Lipid peroxidation and covalent binding in the early functional impairment of liver Golgi apparatus by carbon tetrachloride

The onset of the lipoprotein secretory block provoked by CCl4 in the whole animal was monitored after purification of liver Golgi membranes. Both lipid transit through the apparatus and hexosylation of the lipoprotein are markedly inhibited 5-15 min after poisoning. Pre-treating the animal with alpha-tocopherol, shown to prevent lipid peroxidation without modifying the covalent binding due to CCl4 metabolites, affords little protection against lipid accumulation in the Golgi, but total preservation of galactosyl transferase activity. While haloalkylation therefore appears to be the major mechanism of damage in the early phases of CCl4-induced derangement of lipid secretion, lipid peroxidation is probably more involved later; this is indicated by the marked, though never complete, protection against fatty liver afforded at 24 h after CCl4 poisoning by supplementation of the membrane with alpha-tocopherol.     

Foreword: Protein secretion and the Golgi apparatus

Resonance and nonresonance Raman spectra have been obtained from neoplastically transformed and normal avian lymphocytes. The acyl chains of membrane phospholipids of neoplastic cells are more highly unsaturated than those of normal cells. The observation of prominent carotenoid bands in both cell populations indicates the availability of a sensitive, intrinsic probe of membrane potential and local membrane environment.     

Golgi apparatus and slime secretion in plants: the early implications and recent models of membrane traffic

Summary The function of the Golgi apparatus in the secretion of plant slimes is reviewed. It is shown how the research on slime secretion has increased the knowledge on the structure and dynamics of dictyosomes. Current models on intradictyosomal membrane traffic-anterograde progression of complete cisternae or anterograde movement of lateral vesicles with stationary cisternae-are discussed in the light of old and new results on slime secreting plant cells.Dedicated to Hilton H. Mollenhauer on the occasion of his retirement     

Studies on the Golgi apparatus. Cumulative inhibition of protein and glycoprotein secretion by d-galactosamine

1. The administration of d-galactosamine leads to inhibition of protein and glycoprotein secretion by rat liver. To test the secretory function, the secretion times for galactose-and fucose-containing glycoproteins were determined; they were lengthened from 6 to 9min and from 8 to 13min respectively. 2. The Golgi apparatus was enriched 100-120-fold relative to the homogenate. A new linked-assay system for the marker enzyme, UDP-galactose-N-acetyl-d-glucosamine galactosyltransferase, is presented. The activity of the enzyme was measured spectrophotometrically by following the formation of UDP coupled to nicotinamide nucleotide reduction. The Michaelis constants were calculated to be 0.11mm for UDP-galactose with N-acetyl-d-glucosamine as exogenous acceptor and 19mm for N-acetyl-d-glucosamine itself. 3. The physiological substrate of the galactosyltransferase, UDP-galactose, can be replaced by UDP-galactosamine, which accumulates after d-galactosamine administration. Under conditions in vitro the rate of d-galactosamine transfer to an endogenous acceptor protein of the Golgi fraction reaches 9% of that with d-galactose; this finding is noteworthy, because normally a non-acetylated amino sugar does not occur in glycoproteins. 4. The albumin content of the Golgi-rich fraction was diminished to 55% of the reference value 6h after the injection of 375mg of d-galactosamine hydrochloride/kg body wt. The transfer of d-[1-(14)C]galactose to an endogenous acceptor protein fell to 60% compared with Golgi-rich fractions from untreated animals. Analysis of the Golgi-rich fraction by polyacrylamide-gel electrophoresis showed a decrease or loss of several protein bands. 5. Protein synthesis can be restored by up to 80% if the UTP pool, decreased after d-galactosamine administration, is filled up by several injections of uridine. 6. From the results presented it can be concluded that the disturbed secretion of proteins and glycoproteins was due to a cumulative effect of galactosamine by: (a) inhibition of protein synthesis leading to a diminution of the endogenous acceptor pool of the galactosyltransferase; (b) inhibition of the galactosyltransferase activity by galactosamine metabolites and (c) replacement of UDP-galactose by UDP-galactosamine.     

