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T Mizuguchi

Publications and source records attributed to T Mizuguchi.

At least 37 records · Page 2Linked to original sources
PubMed1996

Changes in cellular distribution of connexins 32 and 26 during formation of gap junctions in primary cultures of rat hepatocytes.

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sections in vivo. Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.

Animals↗
PubMed1996

Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF.

To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The hepatocytes were cultured in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6 x 10(5) cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo, but they never overgrew. Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells possessed hepatic characteristics such as peroxisomes with a crystalline nucleiod and bile-canaliculus structures. When 10% fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could differentitate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells. In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided evidence that the subcultured cells still have the potential to proliferate and to differentiate.

Animals↗
PubMed1996

Distribution of diaphragm blood flow during sevoflurane anaesthesia in dogs.

PURPOSE: The purpose of this study was to determine the effect of increasing the concentrations of sevoflurane anaesthesia on the distribution of diaphragm blood flow (Qdi) in ten dogs during mechanical ventilation. METHODS: Animals were divided into two groups, sevoflurane (n = 6) and time control (n = 4) groups. Blood flow to the crural and the costal diaphragm (Qcru, Qcost) was determined by the hydrogen clearance technique at 0, 0.5, 1.0 and 1.5 minimum alveolar concentration (MAC) of sevoflurane after a 30 min period of steady-state conditions. Cardiac output (CO) and the mean arterial blood pressure (MBP) were also measured. RESULTS: Sevoflurane anaesthesia caused a reduction in CO (L.min-1) from a control value of 1.51 +/- 0.21 to 1.38 +/- 0.1 (0.5 MAC), 1.09 +/- 0.15 (1.0 MAC) and 0.98 +/- 0.12 (1.5 MAC) (Mean +/- SD). Mean blood pressure, Qcru and Qcost also decreased with increasing depth of anaesthesia. In addition, the decrease of Qcru was greater than that of Qcost at all levels of MBP and CO. No change occurred in these variables in the time control group. CONCLUSION: Sevoflurane anaesthesia changes the distribution of Qdi with a greater reduction occurring in Qcru than in Qcost.

Anesthetics, Inhalation↗
PubMed1996

Analysis of circulating hematopoietic progenitors in patients with chronic renal failure under hemodialysis.

Circulating hematopoietic progenitors were analyzed in patients with chronic renal failure (CRF) under hemodialysis (HD) by methylcellulose culture containing interleukin-3 (IL-3) to clarify the differences in hematopoiesis between patients with and without CRF-associated anemia and between good responder whose hematocrit (Ht) was preserved in more than 25% under erythropoietin (Epo) treatment and poor responders whose Ht remained less than 25% even under Epo treatment. The numbers of peripheral blood (PB) erythroid burst-forming units (BFU-E) and granulocyte-macrophage colony-forming units (CFU-GM) in HD patients without Epo treatment, whose Ht levels were greater than 30%, were similar to those in normal subjects. However, these numbers in HD patients who required Epo treatment were significantly lower than those in normal subjects. The number of PB BFU-E in HD patients who showed a poor response to Epo was significantly lower than that in HD patients who showed a good response to Epo. The number of PB BFU-E was well correlated with the number of PB CFU-GM in all groups of HD patients. There also existed a definite correlation between these numbers and the Ht levels in HD patients without Epo treatment, but not those in HD patients with Epo treatment. The sensitivity of PB BFU-E to IL-3 was lower in HD patients who showed a poor response to Epo than in the other HD patients and normal subjects. These findings indicate that hematopoiesis in HD patients with CRF associated anemia is suppressed in both the erythroid and myeloid lineage at primitive stages, and that the lower sensitivity of PB BFU-E to IL-3 in HD patients with a poor response to Epo may be associated with this poor response. In addition, the level of the serum transferrin receptor (sTfR) in HD patients without severe anemia was higher than that in normal subjects and HD patients who required Epo treatment, indicating that erythropoiesis in HD patients who do not require Epo treatment is more active than that in normal subjects and other HD patients.

