Viscoelasticity and function of connectin/titin filaments in skinned muscle fibers.
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Animals↗
Biomedical subjects
H Higuchi
Publications and source records attributed to H Higuchi.
Ca2+/calmodulin-dependent transcriptional activation of neuropeptide Y gene induced by membrane depolarization: determination of Ca(2+)- and cyclic AMP/phorbol 12-myristate 13-acetate-responsive elements.
Membrane depolarization stimuli (high potassium concentration and veratridine) increased neuropeptide Y (NPY) mRNA abundance time-dependently, without a change in beta-actin mRNA level, in NG108-15 and PC12 cells. Although the induction by veratridine was blocked completely by tetrodotoxin, the induction by potassium was suppressed minimally. Voltage-dependent Ca channel blockers and calmodulin antagonists inhibited the increases by both depolarization stimuli completely, suggesting involvement of Ca2+/calmodulin-dependent kinases (CaM kinases). Transient assay using chloramphenicol acetyltransferase reporter genes containing the rat NPY gene promoter indicated that membrane depolarization and Ca entry stimulate transcription of the NPY gene. The depolarization-induced transactivation was also blocked by CaM kinase inhibitors. The 200-bp 5'-upstream region (-344/-145) was localized as a Ca2+/ calmodulin-responsive element (CaMRE), which confers depolarization-induced transactivation. It is interesting that this CaMRE did not contain the canonical Ca-responsive elements such as CRE, SRE, NF-AT, or the C/EBP beta-binding site and was separated from a 64-bp cyclic AMP/ phorbol 12-myristate 13-acetate-responsive element (-144/-81). These findings suggested that membrane depolarization regulates the NPY gene transcription positively through the unique CaMRE by activation of CaM kinases following Ca entry through L-type Ca channels.
Animals↗
Role in nitric oxide in Kupffer cell-mediated hepatoma cell cytotoxicity in vitro and ex vivo.
The metabolic change in tumor cells (AH70, a rat hepatoma cell line) cocultured with isolated rat Kupffer cells were visualized and analyzed by a laser scanning confocal imaging system. When AH70 cells were cocultured with Kupffer cells, fluorescence intensity of rhodamine 123 (Rh123) decreased, indicating the reduction of mitochondrial function. The reduction in Rh123 was eliminated by NG-monomethyl-L-arginine (L-NMMA), an analogue of L-arginine, suggesting the involvement of nitric oxide (NO). Two hour after the cells were cocultured, membrane compromised AH70 cells which were observed as propidium from 2.8% to 25%. This increase was also attenuated by L-NMMA, suggesting that Kupffer cell-mediated injury of tumor cells largely depends on NO. The concentrations of NO-2 + NO-3 in the culture medium markedly increased after coculture of AH70 cells with Kupffer cells. Moreover, NO synthase (NOS) activity in Kupffer cells significantly increased after coculture. These in vitro results suggest that NO mediates Kupffer cell-induced tumor cell damage characterized by reduced mitochondrial function and diminished barrier function. In the ex vivo study of the perfused liver to which AH70 cells were injected via the catheter inserted into the portal vein, some AH70 cells were arrested in the upper stream of sinusoid and the fluorescence intensity of Rh123 in adherent AH70 cells decreased in a time-dependent manner within 2 hours. The number of PI-positive AH70 cells also increased 2 hours after the injection of AH70 cells. These changes were inhibited by either administration of N omega-L-nitroarginine-methylester (L-NAME) to perfusate or pretreatment of the rat liver with GdCl3, which is known to deplete Kupffer cell function. Thus, the present study suggests that NO from Kupffer cells induces mitochondrial dysfunction in tumor cells followed by membrane barrier dysfunction in the liver sinusoid.
Animals↗
alpha 4 integrin plays a critical role in early stages of T lymphocyte migration in Peyer's patches of rats.
