Biomedical subjects

C Uneyama

Publications and source records attributed to C Uneyama.

At least 55 records · Page 3Linked to original sources

PubMed1993

Cytoplasmic pH regulates ATP-induced Ca(2+)-dependent K(+)-current oscillation in rat megakaryocytes.

The effect of cytoplasmic pH (pH(i)) on ATP-induced Ca2+ oscillation of rat megakaryocytes was investigated by the whole-cell patch-clamp technique. Megakaryocytes responded to extracellular ATP and showed the periodic activation of K+ channels, which reflects the oscillation in intracellular free Ca2+ concentration. Intracellular alkalinization by 20 mM NH4Cl resulted in inhibition of the oscillatory response, and intracellular acidification by 20 mM sodium acetate enhanced the response. NH4Cl also inhibited the Ins(1,4,5)P3-induced oscillation. Sodium acetate had no effect on the InsP3-induced oscillation, but enhanced the guanosine 5'-[gamma-thio]triphosphate-induced response. These results indicate that pH(i) modulates the ATP-induced cellular response at least at two points.

Acetates↗

PubMed1993

Intracellular mechanisms of cytoplasmic Ca2+ oscillation in rat megakaryocyte.

Extracellular application of ATP and ADP evoked the oscillatory K+ currents (IKCa) resulting from the periodic rise in cytoplasmic Ca2+ concentration ([Ca2+]i) of megakaryocyte isolated from rat bone marrow (Uneyama, H., Uneyama, C., and Akaike, N. (1992) Jpn. J. Pharmacol. 58, 231). The intracellular mechanism of ATP-induced cytoplasmic Ca2+ oscillation was investigated by the use of nystatin-perforated patch-clamp technique. Caffeine and ryanodine, which release Ca2+ from the Ca(2+)-induced Ca2+ release pool (CICR), and procaine, a blocker of Ca2+ release from CICR, had no effect on the IKCa oscillation in megakaryocyte. Thapsigargin and A23187 activated IKCa irreversibly by mobilizing Ca2+, but under Ca(2+)-free conditions they activated IKCa transiently. Intracellular application of inositol 1,4,5-triphosphate (IP3) also induced IKCa oscillation. Phorbol myristate acetate (PMA), a strong activator of protein kinase C (PKC), inhibited the oscillation completely. The inhibitory action of PMA was reversed by an inhibitor of PKC, staurosporin. The oscillation of ATP-induced IKCa was disrupted by staurosporin or calmodulin (CaM) antagonists such as W-7 and trifluoperazine, resulting in a transient and successive plateau-like IKCa. These results suggest that Ca2+ oscillation in megakaryocyte is caused by the interaction of both the Ca2+ release from IP3-sensitive Ca2+ pool and the Ca2+ uptake stimulated by PKC and Ca2+/CaM complex. Furthermore, forskolin, an activator of adenylate cyclase, and isobutylmethylxanthin, an inhibitor of phosphodiesterase, inhibited the frequency, latency, and current amplitude of the oscillation in a concentration-dependent manner. These reagents inhibited IP3-induced oscillation as well as an ATP-induced oscillation. Thus, the ATP-induced [Ca2+]i oscillation of rat megakaryocyte is also modulated by cAMP.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Cytoplasmic Ca2+ oscillation in rat megakaryocytes evoked by a novel type of purinoceptor.

