Studies on Cellular Autophagocytosis: Cyclic AMP- and Dibutyryl Cyclic AMP-Stimulated Autophagy in Rat Liver

Intraperitoneal injections of either cyclic AMP or dibutyryl cyclic AMP are capable of producing a wave of autophagy in rat liver which resembles closely that stimulated by glucagon. These autophagic vacuoles are acid-phosphatase-positive and appear to arise via the envelopment of organelles by double-walled cisterna. At later times these autophagic vacuoles are usually surrounded by a single membrane. Injection of equimolar amounts of 5′-AMP failed to produce a wave of autophagy. These findings constitute preliminary evidence that glucagon-stimulated autophagy is mediated at least in part by cyclic AMP.     

Electron Microscopic and Histochemical Studies on Vacuolar Alteration of Proximal Convoluted Tubules of Rat Kidney after Hypertonic Sucrose Administration

Changes of the proximal convoluted tubular epithelium of the rats which were sacrificed instantaneously at 5, 15, 30 minutes, 1, 3, 6, and 8 hours after intraperitoneal injections of 40 to 52 cc per kg of a 50 % aquenous solution of sucrose, were observed by electron microscopic and histochemical methods. The results obtained can be summarized as follows:

• 1) 

  The vacuoles begin to appear 15 minutes after the injection in the apical portion of the tubular cytoplasm and become distinct in 30 minutes. After 6 hours, it is observed in the entire cytoplasm which is filled with large vacuoles.
• 2) 

  The vacuoles originate from dilatation of simple pinocytotic vesicles which exist physiologically beneath the brush border microvilli. The small vacuoles of 1 to 1.5 microns in diameter reveal heavy acid phosphatase activity and have temporarily a character of so-called lysosome or cytosome. With the enlargement of vacuoles, the surrounding single limiting membrane reveals discontinuity or rupture at numerous sites. At this stage, the vacuoles aggregate and fuse with each other to form larger ones of 4 to 4.5 microns in diameter and lose their acid phosphatase activity.
• 3) 

  Vacuolar alteration of the proximal convoluted tubules and the role of lysosome and FCD are discussed.

     

The Morphologic Effects of Dieldrin and Methyl Mercuric Chloride on Pars Recta Segments of Rat Kidney Proximal Tubules

This investigation was undertaken to evaluate the morphologic effects in rat kidney resulting from chronic exposure to low doses of the pesticide dieldrin, methyl mercuric chloride (CH₃HgCl) and the combination of dieldrin plus CH₃HgCl. Histologic and ultrastructural changes were confined to the proximal tubules. Alterations in these tubules were consistent and reproducible for each regimen and did not become more severe with duration of exposure. The straight segment of the proximal tubule (pars recta) was more severely affected by dieldrin and CH₃HgCl than the convoluted portion. Female rats were more markedly affected than males. Pars recta tubule cells of male and female rats exposed to dieldrin showed an increase of smooth endoplasmic reticulum (SER). Male rats displayed a greater increase in SER than females. Pars recta tubule cells of animals given CH₃HgCl also exhibited increased amounts of SER, degenerating mitochondria and cell death. Pars recta tubules of females were dilated and contained within the lumens many spherical, hematoxylin-positive staining, cytoplasmic masses, which were visible by light microscopy. These masses were characterized ultrastructurally by the presence of an SER aggregate in an area of material similar to cell matrix. In addition, cells of the pars recta of female animals contained electron-dense membranous cytosomes not present in control animals. Pars recta cells of males showed an increase in SER, but the dense membranous cytosomes observed in the pars recta cells of female rats were not seen. Rats exposed to dieldrin plus CH₃CgCl showed less morphologic alteration of the pars recta tubules than animals given methyl mercuric chloride; however, increased amounts of SER and more degeneration in tubule cells were observed in these animals when compared to control animals. The findings are discussed in relation to the conversion of CH₃HgCl to inorganic mercury in vivo and the known toxicity of inorganic mercury to the pars recta. Decreased tubular alteration in males and dieldrin-treated animals may be explained by sexual differences in renal enzyme levels or activities and the induction of microsomal enzyme systems by dieldrin.     