Lymphocyte recirculation through the blood flow circuit and lymphoid organs is important for the maintenance of immune defense, and is defined as lymphocyte homing. During the homing process, several adhesion molecules have been postulated to play an important role in lymphocyte recruitment from the vascular space. In the present study, we investigated the effect of a novel mAb against rat alpha 4 integrin (MR alpha 4-1) on the interaction of T lymphocytes with postcapillary venules (PCV) and their subsequent migration into the interstitium of Peyer's patches, using intravital video microscopy. T lymphocytes collected from intestinal lymph were labeled with a fluorochrome carboxyfluorescein diacetate succinimidyl ester and were than injected into the jugular vein of recipient rats. The microvasculature in the ileal Peyer's patches of recipient rats was observed sequentially by intravital fluorescence microscopy. In controls, lymphocytes exhibited rolling behavior which was followed by firm adhesion to the endothelium of PCV. The density of sticking lymphocytes gradually increased during the first 30 min. These initial interactions of lymphocytes with the PCV (rolling and adherence) were drastically inhibited by treatment with MR alpha 4-1, both when MR alpha 4-1 was preinfused into rats and when T cells were preincubated in vitro with MR alpha 4-1 before administration. MR alpha 4-1 also significantly inhibited the transendothelial migration of T lymphocytes, associated by the ratio of migration to adherence. However, once T lymphocytes migrated into the interstitium, treatment with MR alpha 4-1 did not affect the pattern of travel of these lymphocytes in the interstitium or their transport into the microlymphatics in the parafollicular area. Therefore, we conclude that alpha 4 integrins play a critical role in the rolling and sticking of T cells and their transendothelial migration in PCV of Peyer's patches.
Animals↗
Kupffer cell-mediated cytotoxicity against hepatoma cells occurs through production of nitric oxide and adhesion via ICAM-1/CD18.
Rat Kupffer cell (KC)-mediated cytotoxicity against both the syngeneic hepatoma cell line AH70 and hepatocytes was evaluated by changes in mitochondrial function, and the possible role of ICAM-1/CD18 in the interaction between the cells was studied. Rhodamine 123 fluorescence, a marker of the mitochondrial membrane potential, decreased in AH70 cells after co-culture with CK, while that in hepatocytes was unchanged by co-culture. This decrease was blocked by anti-ICAM-1 anti-CD18 and the inhibition of nitric oxide synthesis. Cytometric studies demonstrated that ICAM-1 expression on AH70 cells increased after addition of IFN-gamma, IL-1beta, tumor necrosis factor (TNF)-alpha or KC, while in hepatocytes ICAM-1 was not increased. Anti-ICAM-1 pretreatment inhibited the increase in ICAM-1 expression and the decrease in rhodamine 123 fluorescence on AH70 cells after co-culture with KC. CD18 on KC was increased only after co-culture with AH70. TNF-alpha but not IFN-gamma was detected in the supernatant of co-culture between KC and AH70 cells, and this production was partially inhibited by anti-ICAM-1 and anti-CD18. The activity of inducible nitric oxide synthase in Kupffer cells and the levels of nitrites and nitrates in the co-culture supernatant increased over time, and this increase was attenuated either by addition of NO synthesis inhibitors, anti-ICAM-1 or anti-CD18. These results indicate that the rat KC causes mitochondrial dysfunction in cancer cells via the production of NO and cell-to-cell adhesion via ICAM-1/CD18 has an important role in this cytotoxic process.
Animals↗
Functional characteristics of enhanced Fc receptor expression of beta 2 integrin-deficient bovine mononuclear phagocytes.