1. The responses of megakaryocytes isolated from rat bone marrow to externally applied adenosine triphosphate (ATP) were investigated in the whole-cell mode by the use of nystatin perforated patch-clamp technique. 2. ATP at 1-100 microM evoked periodic outward currents at a holding potential of -40 mV. The reversal potential of the currents was close to K+ equilibrium potential (EK) and the K+ channel blockers such as quinine and quinidine suppressed the currents, indicating that the outward currents are predominantly carried by K+. 3. Since it has been reported that adenosine diphosphate (ADP) evoked monophasic K+ current using a conventional whole-cell recording, we compared the results obtained by perforated and conventional patch-clamp techniques. The crucial difference between our results and previous results was due to the intracellular perfusion with internal solution containing a high concentration of EGTA by which both current shape and concentration response were modified. 4. The membrane permeable Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxy methyl ester; BAPTA AM), inhibited the K+ current concentration dependently, suggesting that ATP-induced oscillatory K+ currents are caused by changes in cytoplasmic free Ca2+ concentration ([Ca2+]i). 5. With increasing ATP concentration, the frequency and the maximum amplitude of K+ current oscillation increased and the latency of current, which is the period required to activate the first K+ current after ATP application, decreased. 6. ADP, 2-methylthio-ATP and ATP-gamma-S could also evoke the periodic K+ currents, but adenosine, uridine triphosphate (UTP) and alpha-beta-methylene adenosine 5'-triphosphate (AMP-CPP) failed. 2-Methylthio-ATP was the most potent agonist; next was ADP which showed a 10-30 times stronger effect than ATP. Cross-desensitization was observed between ATP and ADP, but not between ATP or ADP and thrombin. 7. Extracellular Ca2+ was not required for the ATP-induced K+ current activation, indicating that Ca2+ released from intracellular pools induced the oscillatory response. In addition, the agonist potency increased when extracellular Ca2+ concentration ([Ca2+]o) decreased, suggesting that the principal agonists might be ATP4- and ADP3-. 8. The results suggest the presence of a novel subtype of purinoceptor in the megakaryocyte plasma membrane which induces cytoplasmic Ca2+ oscillation and evokes periodic K+ current flux.

Adenosine Diphosphate↗

PubMed1993

Spontaneous oscillations of cytoplasmic free calcium ion concentration in cultured smooth muscle cells from guinea pig ileum.

The cytoplasmic free calcium ion concentration ([Ca2+]i) of cultured guinea pig ileum longitudinal muscle cells loaded with a fluorescent [Ca2+]i indicator, fura-2, was measured by digital ratio imaging microscopy. Spontaneous [Ca2+]i oscillations were observed in 25% to 80% of the cells, which differed with the batches of the cultured cells after 5 to 8 days in culture. The frequency and amplitude of the [Ca2+]i oscillations in each individual cell were usually regular, but heterogeneity between neighboring cells was observed. The spontaneous [Ca2+]i oscillations were also observed even after incubation of the cells under a serum-free condition for 72 hr. Exchange of extracellular solution to Ca(2+)-free solution containing EGTA or BAPTA immediately stopped the [Ca2+]i oscillations. The ratio of the oscillating cells was dependent on the extracellular calcium ion concentration ([Ca2+]o); and heterogeneity in the range of the [Ca2+]o to generate the [Ca2+]i oscillations was observed. An inorganic Ca(2+)-antagonist, LaCl3, immediately suppressed the [Ca2+]i oscillations, but the treatment with verapamil or nicardipine, Ca(2+)-channel blockers, did not have any effect on the [Ca2+]i oscillations. An inhibitor of the intracellular Ca2+ pump, thapsigargin, induced a transient increase in [Ca2+]i and then inhibited the spontaneous [Ca2+]i oscillations. Neomycin, a compound known to inhibit phosphoinositide turnover, inhibited the [Ca2+]i oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Induction of colon adenocarcinomas in CD rats and lung adenomas in ICR mice by 6-nitrochrysene: comparison of carcinogenicity and aryl hydrocarbon hydroxylase induction in the target organs of each species.

Species and organ specificity of 6-nitrochrysene (6-NC)-induced carcinogenicity and the potential correlation with aryl hydrocarbon hydroxylase (AHH) induction in the target organs were investigated in both sexes of ICR mice and CD rats. Animals received total 6-NC doses of 1.4 mumol/mouse and 14.8 mumol/rat. The first i.p. injection was performed within 24 h of birth, then the animals were subjected to 3 and 5 weekly injections in the mouse and rat cases, and the survivors were sacrificed at weeks 24 and 32, respectively. Adenocarcinomas and dysplasias and/or adenomas of the colon in rats and lung adenomas in mice were observed in animals treated with 6-NC. However, no such lesions were observed in animals treated with the vehicle dimethyl sulfoxide alone. AHH activities in the lung, colon, and liver of each animal after treatment with 6-NC or dimethyl sulfoxide were also investigated. Six-week-old animals received a single 6-NC injection i.p. at the dose of 0.8 mumol/mouse or 8.0 mumol/rat. Animals were sacrificed on day 1 or 7 following injections, when AHH levels were measured. The results indicated enzyme levels in all these organs to be elevated by 6-NC treatment, the induction rate in the mouse lung being the highest. These results showed that 6-NC is carcinogenic for the colon of rats, as well as the lung of mice, and that it also induces AHH activity in both target and nontarget organs.

