Relationship between microtubules and Golgi apparatus in hepatocytes: a quantitative study during experimental nephrosis

In many cell types, microtubules are preferentially associated with the Golgi apparatus. However, the existence of a functional link between these two organelles is still hypothetical. To gain insight into this question, the relationships between microtubules and the Golgi apparatus were studied in rat hepatocytes during experimental nephrosis induced by the aminonucleoside of puromycin. This condition is known to cause prolonged stimulation of plasma protein production by the hepatocytes. Rats were studied 2, 4, 5, 10 and 20 days after aminonucleoside injection. The amount of albumin was measured in serum and hepatic microsomes by laser immunonephelometry. The volume densities of microtubules around the Golgi apparatus and in the remaining cytoplasm were measured by ultrastructural morphometry. Changes of the Golgi apparatus were analysed by measuring the volume density of the whole organelle and the respective proportion of saccules and vesicles. Proteinuria began 5 days after aminonucleoside injection and was accompanied by a decrease in serum albumin and a rise in microsomal albumin. These changes were still more striking after 10 days, but protein and albumin levels were almost back to normal after 20 days. Concomitantly, the volume density of the microtubules increased significantly around the Golgi apparatus (32% after 10 days), and not in the remaining cytoplasm. The Golgi apparatus was enlarged (80% after 10 days) with a higher ratio of secretory vesicle to saccule volume densities. These results show that additional microtubules are present around the Golgi apparatus during the enhanced production of plasma proteins which occurs in nephrosis. They suggest that in hepatocytes, microtubules play a part in the Golgi apparatus function of plasma protein processing.     

Immunohistochemical localization of albumin and in situ hybridization of albumin mRNA

Hepatocytes actively involved in albumin synthesis were identified by immunohistochemical method. In sections of perioidate-lysine-2 per cent (w/v) paraformaldehyde fixed normal rat liver, albumin was detected in all hepatocytes. At the ultrastructural level, albumin was localized in the rough endoplasmic reticulum and in Golgi complexes located near the nucleus in only a small subpopulation of hepatocytes, while all other hepatocytes contained albumin only in Golgi complexes located near the bile canaliculi. Stimulation of albumin synthesis by puromycin aminonucleoside-induced nephrosis resulted in an altered intracellular distribution of albumin at the light microscopic level. When examined at the ultrastructural level, albumin was localized in the rough endoplasmic reticulum as well as in Golgi complexes located near the nucleus in nearly all these hepatocytes. Hepatocytes with the potential to synthesize albumin were identified by in situ hybridization of albumin mRNA. In sections of 0.1 per cent (v/v) glutaraldehyde perfusion fixed normal rat liver, albumin mRNA was detected in the cytoplasm of only a few hepatocytes scattered throughout the lobule. Following stimulation of albumin synthesis by the induction of nephrosis, albumin mRNA was detected in the cytoplasm of the hepatocytes. The source of albumin in those hepatocytes which lacked albumin mRNA was identified in analbuminemic rats injected with rat albumin. At 6 h post injection, the light microscopic distribution of albumin in the liver of these animals was virtually indistinguishable from that in normal rat liver. At the ultrastructural level, injected albumin was localized in lysosomes and in Golgi complexes located near the bile canaliculi.     

Evidence that microtubules play a permissive role in hepatocyte very low density lipoprotein secretion