Fc receptor expression, cytoplasmic Ca2+ signaling, chemiluminescent (CL) response, and electron spin resonance (ESR) combined with spin trapping of blood mononuclear phagocytes from control heifers and a heifer with leukocyte adhesion deficiency (LAD) were evaluated to elucidate the relationships between complement receptor type 3 (CR3) and Fc receptor expression and their functional responses. The mean fluorescence intensity of fluorescein isothiocyanate (FITC)-conjugated anti-bovine IgG bound to mononuclear phagocytes from the heifer with LAD was 1.8-fold higher than that of control heifers. The mean increments of cytoplasmic Ca2+ concentrations of mononuclear phagocytes from the heifer with LAD stimulated with OPZ, Agg-IgG, and PMA were 39.4 (P < 0.05), 118, and 71.6% compared with those of control heifers. A 1.27-fold increase in the CL response relative to control heifers was detected when mononuclear phagocytes from the heifer with LAD were stimulated with Agg-IgG. The OPZ-induced CL response of mononuclear phagocytes from the heifer with LAD was significantly (P < 0.05) decreased, whereas the PMA-induced CL response was similar to that of control heifers. The ESR spectrum of mononuclear phagocytes from the heifer with LAD was increased when stimulated with Agg-IgG, and was impaired when stimulated by OPZ compared with that of control heifers. The ESR spectrum of mononuclear phagocytes stimulated with PMA was similar in control heifers and the heifer with LAD. Fc receptors on mononuclear phagocytes from the heifer with LAD were enhanced, and their cytoplasmic Ca2+ signaling, CL response, and ESR-spin trapping when stimulated with Agg-IgG and OPZ appeared to be associated with enhanced Fc receptors.
Animals↗
Adhesiveness for extracellular matrices and lysosomal enzyme release from normal and beta 2 integrin-deficient bovine neutrophils.
The adhesiveness of control and CD18-deficient bovine neutrophils on culture plates precoated with collagen I, collagen IV, fibronectin and laminin was measured to evaluate the possible factors for adherence to extracellular matrices. The release of N-acetyl-beta-D-glucosaminidase (NAGase) from control and CD18-deficient neutrophils stimulated with complement receptor type 3 (CR3) or Fc receptor dependent stimuli was also evaluated. The adhesive activities of CD18-deficient neutrophils to collagen I, collagen IV and fibronectin were significantly diminished (P < 0.05); however, similar adhesion to laminin was observed in CD18-deficient neutrophils and control neutrophils. The adhesive activity of control neutrophils on uncoated plates increased 2.5 times (P < 0.05) with the presence of PMA. The mean activities for NAGase release from CD18-deficient neutrophils stimulated with opsonized zymosan and aggregated bovine immunoglobulin G (Agg-IgG) were 46.7 and 82.7% that of the control neutrophils, respectively. The Agg-IgG-induced NAGase release from control and CD18-deficient neutrophils was eliminated by H7, a protein kinase C inhibitor. These results support that an association between CR3 and Fc receptors on neutrophils appears to play an essential role in neutrophil functions.
Acetylglucosaminidase↗
Effect of chronic ethanol feeding on Kupffer cell-mediated antitumor cell activity.
We have previously reported that the Kupffer cell has antitumor activity through mitochondrial damage to tumor cells by nitric oxide production. In this study, the effect of chronic ethanol feeding on antihepatoma cell activity of the Kupffer cell was examined in rats. Male rats of the Wistar strain were fed ethanol chronically for 8 weeks by liquid diets. Kupffer cells were isolated from the control rat or the ethanol-fed rat, and cocultured with AH 70 cells, a rat hepatoma cell line. Fluorescence of rhodamine 123 or propidium iodide was observed as indicators of the mitochondrial damage or cell membrane injury, respectively, by a laser scanning confocal microscopy. Mitochondrial damage of AH 70 cells as indicated by reduction of rhodamine 123 fluorescence was smaller by the coculture with Kupffer cell from the ethanol rat than that from the control. Cell membrane barrier dysfunction of AH 70 cell was less frequently observed with the Kupffer cell from ethanol-fed rats. A metabolite of nitric oxide (nitrite and nitrate) was less in the cultured medium with the ethanol Kupffer cell than with the control Kupffer cell. Ca2+ mobilization, which induces inducible nitric oxide synthase and observed by the fluorescence of fluo-3, in Kupffer cells cocultured with AH 70 cells was suppressed in ethanol-fed rats. These result suggests that chronic ethanol feeding suppresses antitumor cell activity of Kupffer cell through the impairment of Ca2+ mobilization and nitric oxide production.