Influence of Peritubular Hydrostatic and Oncotic Pressures on Fluid Reabsorption in Proximal Tubules of the Rat Kidney

Proximal tubular reabsorption was measured with the split droplet technique under the following conditions. Group I. Star vessel perfusions with ultrafiltrate in the presence of human serum albumin (HSA) and with ultrafiltrate alone. Group 2. Capillary perfusions, under locally high flow rates, with ultrafiltrate in the absence or presence of HSA. Group 3. Subcapsular perfusions with ultrafiltrate of high HSA concentrations but under low hydrostatic pressure and ultrafiltrate of low HSA concentrations but under elevated hydrostatic pressure. In Group I, the presence of HSA increased the reabsorptive rate, whereas ultrafiltrate alone had no provable effect on the reabsorptive rate. In Group 2, a flow dependent decrease of the reabsorptive rate, that was not influenced by the presence of HSA, was observed. Finally, in Group 3, high concentrations of HSA did not alter the reabsorptive rate, whereas elevation of the hydrostatic pressure decreased the reabsorptive rate. The results cannot be explained on the basis of a simple passive mode of action of colloid osmotic and hydrostatic pressure on the reabsorptive rate. The possibility of a direct tubulae capillary transport route is discussed.     

胸腺在大鼠肝脏自由基代谢中的作用

本文以成年去胸腺大鼠为模型,旨在观察胸腺对肝脏自由基代谢的影响。实验结果表明,雌性去胸腺大鼠和雌、雄性老年大鼠肝匀浆及肝微粒体过氧化脂质增多,肝匀浆超氧化物歧化酶活力和还原型谷胱甘肽含量降低,但雄性去胸腺大鼠上述指标与对照组相比无明显变化。     

Effect of Pollack Liver Oil on Lipid Metabolism in Rat Testes

Rat testes were examined by biochemical and histochemical methods after 1, 3, 6, and 12-month pollack liver fat-supplemented diet (0.1 g/kg). Phospholipid concentration in the testicular tissue increased by 16% and cholesterol concentrations decreased by almost 20% after feeding fish oil for 1-3 months, while after 6-12-month diet further increase in cholesterol level in the testes and degeneration of some gonadal cells were noted. The most pronounced changes were seen in more differentiated elements of the spermatogenic epithelium in convoluted tubules.     

Protein Synthesis in Yolk Sac Tumor: Histochemical Studies of Human and Rat Yolk Sac Tumor

Synthesis of various serum proteins, such as albumin, prealbumin, α₁-antitrypsin, transferrin and α-fetoprotein, by human and rat yolk sac tumors was studied by indirect immunofluorescence and immunoperoxidase techniques. These serum proteins were found to be present in the cytoplasm of tumor cells forming the lining of vitelline cysts, as well as Schiller-Duval bodies and vacuolated meshwork on histological sections. Two of these tumors were cultured. Immunopathological studies revealed that albumin, prealbumin, α₁-antitrypsin, transferrin and α-fetoprotein were found in the cytoplasm of these cultured tumor cells. The immunofluorescence of these proteins was also found on the PAS-positive granules, which existed intra- and extra-cellularly.
The ability to synthesize these serum proteins of these tumor cells was not affected by tissue culture and was maintained during subcloning.
It was demonstrated by electron microscope ferritin antibody technique that α-fetoprotein was mainly synthesized on the ribosomes of the rough endoplasmic reticula of rat yolk sac tumor cells.     

In Vitro Work Load and Rat Heart Metabolism: III. Effect on ribosomal aggregation

In two preceding papers increased pressure load (afterload) was found to accelerate whole heart protein synthesis and amino acid transport in the perfused working rat heart (Hjalmarson and Isaksson 1972, Ahren et at. 1972). To further analyse this effect, sucrose gradient analysis of the postmitochondrial supernatant was performed to evaluate reactions involved in the ribosome cycle. When hearts were perfused in vitro under control conditions with buffer containing normal plasma levels of amino acids and with glucose as substrate, levels of polysomes decreased and levels of ribosomal subunits increased. These findings together with a decreased rate of protein synthesis indicated that a block in initiation of peptide chains had developed during perfusion. Perfusion of hearts with increased afterload increased the levels of polysomes and decreased the levels of ribosomal subunits and accelerated protein synthesis. The effects were obtained both when glucose and palmitate were used as substrate. Insulin further increased the levels of polysomes in pressure overloaded hearts and the ribosome profiles attained by overload in presence of insulin were identical to those obtained from unperfused hearts. Increased levels of polysomes and decreased levels of ribosomal subunits together with an increase in protein synthesis suggested that pressure overload stimulated reactions involved in the initiation of polypeptide chains. It is concluded that increased pressure load increases levels of initiation factors or changes the activity of enzymes regulating initiation processes in the ribosome cycle.     