Case-Control Studies↗
PubMed1996

Effects of oxygen radical scavengers on connexins 32 and 26 expression in primary cultures of adult rat hepatocytes.

Although we recently reported our success in inducing and maintaining the gap junction proteins connexin 26 (Cx26) and connexin 32 (Cx32) in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor, dimethylsulfoxide (DMSO) and glucagon, the mechanisms by which DMSO induces gap junctions are still not clear. It is known that DMSO is not only a differentiation reagent for various cells but also a powerful scavenger of oxygen radicals. In the present study, by using this culture system and the measurement of oxidative stress by the nitro blue tetrazolium (NBT) formazan assay, we have examined the effect of oxygen radical scavengers such as DMSO, dimethylthiourea (DMTU) and alpha-tocopherol on the expression of both Cxs and on gap junctional intercellular communication (GJIC), as compared to another differentiation reagent, hexamethylene-bis-acetamide (HMBA). DMSO and DMTU clearly inhibited the oxidative stress of the cultured hepatocytes, while alpha-tocopherol and HMBA did not. The expression of Cx26 and Cx32 in the cultured hepatocytes was markedly induced by DMSO and DMTU. Furthermore, extensive GJIC was also observed with DMSO and DMTU. These results suggest that the expression of gap junctions in the hepatocytes may be closely related to oxidative stress and that oxygen radical scavengers may be important substances in inducing this expression.

Acetamides↗
PubMed1996

Recovery of mRNA expression of tryptophan 2,3-dioxygenase and serine dehydratase in long-term cultures of primary rat hepatocytes.

Expression of tryptophan 2,3-dioxygenase (TO) and serine dehydratase (SDH) has not previously been maintained or re-induced in long-term cultured hepatocytes. In the present study, we succeeded in inducing expression of TO and SDH mRNAs in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor and 2% dimethyl sulfoxide (DMSO). After the start of culture, the expression of TO mRNA rapidly disappeared and at 96 h it was less than 10% of that at isolation. However, after the addition of 2% DMSO from 96 h, the transcript level gradually increased and reached about 40% of that of the isolated cells at day 14. In addition, the expression of TO mRNA was enhanced in cells treated with both 10(-5) M dexamethasone and 10(-7) M glucagon. In contrast, the expression of SDH mRNA decreased very rapidly and we could not detect it after 24 h of culture. Furthermore, 2% DMSO failed to induce it. However, when both 10(-5) M dexamethasone and 10(-7) M glucagon were added to the culture medium at day 9, we observed dramatic induction of SDH mRNA 24 h later. Primary hepatocytes cultured by this method could express and maintain highly differentiated hepatic functions for a long time. Thus, this in vitro system is suitable for the investigation of hepatic functions.

Animals↗
PubMed1996

High-dose fentanyl does not suppress interleukin-1 beta-induced increases in plasma ACTH and corticosterone in rats.

BACKGROUND: High-dose fentanyl anesthesia is reported to attenuate the metabolic and endocrinal responses to surgery. Interleukin-1 (IL-1) is one of the key mediators in the immunoneuroendocrine system, and may be involved in the stress responses to surgery. We studied whether high-dose fentanyl may influence the IL-1 beta-induced alterations in plasma ACTH and corticosterone in rats. METHODS: Plasma ACTH, corticosterone, blood pressure, heart rate and acid-base status were determined in either awake or fentanyl-anesthetized animals immediately before and after either phosphate buffered saline or IL-1 beta administration. Fentanyl anesthesia was induced by bolus intravenous injections of fentanyl at 50 micrograms/kg and pancuronium bromide at 0.2 mg/kg, and maintained by continuous administrations of fentanyl at 100 or 200 micrograms.kg-1.h-1 and pancuronium bromide at 0.4 microgram.kg-1.h-1. RESULTS: In awake rats, IL-1 beta at incremental doses of 0.25, 0.5 and 1 microgram/kg increased plasma ACTH in a dose-dependent manner, but heat-inactivated IL-1 beta at 4 micrograms/kg did not influence plasma ACTH. A noxious stimulus with tail clamping for 30 min did not significantly alter plasma ACTH in fentanyl-anesthetized rats. Fentanyl reduced the basal plasma corticosterone, but it did not modulate the increases in plasma ACTH and corticosterone after the administration of IL-1 beta at 1 microgram/kg. Fentanyl moderately increased the basal blood pressure and heart rate, but it moderately attenuated the IL-1 beta-induced elevations of blood pressure and heart rate. IL-1 beta moderately decreased PCO2 in awake animals. CONCLUSIONS: Fentanyl anesthesia, which is able to suppress the endocrine responses to noxious stimuli, does not attenuate the IL-1 beta-mediated activation of the pituitary-adrenal axis in rats.