Adenocarcinoma↗

PubMed1992

[Twenty-eight-day repeated dose toxicity test of pentaerythritol in F344 rats].

A twenty-eight-day repeated dose toxicity test of pentaerythritol at dose levels of 1000 or 0 mg/kg/day was carried out in male and female F344 rats. Thirteen animals of each sex were divided into 2 groups with 7 rats receiving pentaerythritol treatment and 6 rats served saline as control. All groups received an i.g. administration daily for 28 days. As to serum biochemical and hematological examinations, there were no serious differences between the pentaerythritol-treated rats and the control rats. On histopathological examination, no specific changes were observed in the pentaerythritol-treated rats. Based on these results, the no-observed-effect level of pentaerythritol can be concluded to be more than 1000 mg/kg/day.

Administration, Oral↗

PubMed1991

Dose-dependent enhancing effects of quinacrine on induction of preneoplastic glutathione S-transferase placental form positive liver cell foci in male F344 rats.

Dose-dependent modifying effects of quinacrine on induction of preneoplastic liver cell foci were investigated in male F344 rats. Six week old animals were injected i.p. with N-nitrosodiethylamine (DEN) at a dose of 200 mg/kg, and starting 2 weeks later, rats were given quinacrine at dietary levels of 20, 100 and 500 p.p.m. for 6 weeks. Groups without either DEN or quinacrine treatment were used as controls. At week 3 following DEN administration, all animals were subjected to two-thirds partial hepatectomy, and after killing the animals at week 8, development of preneoplastic liver cell foci was investigated using the glutathione S-transferase placental form (GST-P) as a marker. The numbers and unit areas of GST-P-positive foci per cm2 were significantly increased in the DEN/quinacrine (500 p.p.m.) group as compared to DEN-alone group values. An increase in number was also evident in the 100 p.p.m. but not the 20 p.p.m. treated group, no lesions being induced by quinacrine alone (500 p.p.m.). Electron microscopic study confirmed that quinacrine dose-dependently induces lipidosis in hepatocytes, i.e. markedly myeloid lamellar cytoplasmic inclusion bodies were observed. The results thus demonstrated that quinacrine treatment enhances GST-P-positive liver cell foci development in a dose-dependent way, this effect presumably being related to the induction of lipidosis.

Animals↗

PubMed1991

Immunohistochemical and biochemical identification of pepsinogen isozymes in the hamster lungs: induction by polychlorinated biphenyls.

Pepsinogens are acid protease enzymes of pepsin usually found in gastric mucosa. In the present study, we demonstrated the presence of pepsinogen isozymes in male Syrian golden hamster lung tissues by a combined immunohistochemical and biochemical approach. Immunohistochemically, using rat pepsinogen 1 antibody, pepsinogen positive cells were observed mainly in the epithelia of the terminal bronchioles. They demonstrated morphological features of Clara cells. The pepsinogen isozyme pattern of lung tissue determined by polyacrylamide gel electrophoresis was similar to that of stomach mucosa. Treatment of hamsters with polychlorinated biphenyls at a dose of 500 mg/kg body weight ip caused a 2.8-fold increase in pepsinogen content (p less than 0.01) as well as increase in numbers of pepsinogen positive cells in the lung.

Animals↗

PubMed1991

[Dose-response relationship of promotion by phenobarbital in rat two-stage hepatocarcinogenesis].

