor. The second one was characterized by medium affinity and medium capacity, and disappeared under the presence of acetazolamide and may be due to the carbonic anhydrase of erythrocytes. The third one was characterized by low affinity, but its binding capacity was extremely high and apparently unsaturable in the HCT concentration range studied (0.5-100 micrograms/ml = 1.68-336 microM). The binding of HCT to erythrocytes seems to be dominated by the second binding site in the therapeutic range (under 1 microgram/ml of plasma).

Adult↗
PubMed1989

Enhancement of gentamicin-induced inhibition of phosphatidylinositol hydrolysis in rabbit renal proximal tubular brush-border membrane by furosemide.

Gentamicin inhibited phosphatidylinositol (PI) hydrolysis catalyzed by rabbit renal phospholipase C in a dose-dependent manner. The inhibition was potentiated by furosemide in a dose-dependent manner. Furosemide may enhance gentamicin nephrotoxicity by potentiating the inhibitory effect of gentamicin on brush-border membrane PI degradation.

Animals↗
PubMed1988

Immunological studies on the respiratory burst oxidase of pig blood neutrophils.

Recently, a flavin enzyme (pI 5.0), that is probably responsible for superoxide (O2-)-generated oxidase activity, was separated by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) from neutrophil membranes in our laboratory [(1987) J. Biol. Chem. 262, 12316-12322]. In the present work, we performed immunological studies on this enzyme derived from pig blood neutrophils. The enzyme extract obtained on IEF-PAGE was injected into guinea pigs to raise antibodies. IgG antibody against the pI 5.0 protein inhibited maximally 54% of the O2- -generating activity of the membrane-solubilized oxidase, whereas the normal serum IgG was not inhibitory at all. Our results further confirmed that the enzyme (PI 5.0) is one of the component(s) of the O2- -generating system. The enzyme gave rise to a band corresponding to a major protein of 72 +/- 4 kDa on both non-denaturing and SDS-PAGE. Immunoblotting after SDS-PAGE demonstrated labelling of peptides of 70-72, 28-32 and 16-18 kDa.

Animals↗
PubMed1988

Albumin gene transcription is enhanced in liver of nephrotic rats.

The level of albumin mRNA and the transcription rate of the albumin gene were studied in the liver of control rats and rats with nephrosis induced by injection of the aminonucleoside of puromycin. Total RNA was extracted from liver by the guanidium thiocyanate method. The albumin mRNA level was measured by cDNA-RNA dot-blot hybridization, and the transcription rate of the albumin gene was measured by the "run-on" transcription assay using isolated nuclei. Urinary protein excretion in nephrotic rats was significantly higher than in control rats (258 +/- 132 vs. 12 +/- 2 mg/day), and the serum albumin concentration in nephrotic rats was significantly lower. There was no difference in body weight, liver weight, serum creatinine, or urea nitrogen between the two groups. Both the level of albumin mRNA and the transcription rate of the albumin gene in the nephrotic liver were about twice as high as those in the control liver. There was no difference in the level of beta-actin mRNA between the two groups. Northern blot analysis showed that both putative precursor RNA and the mature form of albumin mRNA were increased in nephrotic rats. We conclude that albumin synthesis is increased in the liver of nephrotic rats and a transcriptional process is responsible for this increase.

Actins↗
PubMed1988

Pharmacokinetic and pharmacodynamic interactions between furosemide and hydrochlorothiazide in nephrotic patients.

We examined the response of 8 patients with nephrotic syndrome (creatinine clearance 70.4 +/- 16.0 ml/min) to oral furosemide (F; 40 mg) in the absence (control) and in the presence of oral hydrochlorothiazide (HCT; 100 mg). In the 24-hour period after oral F, HCT was shown to increase urine volume and urinary sodium and chloride excretion. Increment was most significant during the 12- to 24-hour period. Enhancement of the diuresis with HCT was associated neither with a significant increase in the area under the curve of plasma F concentration nor an increase in urinary F excretion. Urinary excretion of glucuronidated F, one of the main metabolites of F, however, was decreased with HCT. In summary, HCT significantly enhanced the response to F in nephrotic patients.

