Morphological characteristics of the deep layer of articularis genus muscle.

BACKGROUND: The articularis genus muscle pulls the suprapatellar pouch upwards when the knee joint is extended, preventing mechanical impingement of the joint capsule which theoretically could cause anterior knee pain. However, few anatomical studies have addressed this muscle. Here we present the precise morphology of articularis genus. MATERIALS AND METHODS: A total of 22 (13 male and 9 female) adult cadavers with no pathological conditions in the knee joints were examined during educational dissection at Nagoya City University Medical School in 2012. After exclusion of 4 joints due to their flexion contracture, 40 knee joints (18 right and 22 left) were analysed. We performed statistical analysis on anatomical laterality and the difference of sizes among lateral, medial and central branches and studied the correlation of the length and area of the articularis genus muscle to the lengthand cross-section area of the femur. RESULTS AND CONCLUSIONS: The average number of branches of the deep layer of the articularis genus muscle was 2.7 ± 0.5, the mean length of all brancheswas 5.4 ± 1.3 cm and the mean area of all branches was 5.5 ± 2.6 cm². There was no significant correlation between the length and area of the articularis genus muscle to the length and cross-section area of the femur. There was no significant laterality in central, medial and lateral branches; however we found that the medial branch was statistically longer and larger than the lateral branchon either knee. This could be contributing to prevention of lateral dislocation of the patella.

NADH-generating substrates reduce peroxyl radical toxicity in RL-34 cells.

There is general agreement that oxidative stress may induce apoptotic and necrotic cell death. Recently it has been shown that NADH can be considered an important antioxidant as it reacts with peroxyl and alkoxyl radicals under in vitro conditions. Therefore, in the present study we hypothesized that an increase in intracellular NADH using specific substrates will protect RL-34 cells against cytotoxicity of 2'-azobis (2-amidinopropane) dihydrochloride (AAPH), which is a peroxyl radical generating compound. Cells treated for 24 hours with 6.0 mM AAPH were severely damaged: mitochondria were vacuolated, and the level of free radicals significantly increased. Both apoptotic and necrotic cells were detected (11.1% and 11.4%, respectively) even after 5 hours of treatment. Pretreatment of the cells with substrates which increase the intracellular level of NADH, such as lactate, beta-hydroxybutyrate, and ethanol, distinctly inhibited AAPH-induced reactive oxygen species (ROS) formation and cell death. On the other hand, acetoacetate (AcA), which decrease the intracellular level of NADH, had opposite effects. Interestingly, NADH-generating substrates augment, while AcA reduced superoxide radical formation induced by AAPH. These results may suggest that although NADH generating substrates may exert some deleterious effects within a cell by inducing reductive stress, they diminish alkoxyl or peroxyl radical cytotoxicity. The protection is associated with a decrease in ROS formation measured by dichlorofluorescein, but with an increase in superoxide radical formation.

The co-existence of an aberrant origin of the right subclavian artery and a coronary myocardial bridge.

We encountered the co-existence of an aberrant origin of the right subclavian artery and a myocardial bridge on the left anterior descending coronary artery in the cadaver of an 80-year-old Japanese woman during the course of educational dissection at Nagoya City University Medical School. We document the precise gross anatomical findings with some morphometric measurements. Neither an aberrant origin of the right subclavian artery nor the cardial myocardial bridge is a very rare anomaly, but a case of both anomalies being found in the same body is very rare. We believe this is the first report of the simultaneous occurrence of these two anomalies.

Thymic sarcomatoid carcinoma with skeletal muscle differentiation: report of two cases, one with cytogenetic analysis.