Investigation of the effect of various doses of phenobarbital (PB) in Experiment I (PB dose levels: 0, 38, 75, 150, 300 or 600 ppm) and Experiment II (PB dose levels: 0, 1, 4 or 16 ppm) given to male F344 rats (20 animals/group) in drinking water for 39 weeks after a single intraperitoneal injection of diethylnitrosamine (DEN) was performed using incidence of hepatic tumors and number or area of enzyme-altered foci as end-point lesions. There were no significant differences in the final body weight changes between DEN-initiated PB treatment (DEN+PB) and DEN-initiated (DEN) groups. Dose-dependent increases in the absolute and relative liver weights and in the incidence of hepatic carcinoma were found in the DEN+PB groups treated with 38 ppm PB or above 75 ppm PB or above, respectively. The numbers or areas of gamma-GTP or GST-P positive foci of the liver were increased in the DEN+PB groups treated with 38 ppm PB or above. Additional investigation of 7-ethoxycoumarin o-deethylase (7-ECDE) induction in Experiment III, 7 groups consisting of 3 animals/group being fed water containing PB (0, 1, 4, 16, 75, 300 or 1200 ppm) for 1 week after the initiation of DEN and a further 7 groups (5 rats/group) receiving the same drinking water without DEN treatment, revealed dose-dependent increases of 7-ECDE in the DEN+PB groups and PB groups treated with 16 ppm PB or above. The present studies indicate that the threshold for promotion by PB is 38 ppm.

Administration, Oral↗

PubMed1990

[Measurement of prostaglandin E2 content in lung tissue of mouse].

A radioimmunoassay (RIA) was applied for the determination of PGE2 levels in the lung from 4 groups of male ICR mice 1) fed a standard diet, 2) fed a 20% corn oil-supplemented diet (HFD) for 2 weeks, 3) given a single s.c. injection of 15 mg/kg of 4NQO, a potent lung carcinogen and 4) given a s.c. injection of 4NQO and subsequently fed HFD for 2 weeks. Animals were sacrificed at week 3, and the lung was carefully excised and frozen in liquid nitrogen to prevent the postmortem synthesis of PGE2. PGE2, extracted from the lung tissue, was purified and then measured with or without adding a known amount of PGE2. The lung levels of PGE2 were shown to be significantly higher in the groups treated with HFD and/or 4NQO than the group fed a standard diet. These results show that the modified RIA method can be used for the measurement of PGE2 contents in the tissue of animals.

4-Nitroquinoline-1-oxide↗

PubMed1990

[Lipid peroxidation as a biological marker of mucosal changes in the stomach].

Dose-related increase of malondialdehyde (MDA) was found in both gastric mucosa and urine after 5-week administration of 4.0, 2.0, 1.0, 0.5, 0.25 or 0% NaCl supplemented diet to rats. NaCl, a gastric tumor promoter, is associated with enhanced lipid peroxidation in the gastric mucosa. The urinary MDA level was found to be a good indicator of lipid peroxidation state in the gastric mucosa, higher levels of MDA in the urine clearly correlating with data for stomach glandular tissue. In man, elevation of MDA was also observed in the urine of patients with gastric lesions such as chronic gastritis, precancerous and cancer, as well as in individuals suffering from liver and kidney disease.

Animals↗

PubMed1989

[Twenty-eight day repeated dose toxicity testing of diphenylamine in F344 rats].

A twenty-eight day repeated dose toxicity test of diphenylamine (DPA) was carried out in male and female F344 rats at dose levels of 1000, 333, 111 or 0 mg/kg/day. Thirty-six animals of both sexes were divided into 6 groups of equal number, 4 groups being used for the 28 days dosing study and the remainder for investigation of recovery. Inhibition of body weight gain, increase of liver, spleen and kidney weights, and anemia were observed in the highest dose groups in both sexes. The same groups demonstrated mucosal hyperplasia in the forestomach, dilatation, degeneration or necrosis of renal tubules in the corticomedullary junction, and hyperplasia in the bone marrow histopathologically. Slight increase of spleen, liver and kidney weights as well as slight degeneration of renal tubules were evident in several animals receiving the dose level of 333 mg/kg/day. Repair of histopathological lesions and anemia occurred within 14-day resting period. Based on these findings, under the present experimental conditions, the no observable effect level of DPA was 111 mg/kg/day.