Drug Interactions↗
PubMed1987

The respiratory burst oxidase of neutrophils. Separation of an FAD enzyme and its characterization.

Pig blood neutrophils were briefly activated by various fatty acids and then fractionated into membrane vesicles with different NADPH oxidase activities. Treatment of these membranes with a detergent, octyl glucoside, resulted in a high yield of solubilized oxidase, which was subjected to isoelectric focusing on gels (pI 4.0-8.0). 1) A distinct band staining with NADPH-nitroblue tetrazolium focused at pI 5.0. The enzyme (pI 5.0) showed high specificity for NADPH and similar characteristics to the oxidase involved in the respiratory burst. 2) The enzyme was extracted from gel slices and analyzed. When measured promptly after its extraction, its NADPH oxidase activity was high, but there was apparent superoxide dismutase-insensitive cytochrome c reduction, probably due to direct electron transfer to the heme protein. However, it could produce superoxide anion (O2-) under some micelle conditions. 3) Therefore, the formation of the enzyme-substrate complex of yeast cytochrome c peroxidase was employed for the detection of H2O2. A fresh extract of stimulated cells catalyzed equimolar NADPH oxidation and H2O2 production of 306 and 300 nmol min-1 (mg protein)-1, respectively. The Km value of the enzyme for NADPH was 30 +/- 13 (S.D.) microM. The recovery of the extract (pI 5.0) was 19% of the total activity. 4) The enzyme extract contained 1.1-1.9 nmol of FAD/mg of protein, giving a turnover number of 300-600 min-1 in terms of O2- generation/FAD. No heme protein was found in the enzyme. The enzyme was mainly of 67-kDa molecular mass.

Animals↗
PubMed1987

The urinary excretion of frusemide and its metabolites by kidney transplant patients.

Urine from 5 renal transplant recipients treated with frusemide was analyzed for unchanged frusemide (F), glucuronidated frusemide (G) and 4-chloro-5-sulfamoylanthranilic acid (CSA) by HPLC. In 3 recipients, whose renal function recovered steadily and whose hepatic function was normal throughout, the ratio of frusemide to its metabolites, F/(F + G + CSA), increased steadily in conjunction with the recovery of renal function. In one patient, who received frusemide 200-400 mg/day i.v., the urinary CSA concentration was 64-102 micrograms X ml-1. In 2 patients who experienced shock and/or hepatic dysfunction after transplantation, the F/(F + G + CSA) ratio fluctuated.

Adult↗
PubMed1987

Effects of chronic renal failure on the regulation of pyruvate kinase.

The effects of chronic renal failure on the enzyme activity of pyruvate kinase and the mRNA level of this enzyme were studied in 7 out of 8 nephrectomized rats. The mRNA level was measured by RNA-DNA dot blot hybridization, using cloned pyruvate kinase cDNA as hybridized probe. Neither the activity of M1-type pyruvate kinase nor the level of this enzyme in rat gastrocnemius muscle was affected by chronic renal failure, whereas L-type pyruvate kinase enzyme activity in uremic rat liver was lower than that in control at both fasted and refed states. The levels of L-type pyruvate kinase mRNA were not different between two groups at the fasted state. Induction of L-type pyruvate kinase mRNA after high carbohydrate diet refeeding was suppressed proportionally to the severity of chronic renal failure, which was expressed by the serum creatinine concentrations (r = -.876, P less than .005). These results indicate that the suppression of L-type pyruvate kinase activity in uremia was partly reflected by the decreased accumulation of this enzyme mRNA. There was a significantly negative correlation between L-type pyruvate kinase mRNA levels and plasma glucagon/insulin ratios (r = -.719, P less than .05). Hyperglucagonemia in uremia might play a major role in this suppression.

Animals↗