Characterization of lipoprotein particles isolated from the Golgi apparatus of rat liver

It has been proposed that particles within tubules and vesicles of the Golgi apparatus of liver cells are precursors of very low density lipoproteins in blood plasma. To characterize these particles we isolated a cell fraction rich in Golgi apparatus and associated particles from rat liver in quantities sufficient for analysis. Particles freed from the membranes of the Golgi apparatus and floated at d = 1.006 were studied by chemical analysis, immunodiffusion, and paper electrophoresis. The lipid composition of the Golgi particles was similar to that of very low density lipoproteins from the same rats. The protein content was about 10% of dry weight for both the Golgi particles and plasma very low density lipoproteins. The Golgi particles formed lines of identity with plasma very low density lipoproteins during immunodiffusion against antiserum to plasma very low density lipoproteins. On paper electrophoresis, however, many Golgi particles remained near the origin, with only a few migrating to the pre-beta position. It was concluded that the lipoproteins in the Golgi apparatus are the precursors of plasma very low density lipoproteins.     

The trans-tubular network of the hepatocyte Golgi apparatus is part of the secretory pathway

We have recently demonstrated the presence of sialyltransferase and sialic acid in a trans-tubular network (TTN) continuous with trans Golgi apparatus cisternae of rat liver hepatocytes. Based on these findings, we concluded that this structure, which also exhibited thiamine pyrophosphatase and acid phosphatase activity, is an integral part of the Golgi apparatus and functions in sialylation. In the present study, by comparing the distribution of a major hepatocyte secretory product with that of sialyltransferase, we sought to determine whether the TTN is also part of the secretory pathway. Examination of adjacent serial thin sections labeled for albumin showed its presence throughout the TTN and simultaneously provided new details about the structural complexity of the TTN. Double-immunolabeling with protein A-gold allowed the direct demonstration of albumin throughout the sialyltransferase containing TTN. Additional double staining protocols (combination of preembedding enzyme cytochemistry with postembedding immunolabeling) revealed the presence of albumin in both the thiamine pyrophosphatase and acid phosphatase positive regions of the TTN. These data show that albumin, a nonglycosylated secretory protein, reaches the TTN where terminal glycosylation of glycoproteins occurs. Therefore, it appears that the TTN of rat hepatocytes which functions in terminal glycosylation is also part of the constitutive secretory pathway.     

Formation of secretion granules in the Golgi apparatus of plasma cells in the rat

The three-dimensional structure of the components of the Golgi apparatus was analyzed in plasma cells of rat duodenum. The spheroidal juxtanuclear Golgi apparatus was formed by a continuous ribbonlike structure composed of the following stacked elements. On the cis-face of the Golgi stack, there was a tubular membranous network referred to as the cis-element and/or a slightly dilated saccule perforated with small pores. The two or three subjacent saccules, which showed few pores, were slightly dilated and contained a fluffy granulofilamentous material. They were also perforated in register by cavities or wells containing 80-nm vesicles. The next one or two underlying elements were fenestrated saccules showing flattened portions as well as distended portions containing a homogeneous material denser than that seen in the overlying saccules. The last two or three elements of the stack showed a partially separated or "peeling off" configuration. These last elements consisted of prosecretory granules attached to flattened, empty-looking saccules showing buds at their surface; detached, more-or-less fenestrated, flattened saccules; and shrivelled residual trans-tubular networks. In the trans-region of the stack, in addition to numerous small vesicles, short membranous tubules, detached prosecretory granules, and denser fully formed secretion granules were also seen. These images were interpreted to indicate that secretory material present in the trans-saccules flows toward the dilated portions which become prosecretory granules. The trans-most elements seemingly peel off the stack to yield prosecretory granules and fragmenting trans-tubular networks.     