AIMS: Malignant thymic tumour histologically resembling a soft tissue sarcoma is extremely rare and defined as sarcomatoid carcinoma in the recent World Health Organization (WHO) classification. We report two such cases in which the tumour cells showed a prominent rhabdomyoblastic differentiation and analyse whether these tumours retain an epithelial nature at least in part. METHODS AND RESULTS: One tumour occurred in a 51-year-old man (Case 1) and the other in a 40-year-old woman (Case 2). Microscopically, both tumours consisted essentially of two types of tumour cells: spindle and large round cells, with no apparent epithelial components. Osteosarcomatous small foci were also found in Case 2. Immunohistochemically, desmin and muscle-specific actin were positive in the majority of both types of tumour cells, whereas myogenin was predominant in the spindle cells and myoglobin in the large round cells. Some of both types of cells expressed cytokeratin with co-expression of myoglobin in the large round cells, but with no myogenin in the spindle cells. Some cytokeratin-positive spindle cells were also negative for desmin. Ultrastructural examination of a recurrent tumour in Case 2 revealed some epithelial features among the spindle cells. Cytogenetic study of the same tumour showed a complex abnormality including der(16)t(1;16)(q12;q12.1), an identical pattern previously reported in a case of thymic squamous cell carcinoma. CONCLUSIONS: The findings support the definition in the WHO classification of sarcomatoid carcinoma that includes purely sarcomatous tumour as in the present cases. Occurrence of this type of tumour may indicate a relationship between thymic epithelial cells and myoid cells and/or a potential for divergent differentiation in thymic epithelial tumours.

Gap junctional communication between the satellite cells of rat dorsal root ganglia.

Many studies have described the ultrastructure of the dorsal root ganglia in various embryonic and adult animals, but in spite of the efforts of many investigators the functional role of the satellite cells in this tissue is not clearly understood. In this study, we discuss the function of this cell type based on the concept of cell-to-cell interaction through gap junctions. Five male 60 day-old Wistar strain rats were used. All animals were anesthetized with pentobarbital and perfused with glutaraldehyde fixative, then the dorsal root ganglia in levels L4, L5 and L6 were taken from each rat. After postosmication, the specimens were prepared for observation by transmission electron microscopy. All nerve cells were completely surrounded by satellite cell cytoplasmic expansions. The boundaries between adjacent nerve cells and satellite cells were complicated due to the presence of perikaryal projections of nerve cells. Gap junctions which showed the typical trilamellar structure of plasma membranes were found mainly between satellite cell processes belonging to the same nerve cell. On the other hand, some gap junctions were found between the satellite cell projections belonging to different nerve cells. The size of the gap junctions ranged from 300 to 400 nm. No gap junctions were associated with the plasma membrane of any nerve cell. In conclusion, only satellite cells can share free transcellular exchange of cytoplasmic molecules such as ions, amino acids, sugars and several second messengers including cAMP and inositol 1,4,5-triphosphate by way of gap junctions in dorsal root ganglia.

Calcium oxalate crystal attachment to cultured rat kidney epithelial cell, NRK-52E.

Madin-Darby canine kidney (MDCK) cells have been used in research on crystal adhesion to epithelial cells. Recently, matrix proteins were identified, and studies of the genes and proteins expressed in renal epithelial cells have become active. The present study confirms the usefulness of the NRK-52E cell line, derived from the rat, in the study of attachment with calcium oxalate crystals. The calcium oxalate crystal suspension was distributed on top of the cells. After incubation, the monolayers were rinsed to remove non-associated crystals. After fixation, the association of crystals and NRK-52E cells was visualized using scanning electron microscopy. Calcium oxalate crystals were attached to the surface of NRK-52E cells. Under high magnification, many of the microvilli of the cells had elongated towards the crystals, and microvilli projections appeared to catch the crystals. The NRK-52E cell line is useful in the study of attachment between crystals and urinary epithelial cells in the kidney, especially for the regulation and analysis of genes and proteins.

Effects of hydrocortisone on the formation of gap junctions and the abnormal growth of cilia within the rat anterior pituitary gland: possible role of gap junctions on the regulation of cell development.

We investigated the effects of hydrocortisone on the formation of gap junctions in and the growth of cilia on folliculo-stellate cells. The male rats of experimental groups were given daily intraperitoneal injections of 5 mg/kg of hydrocortisone from Day 20 to 60. Five rats were killed at ages 10, 20, 30 and 40 days after initiation of injections, and the pituitary gland was removed from each rat. Then, the specimens were prepared for observation by transmission electron microscopy. A delay in the formation of gap junctions between folliculo-stellate cells was observed in hydrocortisone treated rats compared with control rats on Day 30, 40 and 50. Another finding in the present study was the increase of ciliated follicles on Day 40 and 50 in the hydrocortisone treated groups, simultaneous with the delay in gap junction formation. The results suggest that hydrocortisone has a suppressive effect on the gap junction formation between folliculo-stellate cells, and loss of intercellular communication by way of gap junctions may lead to alteration of morphological development of the cell.