To determine whether a minimum number of assembled microtubules is required for very low density lipoprotein (VLDL) triglyceride TG) secretion in hepatocytes, antimicrotubule drugs of different concentrations were given to rats. Hepatic VLDL-TG release was subsequently measured by a liver perfusion system, and hepatocyte ultrastructural changes were analyzed by quantitative ultrastructural methods. The results demonstrate a tight coupling between the reduction in hepatocyte microtubule content and the reduction in hepatic VLDL-TG secretion which is related to the dose of colchicine or vinblastine administered. The various estimates imply that a minimum number of microtubules is necessary for hepatic VLDL secretion to proceed normally and that hepatic VLDL secretion rates reach their nadir (10-- 30% of control) when microtubules comprise less than 0.005% of the cytoplasm (or less than 10% of control values) when microtubules comprise less than 0.005% of the cytoplasm (or less than 10% of control values). At this point, hepatocyte Golgi complexes are also greatly altered; Golgi complexes with recognizable dictyosomal membranes are reduced to 15% of control values and the region is filled with large numbers of electron-dense bodies which appear to be lysosomes in the process of digesting VLDL. There is a predilection for the remaining Golgi complexes to be associated with a few segments of microtubules, even when no microtubules can be measured in random samplings of hepatocytes. Clusters of vacuoles containing VLDL are also present throughout the cytoplasm; the limiting membranes of 25% of these vacuoles are studded with ribosomes. These findings demonstrate that the administration of antimicrotubule agents results in decreases in hepatic VLDL-TG secretion which are associated with loss of microtubules and alteration of existing Golgi complexes.     

Development of the fetal rat liver: ultrastructural and stereological study of hepatocytes

Qualitative and quantitative changes in the liver tissue composition have been studied during prenatal development of the Wistar rat by electron microscopy and stereologic methods. The absolute volume of the fetal liver is multiplied by 84 between days 13 and 20 of gestation. In the meantime, the average hepatocyte volume is multiplied by 1.5 between days 12 and 20. The volumetric fraction of hepatocytes increases from 35% of the volumetric fraction of the liver on day 12 to 66% on day 20 of gestation. The non-hepatocyte cells decrease from 49% on day 12 to 25% on day 20. By days 12 and 13, the rough endoplasmic reticulum and the Golgi apparatus are well differentiated, indicating that young fetal hepatocytes are able to synthesize and export plasma proteins. The volumetric fraction of free ribosomes decreases from 38% of the hepatocytic cytoplasm on days 12 and 13 to 6% on day 20. The mitochondrial compartment occupies about 10% of the hepatocyte cytoplasm. The mitochondria, small and round on days 12, 13 and 14, become oblong from day 18 of gestation. The shape of hepatocytes changes during the prenatal development, from potato-like on days 12, 13, 14 to cubic on day 20, with an intermediate, more spheric, stage on day 18.     

Effect of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells. Immunocytochemical observations

We studied the effects of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells using the direct immunoenzyme technique. Livers were perfusion-fixed 0.5, 1, and 2 h after injection of colchicine. Vibratome sections of the fixed liver were stained using peroxidase-conjugated Fab' of anti-albumin or anti-fibrinogen. By light microscopy, reaction deposits showing albumin and fibrinogen were observed in the cytoplasmic granules of hepatocytes. Such stained granules decreased 30 min after injection, but later increased gradually and crowded in the cytoplasm. The Golgi complex stained for the proteins decreased after 30 min but increased in the juxtanuclear region after 60 min. The analysis of serial sections showed that colchicine severely disturbed the spatial relationship between the Golgi apparatus and the bile canaliculus. We obtained similar results by electron microscopy; a positive reaction for albumin and fibrinogen was observed in a small number of the cytoplasmic granules after 30 min. After 1 h of treatment, most of the Golgi complexes were fragmented and lost their stacked cisternae. However, they reappeared accompanied with vacuolated cisternae and secretory granules, which were partially stained for albumin and fibrinogen. After 2 h, the secretory granules positive for both proteins accumulated further. Some of them lined a long the plasma membrane, and others made a cluster in the cytoplasm. The profiles showing exocytosis were very rarely seen. These results showed that in the first 30 min, colchicine primarily disturbs partially the Golgi assembly but does not affect the post Golgi secretory pathway much. Later, the drug affects both the post Golgi pathway and the Golgi assembly, and it causes a marked accumulation of secretory granules.     