Adrenocorticotropic Hormone↗
PubMed1995

Targeted disruption of the tyrosine hydroxylase locus results in severe catecholamine depletion and perinatal lethality in mice.

Tyrosine 3-hydroxylase (TH, EC 1.14.16.2) catalyzes the first and rate-limiting step of the catecholamine biosynthetic pathway in the nervous and endocrine systems. The TH locus was disrupted in mouse embryonic stem cells by homologous recombination. Mice heterozygous for the TH mutation were apparently normal. In these mice, TH activity in the embryos and adult tissues was less than 50% of the wild-type values, but the catecholamine level was decreased only moderately in the developing animals and was maintained normally at adulthood, suggesting the presence of a regulatory mechanism for ensuring the proper catecholamine level during animal development. In contrast, the homozygous mutant mice died at a late stage of embryonic development or shortly after birth. Both TH mRNA and enzyme activity were lacking in the homozygous mutants, which thus explained the severe depletion of catecholamines. These changes, however, did not affect gross morphological development of the cells that normally express high catecholamine levels. Analysis of electrocardiograms of surviving newborn mutants showed bradycardia, suggesting an alteration of cardiac functions in the homozygous mice that may lead to the lethality of this mutation. In addition, transfer of a human TH transgene into the homozygous mice corrected the mutant phenotype, showing recovery of TH activity by expression of the human enzyme. These results indicate that TH is essential for survival of the animals during the late gestational development and after birth.

Animals↗
PubMed1995

Growth and maturation of small hepatocytes isolated from adult rat liver.

Small hepatocytes existed in the supernatant following low-speed centrifugation of the cell suspension after collagenase liver perfusion. The cells proliferated for more than 2 months and formed colonies in the Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide, 10% fetal bovine serum, 1 mM ascorbic 2-phosphate, and 10 ng/ml epidermal growth factor. One small cell finally proliferated to several hundred cells. In addition, some cells in the colonies were shown to differentiate into mature hepatocytes that had a large cytoplasm and sometimes two nuclei. The secretion of albumin in the medium by the hepatocytes increased with time in culture, and the cells possessed connexin 32 in their cell membrane and many peroxisomes. Thus, the small hepatocytes may be "committed progenitor cells" which can further differentiate into mature hepatocytes.

Animals↗
PubMed1995

Immunotoxin-mediated conditional disruption of specific neurons in transgenic mice.

We have developed a transgenic approach, termed immunotoxin-mediated cell targeting (IMCT), to ablate conditionally selective neurons in the brain with the cytotoxic activity of immunotoxins. Transgenic mice were created that express the human interleukin 2 receptor alpha subunit (IL-2R alpha) under the control of the dopamine beta-hydroxylase (DBH) gene promoter. The animals were treated intracerebroventricularly with a recombinant immunotoxin, anti-Tac(Fv)-PE40, which selectively kills animal cells bearing human IL-2R alpha. The immunotoxin caused a characteristic behavioral abnormality only in the transgenic mice. This was accompanied by a dramatic loss of DBH-containing neurons and a significant decrease in DBH activity and norepinephrine levels in various regions of the brain. IMCT should provide a general technique to create animal models of human neurodegenerative disorders by targeting neurons or other cell types.