L-肉碱对NAFLD脂肪肝大鼠肝脏线粒体能量代谢及抗氧化能力的影响

研究L-肉碱对NAFLD脂肪肝大鼠肝脏线粒体能量代谢及抗氧化能力的影响.脂肪肝大鼠48只,随机分为对照组、肉碱组、运训组和肉碱+运训组.运训组和肉碱+运训组进行6周运动,肉碱组和肉碱+运训组每日灌胃一次肉碱(500 mg/kg).6周后各组大鼠进行力竭运动,即刻麻醉取出肝脏提取线粒体,测定线粒体呼吸链酶RCCI~IV活性及自由基代谢相关指标.与对照组比,肉碱组大鼠肝脏线粒体呼吸链RCCII,运训组RCCI、RCCIII,肉碱+运训组RCCI、RCCII、RCCIII活性均显著提高.与肉碱组比,肉碱+运训组RCCI、RCCIII活性显著提高.与运训组比,肉碱+运训组RCCI活性显著提高.与对照组比,肉碱组、肉碱+运训组大鼠肝脏线粒体GSH-Px活性显著提高;与对照组比,三组SOD 活性显著提高,MDA含量降低.与肉碱组比,肉碱+运训组SOD活性显著提高;与运训组比,肉碱+运训组GSH-Px活性显著提高.补充L-肉碱、运动、L-肉碱+运动均可提高非酒精性脂肪肝大鼠肝脏线粒呼吸链酶活性,提高线粒体能量代谢速率及抗氧化损伤能力,补充L-肉碱结合运动训练组效果更加显著.     

Dose- and Time-Dependent Effects of Menadione on Enzymes of Xenobiotic Metabolism in Rat Liver

We studied the effect of synthetic vitamin K analogue menadione on enzymes of xenobiotic metabolism in rat liver (total content of cytochrome P450 and catalytic activities of CYP1A1/2, CYP2B1, CYP2C, NADPH-cytochrome P450 reductase, and glutathione S-transferase). Menadione induced phase I and II enzymes for metabolism of xenobiotics, drugs, and procarcinogens. The effect of menadione depended on its dose and duration of treatment.     

Effect of Lycopine on the Resistance of Rat Liver Microsomes to In Vitro Induced LPO

Lycopine in concentrations of 0.5-50 M suppressed LPO in microsomes induced by NADPH-Fe²⁺ and by ascorbic acid-Fe²⁺. Lycopine in a concentration of 20 M completely prevented the decrease in the rate of benz[a]pyrene hydroxylation and activation of p-nitrophenyl-UDP-glucuronosyl transferase caused by LPO induction in microsomes.     

In Vitro Work Load and Rat Heart Metabolism: II. Effect on amino acid transport

In vitro pressure overload was found to accelerate protein synthesis in the isolated working rat heart (Hjalmarson and Isaksson 1972 a). Since membrane transport of amino acids is considered to be one rate-limiting step in protein synthesis, the amino acid transport in the isolated rat heart was investigated using the non-utilizable amino acids α-aminoisobutyric acid (AIB) and 1-aminocyclopentane carboxylic acid (cycloleucine). Increased pressure load (afterload) accelerated amino acid uptake after a perfusion period of 15 ruin, and a 50—100% increase in the intracellular to extracellular distribution ratio of the amino acids was seen after 60 min of perfusion. This accelerated uptake was not inhibited by cycloheximide, suggesting that the work load effect was not dependent upon a continuous synthesis of proteins. The accumulation rate continued to be stimulated for some time after normalization of the work load and coronary flow, indicating that the work load effect was not directly linked to coronary flow. Increased preload did not stimulate amino acid uptake. Hearts from hypophysectomized rats showed a decreased concentrative uptake but the amino acid uptake was still accelerated by pressure overload. It is suggested that an increased uptake of amino acids could be of physiological significance in relation to the increased protein synthesis under these conditions.     

Expression of the Major Histocompatibility Complex Antigens on Different Liver Cellular Components in Rat and Man

We hint analysed the distribution of the major histocompatibility complex (MHC) antigens on different liver cellular components in rat and man. Two types of antisera were used: monospecific rabbit antisera raised against isolated molecules of class I and class II and ordinary hyperimmune alloantisera directed to their allelic specificities. A modified Staphylococcus aureus rosette test, enabling morphological identification of the rosette-forming cell type, was used. In both species the passenger cells, especially the Kupffer cells, reacted strongly with rabbit anti-class I and II The passenger erythrocytes reacted with anti-class I in rat but not in man. Human liver vascular endothelial and bile duct cells displayed a distinct reaction with anti-class I and II, whereas the hepatocytes reacted very weakly, if at all. In rat liver parenchymal component the expression of these antigens was different and weaker. Rat bile duct cells reacted with anti-class I and II in approximately 50% of the testings, whereas rat vascular endothelial cells and rat hepatocytes were essential non-reactive. The alloantiserum reactivity (tested only in the rat) followed closely that of heterologous antisera, and the reactivity of anti-β₂m that of class I alloantisera. Although these differences are probably quantitative rather than qualitative, the results suggest considerable variation in the expression of the MHC antigens on different liver cellular components. These differences partially explain why liver allografts behave differently than, e.g., renal allografts upon transplantation.     