The Golgi apparatus: defining the identity of Golgi membranes

The Golgi apparatus is a stack of compartments that serves as a central junction for membrane traffic, with carriers moving through the stack as well as arriving from, and departing toward, many other destinations in the cell. This requires that the different compartments in the Golgi recruit from the cytosol a distinct set of proteins to mediate accurate membrane traffic. This recruitment appears to reflect recognition of small GTPases of the Rab and Arf family, or of lipid species such as PtdIns(4)P and diacylglycerol, which provide a unique "identity" for each compartment. Recent work is starting to reveal the mechanisms by which these labile landmarks are generated in a spatially restricted manner on specific parts of the Golgi.     

Carbon tetrachloride-induced inhibition of protein kinase C in isolated rat hepatocytes

Isolated rat hepatocytes exposed to CCl4 showed a dramatic decrease in [32P] incorporation into proteins which was evident as early as 5 min after the haloalkane addition. DEAE cellulose separation of protein kinases present in both particulated and cytosolic fractions of hepatocytes revealed that only the calcium and phospholipids dependent protein kinase C was affected by the treatment with CCl4, while kinases not requiring these factors for their activity were unmodified. Several 4-hydroxyunsaturated aldehydes known to be produced during CCl4-stimulated lipid peroxidation were found to inhibit protein kinase C at micromolar concentrations, suggesting the possibility that peroxidative events might be responsible for the impairment of protein kinase C during CCl4 intoxication.     

Fragmentation of the Golgi apparatus: an early apoptotic event independent of the cytoskeleton

The Golgi apparatus undergoes irreversible fragmentation during apoptosis, in part as a result of caspase-mediated cleavage of several Golgi-associated proteins. However, Golgi structure and orientation is also regulated by the cytoskeleton and cytoskeletal changes have been implicated in inducing apoptosis. Consequently, we have analyzed the role of actin filaments and microtubules in apoptotic Golgi fragmentation. We demonstrate that in Fas receptor-activated cells, fragmentation of the Golgi apparatus was an early event that coincided with release of cytochrome c from mitochondria. Significantly, Golgi fragmentation preceded major changes in the organization of both the actin cytoskeleton and microtubules. In staurosporine-treated cells, actin filament organization was rapidly disrupted; however, the Golgi apparatus maintained its juxtanuclear localization and underwent complete fragmentation only at later times. Attempts to stabilize actin filaments with jasplakinolide prior to treatment with staurosporine did not prevent Golgi fragmentation. Finally, in response to Fas receptor activation or staurosporine treatment the levels of beta-actin or alpha-tubulin remained unaltered, whereas several Golgi proteins, p115 and golgin-160, underwent caspase-mediated cleavage. Our data demonstrate that breakdown of the Golgi apparatus is an early event during apoptosis that occurs independently of major changes to the actin and tubulin cytoskeleton.     

Formation of secretion granules in the Golgi apparatus of pancreatic acinar cells of the rat

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in sections of pancreatic acinar cells by using stereopairs of electron microscope photographs. Pancreatic tissue fixed in glutaraldehyde was postfixed in reduced osmium, and the sections were stained with lead citrate. Tissues were also treated to demonstrate phosphatase activity (i.e., nicotinamide adenine dinucleotide phosphatase, NADPase; thiamine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase). The following stacked components were observed along the branching, anastomotic, continuous, ribbonlike Golgi apparatus. 1) On the cis-face of the Golgi stack there was a tubular membranous network known to be osmiophilic and referred to as the cis-osmiophilic tubular network or cis-element. 2) A first, poorly fenestrated saccule, unreactive for the phosphatases tested, was slightly distended in places and contained a fluffy granulofilamentous material. 3) The subjacent three or four saccules, reactive for NADPase and/or TPPase, showed dilated portions containing a granulofilamentous secretory material similar to that filling the rest of the saccule. They also showed nondilated portions perforated with large fenestrations, some of which were in register and formed wells containing 80-nm vesicles. The dilated portions of these saccules were present at random along the length of the saccules and were not located exclusively at their edges. 4) The remaining one or two elements of the stack, CMPase positive, showed dilated spheroidal portions or prosecretory granules containing a homogeneous secretory material and flattened fenestrated regions free of secretory material and having the appearance of networks of narrow membranous tubules. 5) Lastly on the trans-aspect of the stack there were detached prosecretory granules reactive for CMPase and surrounded by a corona of small vesicles, and smooth-surfaced spherical CMPase-negative granules having a denser content that were identified as fully formed secretion granules; there were also occasional free trans-tubular networks strongly reactive for CMPase that appeared to undergo fragmentation and numerous small vesicles free from acid-phosphatase activity. These various images were interpreted as indicating that prosecretory granules formed in relation to two or three fenestrated saccules on the trans-side of the stack. Such granules, following their detachment from the trans-face of the stack, their separation from trans-tubular networks, and condensation of their content, yielded mature secretion granules.     