ADP Ribose Transferases↗
PubMed1995

Low-dose halothane produces airway dilatation but does not alter parenchymal mechanics in the normal canine lung.

The purpose of this study was to examine whether halothane reduces the contractile tone of the normal lung and to distinguish the effects of halothane on airways from those on lung tissue. We also tested whether a mathematical model was capable of quantitatively describing the mechanical changes in the lung produced by halothane. We measured lung impedence (ZL(omega) a complex function of real (lung resistance) and imaginary (reactance) parts) at low frequencies in dogs using a forced volume oscillation technique before and during 1 MAC halothane anaesthesia. Halothane produced small changes in ZL(omega). The lung resistance tended to decrease during halothane anaesthesia whereas the lung reactance did not show change. Using an alveolar capsule technique to separate the airways from the lung tissue components, these lung mechanical changes were induced mainly by alterations in lung tissue and not in the airways. Our mathematical model featured a single airway leading to an alveolar region surrounded by a viscoelastic lung tissue. In the model analysis, estimates of airway resistance and inertance decreased by the administration of halothane. In contrast, estimates of lung tissue elastance and resistance did not change during halothane anaesthesia. These modeling results were consistent with those obtained by direct alveolar pressure measurements. Our results suggest that a low concentration of halothane dilates the airways but does not alter the parenchymal mechanics in the normal lung, and that the model provides a quantitative tool to assess lung mechanics precisely, if respiratory signals are measured only at the true airway opening.

Airway Resistance↗
PubMed1995

Alteration of catecholamine phenotype in transgenic mice influences expression of adrenergic receptor subtypes.

Agonist-induced regulation of adrenergic receptors (ARs) has an important role in controlling physiological functions in response to changes in catecholamine stimulation. We previously generated transgenic mice expressing phenylethanolamine N-methyltransferase (PNMT) under the control of a human dopamine beta-hydroxylase gene promoter to switch catecholamine specificity from the norepinephrine phenotype to the epinephrine phenotype. In the present study, we first examined changes in catecholamine metabolism in peripheral tissues innervated by sympathetic neurons of the transgenic mice. In the transgenic target tissues, a high-level expression of PNMT led to a dramatic increase in the epinephrine levels, whereas the norepinephrine levels were decreased to 48.6-87.9% of the nontransgenic control levels. Analysis of plasma catecholamines in adrenalectomized mice showed large amounts of epinephrine derived from sympathetic neurons in the transgenic mice. Subsequently, we performed radioligand binding assays with (-)-[125I]iodocyanopindolol to determine changes in binding sites of beta-AR subtypes. In transgenic mice, the number of beta 2-AR binding sites was 56.4-74.9% of their nontransgenic values in the lung, spleen, submaxillary gland, and kidney, whereas the beta 1-AR binding sites were regulated in a different fashion among these tissues. Moreover, northern blot analysis of total RNA from the lung tissues showed that down-regulation of beta 2 binding sites was accompanied by a significant decrease in steady-state levels of the receptor mRNA. These results strongly suggest that alteration of catecholamine specificity in the transgenic sympathetic neurons leads to regulated expression of the beta-AR subtypes in their target tissues.

Animals↗
PubMed1995

OP-1206, a prostaglandin E1 derivative, attenuates the thermal hyperesthesia induced by constriction injury to the sciatic nerve in the rat.