Swelling of free-radical-induced megamitochondria causes apoptosis.

Recently, we have found that cultured cells from various sources exposed to free radicals become apoptotic in the presence of megamitochondria (MG). The purpose of the present study is to answer the following two questions: (1) Do functions obtained from the "MG fraction" isolated from normal mitochondria by a routine procedure represent the functions of MG since the fraction consists of enlarged and normal-size mitochondria? (2) What is the correlation between MG formation and apoptotic changes of the cell? In the present study the heavy fraction rich in mitochondria enlarged to varying degrees and the light fraction consisting mainly of normal-size mitochondria were isolated independently from the livers of rats treated with hydrazine for 4 days (4H animals) and 8 days (8H animals), and some functions related to apoptosis were compared. Results were as follows: (1) Mitochondria in both fractions obtained from 8H animals swelled far less in various media than those obtained from the controls, suggesting that the permeability transition pores had been opened before they were exposed to swelling media. (2) The membrane potential of mitochondria in both fractions obtained from 8H animals was distinctly decreased. (3) The rates of reactive oxygen species generation from mitochondria of both fractions in 4H animals were equally elevated, while those in 8H animals were equally decreased compared to those of controls. These results, together with morphological data obtained in the present study, suggest that enlarged and normal-size mitochondria are a part of MG and that the secondary swelling of MG causes the apoptotic changes in the cell.

Functional aspects of megamitochondria isolated from hydrazine- and ethanol-treated rat livers.

It is essential to analyze functions of megamitochondria (MG) to elucidate the mechanism of the formation of MG induced under various pathological conditions. The MG fraction obtained by a routine isolation procedure for normal mitochondria always consists of a mixed population of mitochondria enlarged to various degrees and also normal-sized ones. The purpose of the present study is to answer the question of whether or not data obtained from the MG fraction consisting of such a heterogeneous population of mitochondria with respect to their sizes really reflect functions of MG. In the present study mitochondria were obtained from the livers of rats treated with a 1% hydrazine diet for 8 days and those given 32% ethanol in drinking water for up to 2 months using various isolation procedures. Results obtained are summarized as follows: (i) mitochondria enlarged to various degrees and normal-sized ones are sometimes connected with each other by a narrow stalk in the hepatocyte of hydrazine-treated animals, and such connections are maintained to some extent when mitochondria are isolated; and (ii) mitochondria obtained from experimental animals by a routine isolation procedure for mitochondria ((700-7000)gR2"') and those obtained by alternative isolation procedure yielding the heavy ((500-2000)gR2"') and light ((2000-7000)gR2"') fractions show some functional similarities: decreases in the content of cytochrome a + a3; decreases in oxygen consumptions and phosphorylating abilities; decreases in monoamine oxidase and cytochrome c oxidase activities; lowered membrane potential of mitochondria; decreases in the rate of the generation of reactive oxygen species. These results may suggest that mitochondria enlarged to various degrees and normal-sized ones are functionally similar to each other and that the MG fraction obtained by a routine isolation procedure for normal mitochondria can be applied to the study of the function of MG.

Postnatal development of synovial capillaries of rats with special reference to permeability.

The size of a substance is a major factor determining whether it can permeate the wall of synovial capillaries. The maximum diameter of particles that can move across the synovial capillary wall has generally been thought to be 50 nm. We studied the permeability of the synovial capillaries of the rat between day 20 and 30 after birth using a polystyrene particle whose diameter was 240 nm. In addition using lecithin-coated polystyrene particles, we studied the maturation of the barrier function supported by endothelial and peripheral cells against foreign bodies. Lecithin-coated particles were found within the fibroblast-like synovial cells near the capillary in the 20 day-old rats, while non-coated particles remained in the endothelial wall and in the peripheral cells of capillaries. In the 30 day-old rats, lecithin-coated particles were present in the peripheral cells and the neighboring synovial cells; however, the non-coated particles were never found in the synovial or perisynovial cells. The present study shows that the size of the transportable substance by transcytosis may be larger than previously thought. Furthermore, the synovial capillaries functionally changed between day 20 and 30 suggesting that active movement of the joint led to the functional maturation of the synovial capillaries.