Ultrastructural location of albumin and fibrinogen in primary cultures of new-born rat liver tissue

In primary cultures of new-born rat liver tissue, albumin and frbrinogen, two proteins normally synthesized by the liver and secreted into plasma were demonstrated by specific antibodies labelled with peroxidase in about 50 and 70% of the hepatocytes; these proteins were not demonstrated in the other types of cells, in particular fibroblasts, present in primary cultures. These two proteins were detected on the ribosomes of the rough endoplasmic reticulum and were also present in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus. It is concluded that

1. 1. In primary cultures of liver tissue, only the hepatocytes synthesize albumin and fibrinogen.

2. 2. Proliferating cultured hepatocytes are able to synthesize albumin and fibrinogen.

3. 3. The presence of detectable albumin and fibrinogen in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in hepatocytes of primary cultures and their absence in the lumina of the rough and smooth endoplasmic reticulum and in the Golgi apparatus in the hepatocytes of adult rat liver might indicate an alteration in the translocation of albumin and fibrinogen through these organelles in cultured hepatocytes.

     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: III. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucose injection

In the first paper of this series (Bennett et al., 1984), light-microscope radioautographic studies showed that colchicine or vinblastine inhibited intracellular migration of glycoproteins out of the Golgi region in a variety of cell types. In the present work, the effects of these drugs on migration of membrane glycoproteins have been examined at the ultrastructural level in duodenal villous columnar cells and hepatocytes. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In duodenal villous columnar cells, 3H-fucose labeling of the apical plasma membrane was reduced by 51% after colchicine and by 67% after vinblastine treatment; but there was little change in labeling of the lateral plasma membrane. Labeling of the Golgi apparatus increased. This suggests that labeled glycoproteins destined for the apical plasma membrane were inhibited from leaving the Golgi region, while migration to the lateral plasma membrane was not impaired. In hepatocytes, labeling of the sinusoidal plasma membrane was reduced by 83% after colchicine and by 85% after vinblastine treatment. Labeling of the lateral plasma membrane also decreased, although not so dramatically. Labeling of the Golgi apparatus and neighboring secretory vesicles increased. This indicates that the drugs inhibited migration of membrane glycoproteins from the Golgi region to the various portions of the plasma membrane. Accumulation of secretory vesicles at the sinusoidal front suggests that exocytosis may also have been partially inhibited. In both cell types, microtubules almost completely disappeared after drug treatment. Microtubules may, therefore, be necessary for intracellular transport of membrane glycoproteins, although the possibility of a direct action of these drugs on Golgi or plasma membranes must also be considered.     

Cytoplasmic organization and quantitation of microtubules in bovine mammary epithelial cells during lactation and involution

Summary Ultrastructural examination of milk secretory cells from lactating bovine mammary gland revealed presence of numerous microtubules in the apical and paranuclear cytoplasm, particularly in the vicinity of Golgi components. Most microtubules were oriented perpendicular to the apical plasma membrane and appeared to form a framework around Golgi dictyosomal elements and secretory vesicles. In comparison, non-secretory cells obtained from involuting glands displayed few microtubules and these were randomly located throughout the cytoplasm with no particular orientation.     

CAMSAP3-dependent microtubule dynamics regulates Golgi assembly in epithelial cells

The Golgi assembly pattern varies among cell types. In fibroblast cells, the Golgi apparatus concentrates around the centrosome that radiates microtubules; whereas in epithelial cells, whose microtubules are mainly noncentrosomal, the Golgi apparatus accumulates around the nucleus independently of centrosome. Little is known about the mechanisms behind such cell type-specific Golgi and microtubule organization. Here, we show that the microtubule minus-end binding protein Nezha/CAMSAP3 (calmodulin-regulated spectrin-associated protein 3) plays a role in translocation of Golgi vesicles in epithelial cells. This function of CAMSAP3 is supported by CG-NAP (centrosome and Golgi localized PKN-associated protein) through their binding. Depletion of either one of these proteins similarly induces fragmentation of Golgi membranes. Furthermore, we find that stathmin-dependent microtubule dynamics is graded along the radial axis of cells with highest activity at the perinuclear region, and inhibition of this gradient disrupts perinuclear distribution of the Golgi apparatus. We propose that the assembly of the Golgi apparatus in epithelial cells is induced by a multi-step process, which includes CAMSAP3-dependent Golgi vesicle clustering and graded microtubule dynamics.     