Nerve ischemia induces wallerian degeneration and peripheral neuropathy, the nerve constriction injury induces thermal hyperesthesia. Nerve ischemia is one possible mechanism in the development of thermal hyperesthesia in the nerve constriction injury model. Prostaglandin E1 increases tissue blood flow. In the present study, the authors examine the role of nerve ischemia in the maintenance of the thermal hyperesthesia induced by nerve constriction injury by orally administering OP-1206, a prostaglandin E1 derivative. A nerve constriction injury model was created by making four loose ligations around the rat sciatic nerve, which induces thermal hyperesthesia in the ligated paw in 2-5 days. OP-1206, was administered six times (Day 7, one time; Day 8, two times; Day 9, two times; Day 10, one time). A single administration of OP-1206 had no effect on the thermal hyperesthesia. Six hours after the sixth-administration of OP-1206, the level of the thermal hyperesthesia was attenuated in a dose-dependent manner, and this effect lasted more than 1 day after the last drug administration. These data indicate that nerve ischemia plays an important role in maintaining the thermal hyperesthesia induced by nerve constriction injury in the rat.

Alprostadil↗
PubMed1995

Modulation of protein kinase C produces glucose-dependent alterations in hemodynamics and metabolism in the perfused liver in fasted rats.

Protein kinase C (PKC) has been suggested to be involved in the regulation of hepatic blood flow and metabolism. To confirm the role of PKC, we studied the effects of active and inactive PKC modulators on hemodynamics and metabolism in the perfused rat liver. In addition, the influence of glucose concentration in the medium was studied. The liver was isolated from fasted Sprague-Dawley rats and perfused through the portal vein at a constant pressure of 12 cm H2O. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester for PKC, slightly decreased hepatic flow only when its initial concentration was raised to 20 microM. In contrast, 4 beta-phorbol 12,13-didecanoate, an active phorbol ester for PKC, at initial concentrations of 80 nM to 1.28 microM decreased hepatic flow and oxygen consumption in a dose-dependent manner, and increased lactate production. HA-1004, a relatively inactive PKC inhibitor, at an initial concentration of 33 microM did not modify the effects of phorbol 12-myristate 13-acetate (PMA), a potent PKC activator. However, H-7, a relatively specific PKC inhibitor, at a concentration of 33 microM attenuated the effects of PMA. The effects of PMA were enhanced by an increase in n-glucose concentration from 10 to 25 mM but not by an increase in L-glucose concentration. These results suggest that modulation of PKC exerts glucose-dependent influences on hepatic flow and metabolism.

Animals↗
PubMed1995

[Cancer-pain management in home care].

Patient was 58 year old female with severe uterus cancer pain who refused to take the oral morphine in hospital because doctor should be hesitated to discharged patient whose pain is particularly difficult to manage in home. A few days later, she returned back home with her family irritably and recovered from hallucination by morphine-intake in home. The oral morphine is the prepared route of analgesic administration in home care. When patient can not take medications orally, continuous intravenous infusion provides the most consistent level of analgesia. In future, transdermal route offers a practical alternative in the hospice and home.

Administration, Oral↗
PubMed1994

Functional and high level expression of human dopamine beta-hydroxylase in transgenic mice.

Dopamine beta-hydroxylase (DBH; EC 1.14.17.1) catalyzes the production of the neurotransmitter and hormone norepinephrine in the third step of the catecholamine biosynthesis pathway. Transgenic mice were generated with multiple copies of a human DBH minigene construct containing the full-length cDNA connected downstream of the 4-kilobase upstream promoter region to achieve overexpression of DBH. Human DBH mRNA and immunoreactivity were detected tissue-specifically in the brain and adrenal gland of these transgenic mice. The transgene products were correctly processed to a glycosylated mature polypeptide with a molecular mass of 72 kDa and existed in the secretory vesicles as both soluble and membrane-bound forms. We detected a marked increase in DBH activity in various catecholamine-containing tissues of the mice that occurred as a consequence of expression of the catalytically active human DBH enzyme. However, in these transgenics the steady-state levels of norepinephrine and epinephrine were normally maintained without the acceleration of the catecholamine turnover rate, suggesting that there are some regulatory mechanisms to preserve a constant rate of norepinephrine synthesis in spite of the increased amount of DBH protein. These transgenic mice with the minigene construct provide one approach to study the mechanisms underlying biogenesis of the DBH polypeptide and regulation of norepinephrine synthesis.

Adrenal Glands↗