大鼠离体海马脑片缺氧耐受性的研究

电刺激大鼠海马脑片的Schaffer氏侧支,记录CA_1区锥体细胞层的细胞外场电位,即群锋电位(PS),观察74个脑片在缺氧后的变化。当浴槽内氮气流速为150ml/min时,PS开始减小时间与完全消失时间分别为3.60±1.01min(均值±标准差)及4.90±1.21min。缺氧10、15、18、20min后再次给氧,PS恢复率分别是85.7、64.3、22.2、17.6％,由此推算50％脑片不能恢复的缺氧时间为15.9min。以上指标可作为脑片缺氧耐受性的可比指标,文中讨论了处理双孔浴槽实验结果时应注意的问题。     

Graft-versus-Host Disease in the Rat: Cellular Changes and Major Histocompatibility Complex Antigen Expression in the Liver

Cellular changes in the liver were studied during an acute lethal graft-versus-host (GVH) disease in relation to the expression of major histocompatibility complex (MHC) antigens on different liver cells. Screening for MHC antigen expression revealed that control livers contained very few Ia+ cells: mainly cells in the portal tract interstitium and a small percentage of the Kupffer cells. The changes during an ongoing GVH reaction could be separated into those related to the sinusoid-associated cells, including the liver parenchyma, and those related to the portal-tract-associated cells, including periportal hepatocytes. In the sinusoids an increase in the number of Kupffer cells was seen, now all expressing Ia antigens. No damage to hepatocytes or other sinusoid-associated cells was observed. It is postulated that the increase in both number and Ia expression of the Kupffer cells is most probably due to an increased phagocytic uptake of blood-borne cellular debris and is not a result of extensive damage to hepatocytes. In the portal tracts expanding infiltrates were found composed of Ia+ T cells and macrophages (ratio 2:1). These infiltrates are probably due to a local accumulation of lymphocytes and macrophages as a result of an interaction of migrating donor-type alloreactive T cells with recipient type Ia+ cells present in the portal tract interstitium, which also interfered with normal recipient lymphocyte and macrophage traffic. Damage to portal-tract-associated cells was slight and confined to bile duct epithelial cells, which now expressed Ia antigens, and to periportal hepatocytes. In conclusion, these data do not indicate that damage to liver parenchyma plays a major role in the pathogenesis of an acute GVH reaction.     

The Interface Between Generating Renal Tubules and a Polyester Fleece in Comparison to the Interstitium of the Developing Kidney

An increasing number of investigations is dealing with the repair of acute and chronic renal failure by the application of stem/progenitor cells. However, accurate data concerning the cell biological mechanisms controlling the process of regeneration are scarce. For that reason new implantation techniques, advanced biomaterials and morphogens supporting regeneration of renal parenchyma are under research. Special focus is directed to structural and functional features of the interface between generating tubules and the surrounding interstitial space. The aim of the present experiments was to investigate structural features of the interstitium during generation of tubules. Stem/progenitor cells were isolated from neonatal rabbit kidney and mounted between layers of a polyester fleece to create an artificial interstitium. Perfusion culture was performed for 13 days in chemically defined Iscove’s Modified Dulbecco’s Medium containing aldosterone (1 × 10⁻⁷ M) as tubulogenic factor. Recordings of the artificial interstitium in comparison to the developing kidney were performed by morphometric analysis, scanning and transmission electron microscopy. The degree of differentiation was registered by immunohistochemistry. The data reveal that generated tubules are embedded in a complex network of fibers consisting of newly synthesized extracellular matrix proteins. Morphometric analysis further shows that the majority of tubules within the artificial interstitium develops in a surprisingly close distance between 5 and 25 μm to each other. The abundance of synthesized extracellular matrix acts obviously as a spacer keeping generated tubules in distance. For comparison, the same principle of construction is found in the developing parenchyma of the neonatal kidney. Most astonishingly, scanning electron microscopy reveals that the composition of interstitial matrix is not homogeneous but differs along a cortico-medullary axis of proceeding tubule development.