Two types of the enlargement of mitochondria related to apoptosis: simple swelling and the formation of megamitochondria.

Our recent finding that free radical-induced formation of megamitochondria (MG) is followed by apoptosis has prompted us to investigate the correlation between the MG formation and the swelling of mitochondria which is considered to play a key role in early stages of apoptotic processes of the cell. Mitochondria of rat hepatocytes or RL-34 cells and those isolated from rat livers became enlarged up to three times in their diameters when they were exposed to a hypotonic medium. MG induced in the liver of rats placed on a 1% hydrazine-diet for 4-5 days or those induced in the liver of mice placed on a 2% chloramphenicol (CP)-diet for 9-10 days were endowed with a dense matrix whereas those fed with the toxic diets for longer periods of time became enlarged further and their matrix became extremely pale indicating that MG in the latter animals became swollen secondarily. The membrane potential, the content of cytochrome c and the rate of the generation of reactive oxygen species (ROS) of MG in the former animals were almost unchanged compared to those of mitochondria in control animals whereas those of MG in the latter animals became distinctly decreased. These results may suggest that free radical-induced MG possibly cause apoptosis via their secondary swelling.

Effects of coenzyme Q10 on changes in the membrane potential and rate of generation of reactive oxygen species in hydrazine- and chloramphenicol-treated rat liver mitochondria.

Effects of CoQ10 and cycloheximide (CHX) on hydrazine- and chloramphenicol (CP)-induced morphological and some functional changes of mitochondria using cultured rat hepatocytes and effects on the process of recovery from CP intoxication using mouse liver were examined. Results obtained are summarized as follows: (1) The formation of megamitochondria induced in the hepatocytes cultured for 22 h in the presence of 2 mM hydrazine or CP (300 microgram/ml) was suppressed by pretreatment of hepatocytes with CoQ10 (1 microM) or CHX (0.5 microgram/ml). This was proved by electron microscopic analysis of mitochondria. (2) Treatment of hepatocytes with hydrazine for 48 h or longer caused decreases in the membrane potential of mitochondria, which were suppressed by CoQ10. (3) Treatment of hepatocytes with hydrazine for 22 h or longer caused remarkable increases in intracellular levels of reactive oxygen species in hepatocytes, which were suppressed by CoQ10. (4) The process of recovery from the CP-induced changes of mitochondria in mouse liver was accelerated by CoQ10 and CHX.

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria.

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.

Anti-rheumatic compound aurothioglucose inhibits tumor necrosis factor-alpha-induced HIV-1 replication in latently infected OM10.1 and Ach2 cells.

NF-kappaB is a potent cellular activator of HIV-1 gene expression. Down-regulation of NF-kappaB activation is known to inhibit HIV replication from the latently infected cells. Gold compounds have been effectively used for many decades in the treatment of rheumatoid arthritis. We previously reported that gold compounds, especially aurothioglucose (AuTG) containing monovalent gold ion, inhibited the DNA-binding of NF-kappaB in vitro. In this report we have examined the efficacy of the gold compound AuTG as an inhibitor of HIV replication in latently infected OM10.1 and Ach2 cells. Tumor necrosis factor (TNF)-alpha-induced HIV-1 replication in OM10.1 or Ach2 cells was significantly inhibited by non-cytotoxic doses of AuTG (>10 microM in OM10.1 cells and >25 F.M in Ach2 cells), while 25 microM of the counter-anion thioglucose (TG) or gold compound containing divalent gold ion, HAuCl3, had no effect. The effect of AuTG on NF-kappaB-dependent gene expression was confirmed by a transient CAT assay. Specific staining as well as electron microscopic examinations revealed the accumulation of metal gold in the cells, supporting our previous hypothesis that gold ions could block NF-kappaB-DNA binding by a redox mechanism. These observations indicate that the monovalent gold compound AuTG is a potentially useful drug for the treatment of patients infected with HIV.

Inhibition of IL-6 and IL-8 induction from cultured rheumatoid synovial fibroblasts by treatment with aurothioglucose.