Animals↗
Effect of chronic ethanol feeding on nitric oxide synthesis by rat Kupffer cells.
Kupffer cells contribute to the important role of the liver defense mechanism through nitric oxide (NO) production. In this study, the effect of chronic ethanol administration on the ability of Kupffer cells to synthesize and release NO was investigated after stimulation with lipopolysaccharide (LPS). Male Wistar rats were chronically fed ethanol for 8 weeks according to the method described by DeCarli and Lieber et al. (J Nutr.91:331-336, 1967). Kupffer cells were isolated and cultured with LPS (1 micrograms/ml) for 24 hr. The levels of nitrite and nitrate, metabolites of NO, were determined in the culture medium, NO synthase (NOS) activity in Kupffer cells was determined by the method that measures conversion of [14C]arginine into [14C]citrulline. In control rats, a significant increase of nitrite and nitrate levels in culture medium was observed after LPS treatment. The magnitude of this increase was significantly smaller in chronic ethanol-fed rats. When the activity of NOS was determined, inducible NOS (iNOS) activity was higher than that of constitutive NOS, and LPS administration produced a significant elevation of iNOS activity in both control and chronic ethanol-fed rats. However, the elevation of iNOS activity by LPS stimulation was diminished by chronic ethanol administration. Distribution of iNOS in Kupffer cells as determined by an immunofluorescence method using a laser scanning confocal image system showed a lower expression of iNOS in chronic ethanol-fed rats even in the presence of LPS. These results demonstrate that the excessive production of NO by increased iNOS activity in Kupffer cells is diminished by chronic ethanol administration.
Animals↗
Ethanol-induced oxidative stress in the liver.
Oxygen stress is well recognized to be a key step in the pathogenesis of ethanol-associated liver injury. Ethanol administration induces an increase in lipid peroxidation either by enhancing the production of oxygen-reactive species and/or by decreasing the level of endogenous antioxidants. Numerous experimental studies have emphasized the role of the ethanol-inducible cytochrome P-450 in the microsomes, as well as the molybdo-flavoenzymes xanthine oxidase in the cytosol. This review shows the putative role of ethanol-induced disturbances in iron metabolism in relation to iron as a prooxidant factor. Ethanol administration also affects the mitochondrial free radical generation. Although many previous studies suggest a role for active oxygens in ethanol-induced mitochondrial dysfunction in hepatocytes, the detailed mechanism of ethanol-induced oxidative stress on mitochondria remains to be clarified further. Studies of our laboratory using a confocal laser scanning microscopic system strongly suggest that active oxidants produced during ethanol metabolism modulate mitochondrial energy synthesis in isolated and cultured hepatocytes. In addition, our investigations implicate endogenous glutathione-glutathione peroxidase system and catalase as important antioxidants and cytoprotective machinery in the hepatocyte mitochondria exposed to ethanol. The fluorographic investigations using the confocal laser scanning microscopy may be useful to extend our knowledge and provide a new view about ethanol-associated oxidative stress and metabolic changes in hepatocytes.
Animals↗
Endotoxin stimulates lymphocyte-endothelial interactions in rat intestinal Peyer's patches and villus mucosa.