Cyclic localization change of Golgi apparatus in Sertoli cells induced by mature spermatids in rats

Previously we reported that the intracellular localization of the Golgi apparatus of rat Sertoli cells changes during the seminiferous epithelial cycle, and that the cyclic changes seem to be correlated to specific generations of germ cells. To ascertain which generations of germ cells are responsible for the cyclic changes, we determined the relative volume of the Golgi apparatus within the basal, mid, and apical cytoplasm of Sertoli cells in testes with and without mature spermatids. In normal adult rats, the Golgi apparatus was usually localized exclusively in the basal cytoplasm, whereas at stages VII-IX it increased remarkably in mid and apical cytoplasm, with a concomitant decrease in the basal cytoplasm. In young adult testes without spermatids at steps 15-19 of spermiogenesis (2nd layer spermatids), the Golgi apparatus was localized in the basal cytoplasm throughout the seminiferous epithelial cycle. Orchiopexy maintained for 35 days following 60 days of cryptorchidism allowed germ cells to regenerate to spermatids at steps 1-14 of sperminogenesis (1st layer spermatids), but failed to change the intracellular localization of the Golgi apparatus in Sertoli cells. At 50 days after orchiopexy, when all generations of germ cells appeared in the tubules, the cyclic changes in localization of the Golgi apparatus were restored similar to those in normal adult testes. These findings indicate that the cyclic change in localization of the Golgi apparatus in Sertoli cells is evoked by the presence of 2nd layer spermatids.     

The role of microtubules in the maintenance of regular localization and arrangement of Golgi apparatus in root cells of Triticum aestivum L.

In plant cells Golgi apparatus organization, maintenance and distribution differ from that in mammalian cells and the mechanisms for this are not clearly understood. Here we investigate the role of microtubules in the positioning and arrangement of Golgi apparatus in the root cells of Triticum aestivum L. by using dual immunofluorescent labeling and laser confocal microscopy to localize both throughout the cell cycle. We observed that Golgi stacks (i) in interphase cells predominantly occupied the perinuclear region, (ii) during mitosis they redistributed to the spindle periphery and/or areas above spindle poles, and (iii) in telophase accumulated around the phragmoplast and the chromosomes/nuclei of daughter cells. Inhibition of microtubule assembly by colchicine resulted in aggregation of Golgi in the cortical cytoplasm of interphase cells and accumulation around the chromosomes in C-mitotic cells, in stark contrast with the distribution in untreated cells. Electron microscopy revealed that in colchicine treated cells many Golgi units became disorganized, yet others were abnormally enlarged. Overall, our results indicate that in plant cells microtubules play a key role in restricting the position and maintaining the arrangement and structural integrity of the Golgi apparatus.     

Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface- expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double- immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.     

Enzyme synthesis and potential during induction synchrony in the fission yeast Schizosaccharomyces pombe

The fine structure of L cells is described at 30 min and 24 h after enucleation by centrifugation in cytochalasin B. The morphology of the 30-min enucleates is the same as that of the cytoplasm of nucleated cells. Centrioles, a normal Golgi apparatus, endoplasmic reticulum, mitochondria, microtubules, and some microfilaments, are present in enucleates. At 24 h after enucleation, the enucleates are extensively vacuolated. The cisternae of the Golgi apparatus are extremely dilated, and the granular ER is sometimes dilated. Microtubules, and, in particular, microfilaments, are still abundant. Nuclei removed from cells by enucleation in cytochalasin B are surrounded by a thin shell of cytoplasm containing numerous ribosomes, an occasional mitochondrion, a few pieces of endoplasmic reticulum, and an enclosing plasma membrane. Continuities between the nuclear envelope and the ER are particularly frequent. These nuclei possesses a normal fine structure.     