Gold compounds have long been used in the treatment of rheumatoid arthritis (RA). However, their actions in RA have not been clarified. In this study, we examined the effect of one of the monovalent gold compounds, aurothioglucose (AuTG), on the IL-1-induced production of IL-6, IL-8 and granulocyte macrophage colony stimulating factor (GM-CSF) from rheumatoid synovial fibroblasts (RSF) isolated from three RA patients. IL-6 and IL-8 induction but not GM-CSF induction was inhibited in most of the RSF after pretreatment with AuTG. Since gene expression of these cytokines is known to be under the control of a common transcription factor, NF-kappaB, the effect of AuTG on the cellular localization of NF-kappaB (p65 subunit) and on NF-kappaB-DNA binding was examined. Although AuTG treatment did not prevent NF-kappaB nuclear translocation, AuTG blocked the DNA-binding activity of NF-kappaB when examined in vitro. Morphologically, both metal-specific cell staining using p-dimethylaminobenzylidene rhodamine and transmission electron microscopic examinations demonstrated the accumulation of metal gold in the cytoplama and some organella (mitochondria and lysosomes) of the AuTG-treated RSF. These results indicate that one of the anti-rheumatic actions of AuTG might be through its inhibitory action on NF-kappaB.

Free radical-induced megamitochondria formation and apoptosis.

Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H2O2, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H2-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G1 phase, and apoptotic sub-G1 populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H2O2 and O2- from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.

Biogenesis and function of lipolysosomes in developing chick hepatocytes.

Proliferation of lipolysosomes is one of the characteristic aspects of embryonic chick hepatocytes. Formation of lipolysosomes is observed in the well-developed trans-Golgi network, with the highest frequency occurring from 11 to 14 days of incubation. The lipolysosomes usually contain a small and electron-dense lipid inclusion; however, during development, they gradually enlarge with an accompanying reduction in the electron density of the inclusion. Lipolysosomes isolated from neonatal chick liver homogenates were mainly composed of esterified cholesterol and showed considerably high activity of lysosomal enzymes. Moreover, the lipolysosome fraction is clearly shown to be a function of intralysosomal lipolysis via acid lipase. This accumulation of esterified cholesterol within lipolysosomes might be attributed to an excessive uptake and conversion of plasma lipoproteins to lipolysosomes. This concept is supported by the appearance of an abundance of coated pits and both "early" and "late" endosomes. The major components of plasma lipoprotein are low density lipoprotein (LDL) and high density lipoprotein (HDL), the cholesterol-rich lipoproteins, whose cholesterol content increases during the last week of incubation when the lipolysosomes quickly enlarge. Plasma lipoprotein particles are produced in the yolk sac epithelium from yolk very low density lipoprotein (VLDL) and transferred via the vitelline circulation to the chick liver. After hatching, when the supply of nutrients from the yolk sac is terminated, lipolysosomes immediately decrease in size and number. The cholesterol and fatty acids released are useful as an energy source and lipid metabolism in general, especially after hatching. Food intake induces the use of and accelerates the disappearance of lipolysosomes. Instead of lipolysosomes, lipid droplets appear and increase in number and size with concomitant increases of triglyceride concentrations in the liver homogenates, suggesting that lipogenesis has begun in the chick hepatocyte.

Folliculo-stellate cells and intercellular communication within the rat anterior pituitary gland.