Although lymphocyte-endothelial cell interactions represent a key step in controlling the recruitment of lymphocytes into gut-associated tissues, its dynamic process in microvessels of lymphoid (Peyer's patches) and nonlymphoid (villus) regions of the small bowel remains poorly understood. We monitored the migration of fluorescence-labeled T lymphocytes into normal and lipopolysaccharide (LPS)-inflamed rat intestinal microvessels using intravital microscopy. In Peyer's patches, T lymphocytes selectively adhered to postcapillary venules, although such selectivity was not observed in submucosal venules of villi. T lymphocytes exhibited rolling behavior followed by firm adhesion in microvessels of both the Peyer's patches and the villi, with both types of adhesive interaction being mediated by alpha 4-integrins. The enhanced rolling and adherence of lymphocytes observed in Peyer's patches and submucosal venules of villi of LPS-treated rats were preceded by a reduction in shear rate and were mediated largely by alpha 4-integrins and partly by beta 2-integrins. In capillaries of intestinal mucosa, lymphocyte adherence occurred without rolling and was independent of alpha 4-integrins. LPS also significantly increased adherence of lymphocytes to villus capillaries, which was not mediated by either alpha 4- or beta 2-integrin. These observations demonstrate significant heterogeneity of lymphocyte-endothelial cell interactions within different regions of the intestinal mucosa.
Animals↗
Roles of ET-1 in endotoxin-induced microcirculatory disturbance in rat small intestine.
The major objective of this study was to investigate whether endothelin-1 (ET-1) plays a significant role in endotoxin-induced microcirculatory disturbances of the intestinal mucosa. Submucosal microvessels of the rat ileum were observed by intravital microscopy with a high-speed video camera system. Preceding the apparent intestinal mucosal damage, red blood cell (RBC) velocity was significantly decreased 30 min after endotoxin treatment in both arterioles and venules. The number of leukocytes sticking to submucosal venules was significantly increased at 30 min. BQ-123, an ETA-receptor antagonist, significantly attenuated the decrease in RBC velocity and also prevented an increase in leukocyte sticking as well as the subsequent mucosal damage induced by endotoxin. The ET-1 concentrations began to be elevated in plasma at 15 min and in the mucosa at 30 min and subsequently further increased in a time-dependent manner. A significant decrease in calcium-dependent nitric oxide synthase activity and significant increases in the concentration of platelet-activating factor (PAF) were demonstrated in the intestinal mucosa after endotoxin treatment. BQ-123 also significantly attenuated these changes. We concluded that the increased ET-1 production in intestinal mucosa induced by endotoxin stimulation could lead to leukocyte sticking and decreased RBC velocity in the intestinal microcirculatory beds via ETA receptors, which are closely related to increased production of PAF and decreased synthesis of constitutive nitric oxide.
Animals↗
Growth hormone receptor gene is expressed in neuropeptide Y neurons in hypothalamic arcuate nucleus of rats.
GH feeds back on the hypothalamus and regulates its own secretion. We have previously shown that systemic administration of GH induces expression of the c-fos gene, a marker of neuronal activity, on the hypothalamic neuropeptide Y(NPY) and somatostatin neurons in rats. We argued that if GH were to act directly on NPY neurons, NPY neurons should express the GH receptor (GHR) gene. To test this hypothesis, coronal sections of the medial basal hypothalamus from adult male Wistar rats were processed by double label in situ hybridization using a 35S-labeled NPY complementary RNA probe and a digoxigenin-labeled GHR complementary RNA probe. In the medial basal hypothalamus, NPY messenger RNA (mRNA) was observed in the arcuate nucleus (ARC) and the dorsomedial nucleus. The majority (95%) of NPY mRNA-containing cells in the ARC expressed the GHR gene, whereas no NPY mRNA-containing cells in the dorsomedial nucleus expressed the GHR gene. These findings suggest that NPY neurons in the ARC mediate the feedback effect of GH on the hypothalamus.
Animals↗
Perivascular coating with fibrin glue of cerebral arteries in patients with aneurysmal subarachnoid hemorrhage; incidence of chronic hydrocephalus.