Carbon tetrachloride-induced liver cell injury in the Mongolian gerbil (Meriones unguiculatus)

1. Male Mongolian gerbils (Meriones unguiculatus) liver activates CCl4 to free radicals that bind covalently to cellular components (CB) and stimulate a lipid peroxidation (LP) process to a larger extent than the rat liver. 2. CCl4 administration results in a less intense necrogenic effect in gerbils than in rats and does not cause fatty liver. 3. CCl4 causes less intense effects on liver ultrastructure or calcium metabolism but more marked depression of glucose 6 phosphatase activity (G6P-ase) in gerbils than in rats. 4. Results suggest that a better ability of gerbil liver to keep calcium homeostasis than rat liver might be the cause of their relative resistance to necrosis. Higher intensity of CB and LP in gerbils than in rats might explain more intense effects on G6P-ase.     

Analysis of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-rich mucilage

Differentiation of the Arabidopsis thaliana seed coat cells includes a secretory phase where large amounts of pectinaceous mucilage are deposited to a specific domain of the cell wall. During this phase, Golgi stacks had cisternae with swollen margins and trans-Golgi networks consisting of interconnected vesicular clusters. The proportion of Golgi stacks producing mucilage was determined by immunogold labeling and transmission electron microscopy using an antimucilage antibody, CCRC-M36. The large percentage of stacks found to contain mucilage supports a model where all Golgi stacks produce mucilage synchronously, rather than having a subset of specialist Golgi producing pectin product. Initiation of mucilage biosynthesis was also correlated with an increase in the number of Golgi stacks per cell. Interestingly, though the morphology of individual Golgi stacks was dependent on the volume of mucilage produced, the number was not, suggesting that proliferation of Golgi stacks is developmentally programmed. Mapping the position of mucilage-producing Golgi stacks within developing seed coat cells and live-cell imaging of cells labeled with a trans-Golgi marker showed that stacks were randomly distributed throughout the cytoplasm rather than clustered at the site of secretion. These data indicate that the destination of cargo has little effect on the location of the Golgi stack within the cell.     

Maturation of the Golgi apparatus in urothelial cells

The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane formation. We have studied the distribution and organization of the GA in normal mouse urothelial cells and in the superficial urothelial cells that undergo differentiation following cyclophosphamide-induced regeneration, in correlation with urothelial cell differentiation. In normal urothelium, immature basal cells have a simple GA, which is small and distributed close to the nucleus. In intermediate cells, the GA starts to expand into the cytoplasm, whereas the GA of terminally differentiated umbrella cells is complex, being large and spread over the whole basal half of the cytoplasm. During early stages of regeneration after cyclophosphamide treatment, the GA of superficial cells is simple and no markers of urothelial differentiation (uroplakins or asymmetric unit membranes, discoidal or fusiform vesicles, apical surface covered with microvilli) are expressed. At a later stage, the GA expands and, in the final stage of regeneration, when cells express all markers of terminal urothelial differentiation, the GA become complex once again. Our results show that: (1) GA distribution and organization in urothelial cells is differentiation-dependent; (2) the GA matures from a simple form in partially differentiated cells to a complex form in terminally differentiated superficial cells; (3) major rearrangements of GA distribution and organization correlate with the beginning of asymmetric unit membrane production. Thus, GA maturation seems to be crucial for asymmetric unit membrane formation. The work was supported by the Ministry of Education and Sport, Government of Republic of Slovenia, Slovenia (grant no. 3311-04-831450).