Folliculo-stellate (FS) cell are agranular and arranged around a follicle. They contain the S-100 protein and beta-adrenergic receptors. It has been suggested that they can act as stem cells, since they show mitotic figures, and could transform into granular or chromophilic cells according to the concept of a "cell renewal system." Cell-to-cell interactions among pituitary cells have been described, and recent progress with freeze-fracture electron microscopy has provided novel observations of the cell surface and gap junctions within the rat or teleost fish pituitary gland, or in cultured rat pituitary cells. In adult rats, the anterior pituitary was composed of lobules incompletely separated by a basement membrane. Follicles consisted exclusively of FS cells. Gap junctions were observed only between adjacent FS cells, in rare cases on the tips of their cytoplasmic processes. Thus, the FS cells, connected by gap junctions, made up a dense cellular network throughout the pituitary. Gap and tight junctions were absent on granular cells. Elongated follicles with columnar FS cells were observed in 10-day-old rats and were separated into smaller units. The number of gap junctions rapidly increased with age until 40-45 days of age. Few S-100 protein positive cells were observed on day 10, along the marginal cell layer and near the so-called postero-lateral wing. The frequency of positive cells increased with age and by day 40; numerous cells were observed throughout the anterior lobe. Gap junction number also varied with the stage of the estrous cycle, and frequency; during diestrus, they were half of that during proestrus or estrus. The number of gap junctions increased in late pregnancy and in lactating rats, probably due to changes in estrogen and progesterone. Hormone (LH-RH and testosterone) treated groups of rats showed accelerated development by almost 10 days, compared with controls. In castrated male rats, the ultrastructure of the pituitary remained immature even at 40 days of age, when the number of gap junctions was a quarter or less than the number in intact rats. Testosterone treatment restored the frequency of gap junctions to a normal level. We conclude that the appearance of gap junctions in the pituitary cells and maturation of the gland are dependent to a large degree upon gonadal steroids.

Effects of ruthenium red on the cellular functions and ultrastructure in intact ferret ventricular muscles.

The effects of ruthenium red (RR) on the cellular functions (intracellular Ca2+ handling and contraction) and permeation of the dye through the cell membrane were investigated in intact ferret papillary muscles. The intracellular Ca2+ concentration ([Ca2+]i), measured using aequorin, was simultaneously recorded with tension. The permeation of the dye through the cell membrane was studied with electronmicroscopy. The preparation was continuously stimulated at 0.2 Hz and treated with 50 microM RR at 30 degrees C. [Ca2+]i was increased by electrical stimulation (0.07 and 2 Hz) and rapid cooling (from 30 to 4 degrees C) (RC). In electrical stimulation, RR time-dependently decreased the peak light of aequorin without a significant change in the time course at 30 degrees C. However, in RC, treatment with RR for about 100 min significantly prolonged the decay time of the light signal and increased the peak light. The peak tension in RC was decreased after treatment with RR for a longer time. The pCa-tension relation of skinned preparations was significantly shifted to the right by 50 microM RR. In the RR (50 microM)-treated specimens, mitochondrial outer membranes were darkly stained if OsO4 was used for fixation. Even though the specimen treated with 500 microM RR was fixed without OsO4 and electron staining, the matrices of mitochondria became electron dense. We concluded that RR could penetrate into intact mammalian cardiac myocytes, and that RR inhibits the release of Ca2+ from the sarcoplasmic reticulum in electrical stimulation, inhibits mitochondrial Ca2+ uptake, and decreases the Ca2+ sensitivity of the myofilaments.

Endotoxin-induced upregulation of type I interleukin-1 receptor mRNA expression in hepatocytes of mice: role of cytokines.

Interleukin-1 (IL-1) signal is transduced through the type I IL-1 receptor (IL-1RI). Although regulation of IL-1R expression has been extensively studied in vitro, little is known about it in vivo. By using RT-PCR analysis, we investigated the regulation of the IL-1RI mRNA expression level in various organs of mice at 2, 6, and 24 h following lipopolysaccharide (LPS) administration. IL-1RI mRNA expression in response to LPS appeared to be different in various organs. As a marked and sustained increase of IL-1RI mRNA expression in the liver was observed, we investigated the mechanism of the upregulation. IL-1, IL-6, and tumor necrosis factor (TNF) all increased the mRNA expression in the liver when administrated in vivo. In situ hybridization revealed that upregulation of IL-1R mRNA was observed in parenchymal liver cells (hepatocytes) in response to LPS administration. When primary cultured hepatocytes were treated in vitro, IL-1, IL-6, conditioned medium from LPS-treated mouse macrophages, and serum from LPS-treated mouse upregulated IL-1RI mRNA expression, but LPS, TNF, and prostaglandin E2 failed to do so. Therefore, these results suggest that the upregulation of IL-1RI mRNA in the hepatocytes by LPS administration is mediated by cytokines, especially by IL-1 and IL-6. The results also indicate that the regulation is different in different organs, and microenvironmental factors may be important.