Dynein supports motility of endoplasmic reticulum in the fungus Ustilago maydis

The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process. However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi. Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis. An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope. Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility. Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein. In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus. Both the gradient and the Golgi apparatus were sensitive to brefeldin A or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi. Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER in U. maydis.     

The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate

The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc) and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vesicle traffic (i.e., microtubules) might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24 hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72 h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells.     

Protein synthesis in the livers of aging mice studied by electron microscopic radioautography.

The application of 3H-leucine results in labeling of the liver cells of mice in which protein is synthesized at various ages of the animals. Quantitative changes of protein synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains in the hepatocytes were mainly located over the rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, cytoplasmic matrix, and a few over the nuclei. The number of silver grains in the cytoplasm and nuclei of the hepatocytes gradually increased after birth, reached the maximum at 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the hepatocyte cytoplasm was more than that in nuclei at various ages. The number of silver grains in the rough surfaced endoplasmic reticulum and mitochondria gradually increased from embryo to 1 month after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the Golgi apparatus showed almost no change from fetal stage to 6 months after birth, thereafter it continued to decrease with aging until the 24th month. The number of silver grains in the cytoplasmic matrix gradually increased from fetal stage to 2 months after birth, then decreased with aging until the 24th month. These changes reflect the quantity of protein synthesized in each cell organelle at various ages of animals.     

Ultrastructural changes in the liver of the sand lamprey,Lampetra reissneri,during sexual maturation

Ultrastructural changes of hepatocytes were examined in the sand lamprey,Lampetra reissneri, during various phases of the life cycle. In hepatocytes of ammocoetes, the rough endoplasmic reticulum was composed of short cisternae and the Golgi apparatus were scarcely developed, showing no sexual differences at this stage of life cycle. In hepatocytes of female lampreys at the metamorphic stages 4 to 5, the rough endoplasmic reticulum was developed to form long parallel cisternae and the Golgi apparatus were well-developed. The rough endoplasmic reticulum developed further to form stacks of long parallel cisternae extending over the cytoplasm in hepatocytes of females at the young adult stage, and became composed of both long parallel and vesicular cisternae in the cells of females at the adult stage. The Golgi apparatus were invariably welldeveloped in hepatocytes of young adult and adult females. No consipcuous development was observed in profiles of the rough endoplasmic reticulum and the Golgi apparatus in hepatocytes of males during and after metamorphosis. The ultrastructural changes of the rough endoplasmic reticulum and the Golgi apparatus observed in hepatocytes of female sand lampreys are considered to have an intimate relation to the activity of vitellogenin synthesis in the liver, and it is suggested that the hepatocytes begin to rapidly synthesize vitellogenin in the sand lamprey at the metamorphic stages 4 to 5.     

ORP10, a cholesterol binding protein associated with microtubules, regulates apolipoprotein B-100 secretion