The incidence of chronic hydrocephalus was studied in 39 patients with subarachnoid hemorrhage, who underwent perivascular coating with fibrin glue of cerebral arteries after clipping of aneurysm. A use was made of this procedure in order to prevent vasospasm by keeping the main cerebral arteries away from direct contact with subarachnoid clots. Most cases in this series belonged to group 3 of Fisher's CT grade (33/39, 84.6%). As a result, despite the high CT grade, the incidence of chronic hydrocephalus was as low as 17.9% (7/39), almost in agreement with those of the previous literature. In conclusion, (1) coating with fibrin glue did not increase the incidence of chronic hydrocephalus and (2) intrathecal application of fibrin glue is a promising method in the field of clinical neurosurgery.
Adhesives↗
Anomalous internal carotid artery-anterior cerebral artery anastomosis associated with fenestration and cerebral aneurysm.
A 69-year-old female presented with a ruptured aneurysm at the distal tip of a fenestrated anomalous artery which originated in the ophthalmic segment of the internal carotid artery (ICA), passed between the optic nerves, ascended along the midline, and formed the fenestration before dividing into the bilateral pericallosal arteries. The anomalous artery manifested as subarachnoid hemorrhage. Four days later, she died from severe pulmonary edema. The angiographic finding was confirmed by postmortem examination. Numerous perforating vessels from the anomalous artery and the distal ICA supplied the optic nerves and chiasm. This finding supports the idea that this anomalous vessel may be an extreme developmental variation of the prechiasmal arterial plexus.
Adult↗
[A case study of nonfunctioning islet cell tumor in patient with splenic tumor and increased AFP].
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Adenoma, Islet Cell↗
Ethanol-induced apoptosis and oxidative stress in hepatocytes.
This short review focuses on ethanol-induced oxidative stress and hepatocyte apoptosis. Apoptosis is increasingly recognized as a fundamental biological process that impacts on an early development, maturation, and acquisition of disease states of multicellular organisms. Although the occurrence of apoptosis has been identified for many decades, relatively recent acceptance of this principle is evidenced by remarkable increases in special conferences and presentations on this topic as well as its rapidly expanding volume of scientific literature. Oxidative stress is well recognized to be a key step in the pathogenesis of ethanol-associated liver injury. Ethanol administration induces an increase in lipid peroxidation either by enhancing the production of oxygen reactive species and/or by decreasing the level of endogenous antioxidants. Studies in our laboratory using a confocal laser scanning microscopic system strongly suggest that agents which inhibit ethanol-induced oxidative stress effectively attenuate hepatocyte death, i.e., apoptosis and necrosis. In addition, our investigations demonstrated that inhibitors of intracellular antioxidants exaggerate ethanol-associated hepatocyte apoptosis. Although the detailed mechanism still remains unknown, it is conceivable that an oxidant-dependent mechanism is largely involved in the process for ethanol-induced hepatocyte apoptosis.
Animals↗
Ethanol-induced leukocyte adherence and albumin leakage in rat mesenteric venules: role of CD18/intercellular adhesion molecule-1.
The adherence and emigration of leukocytes have been implicated as a rate-limiting step in the microvascular disturbance in a variety of pathogenic events. The objective of the present study was to investigate leukocyte-endothelial cell adhesion and endothelial barrier function in rat mesenteric microvessels exposed to ethanol, which is known to cause inflammation and injury in various organs. Mesentery of male Wistar rats was used for intravital microscopic observations. Leukocyte adherence and albumin leakage were monitored in single postcapillary venules using the intravital fluorescence microscope. Superfusion of 50 mM ethanol elicited the leukocyte adherence and albumin leakage within 60 min. Pretreatment with a monoclonal antibody directed against either CD18 or intercellular adhesion molecule-1 (ICAM-1) significantly prevented the ethanol-induced increase in leukocyte adherence and decrease in barrier function of endothelium. These results suggest that ethanol-induced leukocyte adherence is mediated by CD18 on leukocytes and ICAM-1 on endothelial cells. The present study further supports that CD18/ ICAM-1-dependent leukocyte-endothelial adhesive interactions lead to macromolecular leakage in the postcapillary venules exposed to ethanol.
Animals↗