Administration, Oral↗

PubMedDate not supplied

Doxorubicin induces male germ cell apoptosis in rats.

To clarify whether apoptosis is involved in doxorubicin (DXR)-induced testicular toxicity and to identify the target germ cell type, adult Sprague-Dawley rats were treated with a single intravenous dose of DXR (8 or 12 mg/kg) and euthanized at 3, 6, 12, 24, and 48 h subsequently. Histologically, germ cell degeneration was first found 6 h after dosing in meiotically dividing spermatocytes and early round spermatids of seminiferous tubules at stage 1, and subsequently observed in spermatogonia at stages I-VI showing ultrastructural characteristics of apoptosis. Coincident with the appearance of morphological changes, degenerating germ cells were shown to be undergoing apoptosis as revealed by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The frequency of TUNEL-labeled germ cells increased in a stage- and cell type-specific manner, the peak of frequency gradually progressing from stage I of seminiferous tubules to later stages with time after dosing, suggesting that the damaged germ cells, especially spermatogonia, gradually underwent the processes leading to apoptosis. DNA laddering on gel electrophoresis was apparent 24 and 48 h after dosing. The results demonstrate that apoptosis plays an important role in the induction of testicular toxicity caused by DXR with meiotically dividing spermatocytes and type A and intermediate spermatogonia as highly vulnerable target cells.

Animals↗

PubMedDate not supplied

Carcinogenicity study of beta-cyclodextrin in F344 rats.

The carcinogenicity of beta-cyclodextrin, a cyclic, water-soluble carbohydrate comprising seven glucose units, was examined in Fischer 344 (F344) rats. Groups of 50 males and 50 females were given the compound in their diet at concentrations of 0 (control), 2.5 or 5% for 104 wk. Surviving rats were then given a basal diet for a further 5 wk and killed at 109 wk. The dose levels were selected from the results of a 13-wk subchronic toxicity study. Dose-dependent inhibitory effects of beta-cyclodextrin on growth were observed in both sexes of the treated groups. The survival rates, mean survival times and range, however, demonstrated no significant differences between the control and treated groups. A variety of tumours developed in all groups, including the control group, but all the neoplastic lesions were histologically similar to those known to occur spontaneously in this strain of rat, and no statistically significant increase in the incidence of any tumour was found for either sex of the treated groups. Thus, it is concluded that under the present experimental conditions, the high dose, about 340-400 times higher than the current daily human intake from ingestion as a food additive and from pharmaceutical use, does not have any carcinogenic potential in F344 rats.

Administration, Oral↗

PubMedDate not supplied

Lack of carcinogenicity and increased survival in F344 rats treated with 5-fluorouracil for two years.

The carcinogenicity of 5-fluorouracil (5-FU), a compound employed as an antineoplastic drug, was investigated in F344 rats of both sexes. 5-FU was administered to groups of 50 male and 50 female rats ad lib. for 104 weeks, added to drinking water at concentrations of 0 (control), 62 and 125 ppm, these dose levels being selected on the basis of results of a 13-week subchronic toxicity study. Body weight gains were slightly depressed in the 125 ppm group of both sexes. While not statistically significant in females, final survival rates at week 111 in the 125 ppm group of both sexes were higher than those in the control group, suggesting an ability of 5-FU to prolong the lifespan. Histopathologically, a decreased incidence of islet cell adenomas in males and increased incidences of pituitary gland adenomas and pheochromocytomas in females were observed in the 62 ppm group without dose dependence. There was no significant induction of any other neoplastic or non-neoplastic lesions. These results indicate a lack of carcinogenicity of 5-FU under the present experimental conditions using rats.

Adenoma↗

PubMedDate not supplied

Toxic effects of benzyl and allyl isothiocyanates and benzyl-isoform specific metabolites in the urinary bladder after a single intravesical application to rats.