ORP10/OSBPL10 is a member of the oxysterol-binding protein family, and genetic variation in OSBPL10 is associated with dyslipidemias and peripheral artery disease. In this study we investigated the ligand binding properties of ORP10 in vitro as well as its localization and function in human HuH7 hepatocytes. The pleckstrin homology (PH) domain of ORP10 selectively interacts with phosphatidylinositol-4-phosphate, while the C-terminal ligand binding domain binds cholesterol and several acidic phospholipids. Full-length ORP10 decorates microtubules (MT), while the ORP10 N-terminal fragment (aa 1-318) localizes at Golgi membranes. Removal of the C-terminal aa 712-764 of ORP10 containing a predicted coiled-coil segment abolishes the MT association, but allows partial Golgi targeting. A PH domain-GFP fusion protein is distributed mainly in the cytosol and the plasma membrane, indicating that the Golgi affinity of ORP10 involves other determinants in addition to the PH domain. HuH7 cells expressing ORP10-specific shRNA display increased accumulation of apolipoprotein B-100 (apoB-100), but not of albumin, in culture medium, and contain reduced levels of intracellular apoB-100. Pulse-chase analysis of cellular [(35)S]apoB-100 demonstrates enhanced apoB-100 secretion by cells expressing ORP10-specific shRNA. The apoB-100 secretion phenotype is replicated in HepG2 cells transduced with the ORP10 shRNA lentiviruses. As a conclusion, the present study dissects the determinants of ORP10 association with MT and the Golgi complex and provides evidence for a specific role of this protein in β-lipoprotein secretion by human hepatocytes.     

Change of serum L-tryptophan levels following the development and recovery of acute puromycin aminonucleoside nephrosis in rats

Summary It is known that total L-tryptophan (Trp) levels decrease with a decrease in albumin-bound Trp levels and an increase in free Trp levels in the plasma or serum of nephrotic children. We, therefore, examined the change of serum Trp levels following the development and recovery of acute nephrosis in 6-week-old male Wistar rats injected once with puromycin aminonucleoside (100mg/kg body weight) and checked the levels of 16 amino acids including Trp in the serum and the levels of Trp in the liver, kidney, and urine under nephrotic conditions. In this study, the development and recovery of nephrosis were checked by the changes of levels of urinary protein and serum protein and albumin. Total serum Trp and albumin-bound serum Trp levels decreased with the development of nephrosis and these decreased levels returned to the normal level with its recovery. In contrast, free serum Trp levels increased with the development of nephrosis and this increased level returned to the normal level with its recovery. In the serum of nephrotic rats, the decrease of albumin-bound Trp levels and the increase of free Trp levels were well consistent with a decrease in albumin levels and an increase in the level of non-esterified fatty acids which are known to weaken the binding of Trp to albumin and among 16 amino acids studied, only Trp showed a significant change in its levels. Trp levels increased in the liver and kidney but not in the urine under nephrotic conditions. These results indicate that the change of serum Trp levels should be closely related to the condition of nephrosis and that although serum Trp is lost under nephrotic conditions, the lost serum Trp is accumulated in the liver and kidney.     

Morphological effects of somatostatin on rat somatotrophs previously activated by growth hormone-releasing factor

Summary Correlative morphological and physiological analysis was carried out in order to clarify the role of somatostatin in the inhibition of the secretion of growth hormone (GH) from somatotrophs of the rat anterior pituitary gland in vivo. Transmission electron microscopy combined with immunogold labelling showed an increased number of exocytotic GH-containing secretory granules in somatotrophs fixed between 2 and 10 min after injection of GH-releasing factor (GRF). Injection of GRF also induced the appearance of immunopositive material in cisternae of the Golgi apparatus, many coated vesicles and multivesicular bodies. Microtubules were observed more frequently throughout the cytoplasm, particularly in and near the Golgi region. At 2 and 10 min after injection of somatostatin (SRIF), both the number of exocytotic figures in the somatotrophs previously stimulated by GRF and the amount of radioimmunoassayable GH in the plasma were clearly decreased. Undulation of the plasma membrane (PM) induced by GRF rapidly disappeared, and the number of granules just beneath the plasma membrane was significantly reduced. After injection of SRIF, parallel bundles of microfilaments were often observed in the space between the granules and the plasma membrane. SRIF did not cause a noticeable decrease in the amount of immunopositive material, coated vesicles and multivesicular bodies in the Golgi areas or any significant changes in the distribution of microtubules. SRIF therefore appears to inhibit hormone release mainly at the level of the plasma membrane, probably through changes in the distribution of microfilaments.