Allyl isothiocyanate (AITC) is known to be weakly carcinogenic, whereas benzyl isothiocyanate (BITC) has been suggested to exert carcinogenicity toward the rat urinary bladder. To elucidate direct toxic effects of isothiocyanates (ITCs), BITC, AITC, or BITC-metabolites conjugated either with glutathione, cysteinylglycine, cysteine, or mercapturic acid were intravesically instilled into female F344 rats. Exposure to AITC and BITC at 2.8 mg/kg body weight, and the same mol quantity (37 micromol/kg) of BITC-metabolites was for 2 h. Nineteen hours thereafter, the animals were intravenously administered 5-bromo-2'-deoxyuridine (BrdU) and killed 1 h later. BITC caused more profound toxic damage than AITC. Among the BITC-metabolites, cytotoxicity was evident with intermediate glutathione or cysteinylglycine conjugates, whereas the mercapturic acid, considered to be the major final urinary metabolite, exerted little effects. BrdU labeling was essentially dependent on the degree of cytotoxic potential of each compound. Considering the previous study results demonstrating the generation of free BITC from metabolites in urine, the present results support the idea that cytotoxic activity of orally administered ITCs is derived from free forms cleaved from conjugated metabolite(s) in urine.

Acetylcysteine↗

PubMedDate not supplied

Liver tumor-promoting effect of beta-naphthoflavone, a strong CYP 1A1/2 inducer, and the relationship between CYP 1A1/2 induction and Cx32 decrease in its hepatocarcinogenesis in the rat.

Interrelationships among induction of cytochrome P-450 (CYP) 1A1/2, decrease in connexin 32 (Cx32), and liver tumor-promoting activity by beta-naphthoflavone (BNF) in the promotion stage were examined in a 2-stage liver carcinogenesis model. A total of 20 male Fischer 344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone. Starting 2 weeks later, they were fed a diet containing 2%, 1%, or 0% BNF for 6 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Absolute and relative liver weights were significantly increased in the DEN+BNF groups as compared to the DEN-alone group. Diffuse hepatocellular hypertrophy with cytoplasmic eosinophilia, sometimes accompanied by development of adenoma-like hepatic foci, was observed in the BNF-treated rats. Remarkable induction of cytochrome CYP 1A1/2 and significant increase in CYP 2E1 were noted in the DEN+BNF groups, and positive immunohistochemical staining for both was observed diffusely. The areas of Cx32-positive spots per hepatocyte in the centrilobular areas of livers of the BNF-treated rats were significantly decreased, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form were increased in the BNF-treated groups. These results suggest that BNF is a liver tumor promoter that, unlike phenobarbital, does not induce CYP 2B1/2 isozymes, and there seems to be no direct relationship between CYP 1A1/2 induction and Cx32 reduction in BNF hepatocarcinogenesis.

Animals↗

PubMedDate not supplied

Establishment of an animal model for pulmonary fibrosis in mice using monocrotaline.

A preliminary attempt at experimental induction of pulmonary fibrosis in which male ICR mice received 15 weekly sc injections of 200 or 100 mg/kg monocrotaline (MC) revealed that most animals treated with the larger dose died of severe interstitial pneumonia, whereas those given 100 mg/kg exhibited only relatively slight lung injury. Based on these results, male mice were administered sc injections of 200 and 100 mg/kg MC once a week for 9 and 18 times, respectively, and then maintained without any further treatment until week 28 after the start. Mice treated with 200 mg/kg MC showed severe pulmonary damage and died by week 25. Mortalities also occurred in the 100-mg group from week 16, with 11 of 40 animals surviving at the termination of the experiment. Histologically, both dose groups demonstrated severe interstitial pneumonia and/or pulmonary fibrosis. Ultrastructurally, inflammatory edema possibly attributable to injuries of alveolar capillary endothelial cells was observed in the high-dose group at week 8, and there was a remarkable increase in collagen fibers in alveolar septa in this group thereafter. The present study results suggest that lung injuries induced by MC treatment progress to irreversible lung fibrosis and that this animal model may have advantage for studying the pathogenesis of lung cancers in patients with pulmonary fibrosis.

Animals↗